Transcriptomic,metabolomic And Functional Gene Analysis Of The Heat-stress Response In Populus Tomentosa

Plants respond to heat stress at different levels.However,how woody plants response to heat stress is largely unknown at metabolism level.A comprehensive analysis of physiological,metabolomics and transcriptomics has never been reported.In the present study,we exposed Populus tomentosa to control(25?)and heat stress(45?)treatments.We performed an integrated transcriptomic and metabolomic analysis of the response to heat stress in P.tomentosa leaves,evaluated the associated morphological,physiological,biochemical and molecular changes,construct overexpression vectors for key genes,and transformed into Arabidopsis thaliana and 84k poplar.The main results were as follows.(1)Through the observation of SEM,we found that the area of stomatal opening at 45?was smaller than that at 25?,and some ome stomata were almost fully closed under heat stress.Through physiological index measurement,we found that H2O2 concentration and the activity of CAT and SOD significantly increased,and activity of POD no significant change under heat stress.Soluble sugar concentration significantly decreased,while soluble protein contents no significant change under heat stress.(2)Through transcriptome sequencing,we found total of 7,738 DEGs between the control and heat-stressed plants.Among them,3,888 DEGs were upregulated and 3,850 DEGs were downregulated in the heat-stressed relative to the control plants.The differential expressed genes in GO terms response to "abiotic stimulus","response to heat","membrane-bounded organelle","chloroplast thylakoid","chloroplast stroma",and "photosynthesis membrane" were significantly enriched under heat stress.In photosynthetic pathway,the expression levels of most genes(PSB27,PSB28,LHCA5,PETB and PETC)related to the light-harvesting complexes and photosynthetic electron transport system were downregulated by heat stress.The pyruvate metabolism,photosynthesis,pentose phosphate pathway,glycolysis/gluconeogenesis,arginine and proline metabolism,carbon metabolism,and carbon fixation in photosynthetic organisms Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were significantly enriched in DEGs.(3)A large number of metabolites in poplar leaves were identified by metabolome,including 4 lipids,4 carbohydrates,16 inorganic and organic acids.Compared to the control,the levels of proline,raffinose and melibiose were increased in heat stress,whereas the levels of pyruvate,fumarate and myo-inositol were decreased(4)Association analysis between transcriptome and metabolome indicated that glycolysis was enhanced,whereas the tricarboxylic acid(TCA)cycle was suppressed.Particularly,the inositol phosphate;galactose;valine,leucine,and isoleucine;and arginine and proline metabolic pathways were significantly affected by heat stress(5)In our dataset,we found that 553 DEGs were identified as transcription factors(TFs)of 68 TF families.The expression levels of HSFA9,HSFA3,HSFA2,and HSF4 were significantly upregulated by heat stress.However,HSFB4 was significantly downregulated.MYB27 and MYB4R1 were upregulated in heat stress.The genes encoding the WRKY50 and WRKY13 TFs were downregulated by heat stress(6)We verified the expression levels of 16 genes by qRT-PCR.Compared with the control,HSF4 and HSFA9 were significantly upregulated at four time points,the expression of CYP97A3,CYP97B3,DREB1,DREB2,DREB2E,DREB5,HSP21,HSP90-6,HSFA9,HSF4,HSFA3,P5CS,and WRKY50 peaked after 12 h at 45?.However,HSFB4 was significantly downregulated at four time points(7)We constructed overexpression vectors 35S::DREB2-GFP and 35S::Pt-HSFA3-GFP,and transiently transform tobacco leaves.The laser confocal microscopy indicated that DREB2 was mainly located in the nucleus,and HSFA3 was mainly located in cytomembrane and cytoplasm Because of epidemic situations,the future experiment research of Arabidopsis thaliana and 84k poplar transformation have been carried out.