effect of temperature on enzyme activity experiment catalase

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings

Preview improvements coming to the PMC website in October 2024. Learn More or Try it out now .

• Advanced Search
• Journal List
• v.402(Pt 2); 2007 Mar 1

The dependence of enzyme activity on temperature: determination and validation of parameters

Michelle e. peterson.

*Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, 3240, New Zealand

Roy M. Daniel

Michael j. danson.

†Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.

Robert Eisenthal

‡Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by Δ G Dagger cat , and irreversible inactivation defined by Δ G Dagger inact . However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E inact ) that is in reversible equilibrium with the active form (E act ); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, Δ H eq , and a new thermal parameter, T eq , which is the temperature at which the concentrations of E act and E inact are equal; T eq may therefore be regarded as the thermal equivalent of K m . Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T eq and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

INTRODUCTION

The effect of temperature on enzyme activity has been described by two well-established thermal parameters: the Arrhenius activation energy, which describes the effect of temperature on the catalytic rate constant, k cat , and thermal stability, which describes the effect of temperature on the thermal inactivation rate constant, k inact . Anomalies arising from this description have been resolved by the development [ 1 ] and validation [ 2 ] of a new model (the Equilibrium Model) that more completely describes the effect of temperature on enzyme activity by including an additional mechanism by which enzyme activity decreases as the temperature is raised. In this model, the active form of the enzyme (E act ) is in reversible equilibrium with an inactive (but not denatured) form (E inact ), and it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state (X):

Figure 1 shows the most obvious graphical effect of the Model, which is a temperature optimum ( T opt ) at zero time ( Figures 1 A and ​ and1B), 1 B), matching experimental observations [ 2 ]. In contrast, the ‘Classical Model’, which assumes a simple two-state equilibrium between an active and a thermally-denatured state (E act →X), and can be described in terms of only two parameters (the Arrhenius activation energy and the thermal stability), shows that when the data are plotted in three dimensions there is no T opt at zero time ( Figure 1 C).

( A ) Experimental data for alkaline phosphatase. The enzyme was assayed as described by Peterson et al. [ 2 ], and the data were smoothed as described here in the Experimental section; the data are plotted as rate (μM·s −1 ) against temperature (K) against time during assay (s). ( B ) The result of fitting the experimental data for alkaline phosphatase to the Equilibrium Model. Parameter values derived from this fitting are: Δ G ‡ cat , 57 kJ·mol −1 ; Δ G ‡ inact , 97 kJ·mol −1 ; Δ H eq , 86 kJ·mol −1 ; T eq , 333 K [ 2 ]. ( C ) The result of running a simulation of the Classical Model using the values of Δ G ‡ cat and Δ G ‡ inact derived from the fitting described above. The experimental data itself cannot be fitted to the Classical Model.

In addition to the obvious differences in the graphs representing the two models, it has been observed experimentally that at any temperature above the maximum enzyme activity, the loss of activity attributable to the shift in the E act /E inact equilibrium is very fast (<1 s) relative to the loss of activity due to thermal denaturation (shown in Figure 1 by the lines of rate against time) [ 2 ]. This and other evidence to date [ 2 ] suggest that the phenomenon described by the model (i.e. the E act /E inact equilibrium) arises from localized conformational changes rather than global changes in structure. However, the extent of the conformational change, and the extent to which it could be described as a partial unfolding, is not yet established.

The equilibrium between the active and inactive forms of the enzyme can be characterized in terms of the enthalpy of the equilibrium, Δ H eq , and a new thermal parameter, T eq , which is the temperature at which the concentrations of E act and E inact are equal; T eq can therefore be regarded as the thermal equivalent of K m . T eq has both fundamental and technological significance. It has important implications for our understanding of the effect of temperature on enzyme reactions within the cell and of enzyme evolution in response to temperature, and will possibly be a better expression of the effect of environmental temperature on the evolution of the enzyme than thermal stability. T eq thus provides an important new parameter for matching an enzyme's properties to its cellular and environmental function. T eq must also be considered in engineering enzymes for biotechnological applications at high temperatures [ 3 ]. Enzyme engineering is frequently directed at stabilizing enzymes against denaturation; however, raising thermal stability may not enhance high temperature activity if T eq remains unchanged.

The detection of the reversible enzyme inactivation, which forms the basis of the Equilibrium Model, requires careful acquisition and processing of assay data due to the number of conflicting influences that arise when increasing the temperature of an enzyme assay. Determination of T eq to date has used continuous assays, because this method produces progress curves directly and obviates the need to perform separate activity and stability experiments, and has utilized enzymes whose reactions are essentially irreversible (far from reaction equilibrium), do not show any substrate or product inhibition and remain saturated with substrate throughout the assay. However, there are a large number of enzymes that do not fit these criteria, narrowing the potential utility of determining T eq . The present paper describes methods for the reliable determination of T eq under ideal or non-ideal enzyme reaction conditions, using either continuous or discontinuous assays, and outlines the assay data required for accurate determination of T eq and the thermodynamic constants (Δ G ‡ cat , Δ G ‡ inact and Δ H eq ) associated with the model [ 2 ]; it also introduces a method of fitting progress curves directly to the Equilibrium Model and determines the robustness of the data-fitting procedures. The results show directly how the Equilibrium Model parameters are affected by the data.

The methods described in the present paper allow the determination of the new parameters Δ H eq and T eq , required for any description of the way in which temperature affects enzyme activity. In addition, they facilitate the straightforward and simultaneous determination of Δ G ‡ cat and Δ G ‡ inact under relatively physiological conditions. They therefore have the potential to be of considerable value in the pure and applied study of enzymes.

EXPERIMENTAL

Aryl-acylamidase (aryl-acylamide amidohydrolyase; EC 3.5.1.13) from Pseudomonas fluorescens , β-lactamase (β-lactamhydrolase; EC 3.5.2.6) from Bacillus cereus and p NPP ( p -nitrophenylphosphate) were purchased from Sigma–Aldrich. p NAA ( p -nitroacetanilide) was obtained from Merck, wheat germ acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum); EC 3.1.3.2] from Serva Electrophoresis and nitrocefin from Oxoid. All other chemicals used were of analytical grade.

Instrumentation

All enzymic activities were measured using a Thermospectronic™ Helios γ-spectrophotometer equipped with a Thermospectronic™ single-cell Peltier-effect cuvette holder. This system was networked to a computer installed with Vision32™ (version 1.25, Unicam) software including the Vision Enhanced Rate program capable of recording absorbance changes over time intervals down to 0.125 s.

Temperature control

The temperature of each assay was recorded directly, using a Cole-Parmer Digi-Sense® thermocouple thermometer accurate to ±0.1% of the reading and calibrated using a Cole–Parmer NIST (National Institute of Standards and Technology)-traceable high-resolution glass thermometer. The temperature probe was placed inside the cuvette adjacent to the light path during temperature equilibration before the initiation of the reaction and again immediately after completion of each enzyme reaction. Measurements of temperature were also taken at the top and bottom of the cuvette to check for temperature gradients. Where the temperature measured before and after the reaction differed by more than 0.1 °C, the reaction was repeated.

Assay conditions

Assays at high temperature (and over any wide temperature range) can sometimes pose special problems and may need additional care [ 4 – 6 ]. Quartz cuvettes were used in all experiments for their relatively quick temperature equilibration and heat-retaining capacity. Where required, a plastic cap was fitted to the cuvette to prevent loss of solvent due to evaporation (at higher temperatures), or a constant stream of a dry inert gas (e.g. nitrogen) was blown across the cuvette to prevent condensation at temperatures below ambient. Buffers were adjusted to the appropriate pH value at the assay temperature, using a combination electrode calibrated at this temperature. Where very low concentrations of enzyme were used, salts or low concentrations of non-ionic detergents were added to prevent loss of protein to the walls of the cuvette.

Substrate concentrations were maintained at not less than 10 times the K m to ensure that the enzyme remained saturated with substrate for the assay duration. Where these concentrations could not be maintained (e.g. because of substrate solubility), tests were conducted to confirm that there was no decrease in rate over the assay period arising from substrate depletion. In addition, K m values over the full temperature range examined were determined. Since K m values for enzymes tend to rise with temperature [ 7 , 8 ], in some cases dramatically, this is particularly important. Any decrease in rate at higher temperatures that is caused by an increase in K m at higher temperatures is a potential source of large errors.

Assay reactions were initiated by the rapid addition of a few microlitres of chilled enzyme, so that the addition had no significant effect on the temperature of the solution inside the cuvette.

Enzyme assays

Aryl-acylamidase activity was measured by following the increase in absorbance at 382 nm (ϵ 382 =18.4 mM −1 ·cm −1 ) corresponding to the release of p -nitroaniline from the p NAA substrate [ 9 ]. Reaction mixtures contained 0.1 M Tris/HCl, pH 8.6, 0.75 mM p NAA and 0.003 units of enzyme. One unit is defined as the amount of enzyme required to catalyse the hydrolysis of 1 μmol of p NAA per min at 37 °C.

Acid phosphatase activity was measured discontinuously using p NPP as substrate [ 10 ]. Reaction mixtures (1 ml) contained 0.1 M sodium acetate, pH 5.0, 10 mM p NPP and 8 μ-units of enzyme. The assay was stopped using 0.5 ml of 1 M NaOH. The amount of p -nitrophenol released was measured at 410 nm (ϵ 410 =18.4 mM −1 ·cm −1 ). One unit is defined as the amount of enzyme that hydrolyses 1 μmol of p NPP to p -nitrophenol per min at 37 °C.

β-Lactamase activity was measured by following the increase in absorbance at 485 nm (ϵ 485 =20.5 mM −1 ·cm −1 ) associated with the hydrolysis of the β-lactam ring of nitrocefin [ 11 ]. Reaction mixtures contained 0.05 M sodium phosphate, pH 7.0, 1 mM EDTA, 0.1 mM nitrocefin and 0.003 units of enzyme. One unit is defined as the amount of enzyme that will hydrolyse the β-lactam ring of 1 μmol of cephalosporin per min at 25 °C.

Protein determination

Protein concentrations claimed by the manufacturers (determined by Biuret) were checked using the far-UV method of Scopes [ 12 ].

Data capture and analysis

For each enzyme, reaction-progress curves at a variety of temperatures were collected; the time interval was set so that an absorbance reading was collected every 1 s. Three progress curves were collected at each temperature; where the slope for these triplicates deviated by more than 10%, the reactions were repeated.

When required, the initial (zero time) rate of reaction for each assay triplicate was determined using the linear search function in the Vision32™ rate program.

Although earlier determinations of Δ G ‡ cat , Δ G ‡ inact , Δ H eq and T eq used initial parameter estimates derived from the calculation of rates from progress curves (described in [ 2 ]), more recent analysis of results indicates that the method described below is simpler and equally accurate.

Using the values for Δ G ‡ cat (80 kJ·mol −1 ), Δ G ‡ inact (95 kJ·mol −1 ), Δ H eq (100 kJ·mol −1 ) and T eq (320 K) described in the original paper [ 1 ] as initial parameter estimates (deemed to be ‘typical’ or ‘plausible’ values for each of the parameters) and the concentration of protein in each assay (expressed in mol·l −1 ), the experimental data were fitted to the Equilibrium Model using MicroMath® Scientist® for Windows software (version 2.01, MicroMath Scientific Software).

The values for each parameter were first ‘improved’ by Simplex searching [ 13 , 14 ]. The experimental data were then fitted to the Equilibrium Model using the parameters derived from the Simplex search, employing an iterative non-linear minimization of least squares. This minimization utilizes Powell's algorithm [ 15 ] to find a local minimum, possibly a global minimum, of the sum of squared deviations between the experimental data and the model calculations.

In each case, the fitting routine was set to take minimum and maximum iterative step-sizes of 1×10 −12 and 1 respectively. The sum of squares goal (the termination criterion for the fitting routine) was set to 1×10 −12 .

The S.D. values in the Tables refer to the fit of the data to the model. On the basis of the variation between the individual triplicate rates from which the parameters are derived for all the enzymes we have assayed so far, we find that the experimental errors in the determination of Δ G ‡ cat , Δ G ‡ inact and T eq are less than 0.5%, and less than 6% in the determination of Δ H eq .

A stand-alone Matlab® [version 7.1.0.246 (R14) Service Pack 3; Mathworks] application, enabling the facile derivation of the Equilibrium Model parameters from a Microsoft® Office Excel file of experimental progress curves (product concentration against time) can be obtained on CD from R.M.D. This application is suitable for computers running Microsoft® Windows XP, and is for non-commercial research purposes only.

RESULTS AND DISCUSSION

The Equilibrium Model has four data inputs: enzyme concentration, temperature, concentration of product and time. From the last two, an estimate of the rate of reaction (in M·s −1 ) can be obtained. In describing the effect of temperature on catalytic activity, the rate of the catalytic reaction is the measurement of interest. The quantitative expression of the dependence of rate on temperature, T , and time, t , is given by eqn (1) :

where k B is Boltzmann's constant and h is Planck's constant. This is the expression that we have used in our proposal [ 1 ] and validation [ 2 ] of the Equilibrium Model to date. Experimentally, however, rates are rarely measured directly; rather, product concentration is determined at increasing times, either by continuous or discontinuous assay, giving a series of progress curves. The quantitative expression relating the product concentration, time and temperature for the Equilibrium Model can be obtained by integrating eqn (1) , giving eqn (2) :

We find that data processed as enzyme rates using eqn (1) , or as product concentration changes using eqn (2) , give essentially the same results. However, since eqn (2) involves a more direct measurement, the experimental protocol used in the present study involves measuring progress curves of product concentration against time at different temperatures and fitting these data to eqn (2) .

Robustness of the fitted constants

If the enzyme preparation used in the determination of T eq is not pure, then overestimation of the enzyme concentration is likely. Few methods of determining protein concentration give answers that are correct in absolute terms; apart from any limitations in terms of sensitivity and interferences, most are based on a comparison with a standard of uncertain equivalence to the enzyme under investigation. The determination of enzyme concentration is thus a potential source of error.

To determine how dependent the fitted constants are on the accuracy of the enzyme concentration, data for β-lactamase [ 2 ] were fitted against the experimentally determined progress curves with the enzyme concentration reduced 2-, 5- and 10-fold compared with that determined experimentally ( Table 1 ). It is evident that errors in determining enzyme concentration have little effect upon parameter determination, except, of course, in respect of Δ G ‡ cat , which is reduced as the model attempts to relate the reduced enzyme concentration to the observed rates of reaction. Even with changing the enzyme concentration 5-fold, errors in the values for Δ G ‡ inact , Δ H eq and T eq are small.

The experimental data for β-lactamase were used to generate the Equilibrium Model parameters as described in the Experimental section. Changes were then made to the experimentally determined enzyme concentration to determine the dependence of the fitted constants on the accuracy of the protein concentration. Parameter values are ±S.D.

Data sampling requirements: sampling rate

The increasing need for automation in enzyme assays has led to the development of instruments that use sampling techniques to assay enzymes at different times. Additionally, some assays are difficult to carry out continuously. It is therefore important to know whether the fitting procedures described herein are sufficiently robust to deal with discontinuous data collection. To determine the sampling requirement, progress curves for the reaction catalysed by aryl-acylamidase were collected in triplicate at 1 s intervals over a 25 min period at a variety of temperatures. Progress curves were then manipulated by the successive removal of a proportion of the data points to determine the effect of sampling rate on the fitting of the data to the Equilibrium Model and on the resulting parameters ( Table 2 ). Using the 1 s sampling interval as a reference, the absolute values of Δ G ‡ cat , Δ G ‡ inact , Δ H eq and T eq are essentially the same at all sampling rates (up to a 150 s interval), despite the increase in the S.D. values as the sampling interval increases. The results indicate that discontinuous enzyme assays can be used for the determination of T eq . The minimum number of points per progress curve required to give accurate values for the parameters will depend upon the length of the assay and the curvature of the progress curve, but, as expected, the larger number of data points arising from continuous assays give more accurate results. The results also show that accuracy is not dominated by a requirement for ‘early’ data, taken very soon after zero time, and that the S.D. provides a good guide to the accuracy of the parameters.

Progress curves for aryl-acylamidase, collected over 25 min and at ten different temperatures, were used to generate the Equilibrium Model parameters as described in the Experimental section. Experimental data points were then successively removed to give the effect of reduced frequency of data points to determine the effect of various sampling rates on the final parameter values. Parameter values are means±S.D.

The results presented above imply that the parameters can be obtained accurately from as few as ten data points (sampling only every 150 s in the case of the 1500 s aryl-acylamidase assays). We would expect the ‘data sampling’ shown in Table 2 to be a satisfactory proxy for a discontinuous assay. However, this was confirmed using another enzyme. Acid phosphatase was incubated with the substrate p NPP for a total assay duration of 30 min, and the reaction was sampled in triplicate every 60 s, stopped with NaOH, and the absorbance was read at 410 nm. Three progress curves (absorbance against time) at each temperature were generated from the triplicate absorbance values obtained when the reaction was stopped. Product concentrations (expressed in mol·l −1 ) were then calculated for each absorbance reading, and the data set was fitted to the Equilibrium Model as described previously and compared with data obtained in a continuous assay [ 2 ]. Taking experimental error into account, the parameter values generated from fitting these data ( Table 3 ) indicate no significant difference between the two methods, except in the case of Δ G ‡ inact . The increased value of the errors on each parameter determined using the discontinuous data indicate that, as expected, continuous assays give more accurate results.

Acid phosphatase was assayed discontinuously over a period of 30 min with a sampling rate of 60 s and at 5 °C intervals from 20 to 80 °C (13 temperature points). The results of fitting data for the same enzyme over the same temperature range and using the same intervals, but using a continuous assay (effective sampling rate of 1 s) have been included for comparison [ 2 ]. The progress curves generated for both methods were fitted to the Equilibrium Model and the parameters generated as described in the Experimental section. Parameter values are means±S.D.

Data sampling requirements: temperature range

Progress curves at 12 temperatures were collected for acid phosphatase [ 2 ]. Analysis of the initial rate of reaction (i.e. at zero time) shows three points above the temperature at which maximum product is formed ( Figure 2 ). By sequentially truncating the data set from the highest or the lowest temperature point and re-fitting the resulting data sets, we gain some insight into the dependence of the fitting routine and accurate estimation of the parameters on the data points above and below the T opt ( Table 4 ).

Acid phosphatase was assayed continuously as described by Peterson et al. [ 2 ]. For each triplicate progress curve, the initial rate of reaction was determined using the linear search function in the programme, Vision32™. The data are plotted as rate (μM·s −1 ) against temperature (K).

A full set of experimental data for acid phosphatase was used to generate the Equilibrium Model parameters as described in the Experimental section. Temperature points above the T opt ( Figure 2 ) were sequentially truncated from the complete data set to determine the influence of data points above the T opt on the final parameter values. Temperature points were also sequentially truncated from the lowest temperature point to the highest from the complete data set (12 temperature points) to determine how many points, in total, below the T opt are required for the accurate determination of T eq and the other thermodynamic parameters. In this case, each data set included all temperature points above the T opt . Parameter values are means±S.D.

For data truncated from the highest temperature point, the values of Δ G ‡ cat , Δ G ‡ inact and T eq do not vary greatly with the various data treatments. However, for the fit excluding the last three temperature points, there is a substantial loss in accuracy for the Equilibrium Model parameter, Δ H eq . This difference is not reflected in the S.D. values. Figure 3 , which illustrates the differences in each fitting of the truncated data sets to the Equilibrium Model presented as a three-dimensional plot of rate (μM·s −1 ) against temperature (K) against time (s), shows the reason for this. The plots indicate that when only one or two data points are removed, there is little difference in the shape of the plot when the data are simulated in three dimensions, but without a data point above the T opt , the equilibrium model effectively relapses towards the Classical Model ( Figure 1 ), with a sharp decline in Δ H eq , even though a reasonable value for T eq has been obtained. These results suggest that it is possible to obtain acceptable estimates of the parameters with only one temperature point above the T opt .

Acid phosphatase was assayed as described by Peterson et al. [ 2 ]. Temperature points above the T opt (see Figure 2 ) were sequentially truncated from the complete data set to determine the influence of data points above the T opt on the final parameter values. Illustrated here are the results plotted as rate (μM·s −1 ) against temperature (K) against time (s) for the fit of acid phosphatase data to the Equilibrium Model using ( A ) the full data set, ( B ) the data set excluding the last data point, ( C ) the data set excluding the last two data points, and ( D ) the data set excluding the last three data points.

All the foregoing discussion is based on an ab initio presumption that the temperature-dependence of enzyme activity is described by the Equilibrium Model. Of the 50 or so enzymes studied in detail by us, all follow the model. However, data that do not show clear evidence of a T opt when initial (zero time) rates are plotted against temperature may in fact be fitted equally well by the simpler Classical Model. In this situation, it would be foolhardy to carry out the procedure described in the present paper. It must therefore be stressed that if only one or two points above the T opt are determined, the measured initial rates at those temperatures must be sufficiently lower than that at T opt for the assumption of the Equilibrium Model to be justified. Ideally, two or more rate measurements above T opt showing a clear trend of falling rates should be obtained to apply the Equilibrium Model with confidence.

For data sequentially truncated from the lowest temperature point to the highest, a trend in the parameter values was seen ( Table 4 ). Parameter values were maintained close to the ‘complete’ data set level down to eight points; below eight points, values moved outside the S.D. limits, but were still relatively close in all cases.

The results of this data manipulation suggest that data at eight temperatures with two points above T opt (showing a clear downwards trend) are sufficient to yield parameter values for Δ G ‡ cat , Δ G ‡ inact , Δ H eq and T eq with reasonable precision.

Enzymes operating under ‘non-ideal’ conditions: the use of initial rates

To use data from progress curves collected over extended periods of time for valid fitting to the Equilibrium Model requires that any decrease in activity observed is due solely to thermal factors and not to some other process. This means that the enzyme and its reaction be ‘ideal’; that is, the enzyme is not product inhibited, the reaction is essentially irreversible and the enzyme operates at V max for the entire assay. To date, the enzymes that we have fitted to the Equilibrium Model have been chosen to meet, or come very close to meeting, these criteria over the 3–5 min duration of the assay.

However, many enzyme reactions are necessarily assayed under non-ideal conditions. For example, the reaction may be sufficiently reversible that the back reaction contributes to the observed rate during the assay and/or the products of the reaction may be inhibitors of the enzyme. Application of the Equilibrium Model to these non-ideal enzyme reactions can usually be achieved by restricting assays to the initial rate of reaction. Setting t =0 in eqn (1) gives eqn (3) below. Using this, it is possible to fit the experimental data for zero time (i.e. initial rates) to the Equilibrium Model to determine Δ G ‡ cat , Δ H eq and T eq , although the time-dependent thermal denaturation parameter, Δ G ‡ inact , cannot be determined. At t =0,

Another circumstance where ‘non-ideality’ may occur is when the decrease of rate during the assay is partially due to substrate depletion. If the enzyme is saturated at the start of the assay, lowering the enzyme concentration or increasing the sensitivity of the assay may remove this problem. In either case, using initial rates will allow the equilibrium model to be applied. However, if insufficient substrate is present at zero time to saturate the enzyme, either because of, e.g., solubility limitations, or as a result of increases in K m [ 7 , 8 ] as the temperature is altered, then considerable errors may arise. Even here, it may be possible, if the K m is known at each temperature, to obtain reasonable approximations of the initial rates at saturation by calculating the degree of saturation using the relationship v / V max =S/( K m +S), and applying the appropriate corrections.

To simulate the determination of the Equilibrium Model parameters for an enzyme that operates under non-ideal conditions, initial rates of reaction were calculated from each progress curve in the β-lactamase data set [ 2 ] and fitted to the modified zero-time version of the Equilibrium Model using the Scientist® software ( Table 5 ). No significant differences in any of the parameters determined this way were found, suggesting that this manner of determination is potentially as accurate as fitting the entire time course to the Equilibrium Model for the determination of Δ G ‡ cat , Δ H eq and T eq .

To simulate the determination of the Equilibrium Model parameters for an enzyme that operates under non-ideal conditions, initial rates of reaction were calculated from each progress curve in the β-lactamase data set [ 2 ] using the linear search function in the programme, Vision32™, and fitted to the Equilibrium Model via eqn (3) . Parameters calculated for the complete data set (entire time course) have been included for comparison [ 2 ]. Parameter values are means±S.D.

Conclusions

To date, determination of the parameters associated with the Equilibrium Model for individual enzymes has involved continuous assays with collection of data at 1 s intervals over 5 min periods at 2–3 °C temperature intervals over at least a 40 °C range, with each temperature run being carried out in triplicate; i.e. processing approx. 15000 data points gathered in approx. 50 experimental runs [ 2 ]. Using a simple technique of fitting the raw data (product concentration against time) to the Equilibrium Model, we have shown that data collection (and thus labour) can be reduced considerably without compromising the accuracy of the derived parameters. Accurate results require preferably more than one data point taken above T opt and more than eight temperature points in total. Major errors in enzyme determination affect only the determination of Δ G ‡ cat . Although continuous assays will give the most accurate results, Δ G ‡ cat , Δ G ‡ inact , Δ H eq and T eq can be determined accurately using discontinuous assays. Among other things, this will allow the determination of the parameters of enzymes from extreme thermophiles; since T opt for these enzymes may be above 100 °C, and since few continuous assay methods are practical at such temperatures, most such assays will have to be discontinuous [ 4 ]. Finally, we have demonstrated that the use of initial, zero-time rates enables the ready determination of the Equilibrium Model parameters (except Δ G ‡ inact ) of most non-ideal enzyme reactions.

The method described here enables the determination of the new fundamental enzyme thermal parameters arising from the Equilibrium Model. It should be noted that the Equilibrium Model itself enables an accurate description of the effect of temperature on enzyme activity, but does not purport to describe the molecular basis of this behaviour. Evidence so far ([ 2 ], and M. E. Peterson, C. K. Lee, C. Monk and R. M. Daniel, unpublished work) suggests that the conformational changes between the active and inactive forms of the enzyme described by the model are local rather than global, and possibly quite slight. The model, and the work described here, provides the foundation, and one of the tools needed to determine the molecular basis of these newly described properties of enzymes. The focus of future work must now be to apply the appropriate physicochemical techniques to determine the precise nature of this proposed structural change.

Acknowledgments

This work was supported by the Royal Society of New Zealand's International Science and Technology Linkages Fund, and the Marsden Fund.

Loading content

Boost content.

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings
• My Bibliography
• Collections
• Citation manager

Save citation to file

Email citation, add to collections.

• Create a new collection
• Add to an existing collection

Add to My Bibliography

Your saved search, create a file for external citation management software, your rss feed.

• Search in PubMed
• Search in NLM Catalog
• Add to Search

Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates

Affiliations.

• 1 School of Science, University of Waikato, Hamilton 3240, New Zealand; email: [email protected].
• 2 Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom; email: [email protected].
• PMID: 32040931
• DOI: 10.1146/annurev-biophys-121219-081520

We review the adaptations of enzyme activity to different temperatures. Psychrophilic (cold-adapted) enzymes show significantly different activation parameters (lower activation enthalpies and entropies) from their mesophilic counterparts. Furthermore, there is increasing evidence that the temperature dependence of many enzyme-catalyzed reactions is more complex than is widely believed. Many enzymes show curvature in plots of activity versus temperature that is not accounted for by denaturation or unfolding. This is explained by macromolecular rate theory: A negative activation heat capacity for the rate-limiting chemical step leads directly to predictions of temperature optima; both entropy and enthalpy are temperature dependent. Fluctuations in the transition state ensemble are reduced compared to the ground state. We show how investigations combining experiment with molecular simulation are revealing fundamental details of enzyme thermoadaptation that are relevant for understanding aspects of enzyme evolution. Simulations can calculate relevant thermodynamic properties (such as activation enthalpies, entropies, and heat capacities) and reveal the molecular mechanisms underlying experimentally observed behavior.

Keywords: enthalpy–entropy trade-off; enzyme catalysis; enzyme evolution; macromolecular rate theory; molecular dynamics; transition state theory.

PubMed Disclaimer

Similar articles

• Entropy and Enzyme Catalysis. Åqvist J, Kazemi M, Isaksen GV, Brandsdal BO. Åqvist J, et al. Acc Chem Res. 2017 Feb 21;50(2):199-207. doi: 10.1021/acs.accounts.6b00321. Epub 2017 Feb 7. Acc Chem Res. 2017. PMID: 28169522
• Enzyme surface rigidity tunes the temperature dependence of catalytic rates. Isaksen GV, Åqvist J, Brandsdal BO. Isaksen GV, et al. Proc Natl Acad Sci U S A. 2016 Jul 12;113(28):7822-7. doi: 10.1073/pnas.1605237113. Epub 2016 Jun 27. Proc Natl Acad Sci U S A. 2016. PMID: 27354533 Free PMC article.
• Hidden Conformational States and Strange Temperature Optima in Enzyme Catalysis. Åqvist J, Sočan J, Purg M. Åqvist J, et al. Biochemistry. 2020 Oct 13;59(40):3844-3855. doi: 10.1021/acs.biochem.0c00705. Epub 2020 Sep 25. Biochemistry. 2020. PMID: 32975950 Free PMC article.
• Enzyme evolution and the temperature dependence of enzyme catalysis. Arcus VL, van der Kamp MW, Pudney CR, Mulholland AJ. Arcus VL, et al. Curr Opin Struct Biol. 2020 Dec;65:96-101. doi: 10.1016/j.sbi.2020.06.001. Epub 2020 Jul 11. Curr Opin Struct Biol. 2020. PMID: 32659635 Review.
• Protein dynamics and enzyme catalysis: the ghost in the machine? Glowacki DR, Harvey JN, Mulholland AJ. Glowacki DR, et al. Biochem Soc Trans. 2012 Jun 1;40(3):515-21. doi: 10.1042/BST20120047. Biochem Soc Trans. 2012. PMID: 22616861 Review.
• A structural perspective on the temperature-dependent activity of enzymes. McLeod MJ, Barwell SAE, Holyoak T, Thorne RE. McLeod MJ, et al. bioRxiv [Preprint]. 2024 Aug 23:2024.08.23.609221. doi: 10.1101/2024.08.23.609221. bioRxiv. 2024. PMID: 39229032 Free PMC article. Preprint.
• Interplay of structural preorganization and conformational sampling in UDP-glucuronic acid 4-epimerase catalysis. Rapp C, Borg A, Nidetzky B. Rapp C, et al. Nat Commun. 2024 May 8;15(1):3897. doi: 10.1038/s41467-024-48281-6. Nat Commun. 2024. PMID: 38719841
• Cooperative Conformational Transitions Underpin the Activation Heat Capacity in the Temperature Dependence of Enzyme Catalysis. Walker EJ, Hamill CJ, Crean R, Connolly MS, Warrender AK, Kraakman KL, Prentice EJ, Steyn-Ross A, Steyn-Ross M, Pudney CR, van der Kamp MW, Schipper LA, Mulholland AJ, Arcus VL. Walker EJ, et al. ACS Catal. 2024 Mar 8;14(7):4379-4394. doi: 10.1021/acscatal.3c05584. eCollection 2024 Apr 5. ACS Catal. 2024. PMID: 38633402 Free PMC article.
• High temperature delays and low temperature accelerates evolution of a new protein phenotype. Zheng J, Guo N, Huang Y, Guo X, Wagner A. Zheng J, et al. Nat Commun. 2024 Mar 29;15(1):2495. doi: 10.1038/s41467-024-46332-6. Nat Commun. 2024. PMID: 38553445 Free PMC article.
• Streamlined Chemo-Enzymatic Synthesis of Molnupiravir via Lipase Catalyst. Rosangzuala K, Patlolla RR, Shaikh A, Naik KAP, Raveena G, Nemali M, Reddy Mudiam MK, Banoth L. Rosangzuala K, et al. ACS Omega. 2024 Jan 17;9(4):4423-4428. doi: 10.1021/acsomega.3c06872. eCollection 2024 Jan 30. ACS Omega. 2024. PMID: 38313533 Free PMC article.

Publication types

• Search in MeSH

Grants and funding

• BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom
• BB/M000354/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom

LinkOut - more resources

Full text sources.

• Ingenta plc
• Citation Manager

NCBI Literature Resources

MeSH PMC Bookshelf Disclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

Observing earthworm locomotion

Practical Work for Learning

Published experiments

Investigating an enzyme-controlled reaction: catalase and hydrogen peroxide concentration, class practical or demonstration.

Hydrogen peroxide ( H 2 O 2 ) is a by-product of respiration and is made in all living cells. Hydrogen peroxide is harmful and must be removed as soon as it is produced in the cell. Cells make the enzyme catalase to remove hydrogen peroxide.

This investigation looks at the rate of oxygen production by the catalase in pureed potato as the concentration of hydrogen peroxide varies. The oxygen produced in 30 seconds is collected over water. Then the rate of reaction is calculated.

Lesson organisation

You could run this investigation as a demonstration at two different concentrations, or with groups of students each working with a different concentration of hydrogen peroxide. Individual students may then have time to gather repeat data. Groups of three could work to collect results for 5 different concentrations and rotate the roles of apparatus manipulator, result reader and scribe. Collating and comparing class results allows students to look for anomalous and inconsistent data.

Apparatus and Chemicals

For each group of students:.

Pneumatic trough/ plastic bowl/ access to suitable sink of water

Conical flask, 100 cm 3 , 2

Syringe (2 cm 3 ) to fit the second hole of the rubber bung, 1

Measuring cylinder, 100 cm 3 , 1

Measuring cylinder, 50 cm 3 , 1

Clamp stand, boss and clamp, 2

Stopclock/ stopwatch

For the class – set up by technician/ teacher:

Hydrogen peroxide, range of concentrations, 10 vol, 15 vol, 20 vol, 25 vol, and 30 vol, 2 cm 3 per group of each concentration ( Note 1 )

Pureed potato, fresh, in beaker with syringe to measure at least 20 cm 3 , 20 cm 3 per group per concentration of peroxide investigated ( Note 2 )

Rubber bung, 2-holed, to fit 100 cm 3 conical flasks – delivery tube in one hole (connected to 50 cm rubber tubing)

Health & Safety and Technical notes

Wear eye protection and cover clothing when handling hydrogen peroxide. Wash splashes of pureed potato or peroxide off the skin immediately. Be aware of pressure building up if reaction vessels become blocked. Take care inserting the bung in the conical flask – it needs to be a tight fit, so push and twist the bung in with care.

