The Molecular Mechanism Of HsfB4 Involved In Plant Thermomorphogenesis

There are many transcription factors(TFs)in plants,among them,heat shock factors(Hsfs)play key roles in the regulation of plant stress response,growth and development.According to the sequence characteristics,the plant Hsfs family genes can be clustered into A,B,and C classes.Current studies on the plant Hsfs mainly focus on class A,however,there is relatively little research on the biological function and regulation mechanism of class B and C.Previous studies have shown that At Hsf B4 gene is highly expressed in root apical meristem of Arabidopsis and involved in the differentiation of root stem cells.In this study,the expression pattern of At Hsf B4 gene was analyzed and found it also highly expressed in the shoot apical meristem,suggesting that At Hsf B4 may also be involved in shoot development.Therefore,in this study,Arabidopsis and rice were used as materials to explore the function and mechanism of Hsf B4 in plant thermomorphogenesis and drought resistance.The main results are as follows:1.The basic characteristics and biological functions and regulatory mechanism of At Hsf B4 gene were analyzed.(1)The CDS of At Hsf B4(AT1G46264)gene was 1047 bp in length and encoding 348 amino acids.The At Hsf B4 gene was highly expressed in root and shoot meristem during seedling stage but highly expressed inflorescence during reproductive growth stage.The At Hsf B4 protein was located in the nucleus.(2)scz-2 mutant(At Hsf B4 gene deletion mutant)was no significantly change in hypocotyl length under 28 ℃,suggesting that At Hsf B4 gene was required for thermomorphogenesis.(3)Transcriptomic sequencing analysis revealed that the expression levels of phenylpropanoid synthesis,starch and sugar metabolic,ribosome and photosynthetic pathway genes in scz-2 mutant were significantly changed.The monosaccharide composition of scz-2 mutant was significantly higher than that of the wild type,and the synthesis of cellulose and pectin was abnormal at 28 ℃.(4)Some At Hsf B4 target genes were obtained using Ch IP-seq and RNA-seq analysis,which mainly involved in cell wall synthesis and containing HSE elements in promoters.(5)The α-subunit of SNF1 complex(AT3G01090,Sn RK1.1)and catalase 2(AT4G35090,CAT2)were identified by yeast-two-hybrid assay and verified to interacted with At Hsf B4 protein.Suggesting that these proteins may be required for the function of At Hsf B4.(6)b HLH34 and NARS1 proteins were identified by yeast-one-hybrid system to bind to the promoter of At Hsf B4 gene.2.A comprehensive genome-wide analysis of Hsf genes in rice was carried out.The phylogenetic relationships,evolutionary history and expression analysis of Os Hsf genes were investigated.Two class B heat shock factor genes,Os Hsf B4 b and Os Hsf B4 d,were further identified in response to drought stress in rice.(1)25 Hsf family genes were screened and identified at a genome-wide level in rice.Through phylogenetic analysis,Os Hsf family genes could be clustered into three categories: A,B and C,and further divided into 13 subcategories.(2)The results of collinearity analysis showed that Os Hsf family genes expansion was the result of gene tandem duplication and segmental duplication events during evolution.(3)A promoter analysis of the OsHsf genes revealed the presence of various elements related to environmental stress and growth of rice,and promoters of some Os Hsf genes contained transcription factor binding elements.Furthermore,the spatiotemporal expression characteristics of Os Hsf family genes were analyzed by transcriptome data.(4)Two stress-resistant genes Os Hsf B4 b and Os Hsf B4 d in rice were identified by transcriptomic data analysis,RT-q PCR assay and phenotypes of transgenic Arabidopsis thaliana.Subcellular localization analysis showed that Os Hsf B4 b and Os Hsf B4 d proteins were located differently in cells,Os Hsf B4 b was located in the nucleus,while Os Hsf B4 d protein existed in both the nucleus and cytoplasm.(5)Both OsHsfB4 b and OsHsfB4 d could be significant induced under drought stress(PEG).Heterologous expression of Os Hsf B4 b in Arabidopsis can increase the resistance to drought stress.While the Os Hsf B4 d gene transgenic Arabidopsis showed reduced resistance to drought stress.(6)OsHsfB4b and OsHsf B4 d overexpressed transgenic rice lines and mutant lines were constructed.Overexpression of Os Hsf B4 b results in enhanced drought tolerance.