| Cotton is the most important natural textile fiber crop in the world.Cotton fiber,a highly elongated and thickened single cell derived from the ovule epidermis,provides a model system for studying cell elongation and cell wall biosynthesis.Cotton fiber cell walls determine the industrially important fiber quality parameters.Arabinogalactan protein(AGP)is the most highly glycosylated cell wall protein,and it consists of a hydroxyproline(Hyp)-rich core protein and large type-Ⅱarabinogalactan(AG)moieties.AGPs play a pivotal role in the plant growth and development,but the detailed biosynthesis and molecular function of the AG glycan in cotton remains largely unknown.In this study,a putative β-1,3-galactosyltransferase gene involving in biosynthesis of AGP glycan moiety in fiber development was identified and designated GhGalT1,and we systematically characterized the enzyme activity,functions and regulation mechanism of GhGalT1 in cotton fiber development.In addition,the mechanisms of cotton GhDil9-1/-2 in response to high salinity stress have been studied.The main results are as following:1.GhGalTl is a β-1,3-galactosyltransferase and forms homodimers and heterodimerises with other cotton GalT proteinsIn order to investigate the biochemical function of GhGalTl protein,C-terminal-VENUS-tagged full length GhGalT1 fusion protein was transiently expressed in Nicotiana benthamiana,and microsomes in the leaves were extracted.The enzyme assay product was analyzed by reversed-phase(RP)HPLC and ESI-mass spectrometry,and it revealed GhGalT1 is a novelβ-1,3-galactosyltransferase responsible for adding galactose to the β-1,3-galactan backbone of AGPs.Yeast two-hybrid and bimolecular fluorescence complementation(BiFC)assays revealed that GhGalT1 interacted with itself to form homodimer.In addition,GhGalT1 interacted with GhGalT2,but did not interact with GhGalT3 and GhGalT4.These data indicated that GhGalT1 can form homodimers and heterodimers to perform its function in AGP biosynthesis in fibers.2.GhGalTl functions in fiber initiation and elongation as a negative regulatorDuring fiber development,GhGalT1 was expressed at different levels in 0-20 DPA(days post anthesis)fibers.At early developmental stage(0-3 DPA),weak to moderate transcription of GhGalT1 was detected in ovules and fibers.As fibers further developed,GhGalT1 expression was gradually enhanced,and reached its peak in both 6 and 18 DPA fibers.To investigate the functional importance of GhGalT1 in fiber development,constitutive CaMV35S promoter-driven overexpresion and RNA interference(RNAi)constructs were introduced into cotton by Agrobacterium-mediated transformation.Appropriate amount of transgenic cotton plants were obtained.Phenotypic observation and statistical analysis indicated that GhGalTl overexpression plants produced smaller bolls and shorter fibers,whereas GhGalT1-silenced lines displayed increased boll size and fiber length,compared with those of wild type.Furthmore,the mature fiber characteristics and the mechanical properties were compared among the transgenic lines and wild type.The results indicated that fibers of the GhGalT1-RNAi transgenic cotton progenies were still longer,whereas fibers of the GhGalTl overexpression transgenic progenies were shorter than those of wild type.Furthermore,fiber strength and micronaire value were also changed in some transgenic cotton lines,compared with those of wild type.To investigate in more detail,the role of GhGalT1 in regulating morphogenesis of fiber cells,the surface of 0-1 DPA ovules of the GhGalTl transgenic cotton plants and wild type were observed by scanning election microscopy(SEM).The results indicated that the GhGalT1 overexpressed lines had a remarkable reduction in the number of fiber initials relative to wild type.In contrast,the RNAi lines showed a notable increase in the fiber initials number,and fibers elongated in a more synchronous manner.In addition,GhGalTl overexpressors exhibited a delay in fiber expansion and less synchrony in initiation,with fewer and smaller fiber protrusions than wild type.Furthermore,toluidine-blue staining of LR-white sections of ovules and fibers at different developmental stages was performed.At 1-2 DPA,the fiber cells were much fewer and smaller on the surface of the GhGalTl overexpression ovules than wild type,but fiber initials on the surface of GhGalT1-RNAi ovules were more uniform and longer than those on wild type.With further fiber development,GhGalT1 overexpression lines showed severely collapsed,misshaped and irregular fiber cells,whereas fiber cells of GhGalTl-RNAi lines were more