Read our standard health & safety guidance

1 Hydrogen peroxide: (See CLEAPSS Hazcard) Solutions less than 18 vol are LOW HAZARD. Solutions at concentrations of 18-28 vol are IRRITANT. Take care when removing the cap of the reagent bottle, as gas pressure may have built up inside. Dilute immediately before use and put in a clean brown bottle, because dilution also dilutes the decomposition inhibitor. Keep in brown bottles because hydrogen peroxide degrades faster in the light. Discard all unused solution. Do not return solution to stock bottles, because contaminants may cause decomposition and the stock bottle may explode after a time.

2 Pureed potato may irritate some people’s skin. Make fresh for each lesson, because catalase activity reduces noticeably over 2/3 hours. You might need to add water to make it less viscous and easier to use. Discs of potato react too slowly.

3 If the bubbles from the rubber tubing are too big, insert a glass pipette or glass tubing into the end of the rubber tube.

SAFETY: Wear eye protection and protect clothing from hydrogen peroxide. Rinse splashes of peroxide and pureed potato off the skin as quickly as possible.

Preparation

a Make just enough diluted hydrogen peroxide just before the lesson. Set out in brown bottles ( Note 1 ).

b Make pureed potato fresh for each lesson ( Note 2 ).

c Make up 2-holed bungs as described in apparatus list and in diagram.

Investigation

d Use the large syringe to measure 20 cm 3 pureed potato into the conical flask.

e Put the bung securely in the flask – twist and push carefully.

f Half-fill the trough, bowl or sink with water.

g Fill the 50 cm 3 measuring cylinder with water. Invert it over the trough of water, with the open end under the surface of the water in the bowl, and with the end of the rubber tubing in the measuring cylinder. Clamp in place.

h Measure 2 cm 3 of hydrogen peroxide into the 2 cm 3 syringe. Put the syringe in place in the bung of the flask, but do not push the plunger straight away.

i Check the rubber tube is safely in the measuring cylinder. Push the plunger on the syringe and immediately start the stopclock.

j After 30 seconds, note the volume of oxygen in the measuring cylinder in a suitable table of results. ( Note 3 .)

k Empty and rinse the conical flask. Measure another 20 cm 3 pureed potato into it. Reassemble the apparatus, refill the measuring cylinder, and repeat from g to j with another concentration of hydrogen peroxide. Use a 100 cm 3 measuring cylinder for concentrations of hydrogen peroxide over 20 vol.

l Calculate the rate of oxygen production in cm 3 /s.

m Plot a graph of rate of oxygen production against concentration of hydrogen peroxide.

Teaching notes

Note the units for measuring the concentration of hydrogen peroxide – these are not SI units. 10 vol hydrogen peroxide will produce 10 cm 3 of oxygen from every cm 3 that decomposes.( Note 1 .)

In this procedure, 2 cm 3 of 10 vol hydrogen peroxide will release 20 cm 3 of oxygen if the reaction goes to completion. 2 cm 3 of liquid are added to the flask each time. So if the apparatus is free of leaks, 22 cm 3 of water should be displaced in the measuring cylinder with 10 vol hydrogen peroxide. Oxygen is soluble in water, but dissolves only slowly in water at normal room temperatures.

Use this information as a check on the practical set-up. Values below 22 cm 3 show that oxygen has escaped, or the hydrogen peroxide has not fully reacted, or the hydrogen peroxide concentration is not as expected. Ask students to explain how values over 22 cm 3 could happen.

An error of ± 0.05 cm 3 in measuring out 30 vol hydrogen peroxide could make an error of ± 1.5 cm 3 in oxygen production.

Liver also contains catalase, but handling offal is more controversial with students and introduces a greater hygiene risk. Also, the reaction is so vigorous that bubbles of mixture can carry pieces of liver into the delivery tube.

If collecting the gas over water is complicated, and you have access to a 100 cm 3 gas syringe, you could collect the gas in that instead. Be sure to clamp the gas syringe securely but carefully.

The reaction is exothermic. Students may notice the heat if they put their hands on the conical flask. How will this affect the results?

Health and safety checked, September 2008

http://www.saps.org.uk/secondary/teaching-resources/293-student-sheet-24-microscale-investigations-with-catalase Microscale investigations with catalase – which has been transcribed onto this site at Investigating catalase activity in different plant tissues.

(Website accessed October 2011)

Catalase Enzyme Lab

A common enzyme lab for students to measure the impact of temperature and pH on the efficiency of catalase. Catalase is an enzyme is found in almost all living organisms that breaks down hydrogen peroxide (H 2 O 2 ) into oxygen and water. Many teachers use raw chicken liver or potato as the source of the catalase. I’ve done both and frankly potato is less stinky and is easier to clean up after. Here is the gist of the lab:

• Students will need: potato puree, tweezers, a beaker full of hydrogen peroxide, and a stopwatch.
• Peel a raw potato and cut it into pieces. Place the potato in the blender and add a small amount of water. Puree until smooth. (One large potato should be enough for 1 class period).
• Note: The potato will turn brown relatively quickly as it comes in contact with the air. Don’t worry! This does not impact the results of the experiment.
• Collect the paper discs out of your hole puncher (or hit up the copy center at your school).
• Using tweezers, have students dip a paper disc in the potato puree. Place the paper in the bottom of the beaker of peroxide and start the stopwatch. As the catalase on the paper disc breaks peroxide into oxygen and water, the disc will float. Have students time how long it takes for the paper to rise.
• pH: To show students the impact of pH on enzyme efficiency, have them add a few drops of an acid and a base to the potato purees on a spot plate. Vinegar and bleach are great options. Repeat the experiment and have students determine at which pH catalase works best.
• Option 1: Change the temperature of the peroxide. Place a beaker of peroxide in an ice bath, and another in a warm water bath. This option tends to yield the best results.
• Option 2: Change the temperature of the potato puree. This can be done easily by putting some of the puree in the fridge and some in the microwave (or boil it at home ahead of time). This does not always give the best results because the cold potato can warm up pretty quickly, but still works if you don’t have water baths available.

• Have students do multiple trials (at least 3) and take the average. Sometimes they get weird data, so this helps with accuracy.
• If you are testing multiple variables, have students get fresh peroxide before starting the new variable. For example, have students collect all the temperature data, get fresh peroxide, and then collect pH data.
• If the paper disc takes more than 1 minute to rise, tell students that the enzyme is denatured and they can stop timing and move on to the next trial.
• When I first started doing this lab I used petri dishes for all the potato purees and it was a lot of clean up. I recently switched to chemistry spot plates (pictured above) and it made clean up so much easier!

If you have any additional questions, leave me a comment! ​Rock on,

• Read more about: Biochemistry

Hi, I'm Becca!

Search the site, browse by category.

• A list of ALL blog posts
• Back to School
• Biochemistry
• Body Systems
• Classification
• Classroom Decor
• Classroom Management
• Distance Learning
• End of the School Year
• Experiments
• Field Trips
• For NEW Teachers
• Formative Assessment
• Media in the Classroom
• Microscopes
• Photosynthesis & Respiration
• Plate Tectonics
• Sustainability
• Teacher Tips
• Weather and Climate

Get Freebies!

You might also like....

Read Aloud Closure Assignments

Biology Picture Book Recommendations

Earth and Space Science Picture Book Recommendations

Want a fun way to practice science vocabulary? Try out seek and finds!

Your browser is not supported

Sorry but it looks as if your browser is out of date. To get the best experience using our site we recommend that you upgrade or switch browsers.

Find a solution

• Skip to main content
• Skip to navigation

• Back to parent navigation item
• Primary teacher
• Secondary/FE teacher
• Early career or student teacher
• Higher education
• Curriculum support
• Literacy in science teaching
• Periodic table
• Interactive periodic table
• Climate change and sustainability
• Resources shop
• Collections
• Remote teaching support
• Starters for ten
• Screen experiments
• Assessment for learning
• Microscale chemistry
• Faces of chemistry
• Classic chemistry experiments
• Nuffield practical collection
• Anecdotes for chemistry teachers
• On this day in chemistry
• Global experiments
• PhET interactive simulations
• Chemistry vignettes
• Context and problem based learning
• Journal of the month
• Chemistry and art
• Art analysis
• Pigments and colours
• Ancient art: today's technology
• Psychology and art theory
• Art and archaeology
• Artists as chemists
• The physics of restoration and conservation
• Ancient Egyptian art
• Ancient Greek art
• Ancient Roman art
• Classic chemistry demonstrations
• In search of solutions
• In search of more solutions
• Creative problem-solving in chemistry
• Solar spark
• Chemistry for non-specialists
• Health and safety in higher education
• Analytical chemistry introductions
• Exhibition chemistry
• Introductory maths for higher education
• Commercial skills for chemists
• Kitchen chemistry
• Journals how to guides
• Chemistry in health
• Chemistry in sport
• Chemistry in your cupboard
• Chocolate chemistry
• Adnoddau addysgu cemeg Cymraeg
• The chemistry of fireworks
• Festive chemistry
• Education in Chemistry
• Teach Chemistry
• On-demand online
• Live online
• Selected PD articles
• PD for primary teachers
• PD for secondary teachers
• What we offer
• Chartered Science Teacher (CSciTeach)
• Teacher mentoring
• UK Chemistry Olympiad
• Who can enter?
• How does it work?
• Resources and past papers
• Top of the Bench
• Schools' Analyst
• Regional support
• Education coordinators
• RSC Yusuf Hamied Inspirational Science Programme
• RSC Education News
• Supporting teacher training
• Interest groups

• More navigation items

Testing for catalase enzymes

In association with Nuffield Foundation

• No comments

Try this class experiment to detect the presence of enzymes as they catalyse the decomposition of hydrogen peroxide

Enzymes are biological catalysts which increase the speed of a chemical reaction. They are large protein molecules and are very specific to certain reactions. Hydrogen peroxide decomposes slowly in light to produce oxygen and water. The enzyme catalase can speed up (catalyse) this reaction.

In this practical, students investigate the presence of enzymes in liver, potato and celery by detecting the oxygen gas produced when hydrogen peroxide decomposes. The experiment should take no more than 20–30 minutes.

• Eye protection
• Conical flasks, 100 cm 3 , x3
• Measuring cylinder, 25 cm 3
• Bunsen burner
• Wooden splint
• A bucket or bin for disposal of waste materials
• Hydrogen peroxide solution, ‘5 volume’

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout. Students must be instructed NOT to taste or eat any of the foods used in the experiment.
• Hydrogen peroxide solution, H 2 O 2 (aq) – see CLEAPSS Hazcard HC050  and CLEAPSS Recipe Book RB045. Hydrogen peroxide solution of ‘5 volume’ concentration is low hazard, but it will probably need to be prepared by dilution of a more concentrated solution which may be hazardous.
• Only small samples of liver, potato and celery are required. These should be prepared for the lesson ready to be used by students. A disposal bin or bucket for used samples should be provided to avoid these being put down the sink.
• Measure 25 cm 3  of hydrogen peroxide solution into each of three conical flasks.
• At the same time, add a small piece of liver to the first flask, a small piece of potato to the second flask, and a small piece of celery to the third flask.
• Hold a glowing splint in the neck of each flask.
• Note the time taken before each glowing splint is relit by the evolved oxygen.
• Dispose of all mixtures into the bucket or bin provided.

Teaching notes

Some vegetarian students may wish to opt out of handling liver samples, and this should be respected.

Before or after the experiment, the term enzyme will need to be introduced. The term may have been met previously in biological topics, but the notion that they act as catalysts and increase the rate of reactions may be new. Similarly their nature as large protein molecules whose catalytic activity can be very specific to certain chemical reactions may be unfamiliar. The name catalase for the enzyme present in all these foodstuffs can be introduced.

To show the similarity between enzymes and chemical catalysts, the teacher may wish to demonstrate (or ask the class to perform as part of the class experiment) the catalytic decomposition of hydrogen peroxide solution by manganese(IV) oxide (HARMFUL – see CLEAPSS Hazcard HC060).

If students have not performed the glowing splint test for oxygen for some time, they may need reminding of how to do so by a quick demonstration by the teacher.

More resources

Add context and inspire your learners with our short career videos showing how chemistry is making a difference .

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry.

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• 16-18 years
• Practical experiments
• Biological chemistry

Specification

• Enzymes act as catalysts in biological systems.
• Factors which affect the rates of chemical reactions include: the concentrations of reactants in solution, the pressure of reacting gases, the surface area of solid reactants, the temperature and the presence of catalysts.
• Describe the characteristics of catalysts and their effect on rates of reaction.
• Recall that enzymes act as catalysts in biological systems.
• 7.6 Describe a catalyst as a substance that speeds up the rate of a reaction without altering the products of the reaction, being itself unchanged chemically and in mass at the end of the reaction
• 7.8 Recall that enzymes are biological catalysts and that enzymes are used in the production of alcoholic drinks
• C6.2.4 describe the characteristics of catalysts and their effect on rates of reaction
• C6.2.5 identify catalysts in reactions
• C6.2.14 describe the use of enzymes as catalysts in biological systems and some industrial processes
• C5.2f describe the characteristics of catalysts and their effect on rates of reaction
• C5.2i recall that enzymes act as catalysts in biological systems
• C6.2.13 describe the use of enzymes as catalysts in biological systems and some industrial processes
• C5.1f describe the characteristics of catalysts and their effect on rates of reaction
• C5.1i recall that enzymes act as catalysts in biological systems
• B2.24 The action of a catalyst, in terms of providing an alternative pathway with a lower activation energy.
• 2.3.5 demonstrate knowledge and understanding that a catalyst is a substance which increases the rate of a reaction without being used up and recall that transition metals and their compounds are often used as catalysts;
• 7. Investigate the effect of a number of variables on the rate of chemical reactions including the production of common gases and biochemical reactions.

Related articles

How to teach titration post-16

2024-07-08T05:32:00Z By Jo Haywood

Tips for teaching practical titration techniques and the underlying theory

New method grows larger diamonds

2024-05-31T08:24:00Z By Nina Notman

Use this real-world context when teaching about giant covalent structures

Why are some plants poisonous to you and your pets?

2024-05-22T08:16:00Z By Kit Chapman

Dig up the toxic secrets of nature’s blooms

No comments yet

Only registered users can comment on this article., more experiments.

‘Gold’ coins on a microscale | 14–16 years

By Dorothy Warren and Sandrine Bouchelkia

Practical experiment where learners produce ‘gold’ coins by electroplating a copper coin with zinc, includes follow-up worksheet

Practical potions microscale | 11–14 years

By Kirsty Patterson

Observe chemical changes in this microscale experiment with a spooky twist.

Antibacterial properties of the halogens | 14–18 years

By Kristy Turner

Use this practical to investigate how solutions of the halogens inhibit the growth of bacteria and which is most effective

• Contributors
• Email alerts

Site powered by Webvision Cloud

• Publications
• Homepage >
• Resources >
• Chemistry Resources >
• Chemistry Resource List >

Factors Affecting Enzyme Activity (H)

On of the old higher ppas. an experiment looking at the effect of ph or temperature on enzyme activity..

Enzymes are globular protein molecules which catalyse biochemical reactions.

The aim of this experiment is to investigate the effect or pH or temperature changes on enzyme activity.

We will study catalase. an enzyme widely distributed in living organisms. It catalyses the decomposition of hydrogen peroxide into water and oxygen:

Factors Affecting Enzyme Activity – Pupil

Factors Affecting Enzyme Activity – Teacher

Factors Affecting Enzyme Activity – Risk Assessment

SSERC is a Company Limited by Guarantee and a registered educational charity.

Headquarters 1-3 Pitreavie Court, South Pitreavie Business Park, Dunfermline, KY11 8UU

Registered Office 5th Floor, Quartermile Two, 2 Lister Square, Edinburgh EH3 9GL.

Material on this website, including text and images, is protected by copyright. It may not be copied, reproduced, republished, downloaded, posted, broadcast or transmitted in any way except for your own personal, non-commercial use, or for educational use. Prior written consent of the copyright holder must be obtained for any other use of material. Copyright in all materials and/or works comprising or contained within this website remains with SSERC and other copyright owner(s) as specified. No part of this site or sub-domains of this site may be distributed or copied for any commercial purpose.

We use cookies and similar methods to recognize visitors and remember their preferences. To learn more about these methods, including how to disable them, view our Privacy Policy .

Pardon Our Interruption

As you were browsing something about your browser made us think you were a bot. There are a few reasons this might happen:

• You've disabled JavaScript in your web browser.
• You're a power user moving through this website with super-human speed.
• You've disabled cookies in your web browser.
• A third-party browser plugin, such as Ghostery or NoScript, is preventing JavaScript from running. Additional information is available in this support article .

To regain access, please make sure that cookies and JavaScript are enabled before reloading the page.

Genome-wide association scan reveals the reinforcing effect of nano-potassium in improving the yield and quality of salt-stressed barley via enhancing the antioxidant defense system

• Published: 09 September 2024
• Volume 114 , article number  97 , ( 2024 )

Cite this article

• Samar G. Thabet   ORCID: orcid.org/0000-0001-7861-8933 1 ,
• Fatmah Ahmed Safhi 2 ,
• Andreas Börner 3 &
• Ahmad M. Alqudah 4  

Salinity is one of the major environmental factor that can greatly impact the growth, development, and productivity of barley. Our study aims to detect the natural phenotypic variation of morphological and physiological traits under both salinity and potassium nanoparticles (n-K) treatment. In addition to understanding the genetic basis of salt tolerance in barley is a critical aspect of plant breeding for stress resilience. Therefore, a foliar application of n-K was applied at the vegetative stage for 138 barley accessions to enhance salt stress resilience. Interestingly, barley accessions showed high significant increment under n-K treatment compared to saline soil. Based on genome-wide association studies (GWAS) analysis, causative alleles /reliable genomic regions were discovered underlying improved salt resilience through the application of potassium nanoparticles. On chromosome 2H, a highly significant QTN marker (A:C) was located at position 36,665,559 bp which is associated with APX, AsA, GSH, GS, WGS, and TKW under n-K treatment. Inside this region, our candidate gene is HORVU.MOREX.r3.2HG0111480 that annotated as NAC domain protein. Allelic variation detected that the accessions carrying C allele showed higher antioxidants (APX, AsA, and GSH) and barley yield traits (GS, WGS, and TKW) than the accessions carrying A allele, suggesting a positive selection of the accessions carrying C allele that could be used to develop barley varieties with improved salt stress resilience.

Key message

Highlighting the importance of the role of potassium nanoparticles in plant tolerance to abiotic stresses, including salinity is the potential for genetic improvement of barley crop resilience through the enhancement of antioxidant defense systems.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save.

• Get 10 units per month
• Download Article/Chapter or eBook
• 1 Unit = 1 Article or 1 Chapter
• Cancel anytime

Price includes VAT (Russian Federation)

Instant access to the full article PDF.

Rent this article via DeepDyve

Institutional subscriptions

Data availability

All data supporting the findings of this study are available within the paper and its supplementary materials published online.

Abd-Elrahman SH, El-Gabry YAE-G, Hashem FA, Ibrahim MFM, El-Hallous EI, Abbas ZK, Darwish DBE, Al-Harbi NA, Al-Qahtani SM, Taha NM (2023) Influence of nano-chitosan loaded with potassium on potassium fractionation in sandy soil and strawberry productivity and quality. Agronomy 13:1126

Article   CAS   Google Scholar  

Abeed AHA, Saleem MH, Asghar MA, Mumtaz S, Ameer A, Ali B, Alwahibi MS, Elshikh MS, Ercisli S, Elsharkawy MM, Ali S, Soudy FA (2023) Ameliorative effects of exogenous potassium nitrate on antioxidant defense system and mineral nutrient uptake in radish ( Raphanus sativus L.) under salinity stress. ACS Omega 8:22575–22588

Article   CAS   PubMed   PubMed Central   Google Scholar  

Ahmad I, Zhu G, Zhou G, Younas MU, Suliman MSE, Liu J, Zhu YM, Salih EGI (2023) Integrated approaches for increasing plant yield under salt stress. Front Plant Sci 14:1215343

Article   PubMed   PubMed Central   Google Scholar  

Al Abdallat AM, Ayad JY, Abu Elenein JM, Al Ajlouni Z, Harwood WA (2014) Overexpression of the transcription factor HvSNAC1 improves drought tolerance in barley ( Hordeum vulgare L.). Mol Breed 33:401–414

Allaire JJB, MA, (2012) RStudio: integrated development environment for R. Boston, MA 770:165–171

Google Scholar  

Alqudah AM, Sallam A, Stephen Baenziger P, Börner A (2020) GWAS: fast-forwarding gene identification and characterization in temperate cereals: lessons from Barley–A review. J Adv Res 22:119–135

Article   PubMed   Google Scholar  

Al-Tardeh SM, Alqam HN, Kuhn AJ, Kuchendorf CM (2023) In vitro assessment of salinity stress impact on early growth in ten certified palestinian barley cultivars ( Hordeum vulgare L.) potentially suitable for cultivation on former quarry substrates. Water 15:1065

Atta K, Mondal S, Gorai S, Singh AP, Kumari A, Ghosh T, Roy A, Hembram S, Gaikwad DJ, Mondal S, Bhattacharya S, Jha UC, Jespersen D (2023) Impacts of salinity stress on crop plants: improving salt tolerance through genetic and molecular dissection. Front Plant Sci 14:1241736

Bagues M, Neji M, Karbout N, Boussora F, Triki T, Guasmi F, Nagaz K (2024) Mitigating salinity stress in barley ( Hordeum vulgare L.) through biochar and NPK fertilizers: impacts on physio-biochemical behavior and grain yield. Agronomy 14:317

Balasubramaniam T, Shen G, Esmaeili N, Zhang H (2023) Plants’ response mechanisms to salinity stress. Plants 12:2253

Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67:1–48

Article   Google Scholar  

Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287

Article   CAS   PubMed   Google Scholar  

Bian Z, Gao H, Wang C (2020) NAC Transcription factors as positive or negative regulators during ongoing battle between pathogens and our food crops. Int j mol sci 22:81

Chaurasia S, Singh AK, Kumar A, Songachan LS, Yadav MC, Kumar S, Kumari J, Bansal R, Sharma PC, Singh K (2021) Genome-wide association mapping reveals key genomic regions for physiological and yield-related traits under salinity stress in wheat ( Triticum aestivum L.). Genomics 113:3198–3215

Chen YJ, Perera V, Christiansen MW, Holme IB, Gregersen PL, Grant MR, Collinge DB, Lyngkjær MF (2013) The barley HvNAC6 transcription factor affects ABA accumulation and promotes basal resistance against powdery mildew. Plant Mol Biol 83:577–590

Ching LS, Mohamed S (2001) Alpha-tocopherol content in 62 edible tropical plants. J Agric Food Chem 49:3101–3105

Dahnke W (1975) Recommended chemical soil test procedures for the North Central Region

Elakhdar A, Slaski JJ, Kubo T, Hamwieh A, Hernandez Ramirez G, Beattie AD, Capo-Chichi LJA (2023) Genome-wide association analysis provides insights into the genetic basis of photosynthetic responses to low-temperature stress in spring barley. Front plant sci 14:1159016

El-tanahy AMM, Marzouk NM, Soliman MS, Mounir AM (2022) The impact of previously prepared potassium and iron in nano forms using gamma radiation on growth and productivity of green bean %J. Egypt J Chem 65:707–725

Emenecker RJ, Strader LC (2020) Auxin-abscisic acid interactions in plant growth and development. Biomolecules 10:281

Foster JG, Hess JL (1980) Responses of superoxide dismutase and glutathione reductase activities in cotton leaf tissue exposed to an atmosphere enriched in oxygen. Plant Physiol 66:482–487

Gao L, Jia S, Cao L, Ma Y, Wang J, Lan D, Guo G, Chai J, Bi C (2022) An F-box protein from wheat, TaFBA-2A, negatively regulates JA biosynthesis and confers improved salt tolerance and increased JA responsiveness to transgenic rice plants. Plant Physiol Biochem 182:227–239

Guo YH, Yu YP, Wang D, Wu CA, Yang GD, Huang JG, Zheng CC (2009) GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5. The New phytol 183:62–75

Halaji B, Haghighi M, Amiri A, Kappel N (2023) Effects of potassium and nanocapsule of potassium on pepper growth and physiological changes in high-temperature stress. J Soil Sci Plant Nutr 23:6317–6330

Han G, Wang M, Yuan F, Sui N, Song J, Wang B (2014) The CCCH zinc finger protein gene AtZFP1 improves salt resistance in Arabidopsis thaliana . Plant Mol Biol 86:237–253

Han G, Qiao Z, Li Y, Wang C, Wang B (2021) The roles of CCCH zinc-finger proteins in plant abiotic stress tolerance. Int j mol sci 22:8327

Han G, Qiao Z, Li Y, Yang Z, Wang C, Zhang Y, Liu L, Wang B (2022) RING zinc finger proteins in plant abiotic stress tolerance. Front Plant Sci 13:877011

Hasanuzzaman M, Bhuyan MHMB, Nahar K, Hossain MS, Mahmud JA, Hossen MS, Masud AAC, Moumita FM (2018) Potassium: a vital regulator of plant responses and tolerance to abiotic stresses. Agronomy 8:31

Hasanuzzaman M, Bhuyan MHMB, Zulfiqar F, Raza A, Mohsin SM, Mahmud JA, Fujita M, Fotopoulos V (2020) Reactive oxygen species and antioxidant defense in plants under abiotic stress: revisiting the crucial role of a universal defense regulator. Antioxidants 9:681

Havir EA, McHale NA (1987) Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. Plant physiol 84:450–455

Horn PS (1998) Introduction to robust estimation and hypothesis testing. Taylor & Francis, New York

Huang C, He W, Guo J, Chang X, Su P, Zhang L (2005) Increased sensitivity to salt stress in an ascorbate-deficient arabidopsis mutant. J exp bot 56:3041–3049

Huo C, He L, Yu T, Ji X, Li R, Zhu S, Zhang F, Xie H, Liu W (2022) The superoxide dismutase gene family in Nicotiana tabacum : genome-wide identification, characterization, expression profiling and functional analysis in response to heavy metal stress. Front Plant Sci 13:904105

Hussain MI, Al-Dakheel AJ, Chaudhry UK, Khan MI, Alhaithloul HAS, Alghanem SM, Alaklabi A (2022) Morpho-physiological response of barley to assess genotypic differences of salinity tolerance under hyper arid climate. Agric Water Manag 272:107832

Ighodaro OM, Akinloye OA (2018) First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): their fundamental role in the entire antioxidant defence grid. Alexandria J Med 54:287–293

Ijaz M, Khan F, Ahmed T, Noman M, Zulfiqar F, Rizwan M, Chen J, K HMS, Li B, (2023) Nanobiotechnology to advance stress resilience in plants: current opportunities and challenges. Mater today Bio 22:100759

Jackson MJPL, New Delhi (1967) Prentice Hall of India 498

Jamil M, Iqbal W, Bangash A, Rehman SU, Imran QM, Rha ESJPJB (2010) Constitutive expression of OSC3H33, OSC3H50 and OSC3H37 genes in rice under salt stress. Pak J Bot 42:4003–4009

CAS   Google Scholar  

Jan A, Maruyama K, Todaka D, Kidokoro S, Abo M, Yoshimura E, Shinozaki K, Nakashima K, Yamaguchi-Shinozaki K (2013) OsTZF1, a CCCH-tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes. Plant Physiol 161:1202–1216

Javid S, Bihamta MR, Omidi M, Abbasi AR, Alipour H, Ingvarsson PK (2022) Genome-wide association study (GWAS) and genome prediction of seedling salt tolerance in bread wheat ( Triticum aestivum L.). BMC Plant Biol 22:581

Jiang M, Jiang J-J, Miao L-X, He C-MJPC, Tissue Culture O (2017) Over-expression of a C3H-type zinc finger gene contributes to salt stress tolerance in transgenic broccoli plants. Plant cell. Tissue Organ C (PCTOC) 130:239–254

Jing X, Hou P, Lu Y, Deng S, Li N, Zhao R, Sun J, Wang Y, Han Y, Lang T, Ding M, Shen X, Chen S (2015) Overexpression of copper/zinc superoxide dismutase from mangrove Kandelia candel in tobacco enhances salinity tolerance by the reduction of reactive oxygen species in chloroplast. Front Plant Sci 6:23

Kayihan C, Eyidogan F, Afsar N, Oktem HA, Yucel M (2012) Cu/Zn superoxide dismutase activity and respective gene expression during cold acclimation and freezing stress in barley cultivars. Biol Plantarum 56:693–698

Kesawat MS, Satheesh N, Kherawat BS, Kumar A, Kim HU, Chung SM, Kumar M (2023) Regulation of reactive oxygen species during salt stress in plants and their crosstalk with other signaling molecules-current perspectives and future directions. Plants 12:864

Klute AJA (1986) Methods of soil analysis. Part 1:9

Konings EJ, Roomans HH, Beljaars PR (1996) Liquid chromatographic determination of tocopherols and tocotrienols in margarine, infant foods, and vegetables. J AOAC Int 79:902–906

Kumari VV, Banerjee P, Verma VC, Sukumaran S, Chandran MAS, Gopinath KA, Venkatesh G, Yadav SK, Singh VK, Awasthi NK (2022) Plant nutrition: an effective way to alleviate abiotic stress in agricultural crops. Int J Mol Sci 23:15

Li Z, Wang X, Cao X, Chen B, Ma C, Lv J, Sun Z, Qiao K, Zhu L, Zhang C, Fan S, Ma Q (2021) GhTULP34, a member of tubby-like proteins, interacts with GhSKP1A to negatively regulate plant osmotic stress. Genomics 113:462–474

Liu TB, Xue C (2011) The Ubiquitin-proteasome system and F-box proteins in pathogenic fungi. Mycobiology 39:243–248

Liu X, Huang M, Fan B, Buckler ES, Zhang Z (2016) Iterative usage of fixed and random effect models for powerful and efficient genome-wide association studies. PLOS Genetics 12:e1005767

Liu Y, Khan AR, Gan Y (2022) C2H2 zinc finger proteins response to abiotic stress in plants. Int J Mol Sci 23:2730

Mao X, Zhang H, Qian X, Li A, Zhao G, Jing R (2012) TaNAC2, a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis. J Exp Bot 63:2933–2946

McGrann GR, Steed A, Burt C, Goddard R, Lachaux C, Bansal A, Corbitt M, Gorniak K, Nicholson P, Brown JK (2015) Contribution of the drought tolerance-related stress-responsive NAC1 transcription factor to resistance of barley to Ramularia leaf spot. Mol Pathol 16:201–209

Milner SG, Jost M, Taketa S, Mazón ER, Himmelbach A, Oppermann M, Weise S, Knüpffer H, Basterrechea M, König P, Schüler D, Sharma R, Pasam RK, Rutten T, Guo G, Xu D, Zhang J, Herren G, Müller T, Krattinger SG, Keller B, Jiang Y, González MY, Zhao Y, Habekuß A, Färber S, Ordon F, Lange M, Börner A, Graner A, Reif JC, Scholz U, Mascher M, Stein N (2019) Genebank genomics highlights the diversity of a global barley collection. Nature Genet 51:319–326

Mishra N, Jiang C, Chen L, Paul A, Chatterjee A, Shen G (2023) Achieving abiotic stress tolerance in plants through antioxidative defense mechanisms. Front Plant Sci 14:1110622

Nakano Y, Asada KJP, physiology c (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22(867):880

NefissiOuertani R, Abid G, Karmous C, Ben Chikha M, Boudaya O, Mahmoudi H, Mejri S, Jansen RK, Ghorbel A (2021) Evaluating the contribution of osmotic and oxidative stress components on barley growth under salt stress. AoB Plants. https://doi.org/10.1093/aobpla/plab034

Negi NP, Shrivastava DC, Sharma V, Sarin NB (2015) Overexpression of CuZnSOD from Arachis hypogaea alleviates salinity and drought stress in tobacco. Plant Cell Rep 34:1109–1126

Noman M, Ahmed T, Wang J, Ijaz M, Shahid M, Islam MS, Azizullah Manzoor I, Li D, Song F (2023) Nano-enabled crop resilience against pathogens: potential, mechanisms and strategies. Crop Health 1:15

Paradiso A, Berardino R, de Pinto MC, Sanità di Toppi L, Storelli MM, Tommasi F, De Gara L (2008) Increase in ascorbate-glutathione metabolism as local and precocious systemic responses induced by cadmium in durum wheat plants. Plant & Cell Physiol 49:362–374

Piisilä M, Keceli MA, Brader G, Jakobson L, Jõesaar I, Sipari N, Kollist H, Palva ET, Kariola T (2015) The F-box protein MAX2 contributes to resistance to bacterial phytopathogens in Arabidopsis thaliana . BMC Plant Biol 15:53

Qu L, Sun M, Li X, He R, Zhong M, Luo D, Liu X, Zhao X (2020) The Arabidopsis F-box protein FOF2 regulates ABA-mediated seed germination and drought tolerance. Plant Sci : Int Jo Exp Plant Biol 301:110643

Seok HY, Nguyen LV, Park HY, Tarte VN, Ha J, Lee SY, Moon YH (2018) Arabidopsis non-TZF gene AtC3H17 functions as a positive regulator in salt stress response. Biochem biophys Res Commun 498:954–959

Shah WH, Rasool A, Saleem S, Mushtaq NU, Tahir I, Hakeem KR, Rehman RU (2021) Understanding the Integrated pathways and mechanisms of transporters, protein kinases, and transcription factors in plants under salt stress. Int J Gen 2021:5578727

Singh M, Nara U, Kumar A, Choudhary A, Singh H, Thapa S (2021) Salinity tolerance mechanisms and their breeding implications. J, Genet Eng & Biotechnol 19:173

Singh P, Choudhary KK, Chaudhary N, Gupta S, Sahu M, Tejaswini B, Sarkar S (2022) Salt stress resilience in plants mediated through osmolyte accumulation and its crosstalk mechanism with phytohormones. Front Plant Sci 13:1006617

Sun J, Jiang H, Xu Y, Li H, Wu X, Xie Q, Li C (2007) The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis . Plant & Cell Physiol 48:1148–1158

Tarolli P, Luo J, Park E, Barcaccia G, Masin R (2024) Soil salinization in agriculture: mitigation and adaptation strategies combining nature-based solutions and bioengineering. iScience. https://doi.org/10.1016/j.isci.2024.108830

Thabet SG, Alqudah AM (2023) New genetic insights into improving barley cope with salt stress via regulating mineral accumulation, cellular ion homeostasis, and membrane trafficking. Environ Exp Bot 208:105252

Thabet SG, Moursi YS, Karam MA, Börner A, Alqudah AM (2020) Natural variation uncovers candidate genes for barley spikelet number and grain yield under drought. Stress 11:533

Thabet SG, Moursi YS, Sallam A, Karam MA, Alqudah AM (2021) Genetic associations uncover candidate SNP markers and genes associated with salt tolerance during seedling developmental phase in barley. Environ Exp Bot 188:104499

Thabet SG, Sallam A, Moursi YS, Karam MA, Alqudah AM (2021) Genetic factors controlling nTiO(2) nanoparticles stress tolerance in barley (Hordeum vulgare) during seed germination and seedling development. Functional Plant Biol : FPB 48:1288–1301

Thabet SG, Alomari DZ, Börner A, Brinch-Pedersen H, Alqudah AM (2022) Elucidating the genetic architecture controlling antioxidant status and ionic balance in barley under salt stress. Plant Mol Biol 110:287–300

Thabet SG, Alqahtani MD, Jabbour AA, Alqudah AM (2023) Genetic associations underpinning the metabolite-mediated salt stress tolerance in Barley. Plant molecular biology reporter

Wang J, Zhang Z (2021) GAPIT version 3: boosting power and accuracy for genomic association and prediction. Gen, Proteom & Bioinform 19:629–640

Wang W, Liu B, Xu M, Jamil M, Wang G (2015) ABA-induced CCCH tandem zinc finger protein OsC3H47 decreases ABA sensitivity and promotes drought tolerance in Oryza sativa . Biochem Biophys Res Commun 464:33–37

Wang M, Zhao X, Xiao Z, Yin X, Xing T, Xia G (2016) A wheat superoxide dismutase gene TaSOD2 enhances salt resistance through modulating redox homeostasis by promoting NADPH oxidase activity. Plant Mol Biol 91:115–130

Wang Y, Branicky R, Noë A, Hekimi S (2018) Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling. J Cell Biol 217:1915–1928

Wang X, Xie H, Wang P, Yin H (2023) Nanoparticles in plants: uptake, transport and physiological activity in leaf and root. Mater (Basel, Switzerland) 16:3097

Xia L, Sun S, Han B, Yang X (2023) NAC domain transcription factor gene GhNAC3 confers drought tolerance in plants. Plant Physiol Biochem 195:114–123

Xiong L, Ishitani M, Lee H, Zhu JK (2001) The Arabidopsis LOS5/ABA3 locus encodes a molybdenum cofactor sulfurase and modulates cold stress-and osmotic stress-responsive gene expression. The Plant cell 13:2063–2083

CAS   PubMed   PubMed Central   Google Scholar  

Xue Z, Wang B, Qu C, Tao M, Wang Z, Zhang G, Zhao M, Zhao S (2023) Response of salt stress resistance in highland barley ( Hordeum vulgare L. var. nudum) through phenylpropane metabolic pathway. PloS One 18:e0286957

Yadav A, Yadav K, Abd-Elsalam KA (2023) Nanofertilizers: types delivery and advantages in agricultural sustainability. Agrochemicals 2(296):336

Yu CW, Murphy TM, Lin CH (2003) Hydrogen peroxide-induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Functional Plant Biol : FPB 30:955–963

Zhang X, Gonzalez-Carranza ZH, Zhang S, Miao Y, Liu C-J, Roberts JA (2019) F-Box Proteins in Plants. Annu Plant Rev Online 2:307–328

Zhang L, Jiang Q, Zong J, Guo H, Liu J, Chen J (2024) Effects of supplemental potassium on the growth, photosynthetic characteristics, and ion content of Zoysia matrella under salt. Stress 10:31

Zhao C, Zhang H, Song C, Zhu J-K, Shabala S (2020) Mechanisms of plant responses and adaptation to soil salinity. The Innovation 1:100017

Zhou T, Yang X, Wang L, Xu J, Zhang X (2014) GhTZF1 regulates drought stress responses and delays leaf senescence by inhibiting reactive oxygen species accumulation in transgenic Arabidopsis. Plant Mol Biol 85:163–177

Zhou S-M, Kong X-Z, Kang H-H, Sun X-D, Wang W (2015) The involvement of wheat f-box protein gene TaFBA1 in the oxidative stress tolerance of plants. PloS One 10:e0122117

Download references

Acknowledgments

The authors would like to thank Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R318), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia

This study is supported by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2024R318), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Author information

Authors and affiliations.

