Manipulation Of Sucrose Transporter Affects Fiber Initiation, Elongation And Seed Development Of Cotton

Cotton is the most important natural fiber crop in the world.Cottonseeds,in which the oil content is 30%³8%and protein content is over 30%,are also the source of edible oil and protein.Cotton fiber is derived from the epidermal cell of the cotton seed.Carbon partitioning within the cotton seed involves competition between fiber and embryo development.This allocation competition makes us in a dilemma to increase the yield of fiber or seed.Moreover,cotton fiber quality that affects the spinning performance and textile product grade is extremely important for textile industry and cotton producers.However,unfortunately there is negative inheritance relation between fiber yield and quality.Thus,it has become a great challenge for cotton breeding to increase both fiber yield and seed yield,while simultaneously improve fiber quality.To solve the dilemma between fiber and seed yield,a feasible way is the supplying of sufficient photosynthate for both of them.Sucrose,the main form of photosynthetic products transports from"source"to"sink"in higher plants and participating in cell signal transduction,affects the economic yield and quality of crops.Sugar transporter SUTs and SWEETs are responsible for phloem loading,transport and unloading of sucrose,and the important regulators for carbon allocation in plants.In this paper,to increase cotton seed and fiber yield genes of SUTs and SWEETs are used to regulate the sucrose distribution in cotton embryo and fiber.It was demonstrated that,?1?“Sink”specific expression of a maize sucrose transporter,ZmSUT1,promotes the transport of photosynthetic products to the target organ and increases the“sink”strength.As the result,yields of both cotton seed and fiber were increased synchronously.?2?Increase of sucrose concentration promoted mitochondrial oxidative phosphorylation and enhanced the product of ROS in fiber.The increased ROS not only stimulated fiber initiation,but also advances the time of secondary cell wall biosynthesis.The early arrival of secondary cell wall biosynthesis impeded the elongation of the cell and resulted in a shorter mature fiber.?3?GhSWEET15 was specifically expressed in the secondary fiber wall synthesis stage,and controlled the efflux of sucrose in the secondary fiber wall stage.Overexpression of this gene delayed the formation of secondary walls and thus made transgenic fibers became longer and thinner.?4?GhSUT5 accounted for the uploading of sucrose to embryo and seed,affecting the seed embryo development.The transcript of GhSUT5was controlled by WRKY27.Down-regulated GhSUT5 could block the GA signal pathway promoting seed germination.The main results are as follows:1.Senescence-inducible expression of ZmSUT1 in cotton promoted sugar accumulation in leaves and delayed leaf senescence,but decreased fiber yieldThe C4 plant Zea mays exhibits a substantially higher rate of export of photosynthates than many C3 plants.Sucrose transporter ZmSUT1 uploads sucrose in phloem,and retrieves sucrose from the ectoplast as well.Senescence-inducible?pSAG12?expression of this gene in cotton led to an increase of sucrose content in aged leaves.The sucrose content and total soluble sugar content in the aged leaves of transgenic SU-8 and SU-14 lines were increased by 21.6%and 34.4%,and by 5.8%and 10.2%,respectively.The chlorophyll content,SOD and POD activities,the anti-senescence indicators in plants,were significantly higher,while the content of MDA,the senescence indicator,was significantly lower than that in aged leaves of the wild-type cotton.High level of sugars in transgenic leaves might account for the delay of leaf senescence and the decrease of photosynthesis.Consequently,the fiber yield of transgenic cotton declined.Lint yield per plant of SU-8 and SU-14 was decreased by26.2%and 25.6%.2.Seed-coat specific expression of ZmSUT1 promoted sucrose accumulation in cotton fibers and increased seed and lint yieldIn contrast to senescence-inducible expression,seed-coat specific expression of ZmSUT1 promoted sugar into developing fiber and ovule.Sucrose content of pBAN::ZmSUT1 transgenic cotton BU-21 and BU-59 was significantly increased in the ovule epidermis and fibers.The content of total soluble sugars in bolls of BU-21 and BU-59 lines was increased by 4.2%and 20.0%.The decrease of sugar content in turn promoted photosynthesis of leaves.The photosynthetic