Studies On The Functions And Molecular Mechanisms Of GhDUF231L1 And GhSUS2 In Fiber Development Of Cotton

DUF231 is a unique family of proteins containing domain of unknown function(DUF),which is involved in regulating plant growth and development and response to stress.Many studies have shown that plant DUF231 family proteins mainly perform the function of xylan O-acetyltransferases,but the specific molecular mechanism of their involvement remains unclear.Cotton fiber is a single cell trichomes process formed by polar growth and differentiation of ovule epidermal cells.The main components of its cell wall are cellulose,hemicellulose(xylan)and lignin,Xylan is connected with cellulose microfilament by hydrogen bond and lignin by covalent bond,so that the cell wall components are fully connected to form a complete cell wall structure and maintain cell wall hardness.The acetylation of xylan directly affects the connection between xylan and cellulose microfilament,and thus affects cell wall development.To investigate whether the DUF231 family protein of Upland cotton(Gossypium hirsutum)regulates the cell wall development of cotton fiber through the function of xylan O-acetyltransferase,this study systematically screened the DUF231 family gene in the genome of cotton.A GhDUF231L1 gene related to the secondary cell wall(SCW)development of cotton fiber was identified and its function and molecular regulation mechanism were studied.In addition,GhSUS2,a sucrose synthase protein that affects cotton fiber elongation development,was identified by comparing phosphorylated proteomies analysis.The main research results of this paper are as follows:1.Identification and characterization of GhDUF231 family genes related to SCW development of cotton fibersA total of 100 GhDUF231 genes were screened and identified in Gossypium hirsutum L.genome.The conserved motifs of identified GhDUF231 proteins were analyzed by using Hmmscan and pfam tools.It was found that one GhDUF231 protein did not contain GDS and DxxH conserved motifs,while the other proteins contained one or two of them,indicating that this family of proteins had certain conserved and similarity in structure.Chromosomal localization analysis showed that 91 GhDUF231 genes were unevenly distributed on different chromosomes of cotton,and the other 9 GhDUF231 genes were located on scaffolds.Fragment duplication resulted in amplification of GhDUF231.Phylogenetic analysis showed that GhDUF231 proteins were divided into eleven branches in the evolutionary tree.Based on the public transcriptome database,45 GhDUF231 genes with high expression during the SCW development of cotton fibers were identified.GhDUF231Ll was mainly expressed in 15-21 DPA cotton fibers.2.GhDUF231L1 is involved in regulating the fiber SCW development of cottonGhDUF231L1 is localized in Golgi apparatus and can complement the phenotype of reduced xylan acetylation level in tbl3 mutant of Arabidopsis thaliana homologous gene,suggesting that GhDUF231L1 may function as xylan acetyltransferase.GhDUF231L1 overexpression(OE)and RNA interference(RNAi)vectors were constructed to obtain GhDUF231L1 OE and RNAi transgenic cotton plants.Compared with WT,the mature fiber length of both GhDUF231L1 OE and RNAi transgenic cotton was significantly shorter.The phenotype of the SCW of transgenic cotton fibers was observed.It was found that the overexpression of GhDUF231L1 resulted in a significant thickening of the SCW of cotton fiber cells and a significant increase in the content of crystalline cellulose(the main component of SCW).On the contrary,inhibition of GhDUF231L1 resulted in thinning of the SCW of cotton fiber cells and a significant decrease in the content of crystalline cellulose.Furthermore,excessive or inhibited expression of GhDUF231L1 resulted in significant changes in the specific breaking strength and micronucleus value of mature cotton fibers,suggesting that GhDUF231L1 is involved in the regulation of SCW development of cotton fibers.3.GhDUF231L1 plays a role as xylan O-acetyltransferase during cotton fiber developmentThe content of Xyl and Glu in GhDUF231L1 OE transgenic cotton fibers was significantly higher than that in the WT cotton fibers,while the content was opposite in GhDUF231L1 RNAi.The contents of other neutral monosaccharides in cell walls did not change significantly.Xyl is the main component of xylan and Glu is the main component of crystalline cellulose.Immunofluorescence analysis showed that the fluorescence intensity of LM10 and S4B was stronger,that is,the content of xylan and crystalline cellulose was higher in GhDUF231L1 OE transgenic cotton fiber,while the opposite was true in RNAi transgenic cotton fiber,compared to WT.Furthermore,compared with the WT,the acetylation levels of xylan components in GhDUF231L1 OE transgenic cotton fibers were significantly increased,while the acetylation levels of each component in RNAi cotton fibers were significantly decreased,suggesting that GhDUF231L1 may perform the function of xylan acetyltransferase.The main substrate of GhDUF231L1 was xylan,which was modified by 2-O-Ac-xylan and 3-O-Ac-xylan monoacetylation by nuclear magnetic resonance(NMR).These results suggest that GhDUF231L1 mainly plays a role as xylan-O-acetyltransferase during cotton fiber development.4.GhMYBL1 and GhKNL1 can directly bind to the promoter of GhDUF231L1In order to further explore the new molecular pathway that GhDUF231L1 is involved in regulating the fibers SCW development of cotton,using GhDUF231L1 promoter as bait to screen and identify the 18 DPA fiber cDNA library of WT plants.Two transcription factors,GhMYBL1 and GhKNL1,involved in the fibers SCW development of cotton were identified.Yeast one hybridization rotation verification