Department of Botany, Faculty of Science, Fayoum University, Fayoum, 63514, Egypt

Samar G. Thabet

Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh, 11671, Saudi Arabia

Fatmah Ahmed Safhi

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), OT Gatersleben, Corrensstr 3, D-06466, Seeland, Germany

Andreas Börner

Biological Science Program, Department of Biological and Environmental Sciences, College of Arts and Sciences, Qatar University, Doha, Qatar

Ahmad M. Alqudah

You can also search for this author in PubMed   Google Scholar

Contributions

SGT designed the experiment and analyzed the data. SGT wrote the manuscript. SGT, FAS, AB, and AMA edited the manuscript. SGT, FAS, AB, and AMA performed the experiments. SGT conceived the idea and participated in the interpretation of the results.

Corresponding author

Correspondence to Samar G. Thabet .

Ethics declarations

Conflict of interest.

The authors have no competing interests.

Ethical approval

The authors declare that they have no conflicts of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

Additional information

Publisher's note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PPTX 1253 KB)

Supplementary file2 (xlsx 52 kb), rights and permissions.

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Thabet, S.G., Safhi, F.A., Börner, A. et al. Genome-wide association scan reveals the reinforcing effect of nano-potassium in improving the yield and quality of salt-stressed barley via enhancing the antioxidant defense system. Plant Mol Biol 114 , 97 (2024). https://doi.org/10.1007/s11103-024-01489-y

Download citation

Received : 08 April 2024

Accepted : 17 June 2024

Published : 09 September 2024

DOI : https://doi.org/10.1007/s11103-024-01489-y

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Potassium nanoparticles
• Candidate genes
• Linkage disequilibrium
• Find a journal
• Publish with us
• Track your research

• Open access
• Published: 10 September 2024

Promoting salt tolerance, growth, and phytochemical responses in coriander ( Coriandrum sativum L. cv. Balady) via eco-friendly Bacillus subtilis and cobalt

• Sary H. Brengi   ORCID: orcid.org/0000-0003-2241-7061 1 ,
• Maneea Moubarak   ORCID: orcid.org/0000-0002-3986-9294 1 ,
• Hany M. El-Naggar   ORCID: orcid.org/0000-0002-9579-627X 2 &
• Amira R. Osman   ORCID: orcid.org/0000-0001-7497-6426 1  

BMC Plant Biology volume  24 , Article number:  848 ( 2024 ) Cite this article

Metrics details

In plant production, evaluation of salt stress protectants concerning their potential to improve growth and productivity under saline stress is critical. Bacillus subtilis (Bs) and cobalt (Co) have been proposed to optimize salt stress tolerance in coriander ( Coriandrum sativum L. cv. Balady) plants by influencing some physiological activities. The main aim of this work is to investigate the response of (Bs) and (Co) as eco-safe salt stress protectants to resist the effect of salinity, on growth, seed, and essential oil yield, and the most important biochemical constituents of coriander produced under salt stress condition. Therefore, in a split-plot factorial experiment design in the RCBD (randomized complete block design), four levels of salinity of NaCl irrigation water (SA) were assigned to the main plots; (0.5, 1.5, 4, and 6 dS m −1 ); and six salt stress protectants (SP) were randomly assigned to the subplots: distilled water; 15 ppm (Co1); 30 ppm (Co2); (Bs); (Co1 + Bs); (Co2 + Bs). The study concluded that increasing SA significantly reduced coriander growth and yield by 42.6%, which could be attributed to ion toxicity, oxidative stress, or decreased vital element content. From the results, we recommend that applying Bs with Co (30 ppm) was critical for significantly improving overall growth parameters. This was determined by the significant reduction in the activity of reactive oxygen species scavenging enzymes: superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) and non-enzyme: proline by 5, 11.3, 14.7, and 13.8% respectively, while increasing ascorbic acid by 8% and preserving vital nutrient levels and enhancing plant osmotic potential to buffer salt stress, seed yield per plant, and essential oil yield increased by 12.6 and 18.8% respectively. The quality of essential oil was indicated by highly significant quantities of vital biological phytochemicals such as linalool, camphor, and protein which increased by 10.3, 3.6, and 9.39% respectively. Additional research is suggested to determine the precise mechanism of action of Bs and Co's dual impact on medicinal and aromatic plant salt stress tolerance.

Peer Review reports

Introduction

Climate change has recently been linked to a significant increase in salt stress. Thus saline irrigation water has a severe negative impact on agricultural, horticultural, medicinal, and aromatic plant productivity globally and reduced the potential yield of annual crops by around fifty percent [ 1 , 2 ]. Salt stress promotes abiotic stress, which results in water deficiency; a lack of essential nutrients such as K + ; and Na + toxicity inside plants, which causes decreased photosynthetic efficiency and biomass accumulation; PAL gene expression; stomatal closure; reduced leaf expansion; and antioxidant defense systems, all of which influence important molecules involved in medicinal plant synthesis, such as the total phenolic content, lipids, proteins, and DNA; thus, salt stress affects plant production quality [ 3 , 4 ]. As a result, new approaches for dealing with the threat of global salt stress to agricultural production are needed. Several solutions, including the genetic selection of plant cultivars that are most tolerant species to salt stress, plant genetic engineering, and the introduction of plant growth-promoting bacteria (PGPB), have been devised to mitigate the detrimental effects of high salt concentrations on plant growth [ 5 , 6 ].

Bacillus subtilis bacteria are among the best-performing endophytic microorganism eco-safe organisms and may influence plant growth and create systemic resistance to abiotic stresses, such as drought, salinity, and heavy metals, through their participation in antioxidant activities and phenylpropanoid metabolism, optimizing the synthesis of plant hormones such as auxins and gibberellins, or serving as plant protection agents against microbial infections [ 7 ]. The stimulating effect of these plants by endophytic microorganisms may influence the plant to be a limitless source of novel metabolites, the application of which could reduce agrochemicals, thus improving agricultural, horticultural, medicinal, and aromatic plants; furthermore, such plants could be used as base materials in a multitude of categories, such as cosmetics, pharmaceuticals, and food industry production [ 8 , 9 ]. B. subtilis has been proven to protect numerous medicinal and aromatic plant species from various abiotic stressors, including sweet basil [ 10 , 11 ], peppermint [ 12 ], Jew’s Mallow [ 13 ], turmeric [ 14 ], rosemary [ 15 ], and ginger [ 16 ].

Cobalt is an essential micronutrient of cobalamin (vitamin B 12 ) that is a cofactor for a wide variety of enzymes and an essential component of several proteins in prokaryotes and mammals. Reduced activity of this enzyme can cause megaloblastic anemia [ 17 ]. Co can alleviate abiotic stressors such as salt and drought by acting as a component of several enzymes and coenzymes, positively affecting plant development and metabolism, regulating ion homeostasis, and modifying phytohormones at low concentrations [ 18 , 19 , 20 , 21 , 22 ]. However, the impact of Co on redox equilibrium under salt stress has received little attention. Cobalt and salt together enhance the stress tolerance of several medicinal and aromatic plant species, including Salvia officinalis L. [ 23 ], barley and wheat [ 24 ], and Pennisetum divisum [ 25 ].

Coriander ( Coriandrum sativum L.) is an annual herb in the Apiaceae (Umbelliferae) family. It is a very essential medicinal and fragrant herbaceous plant that has been used extensively in folk medicine for centuries as a digestive system regulator, diuretic, antibacterial, vegetable, appetizer, and condiment [ 26 , 27 ]. The plant's green or dry leaves are used in daily human food as a condiment for flavoring salads, soups, various pastries, pickles and seeds (fruit) are used to flavor falafel, which is a traditional Egyptian dish [ 9 ]. Coriander is a medicinal plant that contains a high concentration of essential oils in its roots, leaves, stems, flowers, fruits, and seeds. One of the greatest intriguing options in the culinary, agrochemical, pesticide, cosmetics, and pharmaceutical industries is coriander essential oil, which contains important biological components such as borneol, camphor, cineole, geranyl acetate, coriandrol, cymene, dipentene, pinene geraniol, linalool, phellandrene terpineol, terpinene, and terpinolene [ 28 , 29 , 30 ]. Furthermore, they significantly affect plant tolerance to various stress environments [ 31 ]. It is also famous for its high concentration of medicinal agents, such as anticonvulsants, antidiabetic, antimicrobial, antioxidant, and other compounds, which protect human body cells from sickness [ 32 , 33 ].

Therefore, the current research was designed to investigate the response of Bacillus subtilis (Bs) and cobalt (Co) [CoSO 4 .7 H 2 O] as eco-safe salt stress protectants to resist the effect of salinity, on growth, seed and essential oil yield, and the most important biochemical constituents of coriander produced under salt stress condition.

Materials and methods

Cultivation and plant material.

To determine the efficacy of using various eco-safe salt stress protectants to reduce the deleterious effects of salinity stress on growth parameters, characteristics, chlorophyll content, dry leaf content, K:Na ratio, electrolyte leakage, antioxidant enzyme activity, Asco, proline content in leaves, some biological phytochemicals in seed oil, and seed and oil yield parameters of coriander plants, two pot experiments were conducted on a private farm in Abu Hommus, El-Beheira Governorate, Egypt (31° 5′ 35" north, 30° 18′ 51" east), during two successive winter seasons, from October to April 2022 and 2023. The local variety of coriander seeds ( Coriandrum sativum  L., cv. Balady) was obtained from the Agriculture Administration Abu Hommus, El-Beheira Governorate, Egypt. The seeds were uniformly sized, and five seeds were seeded in black plastic pots (30 cm in diameter × 35 cm in height) filled with 10 kg of soil Fig.  1 . The best three plants were preserved after germination, and the physio-chemical properties of the soil were analyzed before planting [ 34 , 35 ] (Table  1 ).

a The main effect of the four levels of NaCl irrigation water salinity (SA): tap water as a control S0, 0.5; S1, 1.5; S2, 4; and S3, 6 dS m. −1 . b , c , d , and e The interaction between the four salinity levels (S0, S1, S2, and S3) respectively and the six salt stress protectants: distilled water Cont.; 15 ppm (Co1); 30 ppm (Co2); (Bs); (Co1 + Bs); (Co2 + Bs) 50 days after seed sowing of Coriandrum sativum L. cv. Balady. Where: the control (Cont.), B. subtilis (Bs) and cobalt (Co)

Experimental design and treatments

Two factors in a split-plot factorial experiment were arranged by a randomized complete block design with five replications. The main plots were four salinity levels (SA); tap water as a control (Cont.) 0.5, 1.5, 4, and 6 dS m −1 , and subplots were the six eco-safe salt stress protectants (SP): distilled water as a control (Cont.), cobalt [CoSO 4 .7 H 2 O] (Merck Life Science Ltd. Cairo, Egypt) (Co1) at 15 ppm, (Co2) at 30 ppm, and Bacillus subtilis (Bs) (Microbial Culture Network Ain Shams University Cairo, Egypt) at 0.4 L per hectare (8 × 10 8  spores mL −1 ), (Co1 + Bs), and (Co2 + Bs). All possible combinations of two factors were performed (4 × 6 = 24 treatments and 120 experimental units) (Table S1). The treatments were applied with irrigation one week after sowing the coriander seeds and one week after each harvest, using the same amount of water. The Ministry of Agriculture's instructions for cultivating coriander plants in clay loam soil included supplemental fertilizer requirements and agricultural strategies necessary for its growth were applied.

Growth parameters, characteristics, and photosynthetic pigment concentrations in fresh leaves

After 50 days after sowing, the following data were recorded: the number of leaves (NL), leaf length (LL) (cm), shoot fresh weight (SFW) (g), root fresh weight (RFW) (g), root dry weight (RDW) (g), plant fresh weight (PFW) (g), plant dry weight (PDW) (g), plant height (Ph) (cm), and chlorophyll content (SPAD Unit) using a chlorophyll meter (SPAD-502 m, Konica Minolta, Japan). Dry weights were measured after drying the vegetative parts and roots for 72 h at a temperature of 70°C.

Element content, K:Na ratio, electrolyte leakage, and protein content

Nine plants were picked, cleansed with tap water, and subsequently rinsed three times with distilled water. The samples were subsequently dried in a forced-air oven at 70°C until they reached a consistent weight. Finally, the samples were ground in a Willy mill and filtered using a 30-mesh screen. A total of 0.2 g of dried fine powder from the sampled leaves was digested with a combination of hydrogen peroxide and sulfuric acid (El Gomhoria for Medicines and Medical Supplies Trading Alexandria, Egypt.) [ 36 ]. Kjeldahl's approach was used to calculate the percentage of total nitrogen (N) in dry leaves utilizing mineral analysis [ 37 ]. The amounts of potassium (K), phosphorus (P), sodium (Na), and chloride (Cl) were determined using the methods described by Cottenie et al. [ 38 ]. The protein concentration was measured according to Krul [ 39 ]. Electrolyte leakage was utilized to determine the integrity of the cell membranes, and the measurements were performed following the approach of Lutts et al. [ 40 ].

Determination of the activity of antioxidant enzymes, total protein content, Asco, and proline content in leaves

Gao et al. [ 41 ] studied the activity of the enzyme superoxide dismutase (SOD) (EC 1.15.1.1). The ideal amount of enzyme extract required to produce 50% inhibition of photochemical degradation of NBT was found to be one unit of SOD activity. According to Aebi [ 42 ], the elimination of H 2 O 2 from crude extract samples revealed catalase (CAT) activity (EC 1.11.1.6). The activity of lipid peroxidation was determined by measuring the levels of malondialdehyde (MDA) using a technique proposed by Gérard-Monnier et al. [ 43 ]. The entire set of enzymes was obtained using the method provided by Agarwal et al. [ 44 ]. In particular, 100 mg of freshly collected coriander leaves was crushed in an extraction buffer cooled with ice. Bovine serum albumin (Sigma-Aldrich, Inc. St. Louis, MO. United States) was utilized as a standard for determining the total protein content of the raw extract [ 45 ]. A spectrophotometer (Unico W49376 Spectrophotometer 1200, Shanghai, China) with a 525 nm wavelength was used to detect the presence of ascorbic acid in the supernatants. A standard curve was created using ascorbic acid as an analytical reagent from Solarbio. The quantity of ascorbic acid is represented as milligrams per 100 g of fresh weight using the approach given by Srivastava and Singh [ 46 ]. The proline content of the leaves was measured using the approach proposed by Bates et al. [ 47 ].

Determination of biological phytochemicals in seed oil, seed yield per plant, leaf oil, and essential oil yield

After the seeds matured, a representative sample of six random plants was randomly selected from each experimental unit to calculate the seed yield per plant (SYP). The percentage of essential oil yield (EOYS) consumed was measured using a Clevenger apparatus according to Guenther [ 48 ], and the following oil phytochemicals were measured: linalool (%), γ-terpinene (%), α-pinene (%), p-cymene (%), camphor (%), and geranyl acetate (%) using Agilent Gas Chromatography-Mass Spectrometry (GC‒MS) according to El-Kinany et al. [ 49 ]. The phytochemicals of the essential oils were determined by their retention time and correlation of mass spectra with those of the NIST and Wiley library databases. Leaf essential oil (LO) was extracted according to the approach of El-Massry et al. [ 50 ]. Fresh coriander leaves were cut into small fragments and subjected to water distillation for three hours using Clevenger equipment. The essential oils were collected, dehydrated with anhydrous sodium sulfate, and stored in sealed glass vials lined with aluminum foil at 20°C.

Statistical analysis and experimental design

In our experiment, the design was established as a split-plot design in the RCBD (randomized complete block design), where NaCl levels were assigned to the main plots, and the protective treatments were randomly assigned to the subplots. The experiment included 24 treatments that consisted of four NaCl concentrations and six protective treatments, five replicates (five pots for each treatment, with three plans for each pot). All the data were analyzed statistically by the CoStat statistical software program version 6.4; Co Hort, USA [ 51 ] and are presented as the average of the two seasons. At a probability level of p  < 0.05, the means were compared using the least significant difference (LSD) analysis.

Plant growth parameters, morphology, and chlorophyll content

The means, of both seasons (2022 and 2023) for the NL, LL, SFW, RFW, RDW, PFW, PDW, Ph, and chlorophyll content of Coriandrum sativum L. cv. Balady, are presented in Additional file 1: Table S1 and (Fig.  2 A, B, C, D, E, F, G, H, and I) (for details of two seasons, each in Additional file 2: Table S1). Among the studied coriander growth parameters, the chlorophyll content and morphology significantly differed when the salt stress level increased to S3 (6 dS m −1 ), as illustrated in Fig.  1 a. Similarly, the mean values of the two seasons decreased by approximately 38, 37, 55, 37, 22, 55, 44, 26, and 12% for the NL, LL, SFW, RFW, RDW, PFW, PDW, Ph, and chlorophyll content, respectively.

Growth parameters and characteristics of Coriandrum sativum L. cv. Balady; A  number of leaves “NL”, B  leaf length “LL” (cm), C  shoot fresh weight “SFW” (g), D  root fresh weight “RFW” (g), E  root dry weight “RDW” (g), F  plant fresh weight “PFW” (g), G  plant dry weight “PDW” (g), H  plant height “Ph” (cm), and I  chlorophyll content (SPAD Unit) as influenced by six salt stress protectants (SP ) , 15 ppm cobalt (Co1), 30 ppm (Co2), B. subtilis (Bs), (Co1 + Bs), (Co2 + Bs), and distilled water as a control (Cont.) and four levels of NaCl irrigation water salinity (SA); tap water as a control S0 = 0.5, S1 = 1.5, S2 = 4, and S3 = 6 dS m. −1 . Bars with the same lowercase letters are not significantly different at the P  < 0.05 level. The data are presented as the mean ± SE. The statistics are provided in additional file 1: Table S1

Despite the salt stress, the cobalt and B. subtilis treatments had significant effects on all the studied coriander economic growth parameters (Fig.  1 b, c, d and e, and Fig.  2 A, B, C, D, E, F, G, H, and I), where the mean values of the coriander economic growth metrics were found to be the highest (Co2 + Bs), which increased the NL, LL, SFW, RFW, RDW, PFW, PDW, Ph, and chlorophyll content by approximately 30, 21, 28, 17, 18, 28, 29, 7, and 6%, respectively, compared with those of the control. Co1 + Bs had the second most significant effect after Co2 + Bs treatment on the mean average of all the economic growth parameters of the coriander in the two seasons, as shown in Additional file 1: Table S1 (Fig.  2 A, B, C, D, E, F, G, H, and I). However, the lowest mean values in both seasons were achieved with the control treatment.

In general, different salt stress protectants had significant positive effects on the means of all the parameters, NL, LL, SFW, RFW, RDW, PFW, PDW, Ph, and chlorophyll content of the coriander plants in both seasons, the interactions between cobalt at 30 and 15 ppm, and B. subtilis in irrigation water produced the best mean values (NL 17, LL 22 cm, SFW 14 g, RFW 5 g, RDW 0.8 g, PFW 44 g, PDW 5 g, Ph 88 cm, and 44 SPAD). All the treatments increased the growth metrics of the stressed and non-stressed plants compared to those of the control plants (Additional file 1: Table S1) (Fig.  1 b to e and Fig.  2 A, B, C, D, E, F, G, H, and I).

Elemental analysis and electrolyte leakage

In general, Additional file 1: Table S2 and (Fig.  3 A, B, C, D, E, F, and G) present the means of nitrogen, phosphorus, potassium, sodium, and chlorine; the K:Na ratio; and electrolyte leakage of dry leaves in coriander plants for both seasons (2022 and 2023) (for details of two seasons, each alone in Additional file 2: Table S2). The leaf element content of Coriander plants was modulated by irrigation water salinity stress, salt stress protection, and their interaction.

Elemental contents; A  nitrogen “N” (%), B  phosphorus “P” (%), C  potassium “K” (%), D  sodium “Na” (%), E  chlorine “Cl” (%), F  K:Na ratio, and G  electrolyte leakage “EL” (%) of dry leaves in Coriandrum sativum L. cv. Balady as influenced by six salt stress protectants (SP ) , 15 ppm cobalt (Co1), 30 ppm (Co2), B. subtilis (Bs), (Co1 + Bs), (Co2 + Bs), and distilled water as a control (Cont.) and four levels of NaCl irrigation water salinity (SA); tap water as a control S0 = 0.5, S1 = 1.5, S2 = 4, and S3 = 6 dS m. −1 . Bars with the same lowercase letters are not significantly different at the P  < 0.05 level. The data are presented as the mean ± SE. The statistics are provided in additional file 1: Table S2

There were correlations between increased NaCl concentrations and increasing Na + , Cl − , and electrolyte leakage levels. The leaf contents of Na + and Cl − and electrolyte leakage increased with increasing NaCl concentration (Fig.  3 D, E, and G). However, there was a definite inverse relationship between the N, P, and K + contents and between the K:Na ratio and NaCl concentration (Fig.  3 A, B, C, and F) (Additional file 1: Table S2). Separated or combined cobalt and Bacillus subtilis treatments significantly reduced the Na + and Cl- levels as well as electrolyte leakage in coriander leaves (Fig.  3 D, E, and G). Compared to those of the control, the other treatments had significant influences on increasing the N, P, and K + contents (Fig.  3 A, B, and C) and (Additional file 1: Table S2).

Overall, the use of various SP increased the N, P, and K + contents and the K:Na ratio in coriander leaves grown under SA at the S1, S2, or S3 levels compared to those grown under the S0 level in both seasons. S0, S1, S2, and S3, (Co2 + Bs) had the highest mean N, P, and K + contents and K:Na ratio, followed by (Co1 + Bs). Conversely, the lowest means were observed in the (Cont.) with S3, followed by S2, S1, and S0 (Fig.  3 A, B, C, and F) and (Additional file 1: Table S2). The lowest means of Na + , Cl − , and electrolyte leakage in dry leaves of coriander plants in both seasons were recorded for Co2 + Bs, followed by Co1 + Bs under S0, S1, S2, and S3, respectively (Fig.  3 D, E, and G) and (Additional file 1: Table S2).

Antioxidant enzymes, Asco, and proline

Our results showed that irrigation water salinity, salt stress protection agent application, and their interaction significantly ( P  < 0.05) affected the activities of the antioxidative enzymes SOD, CAT, and MDA; Asco; and proline in coriander leaves (Additional file 1: Table S3) and (Fig.  4 A, B, C, D, and E) (for details of two seasons, each alone in Additional file 2: Table S3). Similarly, the activities of SOD, CAT, MDA, and proline increased significantly when the salt stress level increased to S3 (Fig.  4 A, B, C, and E). Conversely, when the Asco decreased significantly (Fig.  4 D).

Determination of the activities of antioxidant enzymes: A  superoxide dismutase “SOD” (U/mg protein); B  catalase “CAT” (U/mg protein); C  malondialdehyde “MDA” (nmol g f wt −1 ); D  ascorbic acid “Asco” (mg 100 g f wt −1 ); and E  proline “Pro” (µg 100 g d wt −1 ) content in the leaves of Coriandrum sativum L. cv. Balady, as influenced by six salt stress protectants (SP ) , 15 ppm cobalt (Co1), 30 ppm (Co2), B. subtilis (Bs), (Co1 + Bs), (Co2 + Bs), and distilled water as a control (Cont.) and four levels of NaCl irrigation water salinity (SA); tap water as a control S0 = 0.5, S1 = 1.5, S2 = 4, and S3 = 6 dS m. −1 . Bars with the same lowercase letters are not significantly different at the P  < 0.05 level. The data are presented as the mean ± SE. The statistics are provided in additional file 1: Table S3

The combination of Co2 with Bs had significant effects on reducing SOD, CAT, MDA and proline levels, with mean values of 5.0, 0.4, 0.94 (mg 100 g f wt −1 ), and 380.70 (µg 100 g d wt −1 ), respectively, compared to those of the control, with mean values of 5.27, 0.45, 1.10 (mg 100 g f wt −1 ), and 441.77 (µg 100 g d wt −1 ), for both seasons (Additional file 1: Table S3) and (Fig.  4 A, B, C, and E). In contrast, the Asco increased significantly (Fig.  4 D) to 30.28 (mg 100 g f wt −1 ). In addition, Co1 or Co2 in combination with Bs had a decrease in SOD, CAT, and MDA activity and proline levels, but (Co2 + Bs) had the greatest reduction in both S2 and S3 (Additional file 1: Table S3) (Fig.  4 A, B, C, and E). Compared to those in the control treatment, the Co2 + Bs treatment had the most significant impact on increasing Asco in S2 and S3 (Additional file 1: Table S3) (Fig.  4 D).

Important phytochemicals in the seed oil of coriander

The means of both seasons (2022 and 2023) for the important biological phytochemicals linalool, γ-terpinene, α-pinene, p-cymene, camphor, geranyl acetate, and protein of coriander are illustrated in Additional file 1: Table S4 and Fig.  5 A, B, C, D, E, F, and G (for details of two seasons, each alone is shown in Additional file 2: Table S4). The findings revealed that the phytochemicals present in the oil varied depending on the treatment and their interactions. The levels of γ-terpinene, α-pinene, p-cymene, camphor, and geranyl acetate significantly increased when the amount of salt stress increased to S3 (Fig.  5 B, C, D, E, and F). However, the linalool and protein levels decreased significantly (Fig.  5 A and G).

Important biological phytochemicals related to the seed oil of Coriandrum sativum L. cv. Balady, A  linalool (%), B  γ-terpinene (%), C  α-pinene (%), D  p-cymene (%), E  camphor (%), F  geranyl acetate (%), and G  protein (%) influenced by six salt stress protectants (SP ) , 15 ppm cobalt (Co1), 30 ppm (Co2), B. subtilis (Bs), (Co1 + Bs), (Co2 + Bs), and distilled water as a control (Cont.) and four levels of NaCl irrigation water salinity (SA); tap water as a control S0 = 0.5, S1 = 1.5, S2 = 4, and S3 = 6 dS m. −1 . Bars with the same lowercase letters are not significantly different at the P  < 0.05 level. The data are presented as the mean ± SE. The statistics are provided in additional file 1: Table S5

Cobalt and B. subtilis treatments alone or in combination had significant influences on the increase in linalool, camphor, and protein levels relative to those in the control (Fig.  5 A, E, and G). However, the same tendency was not observed for the levels of γ-terpinene and p-cymene, as no significant differences were detected for their corresponding (cont.) (Fig.  5 B and D). However, the levels of α-pinene and geranyl acetate decreased significantly (Fig.  5 C and F). The varied concentrations of Co with Bs reversed the negative effect of salinity, causing a reduction in linalool, camphor, and protein levels compared with those of the stressed plants without such application (Cont.) (Fig.  5 A, E, and G). At varying levels of salt stress, the lowest reductions in linalool, camphor, and protein levels were found in the Co2 + Bs treatment group. Furthermore, Co1 performed similarly to the other treatments in terms of reducing the amounts of α-pinene and geranyl acetate under varying levels of salt stress (Fig.  5 C and F).

Coriander yield parameters of seeds and oil

An increase in salinity to the greatest concentration in both seasons significantly reduced the weight of the seed yield per plant and the percentage of essential oil yield of the coriander plants by approximately 42% and 35%, respectively. However, the leaf oil percentage significantly increased with increasing salinity up to the highest concentration, reaching approximately 14% in both seasons under the same amount of salt stress (Additional file 1: Table S5) and (Fig.  6 A, B, and C) (for details of the two seasons, each alone in Additional file 2: Table S5).

Yield parameters of Coriandrum sativum L. cv. Balady seed and oil; A  seed yield per plant “SYP” (g), B  leaf oil “LO” (%), and C  essential oil yield “EOYS” influenced by six salt stress protectants (SP ) , 15 ppm cobalt (Co1), 30 ppm (Co2), B. subtilis (Bs), (Co1 + Bs), (Co2 + Bs), and distilled water as a control (Cont.) and four levels of NaCl irrigation water salinity (SA); tap water as a control S0 = 0.5, S1 = 1.5, S2 = 4, and S3 = 6 dS m. −1 . Bars with the same lowercase letters are not significantly different at the P  < 0.05 level. The data are presented as the mean ± SE. The statistics are provided in additional file 1: Table S3

The combination or combination of Co and Bs had a significantly positive effect on both the weight of the seed yield per plant and the percentage of essential oil yield of the coriander plants, regardless of salt stress. However, both the Co2 and (Co1 + Bs) treatments had the same positive effect on preventing the leaf oil percentage from decreasing. The interaction effect between salinity and salt stress protection agent treatment had significant effects on the coriander yield, percentage of essential oil yield, and leaf oil percentage (Additional file 1: Table S5) (Fig.  6 A, B, and C).

Under low, moderate, and high salinity (S1, S2, and S3), (Co2 + Bs) exhibited the greatest improvement, but at the same level, in terms of seed yield per plant and percentage of essential oil yield but not in leaf oil percentage. The highest seed yield per plant and the percentage of essential oil yield were obtained at (Co2 + Bs), followed by (Co1 + Bs) for the Coriander plants even with the high salinity (S3). However, at the same salt level, the Co2 treatments produced equivalent results in terms of leaf oil percentage after (Cont.) Under the unstressed salt treatment (S0) and (Co2 + Bs), the highest values of seed yield per plant and percentage of essential oil yield were observed, followed by the values in (Co1 + Bs) when compared with the corresponding values (Cont.), Additional file 1: Table S5 and Fig.  6 A, B, and C (for details of two seasons, each alone in Additional file 2: Table S5).