rate of BU-21 and BU-59leaves was enhanced by 5.5%and 5.1%.Consequently,seed yield per plant and lint yield per plant were increased by 32.2%and 14.0%,and 29.6%and 11.8%,respectively,relative to the wild-type control.These data indicated that seed-coat specific expression of ZmSUT1 could promote the accumulation of sugar in bolls and fibers,thus increasing the yield of lint fibers and seeds.However,it was found that the fibers of the transgenic cotton became shorter than that of the wild-type control.3.Increase of sucrose concentration promotes ROS product that stimulates fiber cell initiation and early elongation,and advances the time of secondary wall synthesis resulting in a shorter fiberUmSRT1,a sucrose transporter from maize pathogenic fungous Ustilago maydis displays highly-specific sucrose transport character and much higher sucrose affinity than that from plants.To increase the sugar content in cotton fibers,UmSRT1 was seed-coat specific expressed in transgenic cotton.The content of sucrose,glucose and fructose in 8-DPA and 10-DPA fiber of transgenic cotton lines BUR-18 and BUR-63was higher than that in the wild type.The fiber initiation number at the day of flowering?0-DPA?and mature fiber number per seed of BUR-18 and BUR-63 lines increased significantly.Ovule culture experiment confirmed that a suitable increase of sucrose concentration could promote cotton fiber initiation.Meanwhile,at the early stage of fast elongation,the elongation of transgenic fiber cells was promoted.However,during the late stage of fast elongation and early stage of secondary wall synthesis,the elongation retarded.The length of mature fibers of transgenic cotton became shorter than that of wild-type.Ovule culture experiment showed that the increase of sucrose concentration promoted the elongation of cotton fiber at the early stage,but impeded the elongation at the later stage.In addition to being shortened,the transgenic fibers become thicker.The thickness of secondary walls of transgenic15-DPA and 20-DPA BUR-63 fibers was significantly higher than that of the wild type control.At 12 DPA,the expression of secondary wall biosynthesis related genes,e.g.,GhCesA1,GhCesA2,GhCesA4,GhCesA6,GhCesA7,and GhCTL1,GhCTL2,and GhGluc1 could be detected in transgenic fibers.In contrast,the expression of these secondary wall biosynthesis-related genes became detectable at 15 DPA in wild-type fibers,indicating that the secondary wall accumulation in transgenic cotton fiber was advanced.Ovule culture showed that these secondary wall synthesis-related genes were up-regulated by the increasing of sucrose.It was reasonably speculated that the advanced secondary wall biosynthesis impeded fiber elongation,resulting in the shorter fiber of transgenic cotton.Transcriptomic analysis showed that genes related to mitochondrial oxidative phosphorylation pathway were significantly up-regulated of BUR-63 transgenic cotton fibers.Compared with the wild type,the respiration rate in the 8-DPA ovule of transgenic cotton was significantly promoted.Ovule culture confirmed that increased sucrose concentration promoted cell respiration.Mitochondrial respiratory chain complex I mediated ROS generation.ROS signal in ovules and fibers of transgenic lines is stronger than that in the wild type,and the content of H₂O₂,an important ROS,in ovules and fibers was also higher than that in the wild type,indicating that the increased sucrose in transgenic cotton fiber might stimulate the respiration,thus promoting the accumulation of ROS.Adding DPI,a ROS inhibitor,in cultural medium,the inhibitor could entirely suppress the fiber initiation of wild-type cotton and transgenic line BUR-63 under high sugar concentration culture,confirming that ROS played a crucial role in fiber initiation.When addition with the ROS scavenger,the expression of genes related to secondary wall synthesis were repressed.These results verified the previous study that ROS is a signal molecule to initiate secondary wall biosynthesis.The high-level ROS in transgenic cotton fiber causes the time ahead of the start of secondary wall synthesis,and ultimately the fiber become shorter than the wild type control.In addition to secondary-wall biosynthetic genes,genes related to flavonoid synthesis and phenylalanine metabolism pathway were up-regulated in transgenic cotton fibers.Flavonoid participates in the anti-oxidation of cells and has a function of scavenging ROS.Noticeably,lignin content was