The Evolution And Function Of SWEET Genes In Saccharum

Sugarcane(Saccharum spp.)is the main source of sugar for food and industry,supplying important raw materials for ethanol production in replacement of petroleum.The concentration of sugars contributes mostly to the sugarcane quality,whereas the sugar transporters play key roles for accumulating sugars in sugarcane stem.Understanding the function of sugar transporters in sugarcane is necessary to explore the mechanism of sugar accumulation and the molecular improvement of sugarcane quality.The SWEETs(Sugars Will Eventually be Exported Transporters),a unique new family of sugar transporters,are monosaccharide and disaccharide transporters that are present from Archaea to plants and human.SWEETs play crucial roles in many cellular sugar efflux processes:such as phloem loading,seed filling,pollen nutrition,nectar secretion,and plant-bacterium interactions.In this study,we identified the SWEET genes by comparative genomics and analyzed the gene expresison patterns through transcriptome sequencing,and their functions were explored by transformation in yeast.The main finding were as follows:1.Based on the comparative genomics analysis,the sorghum SWEET genes were employed as queries to search the Saccharum spontaneum BAC library,leading to the identification of 22 S.spontaneum SWEET genes.Conserved domain searching showed that all 22 SsSWEETs harbored two MtN3/saliva domains.The similarities between those protein sequences ranged from 27?95%with an average of 31%.Nonsynonymous to synonymous substitution ratio(Ka/Ks)was analyzed to investigate evolutionary forces acting on orthologous SWEET genes between sorghum and Saccharum.The Ka/Ks ratios of SWEET gene pairs were all under 1,indicating that the SWEET genes were under purifing selection.2.195 SWEET genes from representative monocots(Ananascomosus,Oryzasativa,Zea mays,Sorghum bicolor,and Saccharum spontaneum),eudicots(Vitis vinifera,Arabidopsis thaliana,Populus trichocarpa,and Medicago truncatula),Amborella trichopoda(basal angiosperms),and Chlamydomonas reinhardtii were used to construct a phylogenetic tree.Phylogenetic analysis showed that the SWEET gene family was an evolutionally conserved group of genes existing before the divergence of angiosperm.There were only 8 SWEET genesin the early diverging Angiosperm,Amborella trichopoda,whereas the number of SWEETs in eudicots and monocots varied from 15 to 25,indicating that the SWEET gne family had expanded in both monocot and eudicot lineages.The phylogenetic tree could be divied into 10 eudicot subfamilies and 16 monocot subfamilies.All the eudicot subfamilies except El 1-14 had SWEET genes from the four examined eudicot species,and 5 out of 16 the moncot subfamilies were composed of SWEET genes from all five examined monocot species.The results indicated the progenitor genesin those subfamily may already exist in both ancestors of eudicot and monocot,respectively,and that the evolution of SWEET genes familys howed more diversities in monocotyledon lines than that in eudicotyledon.8 monocotyledonous subfamilies contained SWEET genes from the other monocots excluding A.comosus,demonstrating that the these subfamilies were originated after the rise of Poales from commelinids and before the pan-grass p WGD event.Gene structure analysis revealed that the angiosperm ancestor SWEET genes may have 5 or 6 exons.The comparative analysisof gene structure for SsSWEETs revealed that the variation of SWEET gene structure might be mainly due to the change of intron numbers.3.RNA-seq data showed that SWEETlb and SWEET2b were highly expressedin source tissues(leaves)in both seedling and mature plants of three Saccharum species,while the expression of SWEET4a and SWEET4b was higher in stem(sink)than that in leaves,suggesting that the former two genes may be responsible for the phloem loading of sucrose in parenchyma cells of leaves,and that the latter two genes may play a role in the unloading processin sink tissues.SWEET13a,13b and 13c exhibited predominant expression in seedling leaves and stems of mature plants of all materials,indicating that those three genes may not only be involved in the development of leaves but also affect the quality of sugarcane.In addition,the expression patterns of SWEET genes in continuous gradient leaf segments of the developmental S.spontaneum SES208 showed that SWEET4a,SWEET4b and SWEET11a were expressed at the high levels near the base of the leaf,indicating the involvement of those genes in basic celluar activities such as DNA synthesis,cell wall synthasis and hormone signaling.SWEET16b and SWEET1a exhibited peak expression in region of transition from sink to source tissues,suggesting their association with establishing the photosynthetic machinery.The expresson of SWEET13a was dominant in the tip of the leaf,which led us to speculate that SWEET13a may be the main transporter for the transport of sugar produced by photosynthasis reactions in mesophyll cells.4.Yeast growth complementary experiments showed that SsSWEET1b,SsSWEET4a,SoSWEET4a,and SoSWEET4b could transport glucose,fructose,and sucrose.But SoSWEET13a and SsSWEET11a showed no transport activity for glucose,fructose and sucrose.5.Homozygous T-DNA insert mutants of AtSWEET1,AtSWEET2,AtSWEET8,AtSWEET11,AtSWEET11,AtSWEET12 and AtSWEET13 were identified.AtSWEET8 mutant lines exhibited short silique,the phenotype of other mutants showed no difference with Col-0 Arabidopsis,suggesting that the SWEET genes family may exist functional redundancy.