Our results revealed significant negative responses of all the recorded growth parameters, as well as morphological, biochemical, and physiological processes, in coriander plants exposed to NaCl salt stress, which is more harmful to plants than other salt-induced salinities. The same results were reported by Dustgeer et al. [ 52 ] and Sadak et al. [ 53 ] as they mentioned salt stress inhibits plant growth quality and productivity in maize and white lupine in a variety of ways, such as ionic and osmotic imbalance; a lack of water and nutrient availability, such as N, P, and K; decreased photosynthesis pigment synthesis; and disturbance of photosynthetic activity, plant cells experienced an instant response to salt stress, while osmotic stress was brought on by ions surrounding roots that inhibited their ability to absorb water, which causes limited growth. The number of leaves, leaf length, shoot fresh weight, root fresh weight, root dry weight, plant fresh weight, plant dry weight, plant height, and SPAD index were significantly lower as the salinity concentration increased in coriander [ 7 , 49 ] and Moringa [ 4 ]; these decreases in all growth parameters under salt stress could be attributed to thylakoid enlargement, ion accumulation, and functional abnormalities observed during stoma closure and opening, as well as harm to the photosynthetic machinery and chloroplast structure, reactive oxygen species under salinity stress primarily target chloroplasts, leading to the destruction of photosynthetic pigments and thylakoid membranes. Increased salt ion accumulation not only impaired morphological growth but also had a significant negative response to the percentage of N, P, and K and the K:Na ratio in coriander leaves; earlier research supported similar findings in coriander plants [ 49 , 54 ]; this decrease may be due to the low osmotic potential of the soil solution, overaccumulation of Na and Cl in the cells, and competition with Na, resulting in a significant positive response in the percentage of electrolyte leakage [ 55 ]. Recent findings have shown that increased salt ion accumulation causes an increase in antioxidant defense mechanisms, such as superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA), and the accumulation of osmolytes, such as proline, in all plant cells to maintain turgor pressure, which is vital for cell expansion. However, Asco was reduced, several authors have noted this occurrence [ 56 , 57 , 58 ] in wheat and Hassanein et al. 2022 [ 59 ] in coriander. According to these investigations, one of the reasons why salinized coriander plants were unable to tolerate salt stress was a decrease in Asco. Ascorbic acid is a tiny, water-soluble antioxidant that plays an important role in the cyclic pathway of H 2 O 2 enzymatic detoxification. Furthermore, increased amino acid generation, particularly proline generation, is indirectly associated with the scavenging of oxidative stress and prevents cellular damage by stimulating the production of several antioxidant enzymes, such as SOD, CAT, and MDA, which are vital for osmotic stress resistance in plants under abiotic stress [ 60 , 61 , 62 , 63 ]. Acimovic et al. [ 64 ] studied coriander plants and discovered that increasing the concentration of bio compounds such as γ-terpinene, α-pinene, p-cymene, camphor, and geranyl acetate in fruit essential oils had a significant response to abiotic stress, such as salinity, at the maximum NaCl concentration. These results were found to be in line with the current results. On the other hand, protein levels decreased, which may be attributed to the rate of biosynthesis and degradation associated with N, P, and K accumulation, which decreased as salt ion levels increased because these are important nutrients that play a major role in protein synthesis and metabolism in coriander plants. Additionally, reduced linalool levels are correlated with increased salt stress and abiotic stress [ 64 , 65 ]; these findings are consistent with the present study. Our results illustrated that salt stress adversely affects the seed yield per plant (SYP) and essential oil yield (EOYS %), possibly due to limited macro and micronutrient availability; in particular, the adsorption of N, P, and K under increased salt stress causes a decrease in the biosynthesis of amino acids, which subsequently negatively affects the growth rate; the relative content of leaf water, protein, and chlorophyll contents in coriander plants; the exchange of gases with the net CO 2 acceptance rate; the photosynthesis efficiency; the transpiration rate, all of which are required for flowering and pollination; and even the eventual transfer of assimilates and filling of seeds (SYP and EOYS) [ 66 , 67 , 68 ]. In contrast, when LO levels increase, a greater LO quantity under saline conditions could be connected to an increase in the number and density of oil glands [ 69 ].

In this study, the preventative effects of Bs and Co on coriander plants exposed to salt stress were investigated and compared. When coriander plants growing under salt stress were treated with Co or Bs, whether together or separately, our results revealed a significant positive response of all the recorded growth parameters; the number of leaves, leaf length, shoot fresh weight, root fresh weight, root dry weight, plant fresh weight, plant dry weight, plant height, and SPAD index when compared to those of nontreated plants; however, the concentration of Co applied to the plant also played an important role. Several studies have shown that some halophilic Bacillus species, as dominant bacterial genera under salt stress, act by externally protecting the cell wall, endospore-forming capacity, healthy cell membrane, and enzymatic mechanisms to handle and regulate environmental dangers. Furthermore, Bs exhibit a variety of metabolic and adaptable reactions to varied water supplies, water consumption efficiencies, available N, P, K oxygen, and organic/inorganic materials [ 70 ] in Capsicum [ 71 ] . Species such as B. subtilis potentially minimize soil N loss [ 72 ]; accelerate N uptake and transport [ 73 ]; and produce indole-3-acetic acid (IAA), cytokinins, siderophores, and gibberellins, which tend to promote plant growth characteristics by encouraging significant cell division, proliferation, and differentiation in the meristematic area; light and gravity responses; root and shoot initiation, development and elongation; vegetative growth; and yield development [ 74 , 75 ]. Along with the endogenous IAA content, bacterial IAA loosens plant cell walls; induces cell division in plants; leads to the production of ACC deaminase; decreases plant ethylene levels; and changes in root form and development; increases surface area, volume, and length; and promotes indole-mediated signaling [ 76 ] in Withania somnifera , which allows the plant to obtain nutrients effectively under salt stress conditions and C-N metabolism control; and improves the conversion of inorganic N to amino acids, resulting in significant plant growth stimulation [ 77 , 78 , 79 , 80 ]. Tryptophan is the primary precursor of bacterial indole chemical products detected in root secretions, and it enhances protein synthesis and RNA polymerase activity [ 81 , 82 ]. Jie et al. [ 83 ], preserve ionic equilibrium in plants under salt stress by enhancing the preferential absorption of K over Na into stressed plant root xylem; increasing or regulating K + /Na + ratios; and increasing phosphorus and iron availability, all of which significantly increase plant growth parameters, fresh root weight, dry root weight in blackberry plants [ 84 ], yield, and quality of several medicinal plants [ 85 , 86 ]. Vardharajula et al. [ 87 ] studied Zea mays , Chun et al. [ 88 ] tomatoes, Yasmeen et al. [ 89 ] sunflower plants, Medeiros and Bettiol [ 90 ] reported that Bs not only increased amino acid and carbohydrate uptake and buildup, which may act as essential N and/or C suppliers for increased production of proteins and peptides but also reduced the antioxidant activity of enzymes, such as CAT, SOD, and MDA, in Camellia sinensis and quinoa [ 72 , 84 ]. However, as the Asco concentration increased, the Asco concentration significantly influenced the defense system of the plants. Additionally, Bs significantly reduced proline buildup and electrolyte leakage; these findings are consistent with our present findings. Consequently, proteins trigger protoplasm and vegetative growth, and more nitrogen is provided. Moreover, a plant with several leaves can produce a significant amount of seeds and essential oils [ 91 ] in Coriandrum sativum [ 92 ] Foeniculum vulgare [ 93 ] and chamomile plants as demonstrated by the high fresh and dry weight, and height [ 70 , 94 ]. Furthermore, phosphate-solubilizing bacteria enhance grain yield, herbage and oil yield, chlorophyll, and photosynthetic leaf area but decrease the number, diameter, and distribution of glandular hairs per leaf where oil is produced and preserved, resulting in a decrease in leaf oil in Mentha piperita medicinal plants under stress [ 95 ]. Bacterial growth regulators may control the majority of metabolic and physiological activities, including metabolic rates and essential oil synthesis, in medicinal and aromatic plants. Moreover, increasing concentrations of essential oil can increase the quantity of essential oil [ 96 ] in Ocimum [ 97 ] in Salvia and [ 98 ] in ajwain and change the composition of essential oil, impacting the process of terpenoid biosynthesis to generate a stress factor that triggers defense mechanisms [ 74 ]. Secondary metabolite regulation or accumulation and flavonoid, phenolic, and hydrogen peroxide accumulation in plants are indicators of healthy adaptation to stress, as these compounds coordinate osmotic balance, preserve cytoplasmic macromolecules from water loss, and act as free radical scavengers [ 99 ]. Our results revealed that Bs significantly increased the levels of major essential oil components, which is consistent with the findings of several studies; for example, the levels of linalool in Mentha piperita [ 100 ] and Ocimum basilicum [ 101 ]; the contents of camphor in Salvia officinalis [ 102 ]; and the contents of geranyl acetate [ 103 ], the increase in essential oil components may be correlated with macronutrients; N, P, K availability; uptake [ 104 ]; and plant cells [ 105 ] in Mentha piperita [ 106 ] in basil, which fix free atmospheric nitrogen through leaves and roots [ 31 , 107 ]. However, the amount of α-pinene fluctuates toward lower concentrations and has no significant effect on the levels of γ-terpinene or p-cymene in  O. syriacum [ 108 ].

Our findings revealed that the level of Co had a significant impact on improving the NL, LL, SFW, PFW, RFW, RDW, PDW, Ph, and chlorophyll content during coriander plant growth compared with those of the control, and treatment with 30 ppm Co (Co2) outperformed treatment with 15 ppm Co (Co1). This difference may be attributed to the increase in root N, P, and K + uptake; increase in K + content; and decrease in Na, Cl, and electrolyte leakage in leaves, which were also observed in our study and are important for coriander plant resistance to salt stress. Recent studies have additionally demonstrated that the effect of Co on growth is connected to a rise in the macronutrient levels of stressed plants in Bhendi [ 109 ], stimulating hormone synthesis (increased expression of auxins and gibberellins) and decreasing the activity of several enzymes (catalase and peroxidase), thus enhancing anabolism and reducing catabolism [ 18 , 110 ]. Co is an essential micronutrient for vitamin B 12 [ 17 ] and is necessary for various enzymes and proteins and regulates N2 fixation and cell balance through interactions with other critical micronutrients, such as iron (Fe), nickel (Ni), and zinc (Zn), in plant metabolism [ 24 , 111 ]. Co significantly impacts methionine synthesis, which in turn promotes protein synthesis, ribonucleotide reductase is a vitamin B 12 -dependent enzyme that converts ribonucleotides to deoxyribonucleotides, a process that reduces the rate of DNA synthesis [ 112 ]. Peroxidases are isoenzymes that catalyze redox reactions, including the breakdown of hydrogen peroxide. Han et al. [ 113 ] discovered that Co affects peroxidase efficiency by binding to certain amino acids near the enzyme's active regions in horseradish. Co reduces the activity of the antioxidant enzymes SOD, CAT, and MDA. This difference may be responsible for the decrease in ROS levels resulting from CO application [ 114 , 115 ]. Its application to coriander plants enhances the levels of nonenzymatic antioxidants such as Asco and Pro in response to increasing Co concentrations [ 116 ], which are also useful for decreasing injury to the cell membrane via oxidative stress because of their antioxidative activity in plants [ 117 ]. Co treatments have been shown to increase coriander herb yield; mineral and phytochemical contents; and seed oil constituents, such as linalool, SYP, LO, and EOYS, while also preserving γ-terpinene, p-cymene, camphor, and geranyl acetate compared to those of control plants [ 118 ]. This could be due to numerous stimulating effects on hormonal synthesis and metabolism, which are ascribed to catalase and peroxidase activities that improve all development indices of Lemongrass ( Cymbopogon citratus ) [ 119 ] and peppermint [ 120 ], these results were confirmed to be consistent with the existing results.

Salt stress inhibits plant root growth and development, leading to reduced nutrient uptake. We noticed that, compared with the other treatments of eco-safe salt stress protection agents, the interaction of Co at 30 ppm with Bs had synergistic effects on most of the growth parameters and characteristics of coriander plants, particularly those under significant salt level stress. Several studies have shown that cobalt [CoSO 4 .7 H 2 O] and Bacillus subtilis play independent roles [ 121 ]. The beneficial effects of the combined treatment could be attributed to the fact that particular bacteria demand submicromolar levels of cobalt for growth and have specific systems for cobalt absorption, release, and transcriptional modification that preserve cobalt homeostasis [ 122 ]. Adding cobalt to the Bacillus environment increases the capacity of bacteria to create extracellular alkaline phosphatase [ 123 ]. The concentration of cobalt is associated with its ability to promote the synthesis of alkaline phosphatase, which accounts for a large portion of all proteins produced during the late logarithmic and initial stationary stages of development [ 124 ]. However, the effect of cobalt in combination with Bacillus subtilis on optimizing the productivity of medicinal and aromatic plants under salt stress has rarely been studied. Taken together, the results of the present study showed the combined effects of bacteria and cobalt, especially the combined effects of Co2 + Bs, and the optimal results may be attributed to the rapid release of available nitrogen synthesized by root rhizobia to the plant during vegetative growth; high soil nitrogen fixation; rapid availability of N, P, and K in the rhizosphere; decreased aggregation and absorption of Na + and Cl − ; and osmotic correction of the Na + /K + ratio, which increases photosynthesis pigments, such as total chlorophyll, NL, LL, SFW, RFW, RDW, PFW, PDW, Ph, and chlorophyll content, from improved nodulation; the same results were reported previously [ 125 , 126 ]. In addition, plant phytohormones, indole-3-acetic acid (IAA), cytokinins, siderophores, and gibberellins are modified and produced under environmental stress [ 127 , 128 ], and protein synthesis and RNA polymerase activity are enhanced, encouraging significant cell division, proliferation, and differentiation in the meristematic area [ 129 , 130 ]. cell balance through interactions with other critical micronutrients; decrease in ROS levels; CAT, SOD, MDA, and proline levels; and electrolyte leakage [ 71 , 131 ]. The yield, quality, and therapeutic value of the coriander essential oil are based on its linalool, γ-terpinene, p-cymene, camphor, and protein contents. It is crucial for the cultivation of one of the most economically significant medicinal and aromatic plants, particularly when exposed to salt stress. Our results showed that Co and Bs, either together or individually, increase the amount of these compounds as well as yield. This difference may be related to the superior ability of these materials to reduce Na + and Cl − in coriander leaves. Shahid et al. [ 132 ] and Ha-Tran et al. [ 133 ] reported that reducing Na + and Cl − in plants significantly contributes to yield improvement under salinity stress.

The protective effects of eco-safe Bs and Co under salt stress have been evaluated through comparisons of coriander plant herb growth, yield, essential oil productivity, and physiological and phytochemical responses. Our unique findings revealed that the combination of Bs with Co (30 ppm) can significantly minimize salt stress-induced damage and optimize the growth of coriander plants through the reduction of ROS-scavenging enzymes such as SOD, CAT, and MDA and non-enzyme such as proline while preserving ascorbic acid and vital nutrient levels and enhancing plant osmotic potential to buffer salt stress. Concurrently, the seed yield per plant, leaf oil yield, essential oil yield, and quality have been indicated by the high quantities of the vital biological phytochemicals linalool, γ-terpinene, camphor, geranyl acetate, and protein. Furthermore, the protective effect of the Bs treatment was greater than that of the Co treatment (30 ppm or 15 ppm) in lowering the Na + and Cl − contents, which may lead to greater total yield and quality under excessive salt stress. This investigation provides vital details regarding the role of Co in redox homeostasis and the involvement of Bs in optimizing the quality and quantity of coriander plant productivity under salt stress. Interestingly, further investigations are suggested to clarify the exact mechanism of action of the dual impact of Bs and Co on medicinal and aromatic plant salt stress tolerance.

Availability of data and materials

This published paper and the supplementary data contain all the data created or analyzed during this investigation.

Sadak MS, Dawood MG, El-Awadi MES. Changes in growth, photosynthetic pigments and antioxidant system of Hordeum vulgare plant grown under salinity stress via signal molecules application. Vegetos. 2024. https://doi.org/10.1007/s42535-024-00879-3 .

Article   Google Scholar  

Sadak MS, Dawood MG. Biofertilizer role in alleviating the deleterious effects of salinity on wheat growth and productivity. Gesunde Pflanzen. 2023;75:1207–19. https://doi.org/10.1007/s10343-022-00783-3 .

Article   CAS   Google Scholar  

Sadak MS, Hanafy RS, Elkady FMAM, Mogazy AM, Abdelhamid MT. Exogenous calcium reinforces photosynthetic pigment content and osmolyte, enzymatic, and non-enzymatic antioxidants abundance and alleviates salt stress in bread wheat. Plants. 2023;12:1532. https://doi.org/10.3390/plants12071532 .

Article   PubMed   PubMed Central   CAS   Google Scholar  

Osman A, El-Naggar H. Enhancing salinity stress tolerance and phenylalanine ammonia-lyase gene activity using osmolytes in Moringa seedling production. Ann Agric Sci. 2022;67:127–35. https://doi.org/10.1016/j.aoas.2022.06.002 .

Lastochkina O, Pusenkova L, Yuldashev R, Babaev M, Garipova S, Blagova D, Khairullin R, Aliniaeifard S. Effects of  Bacillus subtilis  on some physiological and biochemical parameters of  Triticum aestivum  L. (wheat) under salinity. Plant Physiol Biochem. 2017;121:80.  https://doi.org/10.1016/j.plaphy.2017.10.020 .

Ke J, Wang B, Yoshikuni Y. Microbiome engineering: synthetic biology of plant-associated microbiomes in sustainable agriculture. Trends Biotechnol. 2021;39:244–61. https://doi.org/10.1016/j.tibtech.2020.07.008 .

Article   PubMed   CAS   Google Scholar  

Dibyajyoti N, Selvi AD, Thiyageshwari S, Anandham R, Venkatesan K. Silicon sources and bacterial inoculants on growth parameters, leaf yield, quality of Coriander ( Coriandrum sativum L.) and soil adsorbed silicon in sandy loamy soil. Int J Plant Soil Sci. 2022;34(22):194–208. https://doi.org/10.9734/ijpss/2022/v34i2231372 .

Ek-Ramos MJ, Gomez-Flores R, Orozco-Flores AA, Rodríguez-Padilla C, González-Ochoa G, Tamez-Guerra P. Bioactive products from plant-endophytic gram-positive bacteria. Front Microbiol. 2019;10:463. https://doi.org/10.3389/fmicb.2019.00463 .

Article   PubMed   PubMed Central   Google Scholar  

Gül V, Öztürk E. The effect of different microbial fertilizer doses on yield and yield components in coriander ( Coriandrum sativum L.). Acta Sci Pol Hortorum Cultus. 2021;20(4):59–67.  https://doi.org/10.24326/asphc.2021.4.5 .

Sayed SA, Atef AS, Soha E. Response of three sweet basil cultivars to inoculation with Bacillus subtilis and arbuscular mycorrhizal fungi under salt stress conditions. Nat Sci. 2011;9(6):31–36. https://api.semanticscholar.org/CorpusID:89408648 .

Mohamed HI, Gomaa EZ. Effect of plant growth promoting Bacillus subtilis and Pseudomonas fluorescens on growth and pigment composition of radish plants ( Raphanus sativus ) under NaCl stress. Photosynthetica. 2012;50(2):263–72. https://doi.org/10.1007/s11099-012-0032-8 .

Para S, Thulasi MS, Penchalaneni J, Challagundla VN. Antibacterial activity and phytochemical analysis of Mentha piperita L. (Peppermint)—An important multipurpose medicinal plant. Am J Plant Sci. 2013;4:77–83. https://doi.org/10.4236/ajps.2013.41012 .

Egamberdieva D, Jabborova D. Efficiency of phytohormone-producing Pseudomonas to improve salt stress tolerance in Jew’s Mallow ( Corchorus olitorius L.). Plant-growth-promoting Rhizobacteria (PGPR) and Medicinal Plants. 2015;201–13. https://doi.org/10.1007/978-3-319-13401-7_9 .

Kumar A, Monika S, Prem PS, Sandeep KS, Pawan KS, Kapil DP. Isolation of plant growth promoting rhizobacteria and their impact on growth and curcumin content in Curcuma longa L. Biocatal Agri Biotechnol. 2016;8:1–7. https://doi.org/10.1016/j.bcab.2016.07.002 .

Abdel-Rahman SSA. Adverse effects of salt stress on root ability of Rosmarinus officinalis cuttings and their alleviation by indole-3-butyric acid (IBA) and Bacillus subtilis . Egypt J Hort. 2019;46(2):279–293. https://doi.org/10.21608/ejoh.2019.18698.1121 .

Jabborova D, Enakiev Y, Sulaymanov K, Kadirova D, Ali A, Annapurna K. Plant growth promoting bacteria Bacillus subtilis promote growth and physiological parameters of Zingiber officinale . Plant Sci Today. 2021;8(1):66–71. https://doi.org/10.14719/pst.2021.8.1.997 .

Osman D, Cooke A, Young T, Deery E, Robinson N, Warren M. The requirement for cobalt in vitamin B12: a paradigm for protein metalation. BBA-Mol Cell Res. 2021;1868: 118896. https://doi.org/10.1016/j.bbamcr.2020.118896 .

Nadia G, Nagwa MKH, El Sayed SAA. Influence of cobalt on tolerating climatic change (salinity) in onion plant with reference to physiological and chemical approach. Plant Archiv. 2020;20(1):1496–1500. https://api.semanticscholar.org/CorpusID:228083184 .

Akeel A, Jahan A. Role of cobalt in plants: Its stress and alleviation. Contaminants in Agriculture. Imp Manag. 2020;339–357. https://doi.org/10.1007/978-3-030-41552-5_17 .

Brengi SH, Abd-Allah EMK, Ibrahim AA. Effect of melatonin or cobalt on growth, yield and physiological responses of cucumber ( Cucumis sativus L.) plants under salt stress. J Saudi Soc Agri Sci. 2022;21(1):51–60. https://doi.org/10.1016/j.jssas.2021.06.012 .

Elshamly AMS, Nassar SMA. The impacts of applying cobalt and chitosan with various water irrigation schemes at different growth stages of corn on macronutrient uptake, yield, and water use efficiency. J Soil Sci Plant Nutr. 2023;23:2770–85. https://doi.org/10.1007/s42729-023-01233-3 .

Elshamly AMS. Cobalt combined with potassium humate as beneficial applications in alleviating water stress impacts on groundnut during sensitive growth stages. J Soil Sci Plant Nut. 2023;23:4505–20. https://doi.org/10.1007/s42729-023-01367-4 .

Hülya T. Cobalt+Salt-Stressed Salvia officinalis : ROS scavenging capacity and antioxidant potency. Intern J Second Metab. 2019;6(1):49–61. https://doi.org/10.21448/ijsm.484954 .

Hu X, Wei X, Ling J, Chen J. Cobalt: an essential micronutrient for plant growth? Front Plant Sci. 2021;12: 768523. https://doi.org/10.3389/fpls.2021.768523 .

Alhammad BA, Saleem K, Asghar MA, Raza A, Ullah A, Farooq TH, Yong JWH, Xu F, Seleiman MF, Riaz A. Cobalt and titanium alleviate the methylglyoxal-induced oxidative stress in Pennisetum divisum seedlings under saline conditions. Metabolites. 2023;13:1162. https://doi.org/10.3390/metabo13111162 .

Shahwar MK, El-Ghorab AH, Anjum FM, Butt MS, Hussain S, Nadeem M. Characterization of coriander ( Coriandrum sativum L.) seeds and leaves: Volatile and nonvolatile extracts. Int J Food Prop. 2012;15:736–747. https://doi.org/10.1080/10942912.2010.500068 .

Kadıoğlu B. Effect of Bacillus sp. application on the germination of coriander ( Coriandrum sativum L.) under salt stress. Muş Alparslan Univ J Agri Nature. 2023;3(2):102–108. https://doi.org/10.59359/maujan.1288669 .

Bhuiyan MNI, Begum J, Sultana M. Chemical composition of leaf and seed essential oil of Coriandrum sativum L. from Bangladesh. Bangladesh J Pharmacol. 2009;4:150–153. https://doi.org/10.3329/bjp.v4i2.2800 .

Nadeem M, Faqir MA, Muhammad IK, Tehseen S, El-Ghorab A, Javed IS. Nutritional and medicinal aspects of coriander ( Coriandrum sativum L.): A Review. Br Food J. 2013;115:743–755. https://doi.org/10.1108/00070701311331526 .

Sarkic A, Stappen I. Essential oils and their single compounds in cosmetics—A critical review. Cosmetics. 2018;5:11. https://doi.org/10.3390/cosmetics5010011 .

Çakmakçı R, Mosber G, Milton AH, Fırat A, Baboo A. The Effect of Auxin and auxin-producing bacteria on the growth, essential oil yield, and composition in medicinal and aromatic plants. Curr Microbiol. 2020;77:564–77. https://doi.org/10.1007/s00284-020-01917-4 .

Aftab A, Haider MA, Ali Q, Malik A. Genetic evaluation for morphological traits of coriandrum sativum grown under salt stress. Biol Clin Sci Res J. 2021;2021:52. https://doi.org/10.54112/bcsrj.v2021i1.52 .

Al-Khayri JM, Banadka A, Nandhini M, Nagella P, Al-Mssallem MQ, Alessa FM. Essential oil from Coriandrum sativum : A review on its phytochemistry and biological activity. Molecules. 2023;28:696. https://doi.org/10.3390/molecules28020696 .

Page A, Miller R, Keeneny D. Methods of soil analysis. Part 2. Chemical and microbiological properties. 2nd edition. Agron. Madison, Wisconsin, USA. 1982;9. https:// https://doi.org/10.2134/agronmonogr9.2.2ed .

Jackson ML. Soil chemical analysis. Prentice Hall of India Pvt. Ltd., New Delhi. 1958;205. https://api.semanticscholar.org/CorpusID:96801453 .

Cottenie A. Soil and plant testing as a basis of fertilizer recommendation. F.A.O. Soil Bull. 1980. https://api.semanticscholar.org/CorpusID:128551876 .

Baker WH, Thompson TL. Determination of total nitrogen in plant samples by Kjeldahl. In plant analysis reference for the southern region of the United States, southern cooperative series, bulletin #368; The University of Georgia: Athens, GA. 1992;368:13–16.‏ http://www.cropsoil.uga.edu/~oplank/sera368.pdf .

Cottenie A, Verloo M, Kiekens L, Velgh G, Camerlynk R. Chemical analysis of plants and soils. lab. Agroch. State Univ. Gent, Belgium. 1982;63:44–45. https://api.semanticscholar.org/CorpusID:134929157 .

Krul ES. Calculation of Nitrogen-to-Protein Conversion Factors: A Review with a Focus on Soy Protein. J Am Oil Chem Soc. 2019;96:339–64. https://doi.org/10.1002/aocs.12196 .

Lutts S, Bouharmont J, Kinet JM. Physiological characterisation of salt-resistant rice ( Oryza sativa ) somaclones. Aust J Botany. 1999;47(6):835–49. https://doi.org/10.1071/bt97074 .

Gao Y, Fang L, Wang X, Lan R, Wang M, Du G, Zhao H. Antioxidant activity evaluation of dietary flavonoid hyperoside using saccharomyces cerevisiae as a model. Molecules. 2019;24(4):788. https://doi.org/10.3390/molecules24040788 .

Aebi H. Catalase in vitro Meth Enzymol. 1984;105:121–6. https://doi.org/10.1016/S0076-6879(84)05016-3 .

Gérard-Monnier D, Erdelmeier I, Régnard K, Moze-Henry N, Yadan JC, Chaudière J. Reactions of 1-Methyl-2- phenylindole with malondialdehyde and 4-Hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation. Chem Res Toxicol. 1998;11:1176–1183. https://doi.org/10.1021/tx9701790 .

Agarwal PK. Role of protein dynamics in reaction rate enhancement by enzymes. J Am Chem Soc. 2005;127(43):15248–56. https://doi.org/10.1021/ja055251s.s001 .

Bradford MM. A rapid and sensitive method for the quantification of protein dye binding. Analy Biochem. 1976;72:248–54. https://doi.org/10.1016/0003-2697(76)90527-3 .

Srivastava A, Singh SK. Determination of vitamin C in chemical, pharmaceutical and biological samples by spectrophotometric titrimetry with o-diacetoxyiodobenzoate. Analysis of mixtures of vitamin C with methionine and cysteine or glutathione. Analyst. 1988;113(2):259–262.‏ https://doi.org/10.1039/AN98813FX005 .

Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant Soil. 1973;39:205–7. https://doi.org/10.1007/BF00018060 .

Guenther E, Kulka K, Rogers JA. Essential Oils and Related Products. Anal Chem. 1961;(4). https://doi.org/10.1021/ac60173a004 .

El-Kinany RG, Berengi SHM, Nassar ANK, El-Batal A. Enhancement of plant growth, chemical composition and secondary metabolites of essential oil of salt-stressed coriander ( coriandrum sativum l.) plants using selenium, nanoselenium, and glycine betaine. Sci J Flowers Ornam Plants. 2019;6(3):151–173. https://doi.org/10.21608/sjfop.2019.84973 .

El-Massry KF, Farouk A, Abou-Zeid M. Free radical scavenging activity and lipoxygenase inhibition of rosemary ( Rosmarinus Officinalis L) volatile oil. J Ess Oil Bear Plants. 2008;11(5):536–43. https://doi.org/10.1080/0972060X.2008.10643663 .

CoStat. CoStat-Statistics Software version 6.400. Monterey: CoHort Software; 2008.

Dustgeer Z, Seleiman MF, Khan I, Chattha MU, Ali EF, Alhammad BA, Jalal RS, Refay Y, Hassan MU. Glycine-betaine induced salinity tolerance in maize by regulating the physiological attributes, antioxidant defense system and ionic homeostasis. Not Bot Horti Agrobot Cluj-Napoca. 2021;49:12248. https://doi.org/10.15835/nbha49112248 .

Sadak MS. Physiological role of arbuscular mycorrhizae and vitamin B1 on productivity and physio-biochemical traits of white lupine ( Lupinus termis  L.) under salt stress. Gesunde Pflanzen. 2023;75:1885–1896. https://doi.org/10.1007/s10343-023-00855-y .

Elhindi KM, El-Hendawy S, Abdel-Salam E, Schmidhalter U, Rehman S, Hassan AA. Foliar application of potassium nitrate affects the growth and photosynthesis in coriander ( Coriander sativum L.) plants under salinity. Prog Nut. 2016;18(1):63–73.

CAS   Google Scholar  

Hniličková H, Hnilička F, Martinkova J, Kraus K. Effects of salt stress on water status, photosynthesis and chlorophyll fluorescence of rocket. Plant Soil Environ 2017;63(8):362–7. https://doi.org/10.17221/398/2017-PSE .

Sadak MS, Sekara A, Al-ashkar I, Habib-ur-Rahman M, Skalicky M, Brestic M, Kumar A, Sabagh AE, Abdelhamid MT. Exogenous aspartic acid alleviates salt stress induced decline in growth by enhancing antioxidants and compatible solutes while reducing reactive oxygen species in wheat. Front Plant Sci. 2022;13: 987641. https://doi.org/10.3389/fpls.2022.987641 .

Akyol TY, Yilmaz O, Uzilday B, Uzilday RO, Turkan I. Plant response to salinity: an analysis of ROS formation, signaling, and antioxidant defense. Turk J Bot. 2020;44:1–13. https://doi.org/10.3906/bot-1911-15 .

Al-Ashkar I, Alderfasi A, Ben RW Seleiman MF, El-Said RA, Al-Doss A. Morphological and genetic diversity within salt tolerance detection in eighteen wheat genotypes. Plants. 2020;9:287. https://doi.org/10.3390/plants9030287 .

Hassanein RA, Hussein OS, Farag IA, Hassan YE, Abdelkader AF, Ibrahim M. Salt-stressed coriander ( Coriandrum sativum L.) responses to potassium silicate, humic acid and gamma irradiation pretreatments. Agronomy. 2022;12:2268. https://doi.org/10.3390/agronomy12102268 .

Sadak MS. Biochemical responses of white termis to pyridoxine and mycorrhizae treatment under salinity stress. Egypt J Chem. 2022;65(10):429–439.  https://doi.org/10.21608/ejchem.2022.118032.5319 .

Gupta A, Mishra R, Rai S, Bano A, Pathak N, Fujita M, Kumar M, Hasanuzzaman M. Mechanistic insights of plant growth promoting bacteria mediated drought and salt stress tolerance in plants for sustainable agriculture. Int J Mol Sci. 2022;23:3741. https://doi.org/10.3390/ijms23073741 .

Ragaey MM, Sadak MS, Dawood MFA, Mousa NHS, Hanafy RS, Latef AAHA. Role of signaling molecules sodium nitroprusside and arginine in alleviating salt-induced oxidative stress in wheat. Plants. 2022;11:1786. https://doi.org/10.3390/plants11141786 .

Abd El-Hameid AR, Sadak MS. Impact of glutathione on enhancing sunflower growth and biochemical aspects and yield to alleviate salinity stress. Biocatal Agric Biotechnol. 2020;29: 101744. https://doi.org/10.1016/j.bcab.2020.101744 .

Acimovic M, Cvetkovic M, Stankovic J. Effect of weather conditions, location and fertilization on coriander fruit essential oil quality. J Ess Oil Bear Plants. 2016;19(5):1208–15. https://doi.org/10.1080/0972060x.2015.1119068 .

Farzad R, Yousef N, Mohammad A, Mohammad BHP, Sina G, Yaghoub P. Fertilizer type and humic acid improve the growth responses, nutrient uptake, and essential oil content on Coriandrum sativum L. Sci Reports. 2022;12:7437. https://doi.org/10.1038/s41598-022-11555-4 .

Arif Y, Singh P, Siddiqui H, Bajguz A, Hayat S. Salinity induced physiological and biochemical changes in plants: an omic approach toward salt stress tolerance. Plant Physiol Biochem. 2020;156:64–77. https://doi.org/10.1016/j.plaphy.2020.08.042 .

Nguyen QH, Talou T, Evon P, Cerny M, Merah O. Fatty acid composition and oil content during coriander fruit development. Food Chem. 2020;326: 127034. https://doi.org/10.1016/j.foodchem.2020.127034 .

Özyazici G. Influence of organic and inorganic fertilizers on coriander ( Coriandrum sativum L.) agronomic traits, essential oil and components under semi-arid climate. Agronomy. 2021;11:1427. https://doi.org/10.3390/agronomy11071427 .

Farouk S, Omar MM. Sweet basil growth, physiological and ultrastructural modification, and oxidative defense system under water deficit and silicon forms treatment. J Plant Growth Regul. 2020;39(3):1307–31. https://doi.org/10.1007/s00344-020-10071-x .

Wang W, Wu Z, He Y, Huang Y, Li X, Ye BC. Plant growth promotion and alleviation of salinity stress in Capsicum annuum L. by Bacillus isolated from saline soil in Xinjiang. Ecotoxicol Environ Saf. 2018;164:520–9. https://doi.org/10.1016/j.ecoenv.2018.08.070 .

El-Shamy MA, Alshaal T, Mohamed HH, Rady AMS, Hafez EM, Alsohim AS, Abd E-M. Quinoa Response to application of phosphogypsum and plant growth-promoting Rhizobacteria under water stress associated with salt-affected soil. Plants. 2022;11(7):872. https://doi.org/10.3390/plants11070872 .

Sun B, Gu LK, Bao LJ, Zhang SW, Wei YX, Bai ZH, Guoqiang Z, Xuliang Z. Application of biofertilizer containing Bacillus subtilis reduced the nitrogen loss in agricultural soil. Soil Biol Biochem. 2020;148: 107911. https://doi.org/10.1016/j.soilbio.2020.107911 .

