Effect Of H₂S Signal Regulating Stomatal Development Of Leaves Via EPFs Peptides In Arabidopsis

Stomata,a unique structure of plant epidermis,whose pore size and density distribution directly determine the photosynthetic efficiency of plant leaves and crop yield,so it is an important morphological indicators in agricultural production.Stomatal development is regulated by the strict "single-cell spacing principle".Epidermal pattern factors?EPFs?are the key links,EPF1 and EPF2 are negative regulators,and EPFL9 is a positive regulator.Hydrogen sulfide?H₂S?signal is involved in growth and development,and stress resistance in plant.The role of H₂S in regulating stomatal movement has attracted more and more attention.In contrast,the effect of H₂S signal on stomatal development has been rarely reported.Therefore,research on the regulation mechanism of leaf stomatal development by H₂S will provide more ideas for understanding the mechanism of H₂S gas signal in the plant response to stress.In this study,the model plant Arabidopsis was used as the material to explore the mechanism of H₂S signal involved in the regulation of stomatal development via EPFs peptides.The main results are as follows:1.H₂S mutants lcd,des1,and lcd/des1,with a major loss of enzyme-encoding genes,whose stomatal density of leaves were significantly reduced compared with wild-type WT;compared with wild-type WT,WT+HA significantly reduced new leaves stomatal density;The stomatal density of new leaves in the WT+Na HS and WT+HT treatment groups did not change significantly compared with wild-type WT;compared with lcd/des1,the stomatal density of new leaves in the lcd/des1+Na HS recovered.Compared with WT,the stomatal index of new leaves in the lcd,lcd/des1 and WT+HA had a tendency to reduce,the stomatal index of new leaves in the des1,WT+Na HS,lcd/des1+Na HS and WT+HT had a tendency to increase,but there was no obvious difference of the stomatal index in the all treatment groups.Compared with WT,there was no significant difference in the size of new leaves guard cells in all treatment groups.The above results indicate thatH₂S signal regulates stomatal development in Arabidopsis.2.Compared with WT,the H₂S content and H₂S production rate of the new leaves in mutants lcd,des1,lcd/des1,WT+HA and WT+HT were significantly reduced.While,the H₂S content of new leaves of WT+Na HS and lcd/des1+Na HS was significantly increased.Compared with WT,the H₂S production rate of the new leaves of lcd/des1+Na HS was significantly reduced,and the H₂S production rate of the new leaves of WT+Na HS had a tendency to increase,but there was no obvious difference.The above results indicate that the changes in H₂S content and H₂S production rate of the new leaves may be the cause of the differences in the development of the stomata of the new leaves.3.When physiologically concentrated Na HS treated WT for 0.5 h,the changes of EPFs-encoded genes were more obvious,EPF1 and EPF2 genes showed a significant downward trend,and EPFL9 showed a significant upward trend.Compared with wild-type WT,the mutant lcd,des1,and lcd/des1 showed a significant increase in the expression of EPF2 in the new leaves;compared with wild-type WT,lcd/des1+Na HS showed a significant increase in the expression of EPFL9 in the new leaves.The above results indicate that the genes encoding EPFs will respond to H₂S signals in a short time,and the participation of endogenous H₂S and exogenous H₂S will have different genetic responses to Arabidopsis EPFs.4.Construction of EPFs prokaryotic expression vector,induction expression and purification of EPFs polypeptides.Using Arabidopsis c DNA as a template,EPF1,EPF2 and EPFL9 genes were cloned to obtain recombinant plasmids p ET28a-EPF1,p ET28a-EPF2 and p ET28a-EPFL9;recombinant plasmids were transferred into E.coli BL21?DE3?for isopropyl?-D-thiogalactoside?IPTG?induction and expression.Optimized expression conditions: IPTG-induced concentrations were 0.5,0.3,and 0.05 m M respectively;optimal induction temperatures were 28 ?,28 ?,and 16 ?;optimal induction times were 16 h,16 h,and 20 h,respectively;The fusion protein was purified by Ni agarose gel column and the size was about 18 k Da,19 k Da and 14.5 k Da,respectively.5.EPFs fusion protein expressed and purified in vitro by H₂S treatment was tested for S-sulfhydration by biotin switch method.The results showed that the S-sulfhydration signals of EPF2 and EPFL9 proteins were significantly higher than those of the control,and there was no significant change in EPF1 protein.It is suggested that H₂S may play an important role in the regulation of stomatal development in Arabidopsis thaliana through the modification of EPF2 and EPFL9 proteins by S-sulfhydration.In summary,H₂S signals are involved in the regulation of stomatal development in Arabidopsis thaliana through EPFs polypeptides,H₂S regulates the transcriptional expression of EPFs,and S-sulfhydration modifies EPF2 and EPFL9 proteins,which in turn affects the development of Arabidopsis stomata and improves Arabidopsis thaliana Stomatal density of leaf epidermis.It provides basic data for further research on the mechanism of the gas signal H₂S on the development of plant stomata,which is of great significance to increase crop yield and enhance plant stress resistance.