Analyses Of Cotton GhGT43A Function And Regulation

Cotton(Gossypium hirsutum L.)is the most important fiber crop in the textile industry.Mature cotton fibers contain more than 90%of cellulose and a small amount of lignin and hemicellulose.Although only little hemicellulose exists in fiber,it can crosslink with cellulose microfibrils through hydrogen bonding,and plays an important role in the integrity and stability of the entire fiber secondary wall framework,which suggests that xylan may affect the quality of cotton fiber.The length and strength of the cotton fiber define the fiber quality.The secondary wall thickening period of cotton fiber development determines the strength and processing properties of mature fibers.The role of xylan in the development of cotton fibers is still unknown.Therefore,the research on the effect of xylan on the secondary wall thickening period of cotton fiber can provide theoretical basis and experimental evidence for the improvement of cotton fiber quality.In our previous study,we isolated two glycosyltransferase genes GhGT43A and GhGT47B that may be involved in cotton fiber xylan synthesis.In this study,we screened four NAC transcription factors genes that may be involved in the regulation of GhGT43A and GhGT47B via co-expression analysis.On one hand,the phenotypic analysis of GhGT43A transgenic cotton revealed its role in plant morphogenesis and fiber development;on the other hand,preliminary functional analyses of NAC transcription factors that may regulate GhGT43A and GhGT47B expression were performed.The main results are as follows:1.GhGT43A affects morphology of cotton plantsCompared with WT,the height of GhGT43ARNAi transgenic cotton plants was significantly reduced,cotton bolls were significantly smaller,while GhGT43A overexpression plants were slightly taller,and cotton boll size did not change significantly.During the early vegetative growth stage,leaves of GhGT43A RNAi showed a yellowish and upwardly curling phenotype,but during the late reproductive growth period,morphology of the GhGT43A RNAi-silenced leaves become identical with those of the wild type.Compared with WT,the leaves located at the similar positions,from either overexpression or RNAi silenced plants were smaller,In addition,wild type plants have longer petioles.2.GhGT43A affects the development of cotton fiberDuring the late stage of secondary wall deposition and mature cotton fiber,the cellulose content of GhGT43A RNAi transgenic cotton fiber decreased,while the GhGT43A OE fibers have increased cellulose level as compared with the wild type.The GhGT43A RNAi transgenic mature cotton fiber is more rounded compared with the broad bean morphology of WT fibers.compared with WT,the cell wall thickness of mature cotton fibers of both the GhGT43A RNAi and GhGT43A OE fibers increased.However,the fiber length of the GhGT43A RNAi and GhGT43A OE plants was significantly shorter than that of WT.3.analysis of NAC transcription factors that may regulate xylan synthesisWe screened four NAC transcription factors GhSND2(GhSND2A and GhSND2B)and GhFSN2(GhFSN2A and GhFSN2D)that may be involved in the synthesis of xylan GhGT43A and GhGT47B via co-expression analysis and phylogenetic analysis.We demonstrated the GhSND2A and GhSND2D both were localized in the nucleus.The two GhSND2 genes were individually expressed in Arabidopsis,in which GhSND2A overexpression increased the number of layers of interfascicular fiber cells,and ectopic deposition of cellulose and lignin was found in the cortex of roots.Moreover,compared with WT,GhSND2A transgenic Arabidopsis showed ectopic deposition of lignin in the hypocotyl when cultured for 7 days in dark.GhSND2B transgenic Arabidopsis inflorescence stem sections were stained with phloroglucinol for lignins and Congo red for cellulose.It was found that the staining intensity was stronger than those of WT,indicating that the content of lignin and cellulose of OE plants increased.Ectopic deposition of lignin and cellulose was also found in root cortex.The RT-qPCR results indicated that the expression of cellulose,lignin and hemicellulose biosynthetic genes in the inflorescence stems and roots of GhSND2 transgenic plants was up-regulated.Transient expression of GhSND2 in tobacco also displayed that the content of cellulose and lignin in tobacco leaves increased.Likewise,the two GhFSN2 genes were individually expressed in Arabidopsis.One is named GhFSN2A from cotton A subgenome and the other is designated GhFSN2D from D subgenome.Both transgenic Arabidopsis plants showed no significant difference in plant morphology compared with WT,but the transgenic Arabidopsis had smaller pods and much less seeds.Moreover,the lignin autofluorescence of the primary roots was more intense in transgenic plants than that of WT;the cellulose-specific dye S4B stained deeper in transgenic plants than WT,which suggests that the content of lignin and cellulose in the main root of GhFSN2 transgenic Arabidopsis increased.4.GhFSN2 directly activates the expression of genes involved in cotton fiber secondary wall synthesisDual luciferase reporter assay showed that both GhFSN2A and GhFSN2D could directly activate the promoters of cotton fiber secondary cell wall cellulose synthase GhCesA4-3 and GhCesA7-1 to regulate cellulose synthesis.They also directly activate the promoters of the hemicellulose xylan synthesis genes GhGT43A,GhGT47A and GhGT47B to regulate the synthesis of xylan.However,They cannot activate the promoters of lignin biosynthetic genes Gh4CL,GhCCR1 and GhCAD6.