Yang L, Zhou B, Hou Y, Wang Q, Chen X. Effects of bacillus subtilis on growth of winter wheat and distribution of soil water and nitrogen under salt stress. J Drain Irrig Mach Eng. 2021;39:517–24. https://doi.org/10.3969/j.issn.1674-8530.19.0267 .

Duca D, Lorv J, Patten CL, Rose D, Glick BR. Indole3-acetic acid in plant–microbe interactions. Antonie Van Leeuwenhoek. 2014;106:85–125. https://doi.org/10.1007/s10482-013-0095-y .

Kreiser M, Giblin C, Murphy R, Fiesel P, Braun L, Johnson G, Wyse D, Cohen JD. Conversion of indole-3-butyric acid to indole-3-acetic acid in shoot tissue of hazelnut (Corylus) and elm (Ulmus). J Plant Growth Regul. 2016;35:710–21. https://doi.org/10.1007/s00344-016-9574-5 .

Anuroopa N, Bagyaraj DJ. Selection of an efficient plant growth promoting rhizobacteria for inoculating Withania somnifera . JSIR. 2017;76:244–248. https://api.semanticscholar.org/CorpusID:56196797 .

Etesami H, Alikhani HA, Mirseyed HH. Indol 3-acetic acid (IAA) production trait, a useful screening to select endophytic and rhizosphere competent bacteria for rice promoting agents. MethodsX. 2015;2:72–8. https://doi.org/10.1016/j.mex.2015.02.008 .

Bhattacharyya D, Garladinne M, Lee YH. Volatile indole produced by rhizobacterium Proteus vulgaris JBLS202 stimulates growth of Arabidopsis thaliana through auxin, cytokinin, and brassino steroid pathways. J Plant Growth Regul. 2015;34:158–68. https://doi.org/10.1007/s00344-014-9453-x .

Çakmakçı R. Screening of multi-trait rhizobacteria for improving the growth, enzyme activities, and nutrient uptake of tea ( Camellia sinensis ). Commun Soil Sci Plan. 2016;47:1680–90. https://doi.org/10.1080/00103624.2016.1206559 .

Parray JA, Jan S, Kamili AN, Qadri RA, Egamberdieva D, Ahmad P. Current perspectives on plant growth-promoting rhizobacteria. J Plant Growth Regul. 2016;35:877–902. https://doi.org/10.1007/s00344-016-9583-4 .

Souza R, Ambrosini A, Passaglia LMP. Plant growth promoting bacteria as inoculants in agricultural soils. Genet Mol Biol. 2015;38:401–19. https://doi.org/10.1590/s1415-475738420150053 .

Lin Y, Zhang H, Li P, Juan J, Zhefei L. The bacterial consortia promote plant growth and secondary metabolite accumulation in Astragalus mongholicus under drought stress. BMC Plant Biol. 2022;22:475. https://doi.org/10.1186/s12870-022-03859-4 .

Liu J, Li H, Yuan Z, Feng J, Chen S, Sun G, Wei Z, Tiantian Hu. Effects of microbial fertilizer and irrigation amount on growth, physiology and water use efficiency of tomato in greenhouse. Sci Horticult. 2024;323: 112553. https://doi.org/10.1016/j.scienta.2023.112553 .

Sánchez-Rojas T, Espinoza-Culupú A, Ramírez P, Iwai LK, Montoni F, Macedo-Prada D, Sulca-López M, Durán Y, Farfán-López M, Herencia J. Proteomic study of response to copper, cadmium, and chrome ion stress in  Yarrowia lipolytica  strains isolated from andean mine tailings in Peru. Microorganisms. 2022;10(10). https://doi.org/10.3390/microorganisms10102002 .

Kang SM, Shahzad R, Bilal S, Khan AL, Park YG, Lee KE. Indole-3-acetic acid and ACC deaminase producing Leclercia adecarboxylata MO1 improves Solanum lycopersicum L. growth and salinity stress tolerance by endogenous secondary metabolites regulation. BMC Microbiol. 2019;19(1):1–4. https://doi.org/10.1186/s12866-019-1450-6 .

Saleem S, Iqbal A, Ahmed F, Ahmad M. Phytobeneficial and salt stress mitigating efficacy of IAA producing salt tolerant strains in gossypium hirsutum . Saudi J Biol Sci. 2021;28(9):5317–24. https://doi.org/10.1016/j.sjbs.2021.05.056 .

Vardharajula S, Zulfkar AS, Grover M, Reddy G, Bandi V. Drought-tolerant plant growth promoting Bacillus spp.: effect on growth, osmolytes, and antioxidant status of maize under drought stress. J Plant Interact. 2011;6(1):1–4. https://doi.org/10.1080/17429145.2010.535178 .

Chun SC, Paramasivan M, Chandrasekaran M. Proline accumulation influenced by osmotic stress in Arbuscular Mycorrhizal symbiotic plants. Front Microbiol. 2018;9:2525. https://doi.org/10.3389/fmicb.2018.02525 .

Yasmeen T, Ahmad A, Arif MS, Mubin M, Rehman K, Shahzad SM. Biofilm forming rhizobacteria enhance growth and salt tolerance in sun-flower plants by stimulating antioxidant enzymes activity. Plant Physiol Biochem. 2020;156:242–56. https://doi.org/10.1016/j.plaphy.2020.09.016 .

Medeiros CAA, Bettiol W. Multifaceted intervention of bacillus spp. against salinity stress and fusarium wilt in tomato. J Appl Microbiol. 2021;131(5):2387–2401. https://doi.org/10.1111/jam.15095 .

Mishra BK, Dubey PN, Aishwath OP, Kant K, Sharma YK, Vishal MK. Effect of plant growth promoting rhizobacteria on coriander ( Coriandrum sativum ) growth and yield under semi-arid condition of India. Indian J Agric Sci. 2017;87:607–612. https://doi.org/10.56093/ijas.v87i5.70126 .

Mishra BK, Meena KK, Dubey PN, Aishwath OP, Kant K, Sorty AM, Bitla U. Influence on yield and quality of fennel ( Foeniculum vulgare Mill.) grown under semi-arid saline soil, due to application of native phosphate solubilizing rhizobacterial isolates. Ecol Eng. 2016;97:327–333. https://doi.org/10.1016/j.ecoleng.2016.10.034 .

Mostafa A, Khalafallah M, Sedera SA, Fathy H, Higazy A. Different methods of bacterial inoculation on the yield of chamomile blossoms and essential oil. Global J Environ Sci Manage. 2019;5:237–248. https://doi.org/10.22034/gjesm.2019.02.09 .

Cappellari LR, Santoro MV, Nievas F, Giordano W, Banchio E. Increase of secondary metabolite content in marigold by inoculation promoting rhizobacteria. Appl Soil Ecol. 2013;70:16–22. https://doi.org/10.1016/j.apsoil.2013.04.001 .

Santoro MV, Cappellari LR, Giordano W, Banchio E. Plant growth-promoting efects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study. Plant Biol. 2015;17:1218–26. https://doi.org/10.1111/plb.12351 .

Hazzoumi Z, Moustakime Y, Joutei KA. Effect of gibberellic acid (GA3), indole acetic acid (IAA) and benzylaminopurine (BAP) on the synthesis of essential oils and the isomerization of methyl chavicol and trans-anethole in Ocimum gratissimum L. Springerplus. 2014;3:1–7. https://doi.org/10.1186/2193-1801-3-321 .

Samani MR, Pirbalouti AG, Moattar F, Golparvar AR. L-Phenylalanine and bio-fertilizers interaction effects on growth, yield and chemical compositions and content of essential oil from the sage ( Salvia offcinalis L.) leaves. Ind Crops Prod. 2019;137:1–8. https://doi.org/10.1016/j.indcrop.2019.05.019 .

Rohamare Y, Nikam TD, Dhumal KN. Effect of foliar application of plant growth regulators on growth, yield and essential oil components of Ajwain ( Trachyspermum ammi L.). Int J Seed Spices. 2013;3:34–41. https://api.semanticscholar.org/CorpusID:85998605 .

Awan S, Ilyas N, Khan I, Raza M, Rehman A, Rizwan M, Rastogi A, Tariq R, Brestic M. Bacillus siamensis  reduces cadmium accumulation and improves growth and antioxidant defense system in two wheat ( Triticum aestivum  L.) varieties. Plants. 2020;9:878. https://doi.org/10.3390/plants9070878 .

Cappellari LR, Santoro MV, Reinoso H, Travaglia C, Giordano W, Banchio E. Anatomical, morphological, and phytochemical effects of inoculation with plant growth promoting rhizobacteria on peppermint ( Mentha piperita ). J Chem Ecol. 2015;41:149–58. https://doi.org/10.1007/s10886-015-0549-y .

Monfort LEF, Bertolucci SKV, Lima AF, de Carvalho AA, Mohammed A, Blank AF, Pinto JEBP. Effects of plant growth regulators, different culture media and strength MS on production of volatile fraction composition in shoot cultures of Ocimum basilicum . Ind Crops Prod. 2018;116:231–9. https://doi.org/10.1016/j.indcrop.2018.02.075 .

Ghorbanpour M, Hatami M, Kariman K, Abbaszadeh DP. Phytochemical variations and enhanced efficiency of antioxidant and antimicrobial ingredients in Salvia officinalis as inoculated with different rhizobacteria. Chem Biodivers. 2016;13:319–30. https://doi.org/10.1002/cbdv.201500082 .

Darzi MT, Hadi MHS. Effects of organic manure and nitrogen fixing bacteria on some essential oil components of coriander ( Coriandrum sativum ). IJACS. 2012;4:787–792. https://api.semanticscholar.org/CorpusID:35842431 .

Velmourougane K, Prasanna R, Chawla G, Nain L, Kumar A, Saxena AK. Trichoderma-Azotobacter biofilm inoculation improves soil nutrient availability and plant growth in wheat and cotton. J Basic Microbiol. 2019;59:632–44. https://doi.org/10.1002/jobm.201900009 .

Kaymak HÇ, Dönmez MF, Çakmakçı R. N2-fixing plant growth-promoting rhizobacteria: potential to increase yield, growth and element contents of Mentha piperita L. leaves. Eur J Plant Sci Biotechnol. 2013;1:38–42. https://api.semanticscholar.org/CorpusID:198990749 .

Abdel-Hakim W, Yasser M, Khalid. Response of Ocimum basilicum plant to some plant growth promoting Rhizobacteria. J Product Dev. 2014;19(3):263–286. https://doi.org/10.21608/jpd.2014.42660 .

Borkar SG. Microbes as bio-fertilizers and their production technology. Woodhead Publishing India, New Delhi. 2015. https://doi.org/10.1201/9780367805500 .

Dalia AA, Haroun SA, Omar MN, Abd-Elgawad AM, El-Shobaky AM, Mowafy AM. Fluctuation of essential oil constituents in Origanum syriacum subsp. sinaicum in response to plant growth promoting bacteria. J Ess Oil Bear Plants. 2019;22(4). https://doi.org/10.1080/0972060X.2019.1661794 .

Kaliyamoorthy J, Aksah P. Soil applied cobalt alters the growth parameters and dry matters production of Bhendi ( Abelmoschus esculentus L.). Inn Agri. 2022;5(0):1–3. https://doi.org/10.25081/ia.2022.v5.1078 .

Muhammad AA, Misbah A, Muhammad ZR. Chapter Ten - Role of inorganic bio stimulant elements in plant growth. Sustain Plant Nut. Academic Press. 2023;229–261. https://doi.org/10.1016/B978-0-443-18675-2.00014-6 .

Orji J, Ngumah C, Asor H, Anuonyemere A. Effects of cobalt and manganese on biomass and nitrogen fixation yields of a free-living nitrogen fixer- Azotobacter chroococcum . Eur J Biol Res. 2018;8:7–13. https://doi.org/10.5281/zenodo.1157098 .

Marschner P. Marschner’s Mineral Nutrition of Higher Plants. Amsterdam: Elsevier/Academic Press. 2012. https://doi.org/10.1016/c2009-0-63043-9 .

Han HY, Xu WA, Lu ZR, Zou F, Li S. Activation and inactivation of horseradish peroxidase by cobalt ions. J Biomol Struct Dyn. 2008;26:83–92. https://doi.org/10.1080/07391102.2008.10507226 .

Babaei K, Moghaddam M, Farhadi N, Pirbalouti AG. Morphological, physiological, and phytochemical responses of Mexican marigold ( Tagetes minuta L) to drought stress. Scientia Horticult. 2021;284: 110116. https://doi.org/10.1016/j.scienta.2021.110116 .

Selim S, Akhtar N, Hagagy N, Alanazi A, Warrad M, El Azab E, Elamir MYM, Al-Sanea MM, Jaouni SKA, Abdel-Mawgoud M, Shah AA, Abdelgawad H. Selection of newly identified growth-promoting Archaea haloferax species with a potential action on cobalt resistance in maize plants. Front Plant Sci. 2022;13: 872654. https://doi.org/10.3389/fpls.2022.872654 .

Azarakhsh MR, Asrar Z, Mansouri H. Effects of seed and vegetative stage cysteine treatments on oxidative stress response molecules and enzymes in Ocimum basilicum L. under cobalt stress. J Soil Sci Plant Nut. 2015;15(3):651–662. https://doi.org/10.4067/s0718-95162015005000044 .

Ghosh UK, Islam MN, Siddiqui MN, Cao X, Khan MAR. Proline, a multifaceted signaling molecule in plant responses to abiotic stress: understanding the physiological mechanisms. Plant Biol. 2022;24:227–39. https://doi.org/10.1111/plb.13363 .

Nadia G, Kandil HM. Influence of cobalt nutrition on coriander ( Cariandrum sativum L.) herbs yield quantity and quality. J App Sci Res. 2012;8(10):5184–5189. https://api.semanticscholar.org/CorpusID:202739809 .

Aziz EE, Nadia G. Physiological and chemical response of lemongrass ( Cymbopogon Citratas L.) to cobalt nutrition. A–Herb yield, Essential oil content and composition. J App Sci Res. 2011;7(21):1732–1736. https://api.semanticscholar.org/CorpusID:209466766 .

Aziz EE, Nadia G, Khaled SM. Effect of cobalt on growth and chemical composition of peppermint plant grown in newly reclaimed soil. Aust J Basic App Sci. 2011;5(11):628–633. https://api.semanticscholar.org/CorpusID:30158584 .

Imran A, Zabta KS, Shomaila S, Shaheen S. Plant beneficial endophytic bacteria: Mechanisms, diversity, host range and genetic determinants. Microbiol Res. 2019;221:36–49. https://doi.org/10.1016/j.micres.2019.02.001 .

Sachi O, Lindsay DE. The biological occurrence and trafficking of cobalt. Metallomics. 2011;3(10):963–70. https://doi.org/10.1039/c1mt00056j .

Vora D, Irani K. Effect of Cobalt on synthesis of extracellular alkaline phosphatase production from Bacillus sp. J Microbiol Exp. 2015;2(6):00069. https://doi.org/10.15406/jmen.2015.02.00069 .

Spencer DB, Chen CP, Hulett F. Effect of cobalt on synthesis and activation of Bacillus licheniformis alkaline phosphatase. J Bact. 1981;145(2):926–33. https://doi.org/10.1128/jb.145.2.926-933.1981 .

Baddour AG, El-Sherpiny MA, Sakara HM. Effect of rhizobium inoculant, nitrogen starter and cobalt on stimulation of nodulation, N fixation and performance of faba bean ( Vicia faba L.) grown under salinity stress. J Soil Sci Agric Eng. 2021;12:61–69. https://doi.org/10.21608/jssae.2021.153322 .

Subhasmita M, Radheshyam Y, Ramakrishna W. Bacillus subtilis impact on plant growth, soil health and environment: Dr. Jekyll and Mr. Hyde. J App Micro. 2022;132(5):3543–3562.  https://doi.org/10.1111/jam.15480 .

Glick BR. Plant growth-promoting bacteria: Mechanisms and applications. Scientifica. 2012;2012: 963401. https://doi.org/10.6064/2012/963401 .

Berg G, Grube M, Schloter M, Smalla K. Unraveling the plant microbiome: Looking back and future perspectives. Front Microbiol. 2014;5:148. https://doi.org/10.3389/fmicb.2014.00148 .

Christopher B, Mathilde NC, Akos TK. Molecular aspects of plant growth promotion and protection by Bacillus subtilis . Mol Plant-Microbe Interac. 2021;34(1):15–25. https://doi.org/10.1094/mpmi-08-20-0225-cr .

Gurkeerat S, Mamta P. Bacillus subtilis as a plant-growth-promoting rhizobacteria: a review. Plantarchives. 2022;22(2):100–109. https://api.semanticscholar.org/CorpusID:257271805 .

Seleiman MF, Semida WM, Rady MM, Mohammad GF, Hemida KA, Alhammad BA, Hassan MM, Shami A. Sequential application of antioxidants rectifies ion imbalance and strengthens antioxidant systems in salt-stressed cucumber. Plants. 2020;9:1783. https://doi.org/10.3390/plants9121783 .

Shahid M, Sarkhosh A, Khan N, Balal R, Ali S, Rossi L, Gómez C, Mattson N, Nasim W, Garcia-Sanchez F. Insights into the physiological and biochemical impacts of salt stress on plant growth and development. Agronomy. 2020;10(7):938. https://doi.org/10.3390/agronomy10070938 .

Ha-Tran DM, Nguyen TTM, Hung SH, Huang E, Huang CC. Roles of plant growth-promoting Rhizobacteria (PGPR) in stimulating salinity stress defense in plants: A Review. Int J Mol Sci. 2021;22(6):3154. https://doi.org/10.3390/ijms22063154 .

Download references

Acknowledgements

The authors express their deep gratitude to the Horticulture Department , Agriculture Faculty, Damanhour University, Egypt, and the Department of Floriculture Faculty of Agriculture, Alexandria University, Egypt, for providing the infrastructure, laboratories to conduct this study, chemicals, nurseries, and all the facilities to help accomplish this research.

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research received no external funding.

Author information

Authors and affiliations.

Department of Horticulture, Faculty of Agriculture, Damanhour University, Damanhour, Beheira, 22516, Egypt

Sary H. Brengi, Maneea Moubarak & Amira R. Osman

Department of Floriculture, Faculty of Agriculture, Alexandria University (El-Shatby), Alexandria, 21545, Egypt

Hany M. El-Naggar

You can also search for this author in PubMed   Google Scholar

Contributions

SHB and ARO visualized, designed, and supervised the study; SHB, HME-N, MM, and ARO performed the experiments and statistically analyzed the data; and SHB, ARO, HME-N, and MM wrote and proofread the final paper. All the authors have read and approved the published version of the manuscript.

Corresponding authors

Correspondence to Hany M. El-Naggar or Amira R. Osman .

Ethics declarations

Ethics approval and consent to participate.

Not applicable. All the authors declare and certify that all the work done in this research is the author’s original work, has not been submitted to any other journal for publication, and that the paper is not considered for publication elsewhere. All the data in this paper are original and reflect the active contributions of the author and coauthor leading to the manuscript.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1., additional file 2., rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Brengi, S.H., Moubarak, M., El-Naggar, H.M. et al. Promoting salt tolerance, growth, and phytochemical responses in coriander ( Coriandrum sativum L. cv. Balady) via eco-friendly Bacillus subtilis and cobalt. BMC Plant Biol 24 , 848 (2024). https://doi.org/10.1186/s12870-024-05517-3

Download citation

Received : 23 April 2024

Accepted : 12 August 2024

Published : 10 September 2024

DOI : https://doi.org/10.1186/s12870-024-05517-3

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Endophytic microorganism
• Micronutrient of cobalamin
• Medicinal and aromatic plant
• Oxidative stress

BMC Plant Biology

ISSN: 1471-2229

• General enquiries: [email protected]

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Open access
• Published: 10 September 2024

Inflammation-free electrochemical in vivo sensing of dopamine with atomic-level engineered antioxidative single-atom catalyst

• Xiaolong Gao 1 , 2   na1 ,
• Huan Wei 1   na1 ,
• Wenjie Ma   ORCID: orcid.org/0000-0003-1268-4959 2 , 3 ,
• Wenjie Wu 2 , 3 ,
• Wenliang Ji 1 ,
• Junjie Mao   ORCID: orcid.org/0000-0003-0223-7119 4 ,
• Ping Yu   ORCID: orcid.org/0000-0002-6096-1933 2 , 3 &
• Lanqun Mao   ORCID: orcid.org/0000-0001-8286-9321 1  

Nature Communications volume  15 , Article number:  7915 ( 2024 ) Cite this article

Metrics details

• Heterogeneous catalysis

Electrochemical methods with tissue-implantable microelectrodes provide an excellent platform for real-time monitoring the neurochemical dynamics in vivo due to their superior spatiotemporal resolution and high selectivity and sensitivity. Nevertheless, electrode implantation inevitably damages the brain tissue, upregulates reactive oxygen species level, and triggers neuroinflammatory response, resulting in unreliable quantification of neurochemical events. Herein, we report a multifunctional sensing platform for inflammation-free in vivo analysis with atomic-level engineered Fe single-atom catalyst that functions as both single-atom nanozyme with antioxidative activity and electrode material for dopamine oxidation. Through high-temperature pyrolysis and catalytic performance screening, we fabricate a series of Fe single-atom nanozymes with different coordination configurations and find that the Fe single-atom nanozyme with FeN 4 exhibits the highest activity toward mimicking catalase and superoxide dismutase as well as eliminating hydroxyl radical, while also featuring high electrode reactivity toward dopamine oxidation. These dual functions endow the single-atom nanozyme-based sensor with anti-inflammatory capabilities, enabling accurate dopamine sensing in living male rat brain. This study provides an avenue for designing inflammation-free electrochemical sensing platforms with atomic-precision engineered single-atom catalysts.

Introduction

Synergistic interactions between various neurochemicals are crucial for maintaining brain homeostasis 1 , 2 , 3 , 4 . Precise quantification of neurochemical variations under physiological or pathological conditions is essential for unravelling the molecular mechanisms underlying brain function 5 , 6 , 7 , 8 . Electrochemical sensors based on implantable microelectrodes provide an effective platform for in vivo tracking of neurochemicals, owing to their exceptional spatiotemporal resolution, high selectivity and sensitivity 9 , 10 , 11 , 12 , 13 , 14 . However, the implantation of microelectrodes into brain tissue inevitably triggers the inflammatory response, leading to the excessive generation of reactive oxygen species (ROS) 15 , 16 , 17 , 18 . Elevated ROS induces secondary injury and exacerbates the inflammatory response, and thereby disrupts the neurochemical microenvironment around the microelectrode, leading to electrode fouling and further inaccuracies and unreliability of long-term neurochemical sensing 19 . Therefore, it is imperative to develop an in vivo sensing platform capable of eliminating ROS to ensure precise monitoring of neurochemicals.

To address the challenge mentioned above, we turn to leveraging the properties of antioxidative enzymes or their mimics that can mitigate the negative effects of ROS and inflammation induced by implanted devices, presenting a viable strategy for maintaining sensor performance in vivo. Natural antioxidative enzymes, such as catalase (CAT), peroxidase, and superoxide dismutase (SOD), are highly efficient and versatile biocatalysts that help balance oxidative stress 20 . However, their practical applications are significantly hindered by poor stability, weak environmental tolerance and high costs. As an alternative, nanomaterials with intrinsic enzyme-like activities, known as nanozymes, has emerged as promising candidates for regulating ROS in biological system due to their high stability, customizable structure, and adjustable performance 21 , 22 , 23 . Recently, single-atom catalysts (SACs), which feature coordination-unsaturated single active metal sites and atomic-precision designability, have been identified as a new type of nanozymes with superior enzyme-mimicking performances 24 , 25 , 26 , 27 . To date, a diverse array of single-atom nanozymes (SAzymes) have been successfully employed in anti-inflammatory treatments, cancer therapy and antibacterial applications 28 , 29 , 30 , 31 . Notably, most of these SAzymes are produced through high-temperature carbonization, resulting in carbon supports with inherent high electrode reactivity, which shows great promise in electrochemical sensing 32 , 33 , 34 . Taken together, the implementation of carbon-based antioxidative SAzymes in developing electrochemical sensing platforms with anti-inflammatory capacity would provide an avenue to in vivo neurochemical sensing.

Herein, we report the atomic-level modulation of metal active site using a temperature-controlled pyrolysis strategy to fabricate Fe SACs with dual functions: mimicking antioxidative enzyme and acting as electrode material for electrochemical in vivo sensing (Fig.  1 ). We employ iron phthalocyanine (FePc)-encapsulated metal-organic framework (MOF) MET-6 (FePc@MET-6) as the precursor to prepare a series of hierarchical porous nitrogen-dopped carbon-supported Fe SACs (i.e., Fe 1 /NC SACs) with different coordination environments (e.g., FeN 5 , FeN 4 , and FeN 3 C) by varying the calcination temperatures. We find that the Fe 1 /NC SAC with FeN 4 active sites shows excellent CAT- and SOD-mimicking property and hydroxyl radical (•OH) eliminating ability for ROS scavenging as well as electrode reactivity for the oxidation of neurochemicals, e.g., dopamine (DA). This finding offers great possibilities in developing platforms with improved reliability for long-term in vivo sensing of neurochemicals.

Schematic illustration of inflammation-free electrochemical in vivo sensing with Fe 1 /NC-900 SAC.

Synthesis and characterization of Fe 1 /NC SACs

In our previous study, we proposed a concept of SAzyme and reported an iron-based antioxidative SAzyme 35 . Since Fe is the active metal center in most natural CATs for catalytic decomposition of H 2 O 2 , we aimed to develop Fe-based SACs with similar catalytic functions by mimicking the active site structure of CAT. In addition, Fe is known to exhibit versatile redox chemistry, allowing it to participate in various reduction-oxidation reactions. This redox activity of Fe is crucial for enabling CAT-like activity and other antioxidative activities in scavenging ROS. Fe 1 /NC SACs were synthesized using an in-situ trapping-pyrolysis method with FePc@MET-6 as precursor at different temperatures, i.e., 800, 900, and 1000 °C, referred to as Fe 1 /NC-T (T represents the specific pyrolysis temperature) (Fig.  2a ). MET-6, composed of Zn clusters and triazolate ligands, was chosen because the volatilization of Zn and decomposition of high-energy triazoles during high-temperature pyrolysis facilitate the generation of gas, forming hierarchical porous carbon network structure that enhances mass transfer and offers accessible active sites for catalysis 36 . Control samples denoted as NC-T were also synthesized with the same procedure but without FePc. Both MET-6 and FePc@MET-6 show similar octahedral morphology and X-ray diffraction (XRD) peaks (Supplementary Figs.  1 and 2 ), indicating that FePc does not significantly affect the structure and morphology of MET-6 or the microporous structure (Supplementary Fig.  3 and Supplementary Table  1 ). However, after high-temperature calcination, all precursors transformed from an octahedral morphology to a hierarchical porous structure. The obtained Fe 1 /NC SACs show no observable particles in scanning electron microscopy (SEM) and transmission electron microscope (TEM) images (Fig.  2b, c and Supplementary Figs.  4 and 5 ). Element mapping images demonstrate the uniform distribution of Fe, N, and C in Fe 1 /NC SACs (Fig.  2d and Supplementary Fig.  6 ). Aberration-corrected high-angle annular dark-field scanning TEM (AC HAADF-STEM) images show that Fe exists as single atoms for all three Fe 1 /NC SACs (Fig.  2e–g ).

a Scheme of the synthesis of Fe 1 /NC SACs. SEM ( b ) and TEM ( c ) images of Fe 1 /NC-900. d HAADF-STEM image and corresponding EDS elemental mapping images of Fe 1 /NC-900. AC HAADF-STEM images of Fe 1 /NC-800 ( e ), Fe 1 /NC-900 ( f ) and Fe 1 /NC-1000 ( g ). Each experiment was repeated independently three times with similar results. Representative images are shown.

In addition, the XRD patterns of Fe 1 /NC SACs and NC samples show a diffraction peak near 23.3°, corresponding to the (002) crystal plane of graphitic carbon, with no other peaks associated with nanoparticles, further indicating the absence of metal particles in Fe 1 /NC SACs (Supplementary Fig.  7 ) 37 . Raman spectra demonstrate that all the catalysts display two peaks around 1335.7 cm −1 and 1575.4 cm −1 , assigned to the D band and G band, respectively (Supplementary Fig.  8 ) 38 . Among the NC samples, NC-900 possesses the lowest intensity ratio of I D / I G , which represents the extent of graphitization and is closely related to the calcination temperature, demonstrating the highest degree of graphitization for NC-900 (Supplementary Table  2 ) 39 . Fe 1 /NC SACs show a similar trend with NC catalysts, indicating that the introduction of Fe has little effect on the graphitization of the catalysts. X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma mass spectrometry (ICP-MS) characterizations show similar Fe content in all three Fe 1 /NC SACs (Supplementary Fig.  9 and Supplementary Tables  3 and 4 ), while the carbon and nitrogen contents of Fe 1 /NC SACs depend on the pyrolysis temperature. With increasing the pyrolysis temperature (i.e., 800, 900, and 1000 °C), the carbon content gradually increases (78.18%, 82.07%, and 83.13%, respectively), and the nitrogen content gradually decreases (9.48%, 6.14%, and 5.54%, respectively). In addition, the binding energy of Fe shifts negatively with increasing temperature, indicating a decrease in the valence state of Fe and the attenuation of the interaction between Fe and N (Supplementary Fig.  9c ) 40 . Moreover, the high-resolution N 1s XPS spectra of Fe 1 /NC can be deconvoluted into three peaks located at around 398.5 eV, 400.0 eV, and 401.1 eV, corresponding to pyridinic nitrogen, pyrrolic nitrogen, and graphitic nitrogen, respectively (Supplementary Fig.  9d ) 41 . Furthermore, the content of pyridinic nitrogen in Fe 1 /NC SACs gradually decreases with the increase of pyrolysis temperature. These results suggest that the Fe-N coordination environment is highly dependent on the pyrolysis temperature.

To further analyze the atomic configuration of the catalysts, we carried out X-ray absorption spectroscopy (XAS) characterization. The Fe K-edge X-ray absorption near-edge structure (XANES) spectra show that the valence states of Fe in Fe 1 /NC SACs range between 0 (Fe foil) and +3 (Fe 2 O 3 ) (Fig.  3a ), and the energy shifts negetively as the pyrolysis temperature increases, implying a decrease in the oxidation state of Fe, consistent with the XPS results 42 . Additionally, the extended X-ray absorption fine structure (EXAFS) spectra of Fe 1 /NC SACs display only one major peak at 1.4 Å in the R space, corresponding to the Fe-N(C) coordination shell, with no Fe-Fe peak similar to that of Fe foil which corresponds to the Fe-Fe coordination shell being observed (Fig.  3 b, c), further confirming that Fe in Fe 1 /NC SACs exists as single atoms. Moreover, the intensity of the Fe-N shell gradually decreases with increasing the pyrolysis temperature, verifying a decrease in the coordination number of N around the Fe center 41 . To obtain the structural parameters of Fe atoms, we performed EXAFS fitting of Fe 1 /NC SACs. According to the fitting curves and parameters (Fig.  3d–f and Supplementary Table  5 ), the coordination numbers of the Fe-N first shell in Fe 1 /NC-800, Fe 1 /NC-900, and Fe 1 /NC-1000 were calculated to be 4.96, 4.09, and 3.26, respectively. In addition to the Fe-N coordination shell, a Fe-C coordination shell with a coordination number of 1.08 was observed in Fe 1 /NC-1000. Taken together, the atomic configurations of the active centers in Fe 1 /NC-800, Fe 1 /NC-900, and Fe 1 /NC-1000 were depicted as FeN 5 , FeN 4 , and FeN 3 C, respectively, as displayed in the insets of Fig.  3d–f .

Fe K-edge XANES ( a ) and FT EXAFS ( b ) spectra of Fe 1 /NC SACs, Fe foil and Fe 2 O 3 . c EXAFS spectra of Fe 1 /NC SACs, Fe foil and Fe 2 O 3 in k space. Fitting plots of EXAFS spectra for Fe 1 /NC-800 ( d ), Fe 1 /NC-900 ( e ) and Fe 1 /NC-1000 ( f ) in R space. Insets: structural models of Fe 1 /NC-800, Fe 1 /NC-900, and Fe 1 /NC-1000, respectively (N, blue; C, gray; Fe, deep red). Source data are provided with the paper.

Antioxidative performance of Fe 1 /NC SACs

Having demonstrated the coordination environment of Fe active sites can be atomically modulated via high-temperature pyrolysis, we moved forward to investigate the relationship between the CAT-mimicking activity of Fe 1 /NC SACs and the atomic configuration of their active centers. The CAT-mimicking activity was evaluated by determining O 2 generation from the catalytic disproportionation of H 2 O 2 43 , 44 , 45 . As shown in Fig.  4a , the as-synthesized Fe 1 /NC SACs exhibit distinct CAT-like activity, among which, Fe 1 /NC-900 with the atomic configuration of FeN 4 shows the highest activity. In contrast, NC controls without Fe show negligible activity, showing the dominating role of Fe single atoms in mimicking CAT. Moreover, the O 2 generation capacities of Fe 1 /NC SAzymes linearly depend on the concentration of the catalysts (Fig.  4b and Supplementary Fig.  10 ), implying that the catalytic reaction of the SAzymes follows first order reaction kinetics like natural enzymes 46 . We next quantitatively determined the specific activities of these three SAzymes, and found that Fe 1 /NC-900 shows much higher activity (28.9 U·mg −1 ), nearly three times that of Fe 1 /NC-800 (10.4 U·mg −1 ) and Fe 1 /NC-1000 (11.4 U·mg −1 ).

a Time-dependent O 2 generation in Britton–Robison (BR) buffer containing 5 mM H 2 O 2 and 5 μg·mL −1 catalysts. b Specific activities (SA, U·mg −1 ) of Fe 1 /NC SACs in BR buffer (pH 7.0) containing 5 mM H 2 O 2 . c Kinetics for CAT-like activity of Fe 1 /NC SACs (5 μg·mL −1 ) with different concentrations of H 2 O 2 . EPR spectra of 25 mM BMPO in methanol containing 5 mM H 2 O 2 without or with 5 μg·mL −1 Fe 1 /NC-800 ( d ), Fe 1 /NC-900 ( e ), Fe 1 /NC-1000 ( f ) and their controls. Blank: no catalyst. g Schematic structure of Fe 1 /NC-900 with intermediates during the catalytic H 2 O 2 disproportionation reaction. N, blue; C, gray; O, red; H, light gray; Fe, deep red. h Free energy diagrams of H 2 O 2 disproportionation on Fe 1 /NC SAzymes. Each experiment was repeated independently three times with similar results ( a – f ). Representative plots are shown. Source data are provided with the paper.

