Integration Omic Analysis Of Brassica Napus In Response To Drought Stress

Rapeseed(Brassica napus L.),which is the third leading source of vegetable oil,is sensitive to drought stress,especially during the early vegetative growth stage.In this study,2AF009,3DH020,and 2AF410 were taken as experimental material in response to drought stress.The molecular mechanisms of drought tolerance in rapeseed are systematic researched by morphological,physiological,RNA-Seq,comparative proteomics,quantitative phosphoproteomics.The main results were as follows: 1.Physiological and biochemical analysis of B.napus in response to drought stressCompared to 2AF410 and 3DH020,2AF009 exhibited lower water loss rates,a smaller decline in RWC,and a higher proline content,chlorophyll a and chlorophyll b content than other cultivars.It was concluded that 2AF009 was a potential drought-tolerant cultivar.Together,these results revealed the changes in the biological process of adaptation to drought stress in early growth stage of rapeseed.2.Transcriptome analysis of B.napus leaf in response to drought stressTranscriptome analysis was used to compare the differentially expression genes(DEGs)between control group and drought stress group.FDR?0.001 and |log2Ratio|?1 were used as thresholds,a total of 7379 genes were defined as DEGs,including 3072 upregulated genes and 4307 down-regulated genes.Functional analysis suggested that a large number of DEGs were related to transcription,post-translational modifications,carbohydrate metabolism and signal transduction.They were mainly located in the cytoplasm and cell membrane,participating in response to stress,phosphorus metabolic process,response to hormone stimulus,response to osmotic stress process.KEGG enrichment analysis results showed that most DEGs were related to starch and sucrose metabolim,plant hormone signal transduction,circadian rhythm-plant phenylpropanoid biosynthesis,glutathione metabolism pathway.In addition,292 transcription factors(TFs)genes including MYB?b HLH?NAC?WRKY?AP2-EREBP?b ZIP?HSF and so on were activated in rapeseed.The results indicated that the upstream regulatory elements also involved in the response of drought stress.3.Comparative proteomics analysis of B.napus in response to drought stress To reveal the drought-tolerant mechanisms of young rapeseed plant at the protein level,a comparative proteomics analysis combining 2D-DIGE and MS was carried out on the highly drought-tolerant cultivar 2AF009 to determine the differentially abundance proteins(DAPs)in leaves.138 DAPs were successfully identified via MALDI TOF/TOF MS,including 107 DAPs in the SD group and 85 DAPs in the MD group.These DAPs represented 98 unique proteins.Functional analysis suggested that a large number of DAPs were related to post-translational modifications,energy conversion and carbohydrate metabolism and were mainly located in the chloroplast and cytoplasm.It is worth noting that 20 unique DAPs including 35 protein spots,with different isoelectric points and molecular weights were identified using experimental values from two to eight protein spots on the same gel.This phenomenon likely resulted from the presence of different protein isoforms,or post-translational modification.Among them,?CA1(GI No.297789439)was the largest group of isoforms,which was identified at eight spots(1327,1331,1328,1339,1343,1360,1361,1370)with the same or very similar molecular weights,and thus was considered to have at least eight protein isoforms.The results suggested that the eight protein isoforms of ?CA1 that might be caused by protein phosphorylation were important for the drought stress response.DAPs were selected for q RT-PCR analysis to determine the relation between genes and encode proteins.The results suggested that drought-induced changes in the abundances of certain proteins were very different from the changes in their cognate transcripts,indicating the importance of post-translational modifications in controlling the final functions of these genes.4.Phosphoproteomics analysis of B.napus in response to drought stressTo analyse the phosphorylation modification of rapeseed in response to drought stress,phosphoproteomics analysis was carried out in rapeseed leaves under different drought treatments.In total,4469 phosphorylated peptides corresponding to 1232 phosphoproteins were determined.To our knowledge,this represents the largest number of phosphopeptides identified to date in B.napus and a significant addition to its phosphoproteome.On the basis of analyses of three biological replicated,only phosphopeptides with a log2 fold change(drought stress/CK LFQ intensity,log2FC)?1 or ?-1(p < 0.05)were considered to be significantly altered at the phosphorylation level.In total,337 phosphoproteins containing 547 phosphorylation sites with phosphorylation level significantly changed(PLSC)were screened out.There were 226,and 268 sample-specific phosphoproteins identified from the MD,and SD samples,respectively.Functional analysis indicated that PLSC phosphoproteins were highly enriched in response to drought stress and signal transduction process through protein kinase activity and phosphotransferase activity.This indicates that the detailed functions of many drought-responsive PLSC phosphoproteins from rapeseed have yet to be elucidated.It is worth noting that the starch and sucrose metabolism was highlighted in drought-responsive PLSC phosphoproteins,2D-DIGE,and DEGs,which contained identified 11 enzymes including starch synthase,beta-amylase,alpha-trehalose-phosphate synthase,sucrose-phosphate synthase,sucrose synthase,phosphoglucomutase.These enzymes by phosphorylation modification could play critical roles coordinate metabolic processes and physiological responses to help plants adapt to drought stress.5.Expression and activity analysis of ?CA1 upon drought stress?CA1(GI No.297789439)was identified from eight different protein spots(1327,1331,1328,1339,1343,1360,1361,and 1370)with a very similar molecular weight but different isoelectric points.These protein isoforms had different abundance levels under different drought conditions.At the gene expression level,?CA1 exhibited an increased relative abundance upon drought treatment.These results demonstrated that protein levels of ?CA1 did not necessarily correlate with gene levels.In contrast to down-regulated phosphorylated site of ?CA1 at Tyr207,CA activity decreased in drought-treated sample.These results demonstrated a strong correlation between protein activity and phosphorylation modification,which suggested that ?CA1 might adjust the protein activity through phosphorylation modification ultimately coordinate specific metabolic processes to help plants adapt to drought stress.The 3D structure was constructed by homology modeling to further analysed the role of phosphorylation site in ?CA1.Interestingly,we found that the phosphorylation site Tyr207 was located in the substrate-binding active centre.The results suggested that changes of phosphorylation/dephosphorylation at Tyr207 might affect the binding ability of ?CA1 and the substrate.From this evidence,we propose that amino acid Tyr207 is one of the most likely phosphorylation target might affect enzyme activity.The ?CA1 was the most homologous to At3g1500 in Arabidopsis thaliana by NCBI sequence alignment.6.Identification of the drought stress-related phenotypes for the ?CA1mutantStress testing suggested that ?CA1mutant showed higher rate of water loss of rapeseed excised leaf and shoter root length,which were sensitive to drought stress.