Functional Characterization Of The FvTst1gene From Strawberry And Their Impact On Plant Growth And Development

Plants require sugar throughout their entire life cycle.Sugar is primarily synthesized photosynthetic organs,such as leaves,and is transported to other parts of the plants as disaccharides,such as sucrose,to the sink tissues.The plant could utilize this sugar in the form of sucrose or after conversion to their respective monosaccharaide,glucose and fructose,in sink tissues including roots,pollen,and fruits.Sugar,unsurprisingly,is crucial for the plant’s productivity and diverse agronomic traits,such as playing a starring role in the quality improvement of the fruits.Sugar partitioning at the whole plant level,as well as intraand intercellularly,is critical for plant growth,development,and fruit quality.Different types and abundances of sugars accumulate in different organelles of mature fruit cells,affecting the flavor quality of the fruit directly.The activity of the sugar transporter promotes the allocation of sugar from cellular to the whole plant level.The sugar transporters mediate the allocation of the sugar within or among the source and sink tissues.The modulation of the sugar by sugar transporters can cause significant changes in sugar distribution,plant growth,and stress response.The major facilitator family(MFS)of sugar transporters,which is responsible for diverse sugar partitioning and allocation,is one of the largest families of transporters involved in plant growth and development.However,its key role in the development of the strawberry fruit sugar abundance,as well as the molecular mechanism underlying the impact of endogenous sugar abundance on flavor quality,growth and development remains largely unknown.Many sugar transporters are involved in the transport and distribution of sugar within and between the cells.Among these,tonoplast sugar transporter(TSTs)are antiporter molecules that import sugar from cytosol to vacuole and help plants to respond to abiotic stresses while also promoting growth and development.During this study,we found 13 sugar transporter genes in the Fragaria vesca genome’s v4.0.a2 protein database.The tempo-spatial expression pattern for all identified sugar transporter was analyzed,which revealed that the gene Fv TST1 has a strong association with strawberry fruit development.Because the transcript level of the Fv TST1 increases with the corresponding stages of the fruit development,and show a higher expression level at the mature stage of the fruit.Moreover,phylogenetic tree revealed that the gene Fv TST1 has a close homology with previously known tonoplast sugar transporters.The gene Fv TST1 has a2217-bp open reading frame that encodes a 738-amino-acid high hydrophobic protein with a MW of 78.90 k Da and an isoelectric value of 4.93 p Is.Similarly,the multiple sequence alignment shows that,Fv TST1 shared the most homology and the closest evolutionary relationship with At TST1,Cm TST1,Cs TST1,and Md TST1.The transmembrane analysis reveals that Fv TST1 has a long hydrophobic loop between transmembrane 6 and 7,which has been documented in previously reported tonoplast sugar transporters.These results suggest that Fv TST1 shares functional similarities with other tonoplast sugar transporters,laying the foundation for elucidating the sugar distribution relationship during strawberry fruit growth and development.The subcellular localization of Fv TST1 in vacuolar membrane(tonoplast)was also indicated by expressing Ca MV35S–Fv TST1–GFP a fusion protein containing GFP reporter gene,and was co-expressed with tonoplast and plasma membrane marker,through a transient expression experiment in Nicotiana benthamiana leaves.The florescent microcopy results suggest that Fv TST1 is mainly targeted to tonoplast.Furthermore,the functional activity of the gene Fv TST1 as a sugar transporter has been demonstrated in hexose deficient yeast mutant strain EBY.VW4000.The EBY.VW4000 expressing gene Fv TST1 can grow on the medium containing,fructose,glucose,sucrose and mannose but the mutant yeast did not grow,which confirms the hypothesis that Fv TST1 from strawberry is functional sugar transporter.We also have transiently expressed the Fv TST1 gene in strawberry fruit.Interesting phenotype such as fast ripening was observed in the Fv TST1 overexpressed strawberry fruit as compared to the control.The expression profile was analyzed using q PCR.High transcript level of the Fv TST1 was observed in overexpressed strawberry as compared to the control.Furthermore,sugar content was analyzed using HPLC to observe the sugar abundance differences in Fv TST1 overexpressed strawberry fruits and their corresponding control.Fv TST1 overexpression strawberry fruit exhibited significantly higher sugar content as compared to control,which confirms its close association with strawberry fruit sugar accumulation and development.Furthermore,Fv TST1 was expressed in tomato driven by the fruit specific promoter of tomato proline rich protein(TPRP-F1).Transgenic tomato seedlings expressing the strawberry Fv TST1 driven by the fruit specific promoter of tomato proline rich protein(TPRP-F1),had higher sucrose and auxin levels,as well as improved seed germination,hypocotyls elongation,root growth,and photosynthetic rate compared to non-transgenic control.Furthermore,Fv TST1-transformed tomato plants flowered and bore fruit significantly earlier than wild-type controls,and had significantly higher sucrose,fructose,and glucose content in seedlings and fruit as compared to their control.RNA-sequence analysis in tomato was used to understand more about the function of Fv TST1 during plant growth and development.A total of 1888 differentially expressed genes(DEGs)were discovered.The RNA-sequence study showed that the auxin signaling pathway was the most significantly upregulated GO term among the 1888 differentially expressed genes(DEGs).A total of 45 auxin activation pathway genes were found to be up-regulated,indicating that the auxin signaling pathway is essential for seedling development.Transgenic tomato seedlings showed increased expression of transcriptional factor genes phytochrome-interacting factor1,-3,and-4,as well as their potential target genes brassinosteroid signaling positive regulator1(BZR1),gibberellin 2-beta-dioxygenase2(GA2ox2),tryptophan aminotransferase of Arabidopsis1(TAA1),and indole-3-acetic acid inducible29(IAA29).Taken together our findings shed light on the function and practical usage of the gene Fv TST1 for future breeding purposes.