Subsequently, we studied the steady-state kinetics of the three SAzymes in mimicking CAT (Supplementary Fig.  11 ). As exhibited in Fig.  4c and Supplementary Fig.  12 , the decomposition reaction catalyzed by Fe 1 /NC SAzymes conforms to the typical Michaelis–Menten kinetics. The kinetic parameters of the SAzymes were determined and listed in Supplementary Table  6 . Fe 1 /NC-900 exhibits a maximum reaction rate ( V m ) of 0.51 mM/min and a reaction rate constant k cat of 2.05 × 10 3  min −1 , which are much higher than those of Fe 1 /NC-800 and Fe 1 /NC-1000 SAzymes, revealing that the highest catalytic activity of Fe 1 /NC-900. The higher Michaelis constant ( K m ) value of Fe 1 /NC-900 indicates a lower binding affinity. In addition, the CAT-like activity of the SAzymes shows an obvious dependence on the pH value of the reaction solution (Supplementary Fig.  13 ), similar to the natural enzymes 47 .

To evaluate the stability of the Fe 1 /NC-900 SAzyme, we conducted post-reaction characterizations. As shown in Supplementary Fig.  14a , b, the used Fe 1 /NC-900 catalyst exhibits a morphology similar to that observed before the catalytic tests. The HAADF-STEM image indicates that no Fe or Fe x O y nanoparticles were formed in Fe 1 /NC-900 after catalysis (Supplementary Fig.  14c ). The corresponding elemental mapping images confirmed the homogeneous distribution of C, N and Fe elements (Supplementary Fig.  14c ). In addition, the XRD spectrum of the used Fe 1 /NC-900 demonstrates the absence of Fe-related nanoparticles (Supplementary Fig.  15 ), showing the high stability of the single-atom configuration.

In addition to the CAT-like activity, we also explored the SOD-mimicking activity of Fe 1 /NC-900. SOD is an antioxidative enzyme for catalyzing the disproportionation of O 2 − into O 2 and H 2 O 2 . To assess the SOD-like activity of Fe 1 /NC-900, we employed SOD assay kits with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H -tetrazolium (monosodium salt) (WST-1), a selective probe that reacts with O 2 − to produce a water-soluble formazan dye, to monitor the disproportionation of O 2 − 48 , 49 . The SOD-mimicking ability of Fe 1 /NC-900 was evaluated and quantified by the inhibition of formazan formation. As shown in Supplementary Fig.  16 , the inhibition of formazan formation increases rapidly with increasing the concentration of Fe 1 /NC-900, indicating that Fe 1 /NC-900 can mimic SOD and displays a concentration-dependent manner. In comparison, NC-900 without FeN 4 active site shows relatively lower SOD-like activity, demonstrating the single Fe atoms in Fe 1 /NC-900 are responsible for the catalytic disproportionation of O 2 − .

Furthermore, we investigated the activity of Fe 1 /NC-900 toward eliminating hydroxyl radicals (•OH), another crucial reactive oxygen species. The •OH elimination capacity of Fe 1 /NC-900 was evaluated and quantified by using terephthalic acid (TA) as a selective fluorescent probe 50 . TA can be oxidized by •OH to generate 2-hydroxyterephthalic acid (TA-OH), which can emit a fluorescent signal at 435 nm 51 . As shown in Supplementary Fig.  17 , no •OH was detected with TA and H 2 O 2 alone. However, upon the addition of Fe 2+ , an obvious fluorescence peak representing the TA-OH appears, indicating the generation of •OH through the Fenton reaction. When Fe 1 /NC-900 was added, the fluorescence intensity of TA-OH significantly decreases, demonstrating that •OH generated from the Fenton reaction can be effectively scavenged by Fe 1 /NC-900.

Given that Fe-based SACs also exhibit peroxidase-like and oxidase-like activities, which may cause cytotoxicity, we systematically evaluated these activities of Fe 1 /NC-900. We used the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (ox-TMB) with a characteristic adsorption at ca . 652 nm as a catalytic model reaction. As shown in Supplementary Fig.  18a , Fe 1 /NC-900 SAC exhibits peroxidase-like activity in BR buffer with a pH value of 4.00. However, under physiological conditions (pH 7.00), Fe 1 /NC-900 SAC shows negligible peroxidase-like activity, even in the presence of 5 mM H 2 O 2 . Similarly, the negligible absorbance of ox-TMB in O 2 -saturated BR buffer (pH 7.00) indicates that Fe 1 /NC-900 possesses low oxidase-like activity under a physiological pH (Supplementary Fig.  18b ). Therefore, Fe 1 /NC-900 is unable to cause undesirable oxidative damage due to the low peroxidase-like and oxidase-like activities under the physiological pH.

Mechanism study of CAT-mimicking activity of Fe 1 /NC SACs

To investigate the CAT-mimicking catalytic process of Fe 1 /NC SAzymes, we conducted electron paramagnetic resonance (EPR) characterization. As shown in Supplementary Fig.  19 , the EPR spectra of 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) and 2,2,6,6-tetramethyl-4-piperidinone hydrochloride (4-oxo-TEMP·HCl), specific probes for •OH and singlet oxygen ( 1 O 2 ), repectively, show no significant difference with or without Fe 1 /NC SAzymes, indicating that •OH and 1 O 2 were not generated during the catalytic process 52 , 53 , 54 . Subsequently, we employed BMPO to capture hydroperoxyl radical (•OOH) in methanol and found that •OOH was generated during the disproportionation reaction of H 2 O 2 catalyzed by the three SAzymes (Fig.  4d–f ), which aligns closely with the mechanism of natural enzymes 55 , 56 . In addition, the EPR intensity for •OOH correlates well with the CAT-mimicking activity of Fe 1 /NC SAzymes, indicating that Fe 1 /NC SAzymes catalyze the decomposition of H 2 O 2 through the generation of the •OOH intermediate.

To further elucidate the catalytic mechanism and the difference in CAT-mimicking activity of Fe 1 /NC SAzymes, we carried out density functional theory (DFT) calculations. Based on the EPR and DFT results, we proposed the reaction pathways for CAT-like activity with Fe 1 /NC SACs (Fig.  4g ), which involve six processes, including a significant •OOH generation step. Whereafter, we calculated the free energies of the reaction steps on the Fe active sites in Fe 1 /NC to identify the rate-determining step (RDS). As displayed in Fig.  4h and Supplementary Table  7 , the adsorption energies of the reactant H 2 O 2 on FeN x (x = 5, 4, and 3) sites in Fe 1 /NC-800, Fe 1 /NC-900, and Fe 1 /NC-1000 are -0.54 eV, 0.11 eV and 0.06 eV, respectively, demonstrating Fe 1 /NC-800 exhibits the highest binding affinity for H 2 O 2 , which is consistent with the result obtained from K m value. Furthermore, by analyzing the Gibbs free energy (Δ G ) of the involved steps, we identified the H 2 O desorption step with a Δ G of 0.41 eV, the H 2 O 2 adsorption step with a Δ G of 0.11 eV, and the •OOH generation step with a Δ G of 0.28 eV as the RDSs for Fe 1 /NC-800, Fe 1 /NC-900, and Fe 1 /NC-1000, respectively. Comparatively, Fe 1 /NC-900 possesses the lowest Δ G value of RDS, suggesting it has the highest CAT-mimicking activity. These findings demonstrate that the CAT-mimicking activity of SACs can be tuned by modulating the adsorption energy of the reactants and intermediates through atomic engineering of local coordination environment of active sites using high-temperature pyrolysis.

Inflammation-free electrochemical sensing with Fe 1 /NC-900

Encouraged by the excellent antioxidative activity of Fe 1 /NC-900 SAC, we further explored its electrode reactivity toward the oxidation of electroactive neurochemicals. We firstly employed K 3 Fe(CN) 6 , a widely used redox probe, to investigate the electrode reactivity of Fe 1 /NC SACs. Supplementary Fig.  20 shows typical cyclic voltammogram (CV) obtained at Fe 1 /NC SACs-modified glassy carbon electrodes (GCEs) in artificial cerebrospinal fluid (aCSF) containing 1 mM K 3 Fe(CN) 6 . Almost no current ascribed to the redox process of Fe(CN) 6 4-/3- was recorded at Fe 1 /NC-800-modified GCE, suggesting the poor electrode activity of Fe 1 /NC-800. In contrast, Fe 1 /NC-900-modified GCE shows obvious redox peaks with an anodic/cathodic peak-to-peak separation (ΔEp) of about 150 mV, which is smaller than that at Fe 1 /NC-1000-modified GCE (170 mV), revealing the good electrode activity of Fe 1 /NC-900.

The good electrode activity of Fe 1 /NC-900 enabled us to develop an in vivo sensing platform for neurochemicals. DA is an important monoamine neurotransmitter that regulates a wide variety of complex neurochemical processes, such as motion, reward, and attention 57 , 58 . We chose DA as the target molecule to evaluate the electrochemical sensing ability of Fe 1 /NC SACs. To do this, we conducted CV measurements of DA at the Fe 1 /NC SACs-modified GCEs. As shown in Fig.  5a , the electrochemical oxidation of DA at Fe 1 /NC-900 commences at a potential of ca . −0.05 V ( vs . Ag/AgCl) and shows an oxidation peak at ca . + 0.12 V ( vs . Ag/AgCl). The onset potential at Fe 1 /NC-800 was more positive than 0.05 V with a tailed current response (Supplementary Fig.  21a ). Fe 1 /NC-1000 and NC-900-modified GCEs show similar onset potentials compared to the Fe 1 /NC-900-modified GCE, but with significantly lower oxidation current responses (Supplementary Fig.  21b, c ). Moreover, the current responses toward DA oxidation were also recorded with different catalysts under a constant potential (+0.20 V vs . Ag/AgCl). As displayed in Fig.  5b and Supplementary Figs.  22 and 23 , the response sensitivity toward DA at Fe 1 /NC-900 was calculated to be 85 nA/μM, which is much higher than those obtained at Fe 1 /NC-800 (22 nA/μM), Fe 1 /NC-1000 (75 nA/μM) and NC-900 (54 nA/μM), suggesting a higher electrode activity of Fe 1 /NC-900 toward DA oxidation.

a CVs obtained at Fe 1 /NC-900-modified GCE in aCSF in the absence (gray) and presence (red) of 1 mM DA. Scan rate, 50 mV/s. b Plot of current response vs . DA concentration obtained with Fe 1 /NCs-modified GCEs. Applied potential, +0.20 V vs . Ag/AgCl. c CVs obtained at Fe 1 /NC-900-modified CFE in aCSF in the absence (gray) and presence (red) of 20 μM DA. Scan rate, 50 mV/s. d Amperometric response recorded at Fe 1 /NC-900-modified CFE toward successive additions of 5 μM DA in aCSF. Applied potential, +0.20 V vs . Ag/AgCl. Each experiment was repeated independently three times with similar results ( a – d ). Representative plots are shown. e Amperometric i-t curve recorded with Fe 1 /NC-900-modified CFE in rat NAc. f Typical amperometric response of Fe 1 /NC-900-modified CFE in rat NAc upon electrical stimulation of rat VTA (3 s at 60 Hz, ±300 μA) before (gray) and 25 min after (red) the rat were injected i.p. with a DA uptake inhibitor (NOM, 12 mg/kg). Applied potential, 0.20 V vs . Ag/AgCl. The in vivo experiments were repeated with 3 animals. Source data are provided with the paper.

Before in vivo analysis, we conducted in vitro experiments on Fe 1 /NC SACs using SH-SY5Y cell line. The SH-SY5Y cell line was chosen due to its neuronal-like properties, making it a commonly used model system to study various aspects of neurobiology, such as evaluating the effects of neurotoxins and the neuroprotective property of different agents 59 , 60 , 61 . First, we conducted the cytotoxicity experiments of Fe 1 /NC SACs. As shown in Supplementary Fig.  24 , the cell viability assay indicates that the Fe 1 /NC SACs exhibit minimal cytotoxicity even at a concentration of 50 μg/mL, demonstrating their high biocompatibility. The negligible cytotoxicity of the Fe 1 /NC SACs suggests their potential application for safe and reliable in vivo neurochemical sensing.

We subsequently compared the efficiency of Fe 1 /NC SACs in protecting cells against oxidative stress using SH-SY5Y cell line. As displayed in Supplementary Fig.  25 , H 2 O 2 can induce obvious cytotoxicity on SH-SY5Y cells. Compared with the NC-900 control, the addition of 10 μg mL −1 Fe 1 /NC SACs to the cell-culture medium effectively attenuates H 2 O 2 -mediated oxidative damage and maintains cell viability. Among the SACs tested, Fe 1 /NC-900 exhibits the highest capability to eliminate H 2 O 2 . Furthermore, we used the xanthine oxidation reaction catalyzed by xanthine oxidase (XOD) to produce O 2 − in situ. After 12-hour exposure to Fe 1 /NC-900, the damage of SH-SY5Y cells caused by O 2 − is mostly reduced when compared with the NC-900 control and other Fe 1 /NC SACs (Supplementary Fig.  26 ), demonstrating Fe 1 /NC-900 also possesses the highest SOD-like activity for scavenging O 2 − . Additionally, we employed the Fenton reaction to produces •OH from the reaction between H 2 O 2 and Fe 2+ . As shown in Supplementary Fig.  27 , Fe 1 /NC SACs reduce apparent Fenton reagent-induced apoptosis, indicating their specific capability to eliminate •OH. All these results validated that the Fe 1 /NC-900 exhibits optimal capability to effectively mitigate oxidative stress and protect cells from ROS induced cytotoxicity.

Combining the excellent electrode activity with the high antioxidative properties of Fe 1 /NC-900, we prepared Fe 1 /NC-900-based carbon fiber electrodes (CFEs) to develop a platform for in vivo DA sensing. As shown in Fig.  5c, d and Supplementary Fig.  28 , the Fe 1 /NC-900-modified CFE exhibits high performance toward DA oxidation with a good linearity between current response and DA concentrations. We implanted the Fe 1 /NC-900-modified CFE into nucleus accumbens (NAc) to test the stability of the microsensor. As displayed in Fig.  5e , the current response keeps constant over 3600-s measurement, demonstrating the high stability of Fe 1 /NC-900-based CFE in in vivo sensing. We further used the microsensor to in vivo record DA release in NAc triggered by electrical stimulation of ventral tegmental area (VTA) 18 . As shown in Fig.  5f , upon electrical stimulation, the current signal ascribed to DA release rises rapidly, reaches its maximal value within few seconds, and quickly decreases to the basal level. To verify the signal comes from DA release, we employed a DA uptake inhibitor, nomifensine (NOM), to block the DA uptake sites, thereby increasing DA overflow 62 . As illustrated in Fig.  5f , upon intraperitoneal injection of NOM, the current signal significantly increases, showing that the signals detected are indeed attributable to DA release. In addition, we assessed the stability of Fe 1 /NC-900 under in vivo conditions through TEM characterization. As displayed in Supplementary Fig.  29 , the TEM images and corresponding elemental mapping images of Fe 1 /NC-900 after in vivo experiments show no significant changes, confirming the high stability of the catalyst under physiological environments. These results demonstrate the reliability of Fe 1 /NC-900 modified CFE for in vivo monitoring of DA dynamics with excellent spatial and temporal resolution.

As reported previously, the implantation of CFE into brain tissue inevitably results in a progressive inflammatory tissue response, including the activation of nearby microglia cells and astrocytes, which migrate to the electrode interface. In addition, the increase of hemoglobin can directly upregulate ROS level, further aggravating the inflammatory response 15 , 16 , 17 , 18 , 19 . To demonstrate the Fe 1 /NC-900, with a high antioxidative performance, can potentially endow the microsensor with anti-inflammatory properties, we performed immunohistochemical analysis of brain slices. We used specific markers for activated microglia (ionized calcium-binding adaptor molecule-1, Iba-1) and astrocyte (glial fibrillary acidic protein, GFAP), as well as diamidio-2-phenylindole (DAPI) to stain nuclei 63 , 64 .

To this end, after the implantation of the microsensor into rat brain for 8 h, brain tissues surrounding Fe 1 /NC-900-modified and bare (i.e., without surface modification with Fe 1 /NC-900) CFEs were collected, sliced, and stained for confocal laser scanning microscopy (CLSM) imaging. As displayed in Fig.  6a , Fe 1 /NC-900 modified CFE elicited a significantly lesser GFAP response toward astrocytes compared to the bare CFE. The markedly reduced intensity and spread of GFAP staining around the Fe 1 /NC-900 modified CFE indicates a lower level of astrocytic activation, suggesting that the Fe 1 /NC-900 effectively minimizes the astrocytic inflammatory response (Fig.  6b ). Furthermore, more microglia were observed adjacent to the bare CFE compared to the Fe 1 /NC-900 modified CFE, as indicated by Iba-1 staining (Fig.  6a ). The higher density of Iba-1 positive cells around the bare CFE signifies a robust microglial activation and clustering. In contrast, the Fe 1 /NC-900 modified CFE shows a significantly reduced microglial presence, suggesting that Fe 1 /NC-900 helps in mitigating microglial activation and the associated inflammatory processes (Fig.  6c ). These histological data collectively demonstrate the high efficacy of Fe 1 /NC-900 in mitigating inflammation, thereby creating an improved microenvironment at neural interfaces adjacent to the CFE.

a Histological comparison of brain tissues from untreated rats and rats after 8-hour acute implantation of bare or Fe 1 /NC-900-modified CFE in NAc (n = 3, for each group). Tissues are labeled for astrocytes (green), microglia (magenta) and nuclei (blue). Scale bar, 50 µm. Fluorescence intensities of astrocytes ( b ) and microglia ( c ) in brain tissues from untreated rats (blue line) and rats after 8-h acute implantation of bare CFE (gray line) and Fe 1 /NC-900-modified CFE (red line) (n = 3, for each group). The distance was measured from the image center for the control group and from the implantation center for the bare and Fe 1 /NC-900-modified CFE groups. The data were presented as mean ± SEM. Quantitative analysis of ELISA for IL-6 ( d ), TNF-α ( e ), and IL-1β ( f ) in brain tissues from untreated rats (blue column) and rats after 8-hour acute implantation of bare (gray column) and Fe 1 /NC-900-modified (red column) CFE (n = 3, for each group). The data were presented as mean ± SEM and p values were provided in the figures using one-way analysis of variance (ANOVA). Source data are provided with the paper.

In addition, we investigated the expression of typical inflammatory cytokines using enzyme-linked immunosorbent assay (ELISA). Specifically, we measured the levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in the brain tissue surrounding the implant site 64 , 65 . The IL-1β level indicates the activation of the inflammatory cascade, while the levels of IL-6 and TNF-α are known to mediate and amplify inflammatory processes. As shown in Fig.  6d–f , the insertion of the bare CFE dramatically upregulated inflammatory cytokines, indicating a pronounced inflammatory response. However, the introduction of Fe 1 /NC-900 gradually restored the levels of IL-1β, IL-6, and TNF-α to near-normal levels, suggesting an effective suppression of the inflammatory response. This anti-inflammatory effect was attributed to the antioxidative properties of the Fe 1 /NC-900, which mitigates the oxidative stress and subsequent cytokine production.

Overall, Fe 1 /NC-900 can simultaneously function both as SAzymes for scavenging ROS and as electrode material for DA oxidation, paving an avenue for accurately probing of neurochemical events in vivo with optimized biocompatibility and reduced local acute neuroinflammatory responses.

To sum up, we developed a multifunctional sensing platform with antioxidative SAzyme for in vivo electrochemical sensing free from inflammation. By modulating the coordination environment through varying the pyrolysis temperature, we obtained Fe 1 /NC-900 SAC with FeN 4 active sites, exhibiting the highest antioxidative activity and optimal electrochemical DA oxidation performance. The atomic-precision engineering of active sites enables the optimization of the adsorption energy of reactants and products, as well as the free energy of the reaction processes, thereby achieving the modulation of antioxidative ability and electrocatalytic activity. By integrating the dual functions of Fe 1 /NC-900 SAzyme, we successfully developed an implantable microsensor for DA sensing with anti-inflammatory capacity, enabling precise quantitative tracking of DA dynamics in vivo.

This study underscores the potential of SACs in biological applications, particularly in the development of implantable devices for reliable neurochemical sensing in vivo. The ability to effectively manage oxidative stress and inflammation presents approaches for enhancing the performance and reliability of neural interfaces, significantly advancing the understanding of brain function and disease mechanisms. Moreover, the successful integration of antioxidative SACs with CFEs highlights the feasibility of using multifunctional SACs in conjunction with in vivo electrochemistry. This integration will not only improve the biocompatibility of the sensing devices but also extend their functional lifespan, reducing the need for frequent replacements and minimizing the risk of complications associated with chronic implantation. Additionally, the highly designable SACs with atomic-level engineered active sites offer the potential to creating new SACs with tailored properties for specific biomedical applications.

Ethics declaration

Our research complies with all relevant ethical regulations: All animal experiments were conducted in accordance with the guidelines of the Animal Advisory Committee at the State Key Laboratory of Cognitive Neuroscience and Learning and were approved by the Animal Care and Use Committee at Beijing Normal University [IACUC(BNU)-NKLCNL 2021-09].

H 2 O 2 (30%) was purchased from Sinopharm. FePc was obtained from Alfa Aesar. Zinc chloride was bought from Acros. 1H-1,2,3-triazole was purchased from Ark Pharm, Inc. Ammonia (25%), phosphoric acid, sodium hydroxide, and ferrous sulfate were purchased from Beijing Chemical Plant. Ethanol, glacial acetic acid, N, N-dimethylformamide and methanol were purchased from Tianjin Concord Technology Co., Ltd. SOD assay kits with WST-1 as a selective O 2 − probe and BMPO were purchased from DOJINDO Molecular Technologies. 4-Oxo-TEMP·HCl was purchased from Sigma-Aldrich. Boric acid was purchased from Beijing Yili Fine Chemicals Co., Ltd. Terephthalic acid, xanthine, and XOD were obtained from Sigma Aldrich. The BR buffer was formulated by mixing 0.04 M phosphoric acid, acetic acid and boric acid, and adjusting the pH to 7.0 by 0.2 M NaOH solution. Electrolyte used for electrochemical measurements was an aCSF prepared by mixing KCl (2.4 mM), NaCl (126 mM), KH 2 PO 4 (0.5 mM), NaHCO 3 (27.5 mM), MgCl 2 (0.85 mM), CaCl 2 (1.1 mM), and Na 2 SO 4 (0.5 mM), with the pH adjusted to 7.4. The experimental water was Mili-Q pure water (18.2 MΩ·cm).

Synthesis of FePc@MET-6

Typically, a mixture of 1.25 g zinc chloride and 5 mg FePc was dissolved into a solution containing 12.5 mL ethanol, 12.5 mL N, N-dimethylformamide, and 18.75 mL water using ultrasonication. Then, 5 mL of ammonia was added to the solution and stirred uniformly, followed by the dropwise addition of 1.565 mL 1H-1,2,3-triazole. Subsequently, the mixture was stirred slowly at room temperature for 24 hours. The precipitate was centrifuged, washed three times with ethanol, and vacuum-dried to obtain FePc@MET-6. MET-6 was synthesized using the same procedure without FePc.

Synthesis of Fe 1 /NC SACs

The prepared FePc@MET-6 was ground into powder with a mortar and calcined in a tube furnace under Ar atmosphere at certain temperatures (800 °C for Fe 1 /NC-800, 900 °C for Fe 1 /NC-900) for 2 h, with a heating rate of 5 °C·min −1 . Fe 1 /NC-1000 was obtained by calcining at 900 °C for 1 h followed by 1000 °C for an additional hour. Control catalysts (NCs) were obtained using similar procedures, but with MET-6 synthesized without FePc as the precursors.

Characterization

SEM images were recorded using a Hitachi S-4800. TEM images were acquired with a Hitachi-7700. XPS spectra were recorded using a PHI Quantum 2000 photoelectron spectrometer with monochromatic Al-K α radiation, produced by an electron beam operating at 15 kV. XRD patterns were acquired using a Rigaku RU-200b X-ray diffractometer with Cu K α radiation ( λ  = 1.5418 Å). Nitrogen adsorption/desorption experiments were performed on a Quantachrome Autosorb iQ one-stop adsorption apparatus (77 K). Raman spectra were acquired using a LabRAM HR Raman Microscope at an excitation wavelength of 633 nm. Fe contents were measured using ICP-MS (Thermo Scientific™ iCAP™ RQ). Briefly, the catalyst was dissolved in aqua regia (HCl/HNO 3   =  3:1, v/v) at 180 °C for 40 min using a microwave digestion system (ETHOS 1, LabTech), followed by ICP-MS analysis. HAADF-STEM images and elemental mapping images were acquired using a Titan G2-600 TEM (FEI) with a spherical aberration corrector. XAS spectra of Fe K edge were gained at BL14W1 station of the Shanghai Synchrotron Radiation Facility. The XAS data were obtained in fluorescence mode and monochromatized by a Si (111) double-crystal, with the energy calibrated using Fe foil.

Tests for CAT-like activity of the catalysts

CAT-like activity was assessed by measuring the O 2 generation during the decomposition of H 2 O 2 using a portable dissolved O 2 meter (Seven2GoTM DO, Mettler Toledo). Typically, O 2 generation was detected every 30 s in BR buffer (pH 7.0) containing 5 mM H 2 O 2 and 5 μg·mL −1 catalyst. The CAT-mimicking kinetic analysis of the catalysts was carried out by using different H 2 O 2 concentration (0.5–5.0 mM) while maintaining a fixed catalyst concentration of 5 μg·mL −1 . Michaelis kinetic curves were obtained by plotting the initial rate of O 2 generation against the H 2 O 2 concentration. The maximum velocity ( V m ) and the Michaelis constant ( K m ) were calculated by using the double reciprocal plot of the Michaelis–Menten curves.

Tests for SOD-like activity of Fe 1 /NC-900

The superoxide scavenging ability of Fe 1 /NC-900 and its comparison with NC-900 was determined using SOD assay kits following the manufacturer’s instructions.

Tests for •OH eliminating activity of Fe 1 /NC-900

The •OH radicals were produced through a Fenton reaction using 2 mM FeSO 4 and 5 mM H 2 O 2 for 10 min. After the reaction, 20 μg·mL −1 of enzyme mimics were added to the mixture. The amount of scavenged •OH radicals was subsequently assessed by measuring the fluorescence of 2-hydroxyterephthalic acid generated through the reaction of 0.5 mM terephthalic acid with •OH radicals at an excitation wavelength of 320 nm.

Theoretical calculation

DFT calculations were performed using the Perdew–Burke–Ernzerhof (PBE) generalized gradient approximation and the projected enhanced wave method, as implemented in the Vienna Ab initio Stimulation Package (VASP). The cut-off energy of the plane wave fundamental set was set to 450 eV. Ultrasoft pseudopotentials were used to describe the interaction between valence electron and ionic nuclei. A special k-point grid of 3 × 3 × 1 Monkhorst–Pack was used for geometric optimization and electronic structure calculations. During geometry optimization, all atoms were freely released until the convergence thresholds for the maximum force and energy were less than 0.01 eV/Å and 1.0 × 10 −5  eV/atom, respectively.

The free energy change (∆ G ) of every elementary step was given by the following equation:

∆ E : the reaction energy

∆ E ZPE : zero-point energy difference

T : temperature

∆ S : entropy difference

U: potential

k B : Boltzmann constant.

Cell culture

The SH-SY5Y cell line purchased from Peking Union Medical College Hospital was used for in vitro experiments. SH-SY5Y cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO 2 atmosphere. The culture medium was partially renewed every 2 days.

Cell viability assay

The cytotoxicity of Fe 1 /NC SACs or NC controls were evaluated using the MTT assay (Beyotime Biotechnology, Shanghai, China). Briefly, SH-SY5Y cells were seeded into each well of 96-well plates at a density of 1 × 10 4 cells and incubated in the culture medium overnight to allow cell attachment. Afterward, the old culture medium was replaced with fresh medium containing Fe 1 /NC SACs or NC controls at final concentrations ranging from 0 to 50 μg·mL −1 . Following an additional 12-h incubation, the cells were treated with MTT assay according to the manufacturer’s protocol for 2 h. Subsequently, relative cell viability was determined by measuring the absorbance of MTT at 490 nm using a Synergy H1M microplate reader (n = 4, for each group).

Protecting cells from H 2 O 2 or •OH-induced oxidative damage

SH-SY5Y cells were seeded into 96-well plates at a density of 1 × 10 4 cells per well and incubated overnight. The old culture medium was then replaced with fresh medium containing 10 μg·mL −1 Fe 1 /NC SACs or NC controls. After an additional 12-h incubation, the old medium was discarded, and the cells were exposed to fresh medium containing 1 mM H 2 O 2 or Fenton’s reagent (1 mM H 2 O 2 and 400 μM FeSO 4 ) for another 4-h incubation. Finally, the cell viability for each group was detected using MTT assay, respectively (n = 3, for each group).

Protecting cells from O 2 − -induced oxidative damage

SH-SY5Y cell were seeded into 96-well plates at a density of 1 × 10 4 cells per well and incubated overnight. The old culture medium was replaced with fresh medium containing 10 μg·mL −1 Fe 1 /NC SACs or NC controls for a 12-hour incubation. Afterward, the old medium was discarded, and the cells were exposed to fresh medium containing XOD (20 mU·mL −1 ) and 250 μM xanthine for another 3-hour incubation. Finally, cell viability for each group was assessed using MTT assay, respectively (n = 3, for each group).

Electrochemical measurements

Electrochemical measurements were conducted on the CHI 660E electrochemical workstation using GCE (diameter: 3 mm) modified with catalyst, Pt electrode, and Ag/AgCl electrode, as the working electrode, auxiliary electrode, and reference electrode, respectively. For CV measurements of K 3 Fe(CN) 6 , GCE was modified with Fe 1 /NC SACs or NC controls (2.5 μL of 1 mg·mL −1 aqueous ink) and dried. The potential window was from −0.2 V to 0.6 V for 5 cycles, which was initiated at ca. 0.3 V cathodically and ended at 0.6 V. For CV measurement of DA, GCE was modified with Fe 1 /NC SACs or NC controls (7 μL of 2 mg·mL −1 aqueous ink) and dried. The potential window was from −0.2 V to 0.6 V for 3 cycles, which was initiated at −0.2 V and ended at 0.6 V. For amperometric measurement of DA, the Fe 1 /NC SACs or NC modified GCE were polarized at 0.2 V and staircase response of anodic current were acquired during DA addition.

Fabrication of Fe 1 /NC-900 modified CFE

CFEs were fabricated as follows 33 . Briefly, the exposed carbon fiber was cut to a length of 200–300 μm using a surgery scalpel under a microscope. The CFEs were then electrochemically activated in 1.0 M NaOH at +1.5 V for 80 s and scanned with CV from 0 to 1.0 V at a scan rate of 0.1 V·s −1 until a stable CV curve was obtained. To modify the CFEs with Fe 1 /NC-900, one drop of the Fe 1 /NC-900 aqueous dispersion (1 mg·mL −1 ) was dropped onto a clean glass slide and the CFE was carefully immersed and rolled in the droplet under a microscopy. The Fe 1 /NC-900-modified CFE was then allowed to dry at ambient temperature.

Electrical stimulation experiment for DA sensing

In vivo analysis was performed using adult male SD rats (300 ± 50 g), which were housed on a 12:12 h light-dark schedule with food and water ad libitum. The animals were anesthetized with isoflurane (4% induction, 2% maintenance) using a gas pump (RWD R520, Shenzhen, China) and positioned on a stereotaxic frame. The Fe 1 /NC-900 modified CFE was carefully implanted into NAc (Anteroposterior (AP) = +2.0 mm, mediolateral (ML) = +1.5 mm, and dorsoventral (DV) = − 6.0 mm from dura) for in vivo DA monitoring. An Ag/AgCl reference microelectrode was positioned into the brain tissue, and a platinum wire served as the auxiliary electrode. The Fe 1 /NC-900-modified CFE was polarized at 0.20 V vs . Ag/AgCl for in vivo measurement of DA. A bipolar stimulating electrode was implanted in VTA (AP = −4.9 mm, ML = +1.0 mm, DV = −8.2 mm from dura). A 180-pulse stimulation at 60 Hz ( ± 300 μA, 2 ms per phase) was applied to trigger DA release in the NAc. Pharmacological investigation of DA release was performed by intraperitoneal injection of a DA uptake inhibitor (nomifensine, NOM, 12 mg/kg), followed by another electrical stimulation 25 min after drug injection. The NOM was dissolved in saline.

Immunofluorescence staining and its quantitative analysis

The rats were perfused transcardially with saline followed by cold 4% paraformaldehyde (PFA) after the removal of bare or Fe 1 /NC-900 modified CFEs (n = 3, for each group). Dissected brains were block-fixed in 4% PFA overnight, then incubated overnight in 20% sucrose solution, and subsequently in 30% sucrose solution until the brains sank. The embedded brains were sectioned at a thickness of 30 μm perpendicular to the implantation path of CFEs using a freezing microtome. The sections underwent the following treatments: (1) three rinses (5 min each) with 0.01 M phosphate buffer saline (PBS) containing 0.2% Triton X-100 (Sigma-Aldrich, T8787), (2) blocking with normal Donkey serum for 30 min, (3) incubation with primary antibodies against Iba-1 (1:200, ab178846, Abcam) and GFAP (1:200, ab302644, Abcam) overnight at 4 °C. (4) three rinses (2 min each) with PBS, (5) incubation with secondary antibodies-Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) (1:400, ab150133, Abcam) and Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 568) (1:400, ab175470, Abcam) for 1 h at room temperature. (6) three rinses (2 min each) with PBS, (7) incubation in DAPI for 5 min, (8) three rinses (2 min each) with PBS, and (9) final mounting onto glass slides with coverslips using Prolong Gold mounting media. Confocal images were collected using a Nikon single-particle microscopy equipped with a 40× objective lens. Immunomarkers were quantified using fluorescent intensity as a function of distance from the implantation site with the ImageJ software. The center of the implantation site was set as x = 0 μm. To obtain the fluorescence intensity curve, the adjacent region around the implantation center at 160 μm (assigned as d = 160 μm) was split into the circular contours of 20 μm increments, and the average intensities of these 20 μm circular contours were calculated. For normal tissue slices, the center of the image was set at x = 0 μm.