analysis verified that GhMYBLl and GhKNLl could directly regulate the GhDUF231L1 expression.Subsequent ChIP-qPCR and EMSA assays also confirmed that GhMYBLl and GhKNL1 could directly bind to the promoter of GhDUF231L1 in vivo and in vitro,respectively.5.GhMYBLl and GhKNLl antagonistically regulate the expression of GhDUF231L1 to maintain the acetylation level of xylan in cotton fibersIn order to further verify the genetic relationship between GhMYBL1 and GhKNLl and GhDUF231L1,first obtained GhMYBL1 RNAi transgenic cotton plants.Phenotypic analysis showed that compared with the WT,the thickness of fiber SCW of GhMYBL1 RNAi transgenic cotton was significantly thinner,the content of crystalline cellulose was decreased obviously,the results showed that GhMYBL1 positively regulate the SCW development of cotton.Compared with WT,the contents of Xyl and Glu monosaccharides of cell wall were significantly reduced,and the acetylation levels of xylan components of fiber cell wall were significantly decreased in GhMYBL1 RNAi transgenic cotton,these results further confirmed that GhMYBLl directly positively regulates GhDUF231L1 expression to enhance the acetylation of xylan in cotton fibers,thereby promoting SCW development of cotton fibers.Similarly,phenotype analysis of GhKNL1 RNAi transgenic cotton plants showed that compared with the WT,the length of mature cotton fiber was significantly longer,the thickness of cotton fiber SCW was increased,and the content of crystalline cellulose was increased,the results showed that GhKNLl negatively regulate the SCW development of cotton.The contents of Xyl and Glu monosaccharides of cell wall were significantly increased,and the acetylation levels of xylan components of fiber cell wall were significantly increased in GhKNL1 RNAi transgenic cotton,compared with WT.These results confirmed that GhKNLl inhibits xylan acetylation in cotton fibers by directly and negatively regulating the expression of GhDUF231L1,thus negatively regulating SCW development of cotton fibers.In conclusion,GhMYBLl acts as a positive regulator and GhKNLl as a negative regulator,and the two factors antagonistically regulate the expression of GhDUF231L1 to stabilize the acetylation level of xylan within an appropriate range,thus forming the normal SCW structure of cotton fiber cells.6.Quantitative comparative phosphorylated proteomics analysis revealed that ABA signaling inhibited cotton fiber elongation through GhCPK84 and GhCPK93 mediated phosphorylation of GhSUS2Morphological analysis showed that the mature fiber length of J7-1 was longer than that of J14-1.A total of 32 differentially expressed proteins(DEPs)were identified by TMT labeled protein quantitative analysis in the 10 DPA fibers of the two cotton mutants.COG/KOG analysis showed that DEPs was mainly enriched in "post-translational modification,protein conversion,chaperone" cellular processes and signal transduction pathways.Quantitative comparative phosphorylated proteomics analysis revealed that 89 phosphorylated DEPs were detected between J14-1 and J7-1.GO analysis showed that phosphorylated DEPs mainly in sucrose synthase,transferase,glucosyltransferase and UDP-glycosyltransferase activity.In J14-1,the phosphorylation of GhSUS2 protein,a key enzyme in sucrose metabolism pathway,was significantly higher than that of J7-1,suggesting that changes in phosphorylation level of GhSUS2 may play an important role in cotton fiber development.GhSUS2 gene silencing cotton plants(TRV:GhSUS2)were obtained by virus-induced gene silencing technique(VIGS).Compared with the control,the mature fiber length of TRV:GhSUS2 plants was shorter,the result indicated that GhSUS2 positively regulates fibers elongation in cotton.GhSUS2 is localized to cell membranes and catalyzes the decomposition of sucrose into UDPG and fructose,providing substrates for the biosynthesis of cellulose and callose required for fiber elongation.During fiber development,GhSUS2 is phosphorylated at Ser11 by Ca2+dependent protein kinases GhCPK84 and GhCPK93.The phosphorylated GhSUS2 locates in the cytoplasm and enhances its sucrose decomposition activity,producing more cytoplasmic targeted UDPG,and the increased cytoplasmic UDPG further synthesizes sucrose.They no longer provide substrates for cellulose and callose synthesis and thus slow down the elongation development of cotton fibers.In addition,exogenous ABA can significantly increase the expression levels of GhCPK84 and GhCPK93,promote the phosphorylation of GhSUS2,enhance the sucrose decomposition activity of GhSUS2,and produce more cytoplasm-located UDPG,which seriously affects the elongation process of cotton fibers,leading to the shortening of mature fibers.Compared with J7-1,the phosphorylation level of GhSUS2 was significantly higher,while the length of mature fiber was significantly shorter,further indicating that phosphorylation of GhSUS2 negatively regulates cotton fiber elongation.In conclusion,quantitative comparison of phosphorylated proteomic analysis revealed the regulate mechanism of how ABA hormone negatively regulate cotton fiber elongation development through GhCPK84 and GhCPK93 mediated phosphorylation of GhSUS2.The elongation of fiber determines the final length of the mature fiber,and the thickening of SCW determines the strength and fineness of the mature fiber,which are the key indexes to measure the quality of cotton fiber.In this study,by studying GhDUF231L1 and GhSUS2 which expressed in different development periods of cotton fiber,affected the synthesis or modification of cell wall components of cotton fiber,and then regulated the quality of cotton fiber,which provides a certain scientific basis for the improvement of cotton fiber quality by genetic engineering.