The Prolyl-4-hydroxylase GhP4H2 Is Involved In Regulating Fiber Development Of Cotton

Cotton is one of the most important cash crops worldwide.Cotton fiber is very widely used in our daily life,it is an important raw material of the textile industry.Cotton fiber cells are differentiated from ovule epidermal cells and represent the one of the longest cells in the plant kingdom.Its development consists of four overlapping stages:initiation,cell elongation(primary wall formation),secondary wall thickening,dehydration and maturation.Cotton fiber cell walls define the fiber quality in the textile industry.Therefore,it is very important to study roles of the cell wall in fiber development.The cell wall of cotton fiber is mainly composed of cellulose,hemicellulose,lignin and glycoproteins.Cell wall composition varied during the whole process of cotton fiber development.Arabinogalactan proteins(AGP s)belong to a subgroup of hydroxyproline(hyp)-rich glycoproteins play important roles in fiber developmen.Arabinogalactan proteins(AGPs)are highly glycosylated proteins which involve two most important post-translational processes,first,the proline residues are hydroxylated by the enzyme prolyl-4-hydroxylases(P4Hs);then the hydroxyprolines(Hyps)were subsequently glycosylated with large arabinogalactan polysaccharides or small arabino-oligosaccharides by glycosyltransferases(GTs).Our previous studies have shown that fiber elongation was repressed when in vitro cotton ovules were treated with inhibitors of P4Hs which implied that GhP4Hs are involved in cotton fiber development.In this study,to further investigate the role of GhP4Hs in plant.One GhP4Hs,which designated GhP4H2,was predominantly expressed during the fiber elongation period.Over expression and RNAi-silencing GhP4H2 vectors were constructed and transformed into cotton.Phenotypic assays were performed,main results are as follows:1?GhP4H2 overexpression(OE)and RNA interference(RNAi)transgenic cotton genetic and phenotypic analysisTo further investigate the role of GhP4Hs in fiber development,over-expression and RNAi-silencing vectors of GhP4H2 were constructed and transferred into wild type cotton.Four independent transgenic lines were obtained:two over-expression(OE)transgenic lines:L38,L10 and two RNAi-silencing lines:RNAi L16 and L28.These lines were used for phenotypic analyses.Quantitative RT-PCR technique was employed to examine the expression level of GhP4H2 in independent transgenic lines.The results revealed that the expression level of GhP4H2 in 5 dpa(day post anthesis)and 10 dpa fiber of OE L38 and OE L10 were significantly higher than that in wild type whereas the expression of GhP4H2 in RNAi lines L16 and L28 were much lower than that in wild type.This expression profile was inherited in the subsequent generations.Mature fiber were harvested and fiber length was compared between the transgenic lines and the wild type.It was found that length of mature fiber from OE lines were markedly shorter than that in wild type control,while fiber length in RNAi-silencing lines was indistinguishable from that of wild type.Fiber quality analyses also exhibited the similar results.In addition,when the lint fibers were stripped,linter fibers remaining in the RNAi-silencing lines were obviously less than those in the wild type.Taken together,alteration of GhP4H2 expression affects fiber development and impacts both lint and linter fiber development.2.Section analyses of 10 dpa fiber cellsTo investigate if GhP4H2 can affect fiber cell morphogenesis and cell wall structure,LR-White resin and paraffin embedded 10 dpa fiber sections were conducted.Preliminary results displayed that fiber cells from OE lines were larger than those from wild type,while cells from RNAi lines were arranged more closely.The experiments are still ongoing.3.AGPs content assay and dot-blot of AGPsTo investigate if GhP4H2 affects AGP biosynthesis,AGPs were extracted from 10 dpa fibers of different transgenic lines.The results showed that the content of AGPs from over-expression lines was higher than that of the wild type,whereas the AGP content from RNAi lines were lower,as compared with the wild type.AGP dot blots using three different monoclonal antibodies(JIM8,JIM13,MAC207)raised against AGP carbohydrate moieties revealed that the signal in the OE lines was stronger than that in the wild type,while the RNAi lines have the opposite results.These results implied that GhP4H2 impacts on polysaccharide chain biosynthesis of AGPs.4.Transcriptome analysis of GhP4H2 fiberTo investigate the potential downstream targets that GhP4H2 acts,10 dpa fiber RNA were extracted from GhP4H2 transgenic lines.The RNA quality was checked by RNA electrophoresis and sent to the company.RNAi L28-35,OE L38-17 and wild-type were selected and repeated twice for transcriptomic analyses.The transcriptome results showed that the most significant differential expressed genes were genes related to cell wall biosynthesis and modification,especially those encoding hydroxyproline-rich proteins when compared OE with the wild type.In OE line,a total of 202 genes were upregulated,160 annotated genes were selected for further research when eliminating those sequences that have no reference gene which were termed as novel genes(42).Among the 160 annotated genes,27 genes(occupy 17%of 160 genes)are related to cell wall biosynthesis and modification,ten of which encode Hyp-rich proteins.In addition,according to the pval value,we analysed the top 30 annotated genes which display the significantly differential expression,among the first 30 annotated genes,seven genes(occupy 23%of 30 genes)are associated with cell wall synthesis,in which four genes encode Hyp-rich proteins.We speculate that Hyp-rich proteins are action targets of GhP4H2.To verify the reliability of transcriptomic results,we designed the gene-specific primers for those genes encoding Hyp-rich proteins.Quantitative PCR results showed that these genes are markedly upregulated in the OE lines compared with the wild type,further supporting the transcriptomic results.In consideration of that the GhP4Hs act upstream of glycosyltransferases,we also checked seven genes encoding GhGalTs,we found that four genes are induced in the OE lines suggesting they might function downstream of GhP4H2.Meanwhile,according to the metabolic process enrichment map and the KEGG pathway,we propose GhP4Hs involves in many metabolic pathways and participate in the development of cotton fiber.5.Yeast two-hybrid assay of GhP4HsTo study if GhP4H2 can form homodimers and form heterodimers with other GhP4Hs to regulate biosynthesis of hydroxyproline.According to the phylogenetic tree,two GhP4Hs which display the highest similarity with GhP4H2 were selected for yeast two-hybrid assays.The pGBKT7-GhP4Hs and pGAD-GhP4Hs vectors were constructed and transformed into yeast AH 109,Y187(pGBKT7-GhP4Hs transformed into Y187,pGAD-GhP4Hs transformed into AH 109)respectively.After the transformation,pGBKT7-GhP4Hs were fused with pGAD-GhP4Hs,and then the fusion protein was screened using the yeast deficient medium.The experiments are still ongoing.