Measurement of inflammatory cytokines

The rats were divided into three groups of control, bare CFE, Fe 1 /NC-900-modified CFE (n = 3, for each group). Brain tissues surrounding the implants were harvested, homogenized on ice, and centrifuged at 8000  g for 10 minutes after 8-h insertion. The supernatant was stored at −80 °C for future use. Inflammatory cytokines were quantified by ELISA kits for IL-6 (Solarbio, SEKR-0005), IL-1β (Solarbio, SEKR-0002), and TNF-α (Solarbio, SEKR-0009). The experimental procedures were conducted according to the manufacturer’s instructions. Each sample was tested in duplicate.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The data generated in this study are present in the main text and Supplementary Information files. The raw data sets are available from the corresponding authors upon request.  Source data are provided with this paper.

Chantranupong, L. et al. Dopamine and glutamate regulate striatal acetylcholine in decision-making. Nature 621 , 577–585 (2023).

Article   ADS   CAS   PubMed   PubMed Central   Google Scholar  

Lezmy, J. et al. Astrocyte Ca 2+ -evoked ATP release regulates myelinated axon excitability and conduction speed. Science 374 , eabh2858 (2021).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Knaus, L. S. et al. Large neutral amino acid levels tune perinatal neuronal excitability and survival. Cell 186 , 1950–1967 (2023).

Article   CAS   PubMed   Google Scholar  

Krok, A. C. et al. Intrinsic dopamine and acetylcholine dynamics in the striatum of mice. Nature 621 , 543–549 (2023).

Article   ADS   CAS   PubMed   Google Scholar  

Zong, W. et al. Large-scale two-photon calcium imaging in freely moving mice. Cell 185 , 1240–1256 (2022).

Wu, Z., Lin, D. & Li, Y. Pushing the frontiers: tools for monitoring neurotransmitters and neuromodulators. Nat. Rev. Neurosci. 23 , 257–274 (2022).

Bednarik, P. et al. 1H magnetic resonance spectroscopic imaging of deuterated glucose and of neurotransmitter metabolism at 7 T in the human brain. Nat. Biomed. Eng. 7 , 1001–1013 (2023).

Zhang, Y., Jiang, N. & Yetisen, A. K. Brain neurochemical monitoring. Biosen. Bioelectron. 189 , 113351 (2021).

Article   CAS   Google Scholar  

Robinson, D. L., Hermans, A., Seipel, A. T. & Wightman, R. M. Monitoring rapid chemical communication in the brain. Chem. Rev. 108 , 2554–2584 (2008).

Wightman, R. M. Probing Cellular chemistry in biological systems with microelectrodes. Science 311 , 1570–1574 (2006).

Schwerdt, H. N. et al. Long-term dopamine neurochemical monitoring in primates. Proc. Nat. Acad. Sci. USA. 114 , 13260–13265 (2017).

Shao, Z., Wilson, L., Chang, Y. & Venton, B. J. MPCVD-grown nanodiamond microelectrodes with oxygen plasma activation for neurochemical applications. ACS Sens. 7 , 3192–3200 (2022).

Forderhase, A. G., Ligons, L. A., Norwood, E., McCarty, G. S. & Sombers, L. A. Optimized fabrication of carbon-fiber microbiosensors for codetection of glucose and dopamine in brain tissue. ACS Sens. 9 , 2662–2672 (2024).

Zhao, C. et al. Implantable aptamer–field-effect transistor neuroprobes for in vivo neurotransmitter monitoring. Sci. Adv. 7 , eabj7422 (2021).

Salatino, J. W., Ludwig, K. A., Kozai, T. D. Y. & Purcell, E. K. Glial responses to implanted electrodes in the brain. Nat. Biomed. Eng. 1 , 862–877 (2017).

Wellman, S. M. & Kozai, T. D. Y. Understanding the inflammatory tissue reaction to brain implants to improve neurochemical sensing performance. ACS Chem. Neurosci. 8 , 2578–2582 (2017).

Kozai, T. D. Y. et al. Ultrasmall implantable composite microelectrodes with bioactive surfaces for chronic neural interfaces. Nat. Mater. 11 , 1065–1073 (2012).

Clark, J. J. et al. Chronic microsensors for longitudinal, subsecond dopamine detection in behaving animals. Nat. Methods 7 , 126–129 (2010).

Kozai, T. D. Y., Jaquins-Gerstl, A. S., Vazquez, A. L., Michael, A. C. & Cui, X. T. Brain tissue responses to neural implants impact signal sensitivity and intervention strategies. ACS Chem. Neurosci. 6 , 48–67 (2015).

Forman, H. J. & Zhang, H. Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nature Rev. Drug Discov. 20 , 689–709 (2021).

Huang, Y., Ren, J. & Qu, X. Nanozymes: classification, catalytic mechanisms, activity regulation, and applications. Chem. Rev. 119 , 4357–4412 (2019).

Wu, J. et al. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II). Chem. Soc. Rev. 48 , 1004–1076 (2019).

Jiang, W. et al. Chiral metal-organic frameworks incorporating nanozymes as neuroinflammation inhibitors for managing Parkinson’s disease. Nat. Commun. 14 , 8137 (2023).

Xu, B. et al. A single-atom nanozyme for wound disinfection applications. Angew. Chem. Int. Ed. 58 , 4911–4916 (2019).

Kraushofer, F. & Parkinson, G. S. Single-atom catalysis: insights from model systems. Chem. Rev. 122 , 14911–14939 (2022).

Wang, A., Li, J. & Zhang, T. Heterogeneous single-atom catalysis. Nat. Rev. Chem. 2 , 65–81 (2018).

Article   ADS   CAS   Google Scholar  

Huang, L., Chen, J., Gan, L., Wang, J. & Dong, S. Single-atom nanozymes. Sci. Adv. 5 , eaav5490 (2019).

Li, G. et al. Dimensionality engineering of single-atom nanozyme for efficient peroxidase-mimicking. J. Am. Chem. Soc. 145 , 16835–16842 (2023).

Ji, S. et al. Matching the kinetics of natural enzymes with a single-atom iron nanozyme. Nat. Catal. 4 , 407–417 (2021).

Lu, X. et al. Single-atom catalysts-based catalytic ROS clearance for efficient psoriasis treatment and relapse prevention via restoring ESR1. Nat. Commun. 14 , 6767 (2023).

Cao, F. et al. Artificial-enzymes-armed Bifidobacterium longum probiotics for alleviating intestinal inflammation and microbiota dysbiosis. Nat. Nanotech. 18 , 617–627 (2023).

Fei, H. et al. Single atom electrocatalysts supported on graphene or graphene-like carbons. Chem. Soc. Rev. 48 , 5207–5241 (2019).

Gao, X. et al. A single-atom Cu–N 2 catalyst eliminates oxygen interference for electrochemical sensing of hydrogen peroxide in a living animal brain. Chem. Sci. 12 , 15045–15053 (2021).

Zhou, M. et al. Single-atom Ni-N 4 provides a robust cellular NO sensor. Nat. Commun. 11 , 3188 (2020).

Ma, W. et al. A single-atom Fe–N 4 catalytic site mimicking bifunctional antioxidative enzymes for oxidative stress cytoprotection. Chem. Commun. 55 , 159–162 (2019).

Gándara, F. et al. Porous, Conductive metal-triazolates and their structural elucidation by the charge-flipping method. Chem. Eur. J. 18 , 10595–10601 (2012).

Article   PubMed   Google Scholar  

Chen, D. et al. Bioinspired porous three-coordinated single-atom Fe nanozyme with oxidase-like activity for tumor visual identification via glutathione. Nat. Commun. 14 , 7127 (2023).

Wei, S. et al. Self-carbon-thermal-reduction strategy for boosting the Fenton-like activity of single Fe-N 4 sites by carbon-defect engineering. Nat. Commun. 14 , 7549 (2023).

Barrio, J. et al. FeNC oxygen reduction electrocatalyst with high utilization penta-coordinated sites. Adv. Mater. 35 , 2211022 (2023).

Fu, H. et al. Machine-learning-assisted optimization of a single-atom coordination environment for accelerated Fenton catalysis. ACS Nano 17 , 13851–13860 (2023).

Xiong, Y. et al. Single-atom Fe catalysts for Fenton-like reactions: Roles of different N species. Adv. Mater. 34 , 2110653 (2022).

Xue, D. et al. Altering the spin state of Fe-N-C through ligand field modulation of single-atom sites boosts the oxygen reduction reaction. Nano Energy 105 , 108020 (2023).

Chen, Y. et al. Atomic-level regulation of cobalt single-atom nanozymes: Engineering high-efficiency catalase mimics. Angew. Chem. Int. Ed. 62 , e202301879 (2023).

Zhou, J. et al. Coordination-driven self-assembly strategy-activated Cu single-atom nanozymes for catalytic tumor-specific therapy. J. Am. Chem. Soc. 145 , 4279–4293 (2023).

Jiang, P. et al. Tuning oxidant and antioxidant activities of ceria by anchoring copper single-site for antibacterial application. Nat. Commun. 15 , 1010 (2024).

Singh, N., Savanur, M. A., Srivastava, S., D’Silva, P. & Mugesh, G. A redox modulatory Mn 3 O 4 nanozyme with multi-enzyme activity provides efficient cytoprotection to human cells in a Parkinson’s disease model. Angew. Chem. Int. Ed. 56 , 14267–14271 (2017).

Feng, K., et al. Breaking the pH limitation of nanozymes: Mechanisms, methods, and applications. Adv. Mater . 36 , 2401619 (2024).

Ma, L. et al. A natural biogenic nanozyme for scavenging superoxide radicals. Nat. Commun. 15 , 233 (2024).

Gao, W. et al. Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme. Nat. Commun. 14 , 160 (2023).

Wang, Z. et al. Biomimetic nanoflowers by self-assembly of nanozymes to induce intracellular oxidative damage against hypoxic tumors. Nat. Commun. 9 , 3334 (2018).

Article   ADS   PubMed   PubMed Central   Google Scholar  

Wang, B. et al. Atomization-induced high intrinsic activity of a biocompatible MgAl-LDH supported Ru single-atom nanozyme for efficient radicals scavenging. Angew. Chem. Int. Ed. 62 , e202307133 (2023).

Liu, Q. et al. Cofactor-free oxidase-mimetic nanomaterials from self-assembled histidine-rich peptides. Nat. Mater. 20 , 395–402 (2021).

Teng, K.-X., Niu, L.-Y., Xie, N. & Yang, Q.-Z. Supramolecular photodynamic agents for simultaneous oxidation of NADH and generation of superoxide radical. Nat. Commun. 13 , 6179 (2022).

Wen, D., Li, K., Deng, R., Feng, J. & Zhang, H. Defect-rich glassy IrTe 2 with dual enzyme-mimic activities for sono-photosynergistic-enhanced oncotherapy. J. Am. Chem. Soc. 145 , 3952–3960 (2023).

Alfonso-Prieto, M., Biarnés, X., Vidossich, P. & Rovira, C. The molecular mechanism of the catalase reaction. J. Am. Chem. Soc. 131 , 11751–11761 (2009).

Campomanes, P., Rothlisberger, U., Alfonso-Prieto, M. & Rovira, C. The molecular mechanism of the catalase-like activity in horseradish peroxidase. J. Am. Chem. Soc. 137 , 11170–11178 (2015).

Garritsen, O., van Battum, E. Y., Grossouw, L. M. & Pasterkamp, R. J. Development, wiring and function of dopamine neuron subtypes. Nat. Rev. Neurosci. 24 , 134–152 (2023).

Liu, C., Goel, P. & Kaeser, P. S. Spatial and temporal scales of dopamine transmission. Nat. Rev. Neurosci. 22 , 345–358 (2021).

Article   PubMed   PubMed Central   Google Scholar  

Kovalevich, J. & Langford, D. Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology. In Neuronal cell culture: Methods and protocols (eds. Amini, S. & White, M. K.) 9−21 (Humana Press, 2013).

Xicoy, H., Wieringa, B. & Martens, G. J. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol. Neurodegener. 12 , 10 (2017).

Wu, D. et al. Pathogenesis-adaptive polydopamine nanosystem for sequential therapy of ischemic stroke. Nat. Commun. 14 , 7147 (2023).

Condon, M. D. et al. Plasticity in striatal dopamine release is governed by release-independent depression and the dopamine transporter. Nat. Commun. 10 , 4263 (2019).

Xu, T. et al. Synergistic charge percolation in conducting polymers enables high-performance in vivo sensing of neurochemical and neuroelectrical signals. Angew. Chem. Int. Ed. 61 , e202204344 (2022).

Liu, S. et al. A nanozyme-based electrode for high-performance neural recording. Adv. Mater. 36 , 2304297 (2024).

Wei, S. et al. Shape-changing electrode array for minimally invasive large-scale intracranial brain activity mapping. Nat. Commun. 15 , 715 (2024).

Download references

Acknowledgements

We acknowledge the financial support from the National Natural Science Foundation of China (Grant Nos. 22134002 for L.M., 22125406 and 22074149 for P.Y.), the Natural Science Foundation of Beijing (2242028 for W.M. and Z230022 for P.Y.), the National Basic Research Program of China (2018YFA0703501, 2018YFA1204503 and 2022YFA1204500 for P.Y.). We are grateful to the photoemission end station beamline BL14W1 in the Shanghai Synchrotron Radiation Facility for XAS characterizations.

Author information

These authors contributed equally: Xiaolong Gao, Huan Wei.

Authors and Affiliations

College of Chemistry, Beijing Normal University, 100875, Beijing, China

Xiaolong Gao, Huan Wei, Wenliang Ji & Lanqun Mao

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS), 100190, Beijing, China

Xiaolong Gao, Wenjie Ma, Wenjie Wu & Ping Yu

University of Chinese Academy of Sciences, 100049, Beijing, China

Wenjie Ma, Wenjie Wu & Ping Yu

College of Chemistry and Materials Science, Anhui Normal University, Wuhu, 241002, China

You can also search for this author in PubMed   Google Scholar

Contributions

L.M. and W.M. conceived the idea. X.G. carried out the synthesis, characterization and performance measurements. H.W. and W.J. conducted the in vivo analysis. J.M. performed the XAS characterization. W.W. analyze the XAS data. P.Y. participated in the discussion of the study. X.G., W.M. and H.W. wrote the manuscript, P.Y. and L.M. revised and finalized it. All authors contributed comments on this work.

Corresponding authors

Correspondence to Wenjie Ma , Ping Yu or Lanqun Mao .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Peer review

Peer review information.

Nature Communications thanks Zhimin Tian, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary information, reporting summary, description of additional supplementary files, peer review file, supplementary data, source data, source data file, rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ .

Reprints and permissions

About this article

Cite this article.

Gao, X., Wei, H., Ma, W. et al. Inflammation-free electrochemical in vivo sensing of dopamine with atomic-level engineered antioxidative single-atom catalyst. Nat Commun 15 , 7915 (2024). https://doi.org/10.1038/s41467-024-52279-5

Download citation

Received : 07 February 2024

Accepted : 02 September 2024

Published : 10 September 2024

DOI : https://doi.org/10.1038/s41467-024-52279-5

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

By submitting a comment you agree to abide by our Terms and Community Guidelines . If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

• Open access
• Published: 08 September 2024

The herbicidal activity of pre-emergence herbicide cinmethylin and its potential risks on soil ecology: pH, enzyme activities and bacterial community

• Haiyan Yu 1 ,
• Hailan Cui 1 ,
• Jingchao Chen 1 &
• Xiangju Li 1  

Environmental Microbiome volume  19 , Article number:  66 ( 2024 ) Cite this article

Metrics details

The herbicide cinmethylin, which was originally registered for use in rice fields, has the potential to control grass weeds in wheat fields before the emergence of wheat. However, its herbicidal activity against various troublesome grass weeds that infest wheat fields in China and its relationships with soil pH, soil enzymes and soil bacteria are not well known. Here, the effects of applying cinmethylin on the soil surface were tested on six grass weeds, and its impacts on soil characteristics, including the soil pH, soil enzymes and bacterial community, were evaluated.

Alopecurus aequalis , A. japonicus and A. myosuroides were highly sensitive to cinmethylin, with GR 50 values of 78.77, 61.49 and 119.67 g a.i. ha − 1 , respectively. The half-lives of cinmethylin at 1-, 10- and 100-fold the recommended rates were estimated at 26.46 − 52.33 d. Cinmethylin significantly increased the soil pH but decreased the activities of soil sucrase and urease. At 10- and 100-fold the recommended rate of cinmethylin, the bacterial abundance and diversity significantly decreased at 30 and 60 days after cinmethylin treatment. Cinmethylin at 100-fold the recommended rates largely promoted bacterial co-occurrence network complexity. Cinmethylin at high concentrations temporarily inhibited the abundance of the Nitrospira genus, as indicated by the copy numbers of the ammonia-oxidising archaea (AOA) amoA and ammonia-oxidising bacteria (AOB) amoA genes. Further analysis revealed that soil pH was negatively related to soil urease, and a significantly positive correlation was detected between soil urease and soil nitrification.

Collectively, the application of cinmethylin at the recommended field dose had nearly no effect on the soil ecosystem, but its potential risks at high concentrations deserve further attention.

Introduction

Cinmethylin was developed by Shell Chemical Company and commercialised in 1982. It disturbs the biosynthesis of fatty acids by inhibiting thioesterase, which releases fatty acids from their carrier protein in plastids and then kills weeds [ 5 ]. Cinmethylin was originally used by mixing it with the soil in transplanted rice fields to control grass weeds, such as Echinochloa crusgalli , Monochoria vaginalis and Cyperus rotundus [ 27 ]. Owing to its high cost and the introduction of some new herbicides to the market at the same time, this herbicide has a small competitive edge over acetolactate synthase (ALS)-inhibiting and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides on the market [ 8 ]. Recently, however, an increasing number of weed species in wheat fields have evolved resistance to ALS- and ACCase-inhibiting herbicides. Additionally, when it is applied to the soil before seedling emergence, cinmethylin has shown excellent inhibitory activity against the troublesome weeds Lolium rigidum and Alopecurus myosuroides , which infest wheat, whereas it is safe for wheat [ 21 , 34 ]. Owing to its different modes of action, it is also effective in controlling the resistant grass weed L . rigidum [ 4 ]. Thus, cinmethylin, a preemergence herbicide, has recently been reused to selectively control annual grass weeds in wheat fields. It has been registered for use in wheat fields in Australia ( http://sitem.herts.ac.uk/aeru/iupac/atoz.htm ). However, whether this herbicide can also effectively control common grass weed species such as Bromus japonicus , A. aequalis and L. perenne , which severely infest wheat fields in China, is still unclear. Under the new application scenario where cinmethylin is directly applied to soil before the emergence of wheat, the environmental risks, particularly to the soil ecosystem, of cinmethylin must be reevaluated.

Soil plays a critical role in crop production, organic matter decomposition, pollutant degradation and groundwater protection [ 23 , 40 , 44 ]. Although herbicides play an important role in weed management, the active ingredients sprayed on soil have diverse impacts on soil biological properties. Soil enzymes produced by microorganisms are among the active elements responsible for biochemical processes in soil ecosystems [ 26 ]. Their activities are sensitive to the soil environment and are related to the quality and productivity of the soil as well as microbiome activity. Herbicide residues in soil can influence the activity of soil enzymes. For example, the activities of invertase and urease in rhizosphere soil are inhibited by low and high doses of the herbicide fomesafen [ 24 ]. Mesotrione significantly inhibited β -glucosidase activity throughout the experimental period, whereas urease and acid phosphatase activities were unaffected by this herbicide [ 13 ]. Soil microorganisms are also important components of the soil ecosystem and are closely related to soil function [ 6 , 9 , 38 , 47 ]. Multiple studies have noted that the application of herbicides has diverse effects on the diversity and composition of the soil microbiome. Pyroxasulfone has been increasingly applied in recent years, and its exposure considerably alters bacterial diversity and composition after 30 days [ 43 ]. Pertile et al. [ 36 ] reported that applying the herbicides imazethapyr and flumyzin in soil influenced the microbial profile, with the potential to affect functions mediated by microbial communities. Du et al. [ 10 ] reported that the ALS-inhibiting herbicide mesosulfuron-methyl influenced the abundance, community and diversity of soil bacteria and fungi. Their abundance was decreased after exposure to mesosulfuron-methyl. Soil N cycling is a key predictor of soil ecological stability and includes three processes: N fixation, soil nitrification and soil denitrification. To date, several microbial groups have been shown to play important roles in the processes involved in producing and immobilising inorganic N [ 3 ]. For example, N 2 -fixing bacteria participate in soil N fixation by reducing N 2 to NH 3 . Ammonia-oxidising bacteria (AOB) and ammonia-oxidising archaea (AOA) are involved in transforming NH 4 + to NO 3 − during the soil nitrification process. However, some studies have shown that these microbial groups are very vulnerable to herbicide pollution [ 12 , 48 ].

Cinmethylin has low toxicity to various aquatic organisms, such as Daphnia magna and Salmo gairdneri [ 30 ]. Its acute mammalian toxicity is also low when it is orally administered to rats [ 31 ]. Although the responses of the soil ecosystem, including soil enzymes and the soil microbiome, to some herbicides have been well investigated, information regarding the potential risk of cinmethylin to soil ecosystems is limited.

Therefore, the objectives of this study were to (1) evaluate the herbicidal activity of cinmethylin on six grass weeds that are widespread in wheat fields in China; (2) determine the degradation dynamics of cinmethylin in soil; (3) assess the impacts of cinmethylin exposure on soil pH and soil enzymes; (4) assess the effects of cinmethylin on the ecology of soil bacteria; and (5) ascertain its impact on soil nitrification. This study helps elucidate the interactions between cinmethylin and the soil ecosystem and provides scientific guidance for the registration of the existing herbicide cinmethylin in a new scenario where it is sprayed on the soil surface before the emergence of wheat seedlings.

Materials and methods

Plant materials and cultivation.

Mature seeds of the grass weeds A. aequalis , A. japonicus , A. myosuroides , L. perenne , Avena fatua and Bromus japonicus were collected from wheat fields through “Z” sampling. Cinmethylin was never used at these collection sites where grass weeds severely infest wheat fields. The detailed geographical information for the six weed species is summarised in Table S1 . The seeds of each species were germinated in Petri dishes containing two layers of filter paper [ 46 ]. When the seeds germinated, they were transferred to pots containing a mixture of soil and organic fertiliser (3:1, v/v) and organic fertiliser prepared from peat and livestock manure (Kai Yin LLC, Beijing). Germinated seeds were sown at a depth of 1 cm, and each pot included ten seeds. All the pots were subsequently placed in a growth chamber under a 14 h light/10 h dark (20 °C/15°C) photoperiod with 500 µmol m − 2 s − 1 light intensity and 60% relative humidity (RH). The seedlings were watered every two days.

Cinmethylin application before the emergence of grass weeds

Cinmethylin (750 g L − 1 emulsifiable concentrate, Luximax, BASF, Melbourne, Australia) at doses of 49.22, 98.44, 196.88, 393.75, 787.5 and 1575 g a.i. ha − 1 was sprayed on the pots 24 h after grass weed sowing. The dose of 393.75 g a.i. ha − 1 was the recommended field rate. Distilled water was used as a control. Herbicide spraying was conducted with an ASS-3 Walking Spray Tower (450 L ha − 1 ; National Engineering Research Centre for Information Technology, Beijing, China). All the plants were subsequently cultivated under the above conditions. The aboveground tissue of each plant was cut at 21 days after treatment (DAT); afterwards, the fresh weights of the seedlings were determined to calculate the herbicide dose resulting in a 50% reduction in weed growth (GR 50 ). Each treatment included four pots, and the experiment was independently repeated three times.

Soil treatment with cinmethylin

Soil at a depth of 0–15 cm was collected from an experimental station located in Shangzhuang, Haidian, Beijing. Pesticides were not used at this sampling site for ten years. According to the chemical and physical properties, the soil sample was a silty clay loam (Table S2). The soil that passed through a 2 mm sieve was used in the experiment. Before treatment with cinmethylin, the soil was placed under controlled conditions at 25 °C and 50% maximum water-holding capacity for two weeks to restore soil microorganisms [ 10 ].

Cinmethylin (97%) was obtained from Beijing Qinchengyixin Technology Development Co., Ltd. (Beijing, China) and dissolved in acetone. Cinmethylin was dissolved in acetone because it is insoluble in water. Cinmethylin solution and 50 g of soil were added to 1 L brown bottles and then mixed adequately [ 11 ]. After being spread out completely in the dark for 24 h to volatilise the acetone, each mixture was mixed with 200 g of soil to reach final concentrations of 0.2625 (C1), 2.625 (C10) and 26.25 mg kg − 1 (active ingredients per soil dry weight, a.i./dw, C100) [ 10 ]. The cinmethylin doses for the soil treatments were set according to GB/T31270.16-2014. C1 is the recommended field dose, assuming that the soil layer depth is 10 cm with a bulk density of 1.5 g cm − 3 [ 19 ]. Soil mixed with the same amount of acetone was regarded as the control (C0), and its soil treatment procedure was conducted as described earlier. The water-holding capacity of the soil was subsequently adjusted to 50% by adding sterile water. All the plants were placed into a dark growth chamber at 25 °C. Sterile water was added to maintain the soil moisture at 50% of the water-holding capacity throughout the experimental period. The soil samples used for pH and enzyme activity assays, cinmethylin residue detection and DNA extraction were collected at 1, 7, 15, 30 and 60 DAT and then stored at -80 °C. Each treatment included four independent replicates.

Extraction and detection of cinmethylin

A total of 5 g of soil was added to 20 mL of 90% acetonitrile aqueous solution and vortexed for 5 min. NaCl (2.0 g) and (Mg) 2 SO 4 (3.0 g) were added to the mixture before vortexing for 2 min and sonicating for 10 min. The supernatant (1 mL) was evaporated until dry with a gentle stream of nitrogen after centrifugation at 8000 × g for 5 min and subsequently redissolved in 1 mL of hexane. The reconstituted solutions were filtered through a 0.22-µm organic membrane and used to detect cinmethylin with gas chromatography‒mass spectrometry (GC‒MS).

Cinmethylin was quantified via a QP2010 SE GC‒MS system (Shimadzu, Japan) according to a previous study with some minor modifications [ 50 ]. Cinmethylin was separated on an Rtx-5MS capillary column (30-m length, 0.25-µm inner diameter (i.d.), 0.25-µm film thickness; Shimadzu, Japan) with helium (99.999%) as the carrier gas, and the injection volume was 2 µL. The gas chromatographic operation conditions were set as follows: the temperature program was started at 60 °C (hold time 1 min), increased to 180 °C at a rate of 30 °C min − 1 and subsequently increased to 250 °C at a rate of 10 °C min − 1 (hold time 5 min). The oven and injector port temperatures were set to 50 °C and 250 °C, respectively. For the mass spectrometer conditions, the ion source and MS interface temperatures were 230 °C and 280 °C, respectively. The solvent delay was 4 min. Cinmethylin was monitored in single ion monitoring (SIM) mode (105 m / z , 123 m / z and 169 m / z ). The ion at 105 m / z was used to quantify the amount of cinmethylin.

To verify the extraction method for cinmethylin in soil, a recovery experiment was performed with spiked concentrations of 0.5, 1.0 and 10 mg L − 1 . Five replicates were included for each spiked concentration. The detailed results are described in Table S3 and indicate that the procedure was suitable for extracting cinmethylin from the soil.

Soil pH assay

The soil pH was assayed following the methods of García-Pérez et al. [ 18 ], with some modifications. A total of 10 g of soil was mixed with 25 mL of deionised water to remove carbon dioxide. This mixture was vortexed for 2 min and then allowed to stand for 30 min. The pH of the supernatant was detected with a FiveEasy Plus pH meter (Shanghai Mettler-Toledo Instrument Co., Ltd., Shanghai, China).

Soil enzyme activity assay

The activities of soil catalase, urease and sucrase were determined following the manufacturers’ instructions for the Soil Catalase Activity Assay Kit, Soil Urease Activity Assay Kit and Soil Sucrase Activity Assay Kit (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), respectively.

DNA extraction, PCR amplification and high-throughput sequencing

The soil microbial DNA was isolated with a MagicPure Stool and Soil Genomic DNA Kit (TransGen Biotech, Beijing, China). The quality and concentration of the DNA were assessed via 1% agarose gel electrophoresis and a NanoDrop 2000 instrument (Thermo Scientific, Massachusetts, USA), respectively. A pair of primers (338-F/806-R) was used to amplify the bacterial 16 S rRNA gene to study the soil bacterial communities (Table S4). PCR amplification was conducted in a total volume of 20 µL containing 10 ng of DNA, 4 µL of 5× FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of forwards and reverse primers (1.0 × 10 − 8 mol L − 1 ), 0.4 µL of FastPfu Polymerase (TransGen Biotech) and a variable volume of double-distilled water (ddH 2 O). PCR began with a predenaturation step at 95 °C for 3 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 55 °C and 45 s at 72 °C, and a final extension at 72 °C for 10 min. PCR products were visually evaluated via 2% agarose gel electrophoresis and then purified with an EasyPure ® Quick Gel Extraction Kit (TransGen Biotech). Purified PCR products were sequenced with an Illumina MiSeq PE300 (Santiago, CA, USA).

The paired-end reads were merged according to the overlap relationship. Strict quality control was conducted on the raw reads following previously reported criteria [ 10 ]. The optimised sequences were used for operational taxonomic unit (OTU) cluster analysis at a 97% similarity level to obtain representative OTU sequences. The RDP classifier (version 2.11) was employed to clarify the representative OTU sequences against the SILVA database at a confidence threshold of 0.7. Sequences annotated as chloroplasts or mitochondria or not classified as bacterial were removed. The obtained sequences were used for further analysis.

Gene copy number assay

The bacterial 16 S rRNA, AOA amoA and AOB amoA genes were amplified with the primers described in Table S4. PCR was performed in a total volume of 25 µL, which comprised 600 ng of DNA, 12.5 µL of 2× Taq PCR Master Mix (Tiangen, Beijing, China), 0.5 µL of forward primer and reverse primer (1.0 × 10 − 8 mol L − 1 ), and 10.5 µL of ddH 2 O. The amplification cycling conditions were set as follows: 4 min at 95 °C; 38 cycles of 30 s at 95 °C, 30 s at the annealing temperature of each gene and 60 s at 72 °C; and a final extension step at 72 °C for 10 min. The evaluation and purification of the PCR products were performed as described in Sect.  DNA extraction, PCR amplification and high-throughput sequencing . The PCR products of each gene were subsequently cloned and inserted into a T1 vector with a pEASY ® -T1 Cloning Kit (TransGen Biotech). The positive clones were subjected to plasmid extraction following the instructions of the EasyPure Plasmid MiniPrep Kit (TransGen Biotech). The plasmid DNA concentration was determined with a NanoDrop 2000. Tenfold dilution of the positive plasmid was conducted with DNase-free water, and a total of seven serial dilutions were obtained and considered plasmid standards. qPCR was performed on the plasmid standards via an ABI 7500 Fast sequencer as reported by Yu et al. [ 45 ] [ 45 ]. The cycle threshold (CT) values of each standard plasmid dilution (y-axis) and the corresponding log of its copy number (x-axis) were used to establish a standard curve. The copy number of the plasmid was obtained on the basis of its concentration [ 20 ]. The primers used for qPCR are listed in Table S4.

The DNA used for high-throughput sequencing was also used for the copy number assay. qPCR was performed on all the DNA samples described above. The copy number of each gene was calculated according to its standard curve and CT values.

Data analysis

The data from all repetitive experiments were pooled for further analysis. The fresh weight relative to the control group was fitted to a four-parameter log-logistic nonlinear regression model via SigmaPlot software (version 12.5). The herbicide concentration required for 50% growth inhibition (GR 50 ) was estimated according to the obtained log-logistic curve [ 39 ]. The residue of cinmethylin in the soil was fit to a first-order kinetic model as follows:

where y is the cinmethylin concentration at time t, y 0 represents the cinmethylin concentration at the initial time, and k represents the first-order rate constant. The half-life (t 1/2 ) represents the time needed to degrade to 50% of the initial concentration of cinmethylin and is calculated on the basis of the formula t 1/2 =(ln2)/ k . Principal coordinate analysis (PCoA) was used to visualise the bacterial community structure. FAPROTAX was employed to further analyse the bacterial functional groups. For bacterial co-occurrence network analysis, the Spearman correlation matrix was obtained on the basis of the relative abundance of OTUs. The significant correlations of each pairwise OTU with r  > 0.60 and P  < 0.05 were used to generate the network. This network was plotted with Gephi (version 0.92). Analysis of variance (ANOVA) and Duncan’s test were conducted via SPSS software (version 21.0) to evaluate the significant differences ( P  < 0.05) among the different treatments. Pearson’s correlation analysis was performed with SPSS software to determine the relationships between cinmethylin, soil characteristics, soil bacterial diversity and soil nitrifying.

Cinmethylin effectively inhibited the growth of A. Aequalis, A. japonicus, and A. myosuroides

To evaluate the potential of the preemergence herbicide cinmethylin to control grass weeds that infest wheat, the sensitivities of six troublesome grass weeds to this herbicide were assessed. The tested grass weeds presented diverse growth responses to cinmethylin (Fig.  1 A). At 21 DAT, more than 80% of the A. aequalis and A. japonicus plants were dead at the recommended field rate of cinmethylin, with GR 50 values of 78.77 and 61.49 g a.i. ha − 1 , respectively. Compared with that of the control group, the fresh weight of A. myosuroides decreased with increasing cinmethylin dose. Its growth was inhibited by an estimated 50% at a dose of 119.67 g a.i. ha − 1 . Approximately 25% of L. perenne plants were killed by cinmethylin at a dose of 393.75 g a.i. ha − 1 , and the GR 50 value was estimated at 535.61 g a.i. ha − 1 . However, no (A) fatua or (B) japonicus plants died at the recommended field concentration of cinmethylin, with GR 50 values over 787.50 g a.i. ha − 1 (twofold greater than the recommended field rate) (Fig.  1 B). Clearly, A. aequalis , A. japonicus and A. myosuroides were more sensitive to cinmethylin, L. perenne was moderately sensitive to cinmethylin, and (A) fatua and (B) japonicus were less sensitive to this herbicide.

