The Regulation Of GRF7 Expression By Arabidopsis SHB1 Under Red Light

Plant growth and development in nature requires external conditions such as light,temperature,and soil.Higher plants respond to the light conditions by absorbing in red and far-red through phytochromes and blue light and ultraviolet-A through cryptochromes.A long hypocotyl mutant shb1-D was previously identified under red light,blue light,and far red light.shb1-D is a gain-of-function mutant and SHB1 is cloned in the nucleus.At present,the functional studies on SHB1 gene mostly focus on its regulation over seed endosperm development,but its involvement in plant light signal transduction remains unclear.PIF4 is a direct target gene downstream of SHB1,and its expression is regulated by the SHB1-CCA1/LHY module,thereby promoting plants to reach a optimum photomorphogenesis.In this study,RNA-Seq analysis was performed in wild-type Ws and shb1-D mutant.Another target gene GRF7 was identified.This study mainly explored the molecular mechanism of SHB1 regulating GRF7 gene expression under red light.The major results of this study are as follows:(1)Through further analysis of RNA-Seq results,823 genes were differentially expressed(?FC?>1.5,P<0.05)in the shb1-D mutant compared with wild-type Ws,of which 586genes were up-regulated and 237 genes were down-regulated.Cluster analysis on these differentially expressed genes showed that these genes were distributed in various molecular biological functions,biological processes,and cell components.These differentially expressed genes are mostly distributed in binding,catalytic activity and electron carrier activity in the molecular biological functions;in participating biological processes,these differentially expressed genes are mostly distributed in biological regulation,cellular process,metabolic process and single organism process;in terms of participating in the formation of cell components,these differentially expressed genes are mostly distributed in cell part,extracellular part and membrane part.(2)The marker gene PIF4 was included in the RNA-Seq analysis,and the expression of some differentially expressed genes such as HEMB2,HEC2,CLE19,GRXS2,CO,and XTH6was verified by qRT-PCR experiments.The qRT-PCR results are consistent with the RNA-Seq data,and the RNA-Seq results are reliable.(3)Among the differentially up-regulated genes,four GRF gene family members(GRF4/5/6/7)were discovered and their expression levels were all up-regulated in the shb1-D mutant.Among them,the expression of GRF7 gene was significantly inhibited by light,but was significantly increased in the shb1-D mutant.Qantitative RT-PCR technology was used to analyze the expression of GRF7 gene under strong(15?mol/m~2/s)and weak(2?mol/m~2/s)red,blue,and far red light in wild-type Ws and mutant shb1-D,and its expression pattern under red light condition was consistent with RNA-Seq results.(4)The hypocotyl phenotypes of grf7 mutants and wild-type Col were examined in the dark and under red light.Compared with Col,grf7 single mutants have significantly shorter hypocotyl under red light,suggesting that GRF7 participates in SHB1 mediated signal transduction pathway.(5)The promoter of GRF7 gene was divided into five fragments from P1 to P5.ChIP-qPCR analysis of pSHB1::SHB1-Myc transgenic plants indicated that SHB1 binds to the P3 and P4 fragments of GRF7 promter,suggesting that GRF7 gene is a direct target gene of SHB1.(6)In this study,wild-type Ws was used as material for Chromatin immunoprecipitation analysis with CCA1 or LHY endogenous antibodies.ChIP-qPCR analysis showed that CCA1/LHY can bind to the P4 fragment of the GRF7 promoter,indicating that CCA1/LHY is a protein factor that can recruit SHB1 to the promoter region of the GRF7 gene.(7)Qantitative RT-PCR technology was used to analyze the expression of SHB1 gene under dark and red light for 3h and 9h in wild-type Col and mutant grf7.The results showed that the expression of SHB1 decreased in the grf7 mutant under red light,indicating that GRF7 can promote the expression of SHB1 gene under ligh.RNA-Seq results suggest that GRF7 expression is regulated by SHB1.And ChIP-qPCR experiments suggest that GRF7 is a direct target gene of SHB1 in responding to the red light signal.This discovery proves for the first time that GRF7 gene is involved in the regulation of plant light signal transduction,which helps us to further understand the role of SHB1 in plant light morphogenesis.