Dynamic Analysis Of Post-Transcriptional Regulatory Effects On The Arabidopsis Proteome In Response To ABA And GA Treatments

According to the central dogma,genes are transcribed to produce mRNA,and mRNA is translated to produce proteins.These two processes are regulated at the transcriptional and post-transcriptional levels,respectively,ultimately affecting changes in protein levels.Post-transcriptional regulation can influence protein synthesis and degradation,allowing for rapid changes in protein levels even when mRNA levels remain unchanged.However,if cells rely solely on transcriptional changes to respond to external stimuli,it may take longer to sufficiently alter protein levels.Under environmental stimuli,organisms undergo both transcriptional and post-transcriptional regulation,thereby influencing changes in protein levels.However,the extent to which these two regulatory mechanisms contribute to protein changes has long been a contentious scientific issue.Investigating this problem requires simultaneous quantification of protein synthesis and degradation.For animal cells,this can be achieved by employing stable isotope labeling methods such as SILAC under cell culture conditions to quantify protein synthesis and degradation simultaneously.However,for plants,which are autotrophic organisms,it is nearly impossible to fully label proteins with stable isotope-encoded amino acids under their natural growth conditions.Therefore,there is an urgent need to establish a system for studying post-transcriptional regulation of plant proteins.GA and ABA are a pair of antagonistically regulated plant hormones.The levels of ABA and GA fluctuate dynamically during plant growth,thereby influencing various physiological changes in plants.These hormones can ultimately affect the changes in various proteins by regulating both the transcriptional and post-transcriptional levels of plant genes,thus enabling responses to various growth stages or environmental changes.However,the post-transcriptional regulatory effects of ABA and GA hormones on the plant proteome have not been systematically studied.This study utilized Luciferase-Assisted Proteome Detection（LAPD）to construct a plant material library for studying post-transcriptional regulation of protein levels in plants.By employing LAPD,the changes in post-transcriptional protein levels in plants under GA or ABA stimulation were detected.This approach aimed to assess the feasibility of the LAPD system for post-transcriptional regulation and to uncover unknown regulatory genes in the ABA signaling pathway.The specific research findings are as follows:1.Construction of the Luciferase-Assisted Proteome Detection（LAPD）library.A total of 5069 genes classified as transcription factors,E3 ligases,RNA-binding proteins,and those categorized as"hormone-and light-regulated genes"in the GO terms were selected.In this study,the open reading frames of these genes were cloned and expressed under the control of the 35S promoter as LUC fusion proteins.A total of2061 vectors expressing single LUC fusion proteins were transformed using the floral dip method,resulting in 3477 transgenic Arabidopsis lines expressing 1088 different LUC fusion proteins.These lines constitute the library of Luciferase-Assisted Proteome Detection（LAPD）.2.Characteristics of LAPD proteome compared to mass spectrometry proteome.The LAPD protein assay utilizes a microplate luminometer to measure the fluorescence changes of LADP Arabidopsis materials treated with ABA and GA every10 minutes.Therefore,the analysis of the LR response curve represents real-time dynamic analysis of LUC fusion protein abundance.Through sensitivity comparison,we found that the LAPD protein assay exhibited sensitivity two orders of magnitude higher than immunoblotting,showing potential for detecting low-abundance proteins.Comparison with proteins quantified by mass spectrometry revealed that 70%of the proteins in the LAPD protein assay were not quantified in the mass spectrometry protein group,including 40.4%transcription factor proteins and 33.3%E3 ubiquitin ligase proteins.This indicates that the LAPD protein assay contains more proteins that cannot be identified by mass spectrometry in Arabidopsis seedlings.Further analysis revealed that the proteins quantified in the mass spectrometry protein group were mostly proteins with high mRNA levels,while the LAPD protein assay contained mostly proteins with low mRNA levels.3.The contribution of post-transcriptional regulation to protein level changes decreases with increasing treatment time of ABA and GA.This study found that the correlation between the transcriptome and proteome of Arabidopsis under ABA and GA treatments was relatively low,ranging from 0.45 to0.55.Moreover,75%to 81%of genes showed mRNA-protein correlations lower than0.75,or even negative correlations.This indicates that the majority of genes are post-transcriptionally regulated under ABA and GA stimuli.Further analysis revealed that as the treatment duration increased,the correlation between changes in mRNA abundance and changes in protein abundance also increased.This suggests that,relative to mRNA-level changes,post-transcriptional regulatory changes weaken with increasing treatment