Herbicidal effects of cinmethylin on six grass weed species infesting wheat. ( A ) Growth status of the six grass weed species at 21 days after treatment (DAT) with cinmethylin. ( B ) Growth dose responses of the six grass weed species to cinmethylin at 21 DAT. GR 50 represents the cinmethylin dose leading to 50% growth inhibition relative to the control

Degradation of cinmethylin in soil

To ascertain the dissipation dynamics of cinmethylin in soil, its residues in soil were measured at different times (Fig.  2 A). The concentration of cinmethylin gradually decreased with time in the soil. At 60 DAT, approximately 22.27%, 29.43% and 44.52% of the initial cinmethylin amount was maintained in the soil of the 0.2625, 2.625 and 26.25 mg kg − 1 treatments, respectively. Its half-life in the C1, C10 and C100 treatments was 26.46, 28.74 and 52.33 days (Fig.  2 B), respectively. Overall, cinmethylin degraded slowly in the soils treated with high amounts of cinmethylin.

Dissipation dynamics of cinmethylin in soil. ( A ) Procedure for cinmethylin extraction and detection. ( B ) Residues of cinmethylin in soil at different times after treatment with cinmethylin

Cinmethylin increases soil pH and decreases sucrase and urease activities

To determine whether cinmethylin affects soil physicochemical properties, the responses of soil pH, sucrase, urease and catalase to this herbicide were determined. Compared with that of the control group, the change in soil pH was relatively stable after treatment with cinmethylin. Throughout the experimental period, the pH of the soils treated with cinmethylin was significantly greater than that of the control soils, especially at 1 DAT ( P  < 0.0001) and 30 DAT ( P  < 0.0001) (Fig.  3 A). Therefore, cinmethylin increased the alkalinity of the soil. The activity of soil sucrase was obviously inhibited by cinmethylin at 30 DAT ( P  = 0.006), with 7.47%, 13.57% and 11.66% reductions in the C1, C10 and C100 groups, respectively. A similar result was also detected at 60 DAT ( P  < 0.0001), with an approximately 15.00% reduction in the treatment groups compared with the control group (Fig.  3 B). The activity of soil urease in the control group was 1101.90 U g − 1 at 15 DAT, which was 1.12-, 1.12- and 1.13-fold greater than that in the C1, C10 and C100 groups, respectively. At 60 DAT, its activity in the control group was also distinctly greater than that in the cinmethylin treatment groups ( P  = 0.001) (Fig.  3 C). However, no significant difference was observed in soil catalase activity between the control and treatment groups (Fig.  3 D). In total, cinmethylin had a significantly inhibitory effect on the activities of soil sucrase and urease, while its impact on soil catalase was slight.

Effects of cinmethylin on soil pH ( A ), sucrase activity ( B ), urease activity ( C ) and catalase activity ( D ). Different letters represent significant differences among different concentrations of cinmethylin at the same time ( P  < 0.05)

Cinmethylin influences bacterial abundance, diversity and composition

To evaluate the relationship between cinmethylin and soil bacteria, the bacterial communities in the control and cinmethylin-treated soils were studied via 16 S rRNA Illumina sequencing. The copy number of the 16 S rRNA gene determined by absolute quantitative real-time PCR was used to estimate bacterial abundance. Throughout the experimental period, the bacterial abundance initially remained stable (1, 7 and 15 DAT), then sharply increased (30 DAT) and then decreased (60 DAT). No significant ( P  = 0.981, P  = 0.938 and P  = 0.974 at 1, 7 and 15 DAT, respectively) difference was observed in bacterial abundance until 30 DAT. Compared with that in the control soil, the bacterial abundance in the C10- and C100-treated soils was obviously lower at 30 DAT ( P  = 0.027) and 60 DAT ( P  = 0.009) (Fig.  4 A). Cinmethylin at high concentrations clearly affected bacterial abundance in the late period.

Effects of cinurine on bacterial abundance, diversity and community composition. ( A ) Copy number of the 16 S rRNA gene under exposure to different cinmethylin application doses. ( B - D ) Sobs index, Shannon index and PD index for 16 S rRNA bacteria over time after cinmethylin treatment. ( E ) Principal coordinate analysis of soil bacteria. Different letters represent significant differences among different concentrations of cinmethylin at the same time ( P  < 0.05). C0, C1, C10 and C100 represent cinmethylin treatment at doses of 0, 0.2625, 2.625 and 26.25 mg kg − 1 , respectively. C0_1, C0_7, C0_15, C0_30 and C0_60 represent samples collected at 1, 7, 15, 30 and 60 days after cinmethylin treatment, respectively

The Sobs index, Shannon index and PD index were used to describe the diversity of bacteria. In the C1-treated soil, the Sobs index first decreased but then increased to the same level as that initially observed. Nevertheless, it gradually declined with time for the soils treated with C10 and C100. The Sobs index was significantly inhibited by C100 treatment at 1 DAT ( P  = 0.04) and 30 DAT ( P  = 0.012) (Fig.  4 B). The responses of the Shannon index and PD index to the C1 treatment were similar to those of the Sobs index throughout the experimental period. Compared with that of the control treatment, the Shannon index distinctly decreased in the soils treated with C10 and C100 at 30 DAT ( P  < 0.0001) and 60 DAT ( P  = 0.019). The PD index was significantly lower in the C100 treatment group than in the control group at 1 DAT ( P  = 0.003). Similar results were also observed at 30 DAT and 60 DAT, but the differences were not significant (Fig.  4 C − D). Bacterial diversity was inhibited by cinmethylin at the 10-fold and 100-fold recommended rates, especially during the late period of cinmethylin degradation. In the PCoA, the differences in bacterial community structure throughout the experimental period were caused mainly by time but not by the concentration of cinmethylin. The bacterial community was clearly divided into five groups according to the sampling time points (Fig.  4 E). The samples collected at 60 DAT were clearly separate from those collected at other time points.

Cinmethylin did not alter the composition of bacteria at the phylum or genus level; however, it changed their relative abundances. At the phylum level, Actinobacteria, Proteobacteria, Acidobacteria and Chloroflexi were the top four dominant phyla, accounting for 80% of the bacterial community abundance (Fig.  5 A). At the genus level, norank_f_Vicinamibacteraceae , norank_f_norank_o_Vicinamibacterales , norank_f_JG30-KF-CM45 , noranl_f_norank_o_Gaiellales and Gaiella were the dominant species in the bacterial communities of each group (Figure S1 ). Among these dominant genera, cinmethylin had significant effects on the abundances of norank_f_Vicinamibacteraceae , norank_f_norank_o_Vicinamibacterales and noranl_f_norank_o_Gaiellales . The abundances of norank_f_Vicinamibacteraceae and norank_f_norank_o_Vicinamibacterales were greater in the cinmethylin-treated soils than in the control soils, and their abundances were positively related to the cinmethylin concentration, suggesting that these two species have the potential to degrade cinmethylin. However, cinmethylin had an inhibitory effect on the abundance of noranl_f_norank_o_Gaiellales (Figure S2 ).

( A ) Relative abundance of 16 S rRNA bacteria in different samples at the phylum level. ( B ) Heatmap of the top 50 dominant functional groups. The functional groups marked with boxes are the groups associated with the soil nitrogen cycle. C0, C1, C10 and C100 represent cinmethylin treatment at doses of 0, 0.2625, 2.625 and 26.25 mg kg − 1 , respectively. C0_1, C0_7, C0_15, C0_30 and C0_60 represent samples collected at 1, 7, 15, 30 and 60 days after cinmethylin treatment, respectively

Bacterial community functions

The most abundant functional groups were those associated with chemoheterotrophy, aerobic chemoheterotrophy, aromatic compound degradation and nitrate reduction. The relative abundances of genes related to chemoheterotrophy, aerobic chemoheterotrophy, aromatic compound degradation and nitrate reduction were comparable among the different treatments (C0, C1, C10 and C100). Among the top 50 dominant functional groups, 13 were associated with the soil nitrogen cycle. In the C10 and C100 groups, nitrite ammonification, nitrate ammonification, nitrite denitrification, nitrate denitrification, nitrous oxide denitrification, denitrification, nitrite respiration, nitrogen respiration, nitrate respiration and nitrogen fixation, which are involved in the soil nitrogen cycle, presented relatively low relative abundances. The lowest relative abundances of nitrification and aerobic nitrite oxidation were observed in the C100 treatment (Fig.  5 B).

Cinmethylin at high concentrations promotes bacterial network complexity

To clarify the effects of cinmethylin on the bacterial community network, co-occurrence networks were established under different treatments. As shown in Fig.  6 , the bacterial networks formed differed among the treatments. The number of nodes in the bacterial network was comparable among the different groups. Compared with that in the control group, the clustering coefficient of the bacterial network formed was greater in the C1 treatment group, slightly lower in the C10 treatment group and obviously greater in the C100 treatment group. Similar tendencies were also detected for the network density, average degree and number of edges (Fig.  6 C). In each network, the composition of the modules was different (Fig.  6 A and B). In the C0 network, Acidobacteria, Proteobacteria and Actinobacteria dominated in Module I, Module II and Module IV, respectively. Acidobacteriota mainly co-occurred with Actinobacteria, and Proteobacteria primarily co-occurred with Chloroflexi. In the C1 network, Actinobacteria, Proteobacteria and Acidobacteria were the dominant members of Module I, Module II and Module IV, respectively. Acidobacteriota mainly co-occurred with Actinobacteria. In the C10 network, Module II and Module VI were composed mainly of Acidobacteriota and Actinobacteriota, respectively. Proteobacteria was the dominant member in Module VII. Proteobacteria primarily co-occurred with Chloroflexi. In the C100 network, Acidobacteriota dominated primarily in Module I. Module II consisted mainly of Actinobacteriota, Proteobacteria and Chloroflexi. Collectively, cinmethylin at 1- and 10-fold the recommended rate had a relatively slight effect on the bacterial network, whereas cinmethylin at 100-fold the recommended rate largely promoted bacterial complexity.

Effects of cinmethylin on the complexity of the bacterial co-occurrence network. ( A - B ) Visual co-occurrence network of 16 S rRNA bacteria under different cinmethylin application rates. ( A ) Nodes are coloured by module. The bacterial modules I-VIII were the eight clusters of closely interconnected nodes. ( B ) Nodes are coloured according to bacterial phyla. The red and green edges represent positive and negative interactions between two nodes, respectively. The sizes of the nodes in A and B are proportional to the number of connections (degree). ( C ) Parameters for the co-occurrence network of 16 S rRNA bacteria. C0, C1, C10 and C100 represent cinmethylin treatment at doses of 0, 0.2625, 2.625 and 26.25 mg kg − 1 , respectively

Effects of cinmethylin on soil nitrification

To determine the influence of cinmethylin on the N-cycling function of soil, the abundance of bacteria involved in soil nitrification was assessed under different treatments. The abundance of nitrifying bacteria Nitrospira assayed by 16 S rRNA Illumina sequencing was significantly influenced by cinmethylin at 1 DAT ( P  = 0.012), 15 DAT ( P  = 0.02) and 30 DAT ( P  = 0.048) (Fig.  7 A). At these time points, it was obviously lower in the soil treated with 100-fold the recommended rate of cinmethylin than in the control group, suggesting that cinmethylin at high concentrations inhibited soil nitrification. To verify this result, the copy numbers of AOA amoA and AOB amoA functional genes involved in soil nitrification were assayed. In contrast with the results of the control group, a visible decrease in the AOA amoA and AOB amoA copy numbers was detected in the cinmethylin-treated soil at 1 DAT, especially at high concentrations. However, at 60 DAT, their copy numbers increased to the maximum numbers, which were significantly greater than that of the control group (Fig.  7 B-C). Clearly, cinmethylin at high concentrations temporarily inhibited the soil nitrification process.

Effects of cinmethylin on soil nitrification. ( A ) Changes in the abundance of the Nitrospira genus following different cinmethylin application rates over time. ( B - C ). Copy numbers of the AOA amoA and AOB amoA genes in soils treated with different concentrations of cinmethylin over time. Different letters represent significant differences among different concentrations of cinmethylin at the same time ( P  < 0.05). C0, C1, C10 and C100 represent cinmethylin treatment at doses of 0, 0.2625, 2.625 and 26.25 mg kg − 1 , respectively

Relationships among cinmethylin, soil characteristics, bacterial diversity and soil nitrifying

To illuminate the relationships among soil characteristics, the soil bacterial community and soil nitrification, Pearson correlation analysis was performed in this study. The results revealed that cinmethylin residues in the soil were negatively related to the soil pH and the relative abundance of Nitrospirota . Soil pH was positively correlated with soil catalase but negatively correlated with soil urease. Soil urease and sucrase activities were positively related to bacterial diversity, whereas soil catalase activity was negatively related to bacterial diversity. The relative abundance of Nitrospirota was positively related to soil urease and sucrase. The copy numbers of AOA amoA and AOB amoA were positively related to soil catalase (Fig.  8 ).

Pearson correlations among cinmethylin residues, soil characteristics, bacterial diversity and soil nitrification. The values in the upper right represent the correlation coefficient. * and ** in the lower left represent significant correlations at P  < 0.05 and P  < 0.01, respectively

Currently, ACC-inhibiting herbicides such as clodinafop-propargyl and fenoxaprop-p-ethyl and ALS-inhibiting herbicides such as mesosulfuron-methyl and pyroxsulam are widely used for grass weed control in wheat fields in China. Owing to their excessive use for a long period, various levels of resistance to these herbicides have evolved in some grass weed species, such as A. aequalis , which is resistant to mesosulfuron-methyl and fenoxaprop-P-ethyl [ 49 ]; Beckmannia syzigachne , which is resistant to mesosulfuron-methyl [ 41 ]d japonicus , which is resistant to flucarbazone-sodium [ 29 ]. The prevalence of weed resistance poses a serious threat to wheat yield. Recently, the commercialised herbicide cinmethylin, which is registered for use in rice fields, has the potential to control grass weeds in wheat fields before the emergence of crops. In this study, cinmethylin had good control effects on the troublesome weeds A. aequalis , A. japonicus , A. myosuroides and L. perenne . Owing to its unique mode of action, cinmethylin may be a good alternative herbicide to solve resistance problems in A. aequalis , A. japonicus , A. myosuroides and L. perenne . Although cinmethylin did not kill (A) fatua or (B) japonicus plants at the recommended field rate, it had an inhibitory effect on the growth of these weed species. Cinmethylin application before the emergence of wheat is a good strategy to synergize the control effect of postemergence herbicides on (A) fatua and (B) japonicus .

In the field, the half-life of cinmethylin was approximately 22.4 days ( http://sitem.herts.ac.uk/aeru/iupac/Reports/1021.htm ), which was lower than our observations in the C1 (26.46 d), C10 (28.74 d) and C100 (52.33 d) treatments. Pesticide degradation in soil is associated with the soil type. In soils containing high levels of organic matter, slow degradation was observed for myclobutanil, clomazone and diazinon [ 12 , 28 , 33 ]. The differences in the cinmethylin degradation rates may be due to the different soil types used in this study and previous studies [ 11 , 22 ]. However, one soil type was used in this research, which is limited to evaluating the dynamics of cinmethylin degradation in soil. Thus, more experimental data regarding cinmethylin dissipation in different soil types are needed in the future. The degradation rate of cinmethylin in the C1 treatment was comparable to that in the C10 treatment, which was obviously greater than that observed in the C100 treatment. Like cinmethylin, some herbicides, such as mesosulfuron-methyl and clomazone, also degrade slowly at high concentrations [ 10 , 12 ]. In the present study, the richness of Sphingomonas significantly decreased in the C100-contaminated soil compared with that in the control soil, whereas no significant difference was observed between the C1 and C10 treatment groups (Figure S2). Sphingomonas can degrade some pesticides, such as chlorpyrifos and ortho -phenylphenol [ 15 , 16 , 35 ]. Therefore, this phenomenon might be due to the adverse effects that high amounts of cinmethylin have on the growth of bacteria related to agrochemical degradation, such as Sphingomonas , ultimately resulting in a slow dissipation rate of cinmethylin in the soil. The bacterial diversity was distinctly decreased in the C100 treatment group, which also suggested that some bacterial groups related to cinmethylin degradation were inhibited under exposure to high amounts of cinmethylin.

Soil acidity–alkalinity is an important soil physiochemical property that directly influences crop growth and soil microbiome activities. In this study, cinmethylin increased the alkalinity of the soil, which differed from the decreased pH in soil contaminated with the herbicide glyphosate [ 18 ]. Cinmethylin can be degraded into hydroxylated metabolites by hydroxylation of the isopropyl moiety in soil (Woodward et al., 1986). The release of hydroxyl ions from hydroxylated metabolites might increase the soil pH. Soil functional enzymes are also closely related to the retention of soil function. Soil urease can hydrolyse urea in soil to generate NH 3 and carbonic acid [ 1 ]. The activity of soil urease is an important indicator reflecting the condition of soil nitrogen. The NH 4 + ions yielded by soil urease can be rapidly transformed into NO 3 − ions and H + ions through the nitrification process, and NO 3 − can leach down with water [ 1 ]. A decrease in urease activity in soil caused by cinmethylin could reduce urea hydrolysis and then inhibit nitrification that leads to the production of NO 3 − ion and H + ion, which indirectly increased soil pH. In addition, cinmethylin obviously increased the alkalinity of the soil but decreased the soil urease activity. In the correlation analysis, a significantly negative relationship was detected between soil pH and soil urease activity. Soil pH is an important factor influencing soil urease activity [ 7 , 14 ]. In galaxolide-contaminated soil, Solanum nigrum affects soil urease activity by regulating the soil pH [ 32 ]. Therefore, it was speculated that cinmethylin contamination inhibited soil urease activity by increasing the soil pH.

Real-time quantitative PCR, 16 S rRNA Illumina sequencing and bioinformatics were used to determine the effects of cinmethylin exposure on the bacterial community. The abundance and diversity of bacteria distinctly decreased in response to high cinmethylin concentrations in the late period of degradation. Several herbicides, including pyroxasulfone [ 43 ], imazethapyr [ 36 ], and fomesafen [ 24 ], have also been reported to affect microbial community structure. Additionally, different bacterial genera responded differently to cinmethylin pollution, indicating that bacterial species might have different levels of sensitivity to the environmental stress caused by cinmethylin. At the genus level, the abundances of norank_f_Vicinamibacteraceae and norank_f_norank_o_Vicinamibacterales were significantly greater in the cinmethylin-treated soil than in the control group. The Vicinamibacteraceae family was first identified in 2018, and some members belonging to this family can degrade chemicals with complex structures [ 25 ]. Thus, norank_f_Vicinamibacteraceae might participate in the biodegradation of cinmethylin. On the basis of an obvious reduction in soil urease activity caused by cinmethylin and the well-established knowledge regarding the relationships between soil urease and soil nitrification as described earlier [ 1 ], we speculated that this herbicide had a decreased effect on the abundance of nitrifiers. To validate these findings, the abundance of the genus Nitrospira and the copy numbers of the AOA amoA and AOB amoA genes were studied via 16 S rRNA Illumina sequencing and qPCR after exposure to cinmethylin, respectively. According to the results of the abundance of Nitrospira , AOA amoA and AOB amoA , cinmethylin imposed a transient decrease in soil nitrification, which was supported by a decrease in the relative abundance of predicted functional groups related to the soil nitrogen cycle under exposure to cinmethylin. The inhibition of soil urease activity by cinmethylin would result in less ammonium being released from organic nitrogen to feed the ammonia oxidisers for nitrification. The soil urease activity was positively correlated with the relative abundance of the Nitrospirota genus. Additionally, the inhibition of nitrifiers by cinmethylin was transient. Therefore, cinmethylin might have an indirect rather than direct toxic effect on soil nitrification. Cinmethylin might impede the soil nitrification process by inhibiting soil urease. Actually, there were many biological factors controlling soil nitrification. For example, the fungal community could also influence the soil nitrification. However, in current study, the response of soil nitrification to cinmethylin were analysed in view of soil bacteria. In the future, it is necessary to evaluate the effect of cinmethylin on soil nitrification based on more experimental data.

Co-occurrence patterns are powerful tools for studying changes in microbial community structure and discovering potential microbiome interactions [ 2 , 37 ]. The microorganisms in one module have strong interactions and similar habitat preferences. As shown in Fig.  5 , the complexity of the bacterial network was obviously promoted by cinmethylin at high concentrations. This finding is inconsistent with previous results showing a decline in microbial complexity in soils treated with the insecticide thiamethoxam [ 47 ], the fungicides fosety-Al and propamocarb-hydrochloride [ 17 ], or the herbicide atrazine [ 42 ]. The increased complexity of the bacterial community in the soil treated with a high dose of cinmethylin might be due to close cooperation among more bacterial species to resist the increasing environmental stress caused by a high dose of cinmethylin.

Overall, the herbicide cinmethylin effectively controlled the grass weeds A. aequalis , A. japonicus and A. myosuroides when applied before the emergence of crops and weeds, suggesting its potential to control grass weeds in wheat fields as a preemergence herbicide. Given the responses of soil pH, soil enzyme activity, the bacterial community and soil nitrification to cinmethylin, the herbicide at the recommended rate hardly influenced the soil ecosystem. However, the potential risks to the soil ecosystem caused by high amounts of cinmethylin deserve further attention. Cinmethylin at high concentrations inhibited soil urease activity by increasing the soil pH; reduced soil urease activity was most likely to cause a temporary decline in soil nitrification. These findings will provide scientific guidance for the use of the existing herbicide cinmethylin in a new scenario where it is directly applied to the soil surface and will help further understand the relationships between cinmethylin and the soil ecosystem.

Data availability

The raw sequence data has been deposited in the NCBI Sequence Read Archive (SRA) database with accession number of PRJNA1141104.

Antonious GF. Impact of soil management and two botanical insecticides on urease and invertase activity. J Environ Sci Heal B. 2003;B38:479–88.

Article   CAS   Google Scholar  

Banerjee S, Walder F, Büchi L, Meyer M, Held AY, Gattinger A, Keller T, van der Charles R. Heijden, M G A. Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots. ISME. 2019;13:1722–36.

Article   Google Scholar  

Bottomley PJ, Taylor AE, Myrold DD. A consideration of the relative contributions of different microbial subpopulations to the soil N cycle. Front Microbiol. 2012;3:373.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Busi R, Dayan FE, Francis I, Goggin D, Lerchl J, Porri A, Powles SB, Sun C, Beckie HJ. Cinmethylin controls multiple herbicide resistant Lolium rigidum and its wheat selectivity is P450-based. Pest Manag Sci. 2020;76:2601–8.

Article   CAS   PubMed   Google Scholar  

Campe R, Hollenbach E, Kämmerer L, Hendriks J, Höffken HW, Kraus H, Lerchl J, Mietzner T, Tresch S, Witschel M, Hutzler. J. A new herbicidal site of action: cinmethylin binds to acyl-ACP thioesterase and inhibits plant fatty acid biosynthesis. Pestic Biochem Physiol. 2018;148:116–25.

Chen T, Nomura K, Wang X, Sohrabi R, Xu J, Yao L, Paasch BC, Kremer LM, Cheng J, Zhang Y, Wang L, Wang N, Xin E, He X. A plant genetic network for preventing dysbiosis in the phyllosphere. Nature. 2020;580:653.

Dalal RC. Distribution, salinity, kinetic and thermodynamic characteristics of urease activity in a vertisol profile. Aust J Soil Res. 1985;23:49–60.

Dayan FE, Romagni JG, Duke SO. Herbicides: cinmethylin. In: Plimmer JR, Gammon DW, Ragsdale, N N, editors. Encyclopedia of agrochemicals. New York: Wiley; 2003.

Google Scholar  

Di Cesare A, Pjevac P, Eckert E, Curkov N, Šparica MM, Corno G, Orli. The role of metal contamination in shaping microbial communities in heavily polluted marine sediments. Environ Pollut. 2020;265:114823.

Article   PubMed   Google Scholar  

Du P, He H, Wu X, Xu J, Dong F, Liu X, Zheng Y. Mesosulfuron-methyl influenced biodegradability potential and N transformation of soil. J Hazard Mater. 2021;416:125770.

Du P, He H, Zhou L, Dong F, Liu X. Different biodegradation potential and the impacted soil functions of epoxiconazole in two soils. J Hazard Mater. 2022;422:126787.

Du P, Wu X, Xu J, Dong F, Liu X, Zhang Y, Zheng Q. Clomazone influence soil microbial community and soil nitrogen cycling. Sci Total Environ. 2018a;644:475–85.

Du ZK, Zhu YY, Zhu LS, Zhang J, Li B, Wang JH, Wang J, Zhang C, Cheng C. Effects of the herbicide mesotrione on soil enzyme activity and microbial communities. Ecotox Environ Safe. 2018b;164:571–8.

Ebrahimi M, Sarikhani MR, Shiri J, Shahbazi F. Modeling soil enzyme activity using easily measured variables: heuristic alternatives. Appl Soil Ecol, 2021, 103753.

Feng FY, Ge J, Li YS, Cheng JJ, Zhong JF, Yu XY. Isolation, colonization, and chlorpyrifos degradation mediation of the endophytic bacterium Sphingomonas strain HJY in Chinese chives ( Allium tuberosum ). J Agric Food Chem. 2017;65:1131–8.

Feng FY, Zhan HL, Wan Q, Wang Y, Li Y, Ge J, Sun X, Zhu H, Yu XY. Rice recruits Sphingomonas strain HJY- rfp via root exudate regulation to increase chlorpyrifos tolerance and boost residual catabolism. J Exp Bot. 2021;15:5673–86.

Fournier B, Pereira Dos Santos S, Gustavsen JA, Imfeld G, Lamy F, Mitchell EAD, Mota M, Noll D, Planchamp C, Heger TJ. Impact of a synthetic fungicide (fosetyl-Al and propamocarb-hydrochloride) and a biopesticide ( Clonostachys Rosea ) on soil bacterial, fungal, and protist communities. Sci Total Environ. 2020;738:139635.

García-Pérez JA, Alarcón E, Hernández Y, Hernández C. Impact of litter contaminated with glyphosate-based herbicide on the performance of Pontoscolex corethrurus, soil phosphatase activities and soil pH. Appl Soil Ecol. 2016;104:31–41.

GB/T31270.1-2014. Test guidelines on Environmental Safety Assessment for Chemical pesticides-Part. Volume 16. Soil Microorganism Toxicity Test; 2014.

Godornes C, Leader BT, Molini BJ, Centurion-Lara A, Lukehart SA. Quantitation of rabbit cytokine mRNA by real-time RT-PCR. Cytokine. 2007;38:1–7.

Goggin DE, Cawthray GR, Busi R, Porri A, Beckie HJ. Enhanced production of water-soluble cinmethylin metabolites by Lolium rigidum populations with reduced cinmethylin sensitivity. Pest Manag Sci. 2022;78:3173–82.

Han LX, Kong XB, Xu M, Nie JY. Repeated exposure to fungicide tebuconazole alters the degradation characteristics, soil microbial community and functional profiles. Environ Pollut. 2021;287:117660.

Hartman WH, Richardson CJ, Vilgalys R, Bruland GL. Environmental and anthropogenic controls over bacterial communities in wetland soils. P Natl Acad Sci USA. 2008;105:17842–7.

Hu H, Zhou H, Zhou S, Li Z, Wei C, Yu Y, Hay. A. Fomesafen impacts bacterial communities and enzyme activities in the rhizosphere. Environ Pollut. 2019;253:302–11.

Huber KJ, Overmann J. Vicinamibacteraceae fam. nov., the first described family within the subdivision 6 Acidobacteria . Int J Syst Evol Microbiol, 2018, 68:2331–2334.

Hussain S, Siddique T, Saleem M, Arshad M, Khalid A. Impact of pesticides on soil microbial diversity, enzymes, and biochemical reactions. Adv Agron. 2009;102:176–88.

Jones RG. In: Grayson BT, Green MB, Copping, L G, editors. Cinmethylin - a new herbicide developed for use in rice. Dordrecht: Springer; 1990.

Chapter   Google Scholar  

Ju C, Xu J, Wu X, Dong F, Liu X, Zheng Y. Effects of myclobutanil on soil microbial biomass, respiration, and soil nitrogen transformations. Environ Pollut. 2016;208:811–20.

Lan Y, Zhou X, Lin S, Cao Y, Wei S, Huang H, Li W, Huang Z. Pro-197-Ser mutation and cytochrome P450-mediated metabolism conferring resistance to flucarbazone-sodium in Bromus japonicus . Plants. 2022;11:1641.

Lee PW, Forbis AD, Franklin L. Bioaccumulation of cinmethylin in bluegill sunfish. J Agric Food Chem. 1990;38:323–7.

Lee PW, Stearns SM, Powell WR, Stoutamire DW, Payne. G B. Metabolic fate of cinmethylin in rats. J Agric Food Chem. 1986;34:162–70.

Lv Z, Ao S, Zhang C, Han XM, Li XG. The effects of Solanum Nigrum L. on soil enzyme activities with pollution of galaxolide and cadmium. J Shenyang Jianzhu Univ (Natural Science). 2022;38:953–60.

Marín-Benito J, Herrero-Hernández E, Soledad Andrades M, Sánchez-Martín M, Rodríguez-Cruz M. Effect of different organic amendments on the dissipation of linuron, diazinon and myclobutanil in an agricultural soil incubated for different time periods. Sci Total Environ. 2014;476–477:611–21.

Messelhauser MH, Saile M, Sievernich B, Gerhards R. Effect of cinmethylin against Alopecurus myosuroides huds. In winter cereals. Plant Soil Environ. 2021;67:46–54.

Perruchon C, Patsioura V, Vasileiadis S, Karpouzas DG. Isolation and characterisation of a Sphingomonas strain able to degrade the fungicide Ortho -Phenylphenol. Pest Manag Sci. 2016;72:113–24.

Pertile M, Sousa RMS, Mendes LW, Antunes JEL, de Souza Oliveira LM, de Araujo FF, Melo VMM, Araujo. A S F. Response of soil bacterial communities to the application of the herbicides imazethapyr and flumyzin. EUR J Soil Biol. 2021;102:103252.

Qiu LP, Zhang Q, Zhu HS, Reich PB, Banerjee S, van der Heijden MGA, Sadowsky MJ, Ishii S, Jia XX, Shao MG, Liu BY, Jiao H, Li HQ, Wei XR. Erosion reduces soil microbial diversity, network complexity and multifunctionality. ISME. 2021;15:2474–89.

Regar RK, Gaur VK, Bajaj A, Tambat S, Manickam N. Comparative microbiome analysis of two different long-term pesticide contaminated soils revealed the anthropogenic influence on functional potential of microbial communities. Sci Total Environ. 2019;681:413–23.

Seefeldt SS, Jensen JE, Fuerst EP. Log-logistic analysis of herbicide dose-response relationship. Weed Technol. 1995;9:218–27.

Tilman D, Knops J, Wedin D, Reich P, Ritchie M, Siemann E. The influence of functional diversity and composition on ecosystem processes. Science. 1997;277:1300–2.

Wang JJ, Chen JC, Li XJ, Li D, Li Z, Cui HL. Pro-197-Ser mutation in ALS and high-level GST activities: multiple resistance to ALS and ACCase inhibitors in Beckmannia syzigachne . Front Plant Sci. 2020;11:572610.

Article   PubMed   PubMed Central   Google Scholar  

Wang P, Cao J, Mao L, Zhu L, Zhang Y, Zhang L, Jiang H, Zheng Y, Liu X. Effect of H 3 PO 4 -modified biochar on the fate of atrazine and remediation of bacterial community in atrazine-contaminated soil. Sci Total Environ. 2022;851:158278.

Wang Y, Men J, Zheng T, Ma YH, Li W, Cernava T, Jin LB. Impact of pyroxasulfone on sugarcane rhizosphere microbiome and functioning during field degradation. J Hazard Mater. 2023;455:131608.

Xu Y, He Y, Tang X, Brookes PC, Xu J. Reconstruction of microbial community structures as evidences for soil redox coupled reductive dechlorination of PCP in a mangrove soil. Sci Total Environ, 2017, 596–7:147–157.

Yu H, Cui H, Chen J, Li X. Regulation of Aegilops tauschii Coss tiller bud growth by plant density: transcriptomic, physiological and phytohormonal responses. Front Plant Sci. 2020;11:1166.

Yu H, Huang S, Chen P, Ji M, Cui H, Chen J, Li X. Different leaf-mediated deposition, absorbed and metabolism behaviors of 2,4-D isooctyl ester between Triticum aestivum and Aegilops tauschii Coss. Pestic Biochem Physiol. 2021;175:104848.

Zhang H, Zhang Z, Song J, Mei J, Fang H, Gui W. Reduced bacterial network complexity in agricultural soils after application of the neonicotinoid insecticide thiamethoxam. Environ Pollut. 2021a;274:116540.

Zhang YQ, Zhang JW, Shi BH, Li B, Du ZK, Wang J, Zhu LS, Wang JH. Effects of cloransulam-methyl and diclosulam on soil nitrogen and carbon cycle-related microorganisms. J Hazard Mater. 2021b;418:126395.

Zhao N, Yan Y, Ge L, Zhu B, Liu W, Wang J. Target site mutations and cytochrome P450s confer resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl in Alopecurus aequalis . Pest Manag Sci. 2019;75:204–14.

Zhu XY, Zhu H, Zheng ZQ, Ding LJ. Study on the determination of cinmethylin residues in cereals. Inspection Quarantine Sci. 1999;9:4–5.

Download references

This work was financially supported by Nanfan special project, CAAS (SWAQ03).

Author information

Authors and affiliations.

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

Haiyan Yu, Hailan Cui, Jingchao Chen & Xiangju Li

You can also search for this author in PubMed   Google Scholar

Contributions

H.Y wrote the main manuscript text and conducted the experiments; H.L. and J.C. analyzed the data; X.J. designed the experiments and reviewed the manuscript.

Corresponding author

Correspondence to Xiangju Li .

Ethics declarations

Ethics approval and consent to participate.

Not applicable.

Consent for publication

Competing interests.

The authors declare no competing interests.

Additional information

Publisher’s note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ .

Reprints and permissions

About this article

Cite this article.

Yu, H., Cui, H., Chen, J. et al. The herbicidal activity of pre-emergence herbicide cinmethylin and its potential risks on soil ecology: pH, enzyme activities and bacterial community. Environmental Microbiome 19 , 66 (2024). https://doi.org/10.1186/s40793-024-00608-y

Download citation

Received : 12 March 2024

Accepted : 26 August 2024

Published : 08 September 2024

DOI : https://doi.org/10.1186/s40793-024-00608-y

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Cinmethylin
• Weed control efficiency
• Soil characteristics
• Bacterial community
• Soil nitrification
• 16S rRNA illumina sequencing

Environmental Microbiome

ISSN: 2524-6372

• Submission enquiries: [email protected]