Study On Cotton NAC Transcription Factors Regulating Secondary Cell Wall Biosynthesis

Cotton fiber is the most important raw material of textile products in the world,and its economic value is self-evident.More than 90%of secondary wall of mature cotton fiber is cellulose,which forms the main load-bearing structure of the cell wall.In addition,a small amount of hemicellulose is cross-linked with cellulose microfilaments through hydrogen bonds to strengthen the cell wall.A small amount of lignin is inserted into the gap between cellulose and hemicellulose to further enhance the toughness and strength of the cell wall.Cotton fiber is derived from ovule epidermis.Its development is divided into four overlapping stages:initiation,primary wall synthesis(cell elongation),secondary wall thickening,dehydration and maturation.In the secondary wall thickening stage,a large number of cellulose are deposited,which directly affects the strength of cotton fiber,while fiber strength is one of the key parameters to determine the quality of cotton fiber.However,the molecular mechanism underlying cotton fiber secondary cell wall synthesis remains largely unknown,so it is imperative to explore the regulation mechanism of secondary wall thickening of cotton fiber.NAC-MYB-mediated network involved in the regulation of secondary cell wall synthesis has been extensively studied in Arabidopsis and poplar.It was found that the NAC-MYB mediated network is conserved in the vascular plants.Among them,NAC family transcription factors act as master switches and play a very important role in the regulation of secondary wall biosynthesis.Previously,we found that GhFSNl-related transcription factors regulate secondary wall synthesis in cotton fibers,but a large number of cotton NAC transcription factors and MYB transcription factors involved in secondary wall synthesis,as well as the specific regulatory pathway are still unknown.In this study,based on RNA-seq data,co-expression analysis combined with phylogenetic analysis,we identified 15 GhNACs that may be involved in the regulation of cotton fiber secondary wall thickening.The functions of these genes were analyzed by complementing the Arabidopsis thaliana nst1nst3 double mutant and transactivation assays.The main results are as follows:1.Expression profile analysis,co-expression analysis and evolutionary analysis of GhNACsIn Arabidopsis thaliana,in addition to NST1/2/3 which are involved in the regulation of secondary wall synthesis,SND2/3,BRN1/2 and several other transcription factors containing NAC domain are also involved in the regulation of secondary wall synthesis.We further screened 15 GhNACs using these proteins as queries to BlastP the cotton protein database.Phylogenetic analysis showed that the GhNACs were homologous to AtSND2/3,AtNST1/2/3,ANAC018/025/056 and other NAC transcription factors.We speculate that these GhNACs may perform the same functions as Arabidopsis SND2/3,NST1/2/3 and so on.Analysis of RNA-seq data showed that these GhNACs were highly expressed in 20 day-post anthesis(dpa),25 dpa fibers which undergo massive secondary wall thickening.Thus,we speculate that the identified 15 GhNACs may be involved in the regulation of secondary wall synthesis in cotton.2.Some GhNA C-overexpressing transgenic Arabidopsis plants became taller and their fertility was impairedIn order to verify the function of these GhNACs,we transformed GhNACs into wildtype Arabidopsis thaliana.Among many GhNAC-overexpressing transgenic Arabidopsis plants,we found that there were significant differences between Gh_D11G0347 overexpression transgenic Arabidopsis and Gh_D01G2346 overexpression transgenic Arabidopsis plants and wild type plants.We found that the plant height of Gh_D11G0347overexpressing transgenic lines was significantly higher than that of wild type.Compared with wild type,the fertility of transgenic plants was impaired.The size of pods and the number of seeds per pod decreased significantly compared with wild type.3.Five GhNACs can rescue the interfascicular fiber deficiency and a pendent stem phenotype of Arabidopsis nst1nst3 double mutantIt is known that the interfascicular fibers of Arabidopsis nst1nst3 double mutant disappeared and the plants cannot stand erect.To see if these GhNACs can regulate secondary cell wall synthesis,we transferred GhNACs into Arabidopsis nst1nst3 double mutant and obtained transgenic plants.We found that Gh_A05G1339,Gh_A02G0977,Gh_D03G0775,Gh_D12G1407 and Gh_A12G2179 could restore the phenotype of interfascicular fiber loss of nst1nst3 double mutant,with different degrees,and the transgenic plants partially or completely recovered to erect growth.Other GhNACs cannot complement the phenotype of nst1nst3 double mutant.It is possible that these GhNACs may not be involved in the regulation of secondary wall synthesis or function as negative regulatory factors.4.Eight GhNACs can activate the promoter activity of cotton fiber secondary wall cellulose synthasesWe previously isolated the promoters of cotton fiber secondary wall cellulose synthase genes,and further used the dual luciferase system to examine the transactivation activity of these GhNACs in tobacco leaves.Our results showed that Gh_D11G0347,Gh_A04G1299 and Gh_D05G3475 activated the GhCesA 4-2 promoter,Gh_A05G1339 activated the GhCesA7-3 promoter,and Gh_A11G0290 activated the GhCesA4-2 and GhCesA7-3 promoter.Gh_A12G2179 activated the promoters of GhCesA 4-2,GhCesA73 and GhCesA8-2.Gh_A02G0977 and Gh_D03G0775 strongly activated the promoters of GhCesA 4-2,GhCesA7-3 and GhCesA8-2.We infer that the above genes may be involved in regulating the synthesis of secondary wall of cotton fiber.5.Expression of five GhNACs in tobacco leaves can induce the ectopic deposition of secondary wallsCombined with the phenotypic analysis and transcriptional activation assays,we preliminarily concluded that Gh_A05G1339,Gh_A11G0290,Gh_D 11G0347,Gh_A04G1299,Gh_D05G3475,Gh_D12G1407,Gh_A02G0977,Gh_D03G0775 and Gh_A12G2179 were involved in the regulation of secondary wall synthesis.To further verify that these GhNACs function as regulators of secondary wall biosynthesis.We transiently expressed these genes in tobacco leaves.It was found that Gh_A11G0290,Gh_D11G0347,Gh_A04G1299,Gh_D05G3475 can’t induce secondary wall deposition like wild type tobacco;Gh_A05G1339,Gh_D12G1407,Gh_A02G0977,Gh_D03G0775 and Gh_A12G2179 indeed induce secondary wall deposition in tobacco epidermal cells.indicating that these genes can regulate secondary wall synthesis.In summary,five GhNACs of Gh_A05G1339,Gh_D12G1407,Gh_A02G0977,Gh_D03G0775 and Gh_A12G2179 were identified as regulators of secondary wall synthesis in this study.The upstream and downstream regulatory relationship between mem remains to be further studied.Our results lay a solid foundation for further dissecting the functions of these GhNACs in cotton fibers.