duration.Contribution analysis further demonstrated that with increasing treatment duration,the contribution of post-transcriptional regulation to protein changes decreases,while the contribution of mRNA changes to protein changes increases.4.The rate of change in the Arabidopsis proteome after ABA and GA treatments is significantly influenced by post-transcriptional regulation.By comparing the proportion of proteins showing the highest rate of change in response to ABA and GA treatments within the time intervals of 0-1 h,1-3 h,and 3-5h,it was found that 86%to 98%of LAPD proteins exhibited the highest rate of change within 3 h.In contrast,only 56%to 64%of proteins in the transcriptome and 78%to91%of proteins in the proteome showed the highest rate of change within 3 h.The proportion of proteins with the highest rate of change in different time intervals was closer to the LAPD group in the proteome,indicating that the rate of protein changes is more influenced by post-transcriptional regulation.Through GO enrichment analysis of proteins showing the highest rate of change in different time intervals in the LAPD group,it was found that proteins undergoing high-rate changes at 0-1 h and 1-3 h under ABA treatment,as well as at 0-1 h under GA treatment,were enriched in proteins associated with ABA,GA,or seed development regulatory pathways（referred to as"functionally regulated proteins"in this study）.This suggests that these post-transcriptionally regulated proteins are more likely to influence the response of Arabidopsis seedlings to ABA and GA.5.Proteins negatively correlated with LAPDABA-GA（early-stage）and LAPDABA-GA(mid-_stage)are potential functional regulatory proteins.Through WGCNA analysis,the proteins in the LAPD protein group after ABA and GA treatments could be divided into six modules.By dividing the hormone treatments into time periods of 100 minutes,this study divided the 5-hour treatment into early,middle,and late stages.Correlation analysis revealed that proteins in the middle stage of ABA treatment and the early stage of GA treatment were highly correlated with most modules,indicating varying degrees of post-transcriptional regulation during different time periods of ABA and GA treatments.Subsequently,correlation analysis was performed on the 182 proteins in the LAPD protein group that responded to both ABA and GA treatments.It was found that proteins negatively correlated with LAPD_ABA-GA（early-stage）and LAPDABA-GA（mid-stage）were enriched in"functionally regulated proteins",suggesting that proteins negatively correlated with LAPD_ABA-GA in the early and middle stages are potential functional regulatory proteins6.Proteins negatively correlated with LAPDABA-GA（early-stage）and LAPDABA-GA(mid-_stage)are involved in ABA signaling pathway regulation.Most of the proteins negatively correlated with LAPD_{ABA-GA（early-stage）}and LAPD_{ABA-GA（mid-stage）}have not been studied in relation to the ABA signaling pathway.Through analysis of seed germination rates under ABA treatment,we identified four proteins,ERF34,WAG1,PUCHI,and AT5G56690,among these negatively correlated proteins,involved in the ABA-mediated inhibition of seed germination pathway.Additionally,using protein-protein interaction prediction,we identified the interaction between ERF34,the protein with the highest negative correlation with LAPD_ABA-GA,and ABI3,along with their common downstream gene,ADH1.Further validation through HEK293T Co-IP and BIFC experiments confirmed the interaction between ERF34 and ABI3.Subsequent experiments using DNA pull-down and in vivo and in vitro dual fluorescence reporter gene assays validated the direct binding of ERF34 to the ADH1 promoter and its transcriptional upregulation of ADH1 through interaction with ABI3.This provides a new perspective for filling in the network of the ABA signaling pathway.In summary,this study systematically investigated the impact of post-transcriptional regulation on the proteome under ABA and GA stimuli for the first time by constructing the LAPD material library.Analysis of the LAPD proteome is not limited by the natural abundance of proteins.Coupled with the high sensitivity of the LAPD method,it is particularly suitable for dynamic studies of low-abundance proteins,which are often difficult to study using other high-throughput methods.It was found for the first time that the contribution of post-transcriptional regulatory changes to protein changes outweighed that of mRNA changes during the early stages of ABA and GA treatments,while the contribution of post-transcriptional regulatory changes to protein changes decreased with increasing treatment duration.Additionally,correlation analysis of the LAPD proteome under ABA and GA treatments can be used to identify proteins involved in the ABA signaling pathway.Furthermore,compared to other proteomic libraries reported in similar model organisms,the LAPD proteome can be more conveniently provided to other researchers in the form of Arabidopsis seeds,which can be easily stored and amplified,enabling most laboratories to conduct high-throughput dynamic proteomic studies.This facilitates the study of post-transcriptional regulatory effects of other environmental stimuli on the proteome.