A Cotton NAC Domain Transcription Factor,GhFSN5,negatively Regulates Secondary Cell Wall Biosynthesis And Anther Development In Transgenic Arabidopsis

NAC proteins comprise one large family of plant specific transcription factors(TF)which play important roles in many biological processes.Cotton is one of the most important fiber crops in the world.The secondary wall of mature cotton fiber contains more than 90%of cellulose and a very small amount of hemicellulose and lignin,but the molecular mechanism underlying the cotton fiber secondary cell wall synthesis remains largely unknown.Our previous study identified seven potential fiber secondary cell wall related NAC TFs,which we named GhFSN1-7,experimental data has revealed that GhFSN1 positively regulate fiber secondary cell wall formation,but for the resting six genes,their functions are unknown.In the present study,we focused on GhFSN5 and its three possible downstream MYB genes,functional characterization was performed.The main results are as follows:1.GhFSN5 shares high amino acid identity with AtNST1,AtNST2 and AtNST3GhFSN5 gene ORF is 1218 bp long and encodes a 405 amino acid protein.The protein has a typical structural feature of the NAC TF family,with a conserved NAC-domain at the N-terminal region.The full-length sequence of the GhFSN5 cDNA contains three exons and two introns.SALAD analysis reavealed that GhFSN5 are closely related to AtNST1,AtNST2 and AtNST3,three master regulators of secondary wall synthesis in Arabidopsis thaliana;coexpression analysis shows that GhFSN5 was co-expressed with a number of secondary wall synthesis-related genes.2.GhFSN5 was highly expressed during cotton fiber secondary wall thickening phaseqRT-PCR analysis showed that GhFSN5 was strongest expressed in 15DPA fiber,and also highly in 18 DPA and 20 DPA fiber.To further explore the spatiotemporal expression of GhFSN5,its 2023-bp promoter upstream of initiation codon was isolated and GhFSN5 promoter::GUS vector was constructed and transformed into Arabidopsis thaliana.Histochemical analysis showed that GUS staining was strongest in stems and anthers in 7 week old transgenic plants,implying that this gene might be involved in secondary wall synthesis.3.GhFSN5 overexpressing transgenic Arabidopsis have reductions in stem secondary wall thickening and defects in anther developmentTo exploit the roles which GhFSN5 might play,the GhFSN5 overexpression vector was constructed and introduced into Arabidopsis.Two independent transgenic lines with strong expression of GhFSN5 were selected for phenotype analyses.Overall,the transgenic plants were indistinguishable from the wild type,except for the much smaller siliques in overexpression plants.To investigate whether GhFSN5 is involved in regulating secondary cell wall formation.Cross sections of 7 week-old stems from transgenic lines were stained with S4B and Calcofluor White(two dyes specifically stained cellulose)and visualized with confocal laser scanning microscopy.No ectopic deposition of secondary cell wall was observed in overexpression lines.Compared with the wild type,the fluorescence intensity was much weaker in overexpression stems,suggesting that cellulose content was less.Similar results were obtained for roots.In addition,lignin was displayed with phloroglucinol staining,lighter staining was observed in overexpression lines compared to wild type which suggest that overexpression lines contained less lignins.Analysis of genes related to secondary wall synthesis by qRT-PCR showed that genes involved in secondary wall cellulose,hemicellulose and lignin biosynthesis were down-regulated in overexpression lines.We also constructed complementation vector and introduced into Atnst1/nst3 double mutant,the Atnstl/nst3 double mutant can not stand upright due to the absence of secondary cell wall formation in interfascicular fibers.Expression of GhFSN5 in Atnst1/nst3 double mutant can not recover the mutant phenotype suggesting that GhFSN5 did not play a similar role as its Arabidopsis counterpart AtNST1/2/3 does.Altogether,the above results showed that GhFSN5 might negatively regulate secondary cell wall biosynthesis in transgenic Arabidopsis.To dissect what caused less siliques,flower was observed under a stereomicroscope,no apparent difference was found for the morphology of pistil or stamens,however,lots of pollen grains were attached to the surface of the style part after stigma in wild type flower,negligeble pollens could be observed for transgenic styles.Further scanning electron microscopy of anthers showed that anther dehiscence was not affected in transgenic lines,but pollen grains were collaped and shrunken which could attribute to less release of pollens and finally less seeds.4.Preliminary analyses of three MYB TFs which possibly function downstream NACTFConsidering NAC-MYB gene regulatory network is somewhat conserved in vascular plants,we isolated three MYB genes homologous to AtMYB85 which might act downstream of GhFSN5,which we designated as GhMYB85A,GhMYB85B and GhMYB85C.Self-activation assay showed that GhMYB85A had self-activation activity,whereas GhMYB85B and GhMYB85C had no self-activation activity.Expression analysis showed that GhMYB85A and GhMYB85B were highly expressed in 20DPA and 25DPA fibers,which may play a role in cotton fiber secondary synthesis.To study the function of these MYBs,overexpression vectors were constructed and transformed into Arabidopsis thaliana.Three independent transgenic lines for each transgene were selected,no obvious morphology difference was observed between transgenic lines and the wild type.To find if GhMYB85A was involved in secondary cell wall formation,transverse sections of 7 week-old stems and roots were stained with phloglucinol,the most striking alteration was observed in xylem vessels,As much stronger staining was observed in xylem vessels of transgenic lines,compared with wild type.UV illumination of lignins also has similar results.further analyses are underway.