Functional Characterization Of GhKSR1 Gene In Fiber Development

Cotton is a major textile industry crop worldwide.China is the major cotton producer and consumer in the world.With the technological innovation of the textile industry and the improvement of consumption level,people put forward higher requirements for the quality of cotton fiber.Cotton fiber cell is a highly elongated single cell which is formed by the epidermal cells of the ovule.The development of cotton fiber includes four periods: initiation,rapid elongation,maturation(secondary cell wall deposition,maturation)and dehydration.It’s an ideal material for studying plant cell growth.The membrane microdomain is an important part of the membrane,which is composed of sphingolipids and steroids,and is a platform for cell signal transduction.Sphingolipids are the structurally lipid molecule with a complex structure,which have large varieties and are widely distributed.Sphingolipids have been studied extensively in animals and yeast,but less in plants.Sphingolipids and its derivatives are important regulatory molecules,particapate in a variety of cellular processes,including programmed cell death,hormone signal transduction(ABA,JA,etc),and responses to biotic and abiotic stresses.In order to explore the relationship between sphingolipids and fiber cell growth,we have cloned a cotton sphingolipid synthase gene GhKSR1.Previously,we have analyzed the biological function of the gene by transgenic Arabidopsis thaliana.However,the roles of this gene in cotton fiber development are still unclear.In this study,we generated the transgenic cotton for this gene and analyzed the function of GhKSR1 in fiber cell development.The main results are as follows:1.Content and composition changes of sphingosine during cotton fiber development:In order to clarify the dynamic changes of sphingolipid content and components during the development of cotton fiber,we analyzed the sphingolipid groups in cotton fibers at different developmental stages.The results showed that mainly 6 types of sphingolipids were detected in cotton fiber,including ceramide(Cer),glucosylceramide(Glu Cer),phytoceramide(Phyto Cer),glucosinositol phosphate ceramide(GIPC),sphingosine Alcohol(Sph)and sphingosine-1-phosphate(S-1-P).The content of sphingosine in fiber is the highest.With the development of fiber cell,the content of sphingosine exhibited a typical low-high-low pattern.The content is higher during the elongation of fiber cells,and the content is lower when the cell elongation terminated.The results imply that plant sphingosine might plays important roles in the fiber cell elongation.2.Effect of sphingolipid on cotton fiber growth and development:There are two enzymes that mediate sphingosine synthesis in the sphingolipid synthesis pathway: serine-palmitoyltransferase(SPT)and 3-ketodihydrosphingosine reductase(KSR).Myriocin is a specific inhibitor of SPT,and there is no specific inhibitor of KSR.We applied Myriocin to in vitro cultured cotton ovules.The results showed that 10 μM,and 20 μM myriocin could significantly inhibit the growth of wild-type cotton fibers.The results suggest that sphingosine synthesis-related genes may be important genes in fiber elongation.3.Effect of over-expression of GhKSR1 on the development of cotton plant and fiber cell:The GhKSR1-overexpression cottons exhibited shorter fruit branches,larger leaves,and larger petals and sepals.The up-regulation of GhKSR1 gene inhibited the initiation of fiber cells to some extent,and significantly promoted the elongation of fiber cells.The average length of mature fibers in transgenic lines increased by 13.7%～16.4%compared with that in the wild type.Meanwhile,it also promoted the growth of seeds,the seed width of transgenic lines increased by 21.1 %～25.5 %,and the seed index increased by 9 %～24.6 %.The up-regulation of GhKSR1 gene also decreased the sensitivity of the fibers to myriocin by application of myriocin to in vitro cultured ovules.Overexpression of GhKSR1 gene decreased the thickness of fiber secondary cell wall.These results suggest that overexpression of GhKSR1 gene promotes plant growth and fiber cell elongation.4.Effect of antisense-GhKSR1 on the development of cotton plant and fiber cell:The antisense-GhKSR1 transgenic lines exhibited increased plant height,smaller flowers and sepals,and the fertility of the plant was seriously affected,almost no fruit was produced.Inhibition of the expression of GhKSR1 significantly reduced the length of hypocotyl and main root of transgenic seedlings.When used wild-type pollen for pollination,a small amount of seeds could be harvested for the antisense lines.Compared to wild-type plants,the mature fiber length of antisense lines decreased by16.1 %～23.4 %,seed index increased by 10.7～15.5 %,seed index decreased by42.5 %～54.0 %,and the secondary wall became thinner.These results suggest that the down-regulation of GhKSR1 gene has an inhibitory effect on the growth and development of fiber cells.5.Explore the molecular mechanism of GhKSR1 gene to promote fiber cell elongation:In order to explore the molecular mechanism of GhKSR1 gene promoting the elongation of fiber cells,transcriptome analysis was performed on fiber cells of transgenic lines.The results showed that the expression levels of genes related to sphingolipid biosynthesis pathway in transgenic fiber cells were significantly changed.The most varied metabolic pathways were the s-1-p metabolic pathwa and the ceramide pathway,in which several genes were significantly up-regulated or down-regulated.These results suggest that the overexpression of GhKSR1 gene may affect the metabolism of s-1-p pathway and ceramide pathway by changing the components and contents of sphingolipid in the fiber cells,and thus regulate the growth and development of the fiber cells.