Identification And Functional Analysis Of GhSWNs And GhGLU18 Genes Related To Secondary Cell Wall Biosynthesis In Cotton

Plant secondary cell wall(SCW)is mainly composed of cellulose,lignin and xylan,and has strong impacts on plant growth and adaptation to the environments,which is involved in contributing to anther dehiscence,providing mechanical support and water transport,and resisting the invasion of pathogens.SCW is mainly deposited in specific plant tissues,such as stems and anthers.Different from other plants,cotton fibers,as a tissue with important economic value,also experience the thickening of SCW.Previous studies have shown that NAC transcription factors play important roles in the regulation of SCW development,however,little is known about the regulation mechanism in cotton.In-depth exploration of genes related to cotton SCW development and investigation of their regulatory mechanisms not only increase the fundamental knowledge on SCW development,but also provide key candidate genes and theoretical guidance for cotton fiber quality improvement.Fiber development consists of four stages: initiation,rapid elongation,SCW thickening,and maturation.Cotton fiber development is a complex process,and polysaccharide metabolism plays pivotal roles in fiber development.Callose is deposited in the cell wall during cotton fiber cell elongation,and the content of callose reaches a peak at the end of fiber elongation.At the onset of SCW formation,the callose is gradually degraded,dropping to less than 1 % in mature fiber.However,how β-1,3-glucanase regulates fiber cell development via degrading callose in cell wall remains largely unknown.In this study,the SWNs(secondary cell wall NACs,SWNs)regulating SCW development in cotton stems were identified and the functions and molecular regulatory mechanisms were verified.Moreover,a fiber-specifically expressed β-1,3-glucanase gene GhGLU18 was identified,and its functions were investigated.The main results were as follows:1.Identification and functional analysis of NAC genes related to SCW development in cottonWe identified a class of NAC transcription factors involved in SCW development in cotton by phylogenetic analysis of NAC proteins in Arabidopsis thaliana and G.raimondii,and named as SWNs.Phylogenetic analysis showed that SWNs could be divided into two subgroups,named SND1(secondary wall-associated NAC domain protein1,SND1)and NST1(NAC secondary wall thickening promoting factor 1,NST1),respectively.RNA-seq and q RT-PCR results in G.hirsutum TM-1 indicated that SWNs were preferentially expressed in tissues that underwent SCW thickening,including stem,stamen,and cotton fiber.Subcellular localization results suggested that all the seven SWN proteins located in the nucleus,which were consistent with their functions as transcription factors.Transcriptional activity detection indicated that SWNs function as transcriptional activators,and their Cterminus were the transactivation domain.The function of SWNs genes was further verified by VIGS(virus-induced gene silencing)experiments in TM-1 and Hai7124.Plants with SND1 or NST1 silencing alone showed no obvious phenotype in TM-1 or Hai7124.Simultaneous silencing of SWNs resulted in a drastic reduction in stem strength in TM-1 and Hai7124.SWNs-silenced Hai7124 plants exhibited a more severe phenotype,resulting in significantly curved hypocotyls.To investigate the functions of SWNs in SCW biosynthesis,the contents of cellulose and lignin,two main components of SCW,were measured.The amount of cellulose and lignin was significantly decreased in SWNs-silenced stems,whereas no significant alteration was detected in NST1-silenced or SND1-silenced plants.Cross-sections showed that xylem and phloem development was significantly inhibited in SWNs-silenced plants,while they were normal in NST1-silenced or SND1-silenced plants.Taken together,SWNs were involved in the regulation of SCW development in cotton stem,and the two phylogenetically different subgroups of NST1 and SND1 exhibited functional redundancy.To gain insight into the molecular basis of the decreased SCW formation,a comparative transcriptome analysis in TM-1 stems was performed.A total of 1359 differential expression genes(DEGs)were detected between SWNs-silenced and control.GO analysis showed that a subset of genes was further enriched,including those involved in the xylan,lignin,and cellulose biosynthetic processes.A total of 520 DEGs were found between SND1-silenced and TRV: 00 plants,and 56 DEGs were found between NST1-silenced and TRV: 00 plants.No any items were related to SCW formation,no matter in NST1 or SND1-silencing plants.RNA-seq expression data and q RT-PCR results showed that downregulation of SWNs led to reduced expression of the genes related to SCW biosynthesis,including cellulose synthesis(Ces A4,Ces A7,Ces A8,COBL4),lignin synthesis(PAL1,C4 H,4CL1,C3H1,HCT,CCo AOMT,CCR1,F5H1,CAD6)as well as xylan synthesis(IRX7,IRX8,IRX9,IRX10,IRX14,IRX15),while no significant changes were found in NST1-silenced or SND1-silenced plants.Overexpression of SWNs induces ectopic SCW formation in tobacco leaves.Transcriptome analysis found that the expression levels of GhMYB46 and GhMYB83 genes decreased significantly in SWNs-silenced plants,reaching a maximum of 87 %reduction.Meanwhile,co-expression analysis showed that GhMYB46 and GhMYB83 had similar expression patterns with SWNs.We discovered three types of SNBE sites on the upstream region of GhMYB46 