Systemtic Identification Of Phytohormone-binding Proteins And Functional Characterization

Phytohormones are signal molecules produced within the plants.These molecules involve in many physiological processes,including modulating plant growth and development,mediating plant immune response,and many other important biological processes.It is important to note that phytohormones play key roles under various environmental stresses and also play a momentous influence on the accumulation of secondary metabolites.Meanwhile phytohormones are important for medicinal plants to survive under stress conditions and the underlying molecular mechanisms are of keen interest.Even though phytohormones function on the premise of binding to receptor proteins in plants,systematic identification of phytohormone-binding proteins and investigating the molecular functions of such interactions remain challenging.Objectives:Because the molecular functions of phytohormones are highly conserved in higher plants,this project employed Arabidopsis as a model system to establish a platform to systematically profile phytohormone-binding proteins and study the related functions.Using salicylic acid and abscisic acid as two representative examples,this project set up a chemical proteomic approach to identify the binding proteins of these two phytohormones and explore the molecular mechanisms.These results may provide insights into the cultivation of medicinal plants under stress conditions.Methods:(1)Characterizing the molecular mechanisms of salicylic acid using a chemical proteomic approach.Salicylic acid(SA)is a defense hormone that primes the plants for better survival under biotic and abiotic stresses.I used a chemical proteomic approach to uncover SA-mediated proteomic remodeling in Arabidopsis thaliana,focusing on the effects of SA on newly synthesized proteins as well as SA binding proteins(SABPs).First,Bioorthogonal noncanonical amino acid tagging(BONCAT)combined with Click Chemistry was used to label newly synthesized proteins in Arabidopsis thaliana,and the azide group on the AHA probe can selectively modify and enrich the labeled newly synthesized protein through the click reaction.In this study,the probe is the methionine surrogate L-Azidohomoalanine(AHA),which carries a reactive azide moiety in its amino acid side chain.The azide group in Aha can be selectively modified and enrichment of the labeled newly synthesized protein through the click reaction.In addition,salicylic acid probe(SA-P)was designed and synthesized based on the structure of SA.Combined with click chemistry reaction,the optimal SAP concentration in Arabidopsis lysates was determined.Based on ABPP and LCMS/MS,SABPs in Arabidopsis lysates were identified.Through bioinformatics analysis and functional cluster analysis,target protein and its related signal pathway were screened out.The target protein was purified by constructing gene expression vector and exogenous expression.Surface plasmon resonance(SPR)and differential scanning fluorimetry(DSF)were used to verify whether SA binds to these proteins.The potential binding sites between SA and target proteins were detected by mass spectrometry.The effects of SA treatment on expressions and activities of target proteins were investigated by Western blotting(WB).(2)Characterizing the molecular mechanisms of abscisic acid(ABA)using a chemical proteomic approach.According to the structure of ABA,Abscisic acid probe(ABA-P)was designed and synthesized.Using the model Arabidopsis thaliana,the suitable concentration of ABA-P in Arabidopsis lysate was screened by chemical reaction.At the same time,the binding proteins of ABA were analyzed qualitatively and quantitatively by protein mass spectrometry.By means of bioinformatics analysis combined with biological questions,the target proteins with high reliability were screened out.The target protein was purified by constructing gene expression vector and exogenous expression.The labeling and competition experiments by using recombinant proteins and surface plasmon resonance(SPR)techniques were carried out to verify whether ABA binds to these target proteins.The effects of ABA on the activities of target proteins were also assessed by using GST activity assay kit.The specific binding sites between ABA and target protein were identified by mass spectrometry.In addition,we compared the expression levels of ABA-related marker genes in WT and mutants by RT-qPCR experiments.Results:(1)The results of bio-orthogonal BONCAT experiment showed that AHA probe can effectively label the newly synthesized proteins in Arabidopsis and SA could inhibit the synthesis of newly synthesized proteins in a dose-dependent manner.Combined with the results of mass spectrometry,we found that S A rebuilds Arabidopsis proteome to adapt to environmental stress,mainly by inhibiting the ribosomal protein required for protein synthesis.In the search for SABPs,salicylic acid probe(SA-P)was designed and synthesized.SA-P has the similar function as SA which could induce the expression of pathogen-associated genes(PR1 and WRKY51)and bind to the known SABPs,glyceraldehyde-3-phosphate dehydrogenase 2(GAPC2).Base on ABPP,SA-P could label the binding proteins in Arabidopsis lysates in a dose-dependent manner and the higher concentration of SA,the less protein bound by SA-P.Subsequently,through ABPP combined with LC-MS/MS,126 SABPs were identified.We performed functional clustering analysis on the target protein,and the results showed that the proteins involved in biosynthesis of amino acid were among the most notable pathways.Furthermore,it was noteworthy that interacting proteins involved in translation were targeted by SA-P and occupy an important position in protein-protein interactions(PPI)analysis.We screened translation-related proteins and tested the interaction between SA and its interacting proteins in vitro.Consistent with our MS results,we found that SA could directly bind to eIF2α and RPS8A protein directly by pure protein competition,DSF and SPR experiments.Furthermore,it was found that SA-P could bind to the lysine of eIF2α protein as a potential binding site of SA.The surface plasmon resonance assay found that the KD between SA and eIF2α was 3.53 μM,suggesting that SA might directly modulate the activity of eIF2α through relatively high-affinity binding.In addition,phosphorylation experiments showed that SA treatment promoted eIF2αprotein phosphorylation without affecting protein levels.(2)According to the chemical structure of ABA,ABA-P was successfully synthesized.ABA-P had abscisic acid-like activity and up-regulated the expression level of ABAresponsive gene RD29A,which was similar to that of ABA treatment.Base on ABPP,ABA-P could label the binding proteins in Arabidopsis lysates in a dose-dependent manner,and the higher concentration of ABA,the less protein bound by AB A-P.After determining the probe labeling concentration,we performed subsequent ABPP in combination with LC-MS/MS analysis.Bioinformatics analysis showed that GSTF9 and GSTF10 were highly credible target proteins related to Glutathione metabolism pathways and they were important stress-resistant proteins in plants.Then,it was confirmed that ABA could bind GSTF9 and GSTF10 directly and inhibit their catalytic activities by pure protein competition and SPR experiments.It was further proved that ABA could bind to Lys152 of GSTF9 and GSTF10 proteins by mass spectrometry.In addition,GSTF10 can affect the expression of ABA signaling pathway-related marker genes.The results indicated that GSTF10 can directly bind to ABA and might be involved in ABA metabolic pathway.Conclusion:Taken altogether,these results suggest that SA alters the biochemical landscape of the Arabidopsis proteome for stress adaptation by shutting down protein synthesis.Phytohormones SA can directly bind the eukaryotic translation initiation factor eIF2α and promote its phosphorylation,while potentially affecting the amino acid biosynthesis pathway leading to protein translation inhibition and immune function.In addition,ABA can directly bind GSTF family proteins,and further inhibit their enzymatic activities,which provides a possible reference for explaining the mechanism of ABA involved in plant stress resistance.The findings of this study can provide important theoretical basis for further exploring the role of plant hormones in regulating plant homeostasis and understanding the mechanisms of plant survival in adversity.