Study On The Function Of Pectin Synthetase Gene GhGAUT9 Regulating Fiber Length In Upland Cotton

Cotton is the most important fiber economic crop in China and the world,providing the main raw material for the textile industry.Pectin plays a crucial role in the growth and development of cotton.A candidate gene GhM＿D07G2211 was identified by genome-wide association study(GWAS)based on the previous research of our team,which was significantly associated to fiber length.It annotated galacturonosyltransferase9(GAUT9),an enzyme involved in catalytic glycosylation reactions,as key enzyme in the pectin metabolic pathway.However,it remains unclear about function of fiber development in cotton.In this study,we firstly identified and analyzed the family members in the modern cultivars Gossypium hirsutum L.NDM8 and Gossypium barbadense L.Pima90.The functional of Gh GAUT9 were further investigated in cell elongation,fiber development and the expression mechanism by overexpression in the model plant Arabidopsis thaliana and virus-induced gene silencing in cotton.1.We identified 40 and 41 GAUT members in the genomes of NDM8 and Pima90,respectively.A homologous gene pair(Gh GA UT22 and GbGA UT23)has undergone positive selection during evolution through Ka/Ks analysis.Some GhGAUT family members were induced by drought stress,and the promoter regions of up-regulated expression members contained a GT1 cis-acting element.GhGAUT30(GhM＿D07G2211)was significantly expressed higher levels during fiber development of short-fiber varieties than long-fiber varieties,especially fiber elongation.2.GhM＿D07G2211 was isolated by cloning and sequencing,and the gene was homologous with AtGA UT9,so it was named GhGA UT9.qRT-PCR revealed that Gh GA UT9 was highly expressed in the fiber elongation period of short-fiber variety(Deltapine short branch),but almost not expressed in long-fiber variety(NDM13).The homozygous mutants gaut9-1 and gaut9-2 of AtGA UT9 were obtained;Arabidopsis was transformed with pGreenGhGAUT9-GFP overexpression vector,and homozygous overexpression transgenic lines were obtained by screening.In the hypocotyl of overexpressing Arabidopsis,Gh GA UT9 was localized on the Golgi apparatus and acted on both ends of cell wall elongation.Overexpression of GhGAUT9 promoted pectin synthesis and improved tolerance to excess galactose.Leaf trichomes of transgenic Arabidopsis were significantly shorter than those of the wild type,while those of the mutant were significantly longer than those of the wild type.Overexpression of GhGAUT9 resulted in shorter hypocotyl cell lengths in Arabidopsis,while hypocotyl cell lengths were longer in the mutant.Further,silencing of GhGAUT9 resulted in significantly longer fiber length in cotton,significantly lower pectin content but no significant difference in pectin methylation.3.The promoter of GhGAUT9 was isolated in short-and long-fiber varieties.One difference in short-fiber varieties led to the production of TATA-box(TATA/ATATAT),while this locus(ATTTAT)had no functional element in long-fiber varieties.The promoter fragment carrying TATA-box could drive more reporter gene expression.Further combination of transcriptome data from different fiber development periods of long-and short-fiber varieties revealed that the gene GhM＿A03G0476(encoding TFIID transcription factor)showed consistent expression trends and significant positive correlation with GhGAUT9 in short-fiber varieties.Fiber length existed differences among the varieties carrying different genotypes of the above variant loci.In addition,the genotypes of this mutation site were significantly different in different subspecies and geographical distribution of cotton.In summary,we identified the distribution and physicochemical characteristics of GAUT family members in modern cultivated cotton species.At the transcriptional level,the potential functions of GAUT family members were revealed growth and development and response to drought stress in cotton.It was further verified that GhGAUT9,which is highly expressed in the fiber elongation period of short-fiber variety,can participate in the metabolic pathway of galacturonic acid and promote pectin synthesis,and then negatively regulate the length of Arabidopsis leaf trichomes,hypocotyl cell and cotton fiber.We revealed that natural variation alters the cis-elements of Gh GA UT9 promoter to confer negative regulation on fiber length.The excellent genotypes selected actively in the process of evolution have the potential to improve cotton fiber quality.