Functional Study Of Key Members From Sugar Transporter Superfamily In Sugarcane

Sugarcane(Saccharum spp.)is one of the most important sugar crops in China and even in the world.Sugar content is the most important indicator for quality of sugarcane.Saccharum officinarum and Saccharum spontaneum are the two most important original species of Sacchahum,and the hybridization of these two species has generated the modern cultivars.S.officinarum has the characteristics of high sugar content,while the S.spontaneum has low sugar content but strong resistance to stress.The rencent avaiaible of the whole genomes of S.spontaneum and S.officinarum provided the genomic resource for the sugarcane foundation research.Based on the previous sugar transporter gene superfamily in S.spontaneum in our group.Here,we study used comparative genomics to identify sugar transporter genes in S.officinarum.Further more,selected key members of the subfamily were functional analysised and its possible regulatory protein genes were analyzed through functional complementation experiments and using yeast single-hybrid technology.The main findings are as follows:(1)Indentification of sugar transporters in S.officinarum.Using comparative genomics and bioinformatics methods,414 allelic sequences for 189 sugar transporter genes(STs)were identified in S.officinarum.The sugar transporters could be divided into 8 subfamilies based on phylogenetic distribuction and functional domain.Of note,the SUT(sucrose transporter)subfamily has 18 members and INT(inositol transporter),TMT(tonoplast monosaccharide transporter),VGT(vacuolar glucose transporter),p Glc T(plastidic glucose transporter),and SFT(early-responsive to dehydration)subfamilies have 13,15,7,4,and 15 members,respectively.PMT(polyol/monosaccharide transporter)and STP(hexose transporter)subfamilies are larger,with 55 and 62 family members,respectively.Compared with S.spontaneum,S.officinarum has a significant expansion of the sugar transporter genes in the total number and the number of all subfamily members,and this expansion also appears between sugarcane with other plants such as sorghum and rice.In general,S.officinarum sugar transporter genes shared highly similar in sequence with both S.spontaneum and sorghum,and the sequences and domains of homologous genes are highly conserved.The sucrose transporters harbour a GPH?sucrose family conserved domains,all monosaccharide transporters harbour MFS superfamily conserved domains,and the TMT subfamily has two shortest MFS superfamily conserveddomains.(2)Analysis of the transcriptome found that,in the leaf development gradients,So SUT1,So VGT3,So STP13,etc.are expressed at higher levels in mature leaves than young ones,indicating that these gene may be related to the transport of sugar produced by photosynthesis in mature regions of leaves;So STP4 and its six paralog genes were highly expressed at the mature part and the base of the leaf,indicating that these gene may relate to the transport of sugar from the leaf to the stem.The transcriptome data of leaves and stems at different growth stages showed that So VGT3,etc.were significantly more expressed in leaves than in stems,and showed a downward trend with the maturity of plants,indicating that these genes may be mainly responsible for sugar transport in leaves,and related to the growth period of the plant.Conversely,So TMT3 and other genes are more expressed in stems than in leaves,and they may be more involved in sugar transport in the stem of sugarcane.In addition,in the analysis results of circadian rhythm transcriptome data,So SUT1,So INT2 and other genes were significantly more expressed at night than during the day,indicating that their participation in the process of sugar output from chloroplasts at night,and may be related to sugar storage or consumption at night.On the contrary,So SUT4?T1,So TMT4 and other genes havd higher expressional level in the day time than at night time,indicating that they are more involved in the transportation of sugar in the sugarcane during the day,which may be related to the sugar consumed by the respiration of sugarcane.The analysis of transcriptome data showed that each sugar transporter has different expression at different locations and periods in S.officinarum,and they completed the sugar transport,utilization and storage jointly of sugarcane at various stages and locations.In addition,9 genes with abundant transcripts in the 8 ST subfamilies of S.officinarum,7 genes have higher expression in leaves than the highly expressed homologous genes of S.spontaneum,indicating that most STs in S.officinarum may have higher expression abundance than in S.spontaneum,it forms the difference in sugar content between species with its quantitative expansion.(3)The analysis of gene function for the sugar transproters in sugarcane.Hexose absorption-deficient yeast EBY.VW4000 was used to perform functional complementation experiments on highly expressed members of major ST subfamilies of S.officinarum and S.spontaneum.The result revealed that,monosaccharide transporters including STP,VGT,p Glc T have the ability to transport monosaccharides(glucose and fructose),while the monosaccharide transporters TMT genes,not only have transport capacity of glucose and fructose,but also have a transsucrose capacity.In addition,members of the SUT subfamily show only specific sucrose transport capabilities.Moreover,homologous genes of S.officinarum and S.spontaneum diplayed similar function for the transportation of sugar substrates.(4)Indentification of transcription factors for the sugar transporter in sugarcane.Using the yeast one-hybrid system,33 interacting protein genes with high expression sucrose transporter gene Ss SUT1?T1 were screened,and further annotated to be 15 genes of.Among them,kafirin PSKR2-like and histone H3.3 are the most common ones,as well as some asparaginases AED3,ru Bis CO large subunit binding protein,alpha coenzyme 7 and so on.The 15 genes of S.spontaneum were compared in the Peking University plant transcription factor database,and 3 transcription factors of other species were compared,indicating that they may be transcription factors that can interact with Ss SUT1?T1 in S.spontaneum.The three putative interaction transcription factors of Ss SUT1?T1 are heat stress transcription factor(HSF),Trihelix transcription factor(GT transcription factor)and CO-like transcription factor.Further analysis of the expressional patterns of these 15 genes revealed that most genes including the putative Trihelix transcription factor and the decoy gene Ss SUT1?T1 had opposite expression trends in leaf segments,and only HSF transcription factors had similar expression trends.In addition,the analysis of their circadian rhythm transcriptome showed that most genes such as HSF,Trihelix,and CO-like transcription factors had a significant opposite trend to the bait gene Ss SUT1?T1 in circadian rhythm.These results indicates that these transcription factors may be related to light and negatively regulate Ss SUT1?T1,and the trend of HSF transcription factors(heat stress transcription factors)on circadian rhythm may be related to temperature.However,Ss SUT1?T1,Trihelix,and CO-like transcription factors have similar protein content in each leaf and stem segment,while HSF transcription factor has no protein content detected.