Functional Identification Of GhMYB4,GbTCP4 And GbTCP5 In Response To Drought And Salt Stress In Cotton

Cotton is one of the important economic crops in the world.Xinjiang is the main cotton producing area in China.However,Xinjiang is located in arid desert areas and has a dry climate,which restricts the development of cotton in Xinjiang.Therefore,it is crucial to understand the molecular mechanism of cotton response to drought and salt stress and to cultivate cotton varieties with drought and salt tolerance.In this study,the functions and molecular mechanisms of previously cloned GbTCP4,GbTCP5 and GhMYB4 genes in cotton drought and salt stress responses were investigated.The research content mainly includes the analysis of the expression patterns of 3 genes GbTCP4,GbTCP5 and GhMYB4 by real-time PCR.The functions of GbTCP4 and GbTCP5 genes were investigated using transgenic Arabidopsis,VIGS-silenced cotton and transcriptome sequencing.The function of GhMYB4 was further analyzed by transgenic Arabidopsis,VIGS-silenced cotton,genetically transformed cotton,yeast library screening and transcriptome sequencing.The main findings are as follows:1.The transcript level of cotton GbTCP4 was increased under ABA,drought and Na Cl treatment conditions.Under drought conditions,Arabidopsis overexpressing GbTCP4 increased seed germination rate,germinated root length,survival rate and decreased leaf water loss rate.Compared with Col,35S:GbTCP4 was insensitive to ABA at the seed germination stage of Arabidopsis,but on the contrary at the seedling stage,reducing stomatal opening and increasing ABA content.Under salt conditions,Arabidopsis overexpressing GbTCP4 increased seed germination,survival and SPAD content,but decreased MDA content.GbTCP4 upregulates the transcription level of At CAD5 by binding the TCP cis-acting element in the At CAD5 promoter,thereby increased the lignin content of Arabidopsis.Reduction of GbTCP4 expression in cotton by VIGS.Compared with p TRV2,p TRV2-GbTCP4 reduced root length,root area,root volume,survival rate,SPAD and increased water loss rate and MDA content.RNA-seq and q PCR analysis revealed that GbTCP4 affected the transcription levels of genes in multiple metabolic pathways associated with abiotic stress responses.2.Transcript levels of cotton GbTCP5 increased under ABA,drought and Na Cl treatments.Under drought conditions,Arabidopsis overexpressing GbTCP5 increased the survival rate and decreased the rate of leaf water loss.Under salt conditions,the Arabidopsis overexpressing GbTCP5 increased seed germination,germinated root length,survival rate,SPAD content and decreased MDA content.In addition,35S:GbTCP5 was insensitive to ABA during Arabidopsis seed germination.GbTCP5 not only upregulates the transcription levels of At CAD5 and At CCOMT1 by binding the TCP cis-acting elements in the promoters of At CAD5 and At CCOMT1,thereby increased the lignin content of Arabidopsis,but also combine with the TCP cis-acting elements in the promoters of At MYB46,At LBD30,At Ces A4 and At VND7 to up-regulate the transcription levels of At MYB46,At LBD30,At Ces A4 and At VND7,thereby increased the expression of Arabidopsis in the late stage of secondary wall hyperplasia.Using VIGS to reduce the expression of GbTCP5 in cotton.Compared with p TRV2,p TRV2-GbTCP5 decreased root length,root area,root volume,survival rate,SPAD content.But increased leaf water loss rate and MDA content.q PCR experiments showed that GbTCP5 enhanced drought resistance by affecting the transcription levels of At RD29 A,At COR15 A,At COR414,At PUB22 and At LTP4 genes in Arabidopsis.q PCR and RNA-seq experiments showed that GbTCP5 reduced cotton drought resistance by affecting the transcription levels of Gb GLOS2,Gb UBC19 and Gb ERD7 genes.3.Overexpression of GhMYB4 increased root hair length and stem epidermal hair density in Arabidopsis,but the number of leaf epidermal hairs did not change significantly.GhMYB4 up-regulates the transcription level of At CCR by binding the MYB cis-acting element in the At CCR promoter and down-regulating the transcription level of At C4 H by binding the MYB cis-acting element in the At C4 H promoter.The transcript level of cotton GhMYB4 was increased under ABA,drought and Na Cl treatment conditions.Under drought conditions,Arabidopsis overexpressing GhMYB4 increased seed germination rate,germinated root length and survival rate,but decreased leaf water loss rate and MDA content.Compared with Col,35S:GhMYB4 was insensitive to ABA at seed germination stage of Arabidopsis,but the opposite was observed at seedling stage,reducing stomatal opening and increasing ABA content.Under salt conditions,Arabidopsis overexpressing GhMYB4 increased seed germination,survival,SPAD content,but decreased MDA content.Compared with Col Arabidopsis,transgenic Arabidopsis overexpressing GhMYB4 showed no significant change in lignin content.The metabolome showed that overexpression of the GhMYB4 gene increased the content of the lignin metabolites coumarin and 4-coumaric acid in the stems of transgenic Arabidopsis.q PCR experiments showed that GhMYB4 enhanced drought resistance by affecting the transcription levels of At RD29 A,At COR414,At PUB22 and At LTP4 genes in Arabidopsis.VIGS reduced GhMYB4 expression in cotton.Under drought and salt stress,p TRV2-GhMYB4 reduced root length,root area,root volume,survival rate,SPAD content,lignin content and xylem layer number,but increased water loss rate and MDA content compared with p TRV2.The results of genetically transformed GhMYB4 cotton showed that under drought and salt stress,compared with the control CQJ-5,the overexpression GhMYB4 cotton increased the survival rate,chlorophyll content,xylem layer number and decreased the water loss rate;the silenced GhMYB4 cotton showed the opposite results.GhMYB4 does not have transcriptional autoactivation activity and the yeast two-hybrid library screen showed that GhMYB4 interacts with Gh MYB6,Gh LBD42 and Gh WRKY44.Transcript levels of Gh MYB6,Gh LBD42 and Gh WRKY44 were increased under ABA,drought and Na Cl treatment conditions.VIGS results showed that Gh MYB6,Gh LBD42 and Gh WRKY44 reduced the drought and salt tolerance of cotton.q PCR,RNA-seq,yeast one-hybrid and VIGS experiments showed that GhMYB4 affects cotton drought and salt tolerance by binding to the MBS cis-acting element in the Gh ERF109 promoter.