and GhMYB83 genes.Yeast one-hybrid assay and luciferase reporter assay demonstrated that SWNs were able to regulate the expression of GhMYB46 and GhMYB83 genes by binding to their different SNBEs.2.Functional analysis of GhGLU18 in cotton fiber developmentWe identified a β-1,3-glucanase gene,GhGLU18,which was specifically expressed in cotton fiber based on the RNA-seq and q RT-PCR data in G.hirsutum TM-1.The expression of GhGLU18 in fibers gradually increased from the elongation stage and reached the peak at SCW formation stage,suggesting that GhGLU18 may contribute to fiber elongation and SCW deposition.GO enrichment analysis showed that GhGLU18 co-expressed genes are mainly involved in cell wall development,which suggests that GhGLU18 may regulate the process of fiber development by participating in cell wall synthesis.The protein structure analysis showed that GhGLU18 contains an N-terminal signal peptide sequence and glycosyl hydrolase 17 domain(GH17)without GPI anchor site.Subcellular localization indicated that GhGLU18 was a cell wall localized protein.To investigate the function of GhGLU18 in fiber development,we constructed overexpression and antisense constructs both driven by the fiber-specific RDL promoter to up-and down-regulate the expression of GhGLU18 in cotton.Two overexpression lines with the highest GhGLU18 expression level(OE-1 and OE-2),and two GhGLU18-suppressed lines that exhibited markedly reduced GhGLU18 expression(AS-2 and AS-4)were selected for further functional analysis.Compared with the wild-type,mature fiber length was significantly decreased in antisense lines while increased in the overexpression transgenic lines.Fiber quality test with HVI9000 system further confirmed these results.Fiber length and strength were remarkably increased in the GhGLU18 overexpression transgenic lines,while the antisense lines displayed shorter fiber length and decreased fiber strength.Microscopic analysis of 20 DPA,25 DPA and mature fiber demonstrated that overexpression of GhGLU18 increased the thickness of fiber cell wall,while suppression of GhGLU18 reduced the cell wall thickness,compared with the W0.Scanning electron microscopy analysis showed that the GhGLU18-overexpressing lines had higher degree of helicalization and shorter helical pitch,while the GhGLU18-suppressed lines exhibited the opposite phenotype.In sumarry,these results indicated that GhGLU18 is involved in fiber elongation and SCW thickening.To explore the molecular mechanism of GhGLU18 in fiber development,we performed RNA-seq analysis on 15 DPA and 20 DPA fibers from the wild type and transgenic lines(OE-1,AS-2).In 15 DPA fibers,a total of 2115 DEGs were identified in the OE-1 line compared with the control of W0,including 1478 up-regulated and 637 down-regulated genes.In the antisense line of AS-2,a total of 1307 DEGs were identified,including 433 up-regulated and870 down-regulated genes.In 20 DPA fiber,2949 DEGs were obtained in the OE-1,among which 2846 genes were upregulated and 103 genes were downregulated.By contrast,6125 genes were differentially expressed in the GhGLU18-suppressed line,of which 82 genes were upregulated and 6043 genes were downregulated.GO enrichment analysis of up-regulated genes in OE-1 and down-regulated genes in AS-2 in 15 DPA and 20 DPA fibers showed that cell wall development and polysaccharide metabolism pathways were significantly enriched in both periods.These results demonstrated that GhGLU18 may regulate polysaccharide metabolism and cell wall development processes,and ultimately participates in fiber elongation and SCW synthesis.Based on the above RNA-seq data,we first determined the content of sucrose,fructose,glucose,and total soluble sugar in fibers at different developing stages.The results showed that the soluble sugar content was significantly higher in the GhGLU18-overexpressing lines while lower in the GhGLU18-suppressed lines from 15 DPA onwards.In addition,the expression of some key genes involved in sucrose metabolism was also changed significantly,which indicated that GhGLU18 affected the carbohydrate metabolism of fiber cells.To verify the effect of GhGLU18 on cell wall development,the cellulose content of fibers was determined.From 15 DPA,the cellulose content was significantly increased in GhGLU18-overexpressing lines and decreased in the GhGLU18-suppressed lines.The expression levels of a series of key genes involved in fiber cell wall development were significantly up-regulated in GhGLU18 overexpression lines and down-regulated in suppressed lines,including EXP,XTH,and Ces A etc.These results suggest overexpression of GhGLU18 promotes cell wall development,and finally promotes fiber elongation and thickening.The upstream regulators of GhGLU18 were further identified.Analysis of the 1500 bp promoter of GhGLU18 revealed that multiple SNBE motifs of the promoter were bound by the SCW-associated NAC transcription factors.Co-expression analysis showed that GhFSN1 had similar expression patterns with GhGLU18,and predominantly expressed in SCW thickening fibers.Dual luciferase fluorescence assay suggested that GhGLU18 could be directly activated by GhFSN1.Taken together,we identified GhSWNs and GhGLU18 are important genes for SCW development in cotton,and clarified their roles and molecular mechanisms during SCW formation.These results enriched the theoretical knowldege on SCW development,and provided candidate genes for breeding high-quality cotton varieties.