increased in transgenic cotton fibers,suggesting that the increase of sucrose level not only increased ROS generation,but also promoted the synthesis of flavonoids that is involved in ROS scavenge.Besides,as a component of cell wall,the increased carbohydrate flow to lignin synthesis might decrease the allocation to cellulose.4.Secondary-wall-synthesis-specific GhSWEET15 is responsible for the efflux of sucrose in fiber and up-regulation of it results in longer and thinner fibersDown-regulation of clade?SWEETs in cotton increased the soluble sugar accumulation in the transgenic fibers.Meanwhile,the seed index?weight of hundred cottonseeds?and the lint index?fiber weight produced by hundred seeds?increased,indicating that clade?GhSWEETs were responsible for the efflux of sucrose.Among them,GhSWEET15 showed specific expression during secondary wall thickening.Overexpression of this gene,sucrose content in the fiber of transgenic lines was decreased,and genes related to secondary wall synthesis were down-regulated.The fiber cell wall of transgenic fibers became thinner,and mature fiber became longer than that of the wild-type control.These results indicated that moderately up-regulating the expression of GhSWEET15 could improve the fiber fineness and length of transgenic fibers.5.GhSUT5 accounts for the influx of sucrose in ovule/seed and down-regulation of it retards seed germinationRepression of the expression of sucrose transporter SUT family in cotton resulted in a decrease in the soluble sugar content in the fiber of transgenic cottons.The seed index and lint index of transgenic lines were also decreased,indicating that GhSUTs were mainly responsible for upload sucrose into fiber cells.Among them,GhSUT5showed high-level expression in the later stage of ovule development,and low-level expression in other tissues.Down-regulation of GhSUT5 leads to an increase in the total soluble sugar content in 20-DPA ovules of the transgenic lines,while a decrease in 20-DPA fibers.Meanwhile,the seed index of transgenic cottons was enhanced and lint index was decreased,suggesting that down-regulation of GhSUT5 reduced carbohydrate efflux to fiber,leaving more carbohydrate in ovule thus increasing seed weight and reducing fiber yield.6.WRKY27 regulates AtSUC5/GhSUT5 expression and seed germinationGhSUT5 is a homologous gene of Arabidopsis thaliana AtSUC5.Down-regulation of AtSUC5/GhSUT5,the embryo development and seed germination were delayed.Moreover,GA was unable to relieve the seeds from inhibition of germination.Interestingly,in the late stage of embryo development,GA was capable of inhibiting the expression of GhSUT5/AtSUC5.Furthermore,AtWRKY27 was found to bind to the upstream element of AtSUC5 and activate AtSUC5 transcription.It was suggested that at the late stage of embryo development the presence of GA promoted DELLA protein degradation,leading to a down-regulation of WRKY27 and suppressing the expression of the downstream AtSUC5;the down-regulation of AtSUC5 in turn inhibited the sucrose transport to embryo,and then delayed embryo development.Meanwhile down-regulation of AtSUC5 caused sucrose accumulation in the inner integument and blocked the GA signaling through T6P/SnRK1 pathway,thus inhibiting the early germination of seeds.In conclusion,sucrose transporters play an essential role in cotton fiber initiation,elongation and seed germination.Genetic manipulation of sucrose transporters to promote the transport of photosynthetic products to the harvest organs/tissues can increase both seed and fiber yield of cotton.The increased sucrose in fiber cells would promote mitochondrial oxidative phosphorylation and cell respiration,the result of which is the promotion of ROS accumulation.ROS signal in turn stimulates secondary walls synthesis of fiber cells which impedes the elongation of fibers and leads to shorter mature fibers.Thus,to make a feasible strategy for the utilization of sucrose transporter in cotton improvements,the developmental feature of cotton fiber cell should be taken into consideration.For example,we can moderately elevate the sugar content in the ovule epidermal layer during the fiber initial and early elongation stage to promote the fiber initiation and elongation,and then appropriately reduce the level in the cell during the overlapping stage of fiber rapid elongation and secondary wall accumulation to retard the synthesis of secondary wall and increase the fiber length and fiber fineness.