coherent and more round,compared with those of wild type.It revealed that low level of GhGalT1 expression is required for normal morphogenesis of cotton fiber cells,possibly by affecting cell wall structure in cotton.The above results indicate that GhGalT1 acts as a negative regulator on fiber initiation and elongation.3.GhGalT1 modulates AG glycans of AGPs in fibersTo verify if the carbohydrate moieties of AGPs are changed,higher AGP content was detected in GhGalT1 overexpression fibers,while GhGalT1-RNAi fibers contained less AGPs compared with wild type.In addition,a number of AGP epitopes recognized by JIM8,JIM 13 and MAC207 were enhanced in the GhGalT1 overexpression fibers but reduced in the GhGa/T1-silenced fibers by Western blot,dot blot and immunohistochemical localization analyses,demonstrating AG glycan structures were altered in the transgenic fibers.It was previously suggested that AGPs/AGs could interact with pectin.To investigate the correlation between AGPs and pectin,two methyl-esterified homogalacturonan(Me-HG)-recognized mAbs(JIM5 and JIM7)were used to check the pectin distribution and abundance in developing fibers at different stages,similar labeling patterns as anti-AGP mAbs were observed.This finding suggested that GhGalTl might also affect pectin rearrangement in the cell wall.To further investigate if GhGalT1 affects the biosynthesis of AG glycans,we measured the sugar composition of fiber AGPs from different genotypes.We conclude that the monosaccharide composition(including the galactose(Gal)and arabinose(Ara)level),especially the relative amounts of Gal and Ara(the rate of Gal%/Ara%),of AGPs were substantially altered in the transgenic fibers,suggesting that GhGalT1 is involved in controlling the glycosylation of AGPs and thereby modifying monosaccharide composition of AGPs in cotton fibers.4.GhGalTl influences monosaccharide composition of cell wall fractions in fibersTo know if changes of AG glycan of AGPs affect biosynthesis of other cell wall components,we also analyzed cell wall monosaccharide composition of alcohol insoluble residues(AIR)and measured the monosaccharide contents of pectin-rich and xylan-rich fractions in fiber cell walls.Compared with wild type,GhGaIT1 overexpression cell walls contained relative increased Ara and Gal,but reduced glucose(Glc)in total AIR sugar.Furthermore,GhGalTl overexpression lines exhibited substantial increase in Ara,and Gal and a relative decrease in Glc in the pectin-rich fraction,and an increase in xylose(Xyl)and a decrease in Glc in xylan-rich fractions.In contrast,the opposite pattern was observed in the GhGalT1-silenced lines.The above results demonstrated that GhGalT1 can influence the overall cell wall components.5.GhGalT1 affects expression of cell wall-related genes(especially GhFLAs)in fibersTranscriptomic analysis revealed among 112 up-regulated cell wall genes,noticeably,FLA genes represented the most abundant gene category in the GhGalT1 overexpression fibers.Furthermore,GhFLA2,GhFLA3,GhFLA8 and GhFLA9 were coexpressed with GhGalT1 during fiber development by qRT-PCR analysis.It indicated that GhGalT1 predominantly glycosylates GhFLAs.6.Study on the mechanism of cotton GhDil9-1/-2 responding to high salinity stress and ABA signalingDi19(drought-induced protein 19)family is a novel type of Cys2/His2 zinc-finger proteins.In this study,we demonstrated that cotton Dil9-1 and Di19-2(GhDil9-1/-2)proteins could be phosphorylated in vitro by the calcium-dependent protein kinase(CDPK).Mutation of Ser to Ala in N-terminus of GhDil9-1/-2 led to the altered subcellular localization of the two proteins.GhDi19-1/-2 overexpression transgenic Arabidopsis seedlings displayed the hypersensitivity to high salinity and abscisic acid(ABA).However,Ser site-mutated GhDi19-1(S116A)and GhDi19-2(S114A),and Ser and Thr double sites-mutated GhDi19-1(S/T-A/A)and GhDi19-2(S/T-A/A)transgenic Arabidopsis did not show the salt-and ABA-hypersensitive phenotypes.In contrast,overexpression of Thr site-mutated GhDil9-1(T114A)and GhDil9-2(T112A)in Arabidopsis still resulted in salt-and ABA-hypersensitivity phenotypes,like GhDil9-1/-2 transgenic lines.Overexpression of GhDil9-1/-2 and their constitutively activated forms in Atcpkll background could recover the salt-and ABA-insensitive phenotype of the mutant.Thus,our results demonstrated that Ser phosphorylation(but not Thr phosphorylation)is crucial for functionally activating GhDi19-1/-2 in response to salt stress and ABA signaling during early plant development,and GhDi19-1/-2 proteins may be downstream targets of CDPKs in ABA signal pathway. |
