Vacuolar Phosphate Transporter1 (VPT1) may transport sugar in response to soluble sugar status of grape fruits

Abstract

Vacuolar Phosphate Transporter1 (VPT1)-mediated phosphate uptake in the vacuoles is essential to plant development and fruit ripening. Interestingly, here we find that the VPT1 may transport sugar in response to soluble sugar status of fruits. The VvVPT1 protein isolated from grape (Vitis vinifera) berries was tonoplast-localized and contains SPX (Syg1/Pho81/XPR1) and MFS (major facilitator superfamily) domains. Its mRNA expression was significantly increased during fruit ripening and induced by sucrose. Functional analyses based on transient transgenic systems in grape berry showed that VvVPT1 positively regulated berry ripening and significantly affected hexose contents, fruit firmness, and ripening-related gene expression. The VPT1 proteins (Grape VvVPT1, strawberry FaVPT1, and Arabidopsis AtVPT1) all showed low affinity for phosphate verified in yeast system, while they appear different in sugar transport capacity, consistent with fruit sugar status. Thus, our findings reveal a role for VPT1 in fruit ripening, associated to its SPX and MFS domains in direct transport of soluble sugar available into the vacuole, and open potential avenues for genetic improvement in fleshy fruit.

Acknowledgements

We thank Prof. Zhenxian Zhang, Lailiang Cheng and Dong Meng for providing the yeast stain SUSY7/ura3; Xuexian Li for providing the yeast stain EBY.VW4000; Yifang Chen and Erin K. O’Shea for providing the yeast stain YP101. This work was supported by the National Natural Science Foundation of China (Projects 32030100; 32102362), Natural Science Foundation of Beijing (6222004), National Key Research and Development Program (2018YFD1000200), Science and Technology Innovation Support Program (BUA-HHXD2022005), and Research and Innovation Ability Improvement Program for Young Teachers of Beijing University of Agriculture.

Author contributions

Y.H. and Y.S. conceived the study and managed the projects; Q.B., X.C., and Z.Z. performed the experiments. Q.B., X.C., Z.Z., and Y.H. performed data analysis. Q.B., X.C., Z.Z., Y.H., and Y.S. wrote and revised the article.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Conflict of interests

The authors declare that no competing interests exist.

References

[1] Wang Z, Kuo HF, Chiou TJ . Intracellular phosphate sensing and regulation of phosphate transport systems in plants. Plant Physiol. 2021; 187: 2043-55.

[2] Huang Y, Xu PH, Hou BZ, Shen YY . Strawberry tonoplast transporter, FaVPT1, controls phosphate accumulation and fruit quality. Plant Cell Environ. 2019; 42: 2715-29.

[3] Liu JH, Yang L, Luan M et al. A vacuolar phosphate transporter essential for phosphate homeostasis in Arabidopsis. Proc Natl Acad Sci U S A. 2015; 112: E6571-8.

[4] Liu TY, Huang TK, Yang SY et al. Identification of plant vacuolar transporters mediating phosphate storage. Nat Commun. 2016; 7: 1-11.

[5] Xu L, Zhao H, Wan R et al. Identification of vacuolar phosphate efflux transporters in land plants. Nat Plants. 2019; 5: 84-94.

[6] Baena-González E, Lunn JE . SnRK1 and trehalose 6-phosphate - two ancient pathways converge to regulate plant metabolism and growth. Curr Opin Plant Biol 2020; 55: 52-9.

[7] Li YM, Forney C, Bondada B et al. The molecular regulation of carbon sink strength in grapevine (Vitis vinifera L.). Front Plant Sci. 2021; 11: 606918.

[8] Kuang L, Chen S, Guo Y, Ma H . Quantitative proteome analysis reveals changes in the protein landscape during grape berry development with a focus on vacuolar transport proteins. Front Plant Sci. 2019; 10: 641.

[9] Niño-González M, Novo-Uzal E, Richardson DN et al. More transporters, more substrates: the Arabidopsis Major facilitator superfamily revisited. Mol Plant. 2019; 12: 1182-202.

[10] Miyaji T, Kuromori T, Takeuchi Y et al. AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis. Nat Commun. 2015; 6: 5928.

[11] Yan N. Structural biology of the major facilitator superfamily transporters. Annu Rev Biophys. 2015; 44: 257-83.

[12] Yang B, Wang J, Yu M et al. The sugar transporter ZmSUGCAR1 of the nitrate transporter 1/peptide transporter family is critical for maize grain filling. Plant Cell. 2022; 34: 4232-54.

[13] Grierson C, Du JS, de Torres Zabala M et al. Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene . Plant J. 1994; 5: 815-26.

[14] Zheng X, Li Q, Liu D et al. Promoter analysis of the sweet potato ADP-glucose pyrophosphorylase gene IbAGP1 in Nicotiana tabacum. Plant Cell Rep. 2015; 34: 1873-84.

[15] Gao Z, Li Q, Li J et al. Characterization of the ABA receptor VlPYL1 that regulates anthocyanin accumulation in grape berry skin. Front Plant Sci. 2018; 9: 592.

[16] Wykoff DD, O’Shea EK . Phosphate transport and sensing in Saccharomyces cerevisiae. Genetics. 2001; 159: 1491-9.

[17] Wieczorke R, Krampe S, Weierstall T et al. Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett. 1999; 464: 123-8.

[18] Etxeberria E, González P, Tomlinson P, Pozueta-Romero J . Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells. J Exp Bot. 2005; 56: 1905-12.

[19] Yamada K, Saito M, Matsuoka H, Inagaki N . A real-time method of imaging glucose uptake in single, living mammalian cells. Nat Protoc. 2007; 2: 753-62.

[20] Chen LQ, Hou BH, Lalonde S et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature. 2010; 468: 527-32.

[21] Riesmeier JW, Willmitzer L, Frommer WB . Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast. EMBO J. 1992; 11: 4705-13.

[22] Kühn C, Frommer WB . A novel zinc finger protein encoded by a couch potato homologue from Solanum tuberosum enables a sucrose transport-deficient yeast strain to grow on sucrose. Mol Gen Genet. 1995; 247: 759-63.

[23] Gora PJ, Reinders A, Ward JM . A novel fluorescent assay for sucrose transporters. Plant Methods. 2012; 8: 1-6.

[24] Li C, Meng D, Piñeros MA et al. A sugar transporter takes up both hexose and sucrose for sorbitol-modulated in vitro pollen tube growth in apple. Plant Cell. 2020; 32: 449-69.

[25] Wild R, Gerasimaite R, Jung JY et al. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science. 2016; 352: 986-90.

[26] de Freitas Laiber Pascoal G, de Almeida Sousa Cruz MA, de Abreu J et al. Evaluation of the antioxidant capacity, volatile composition and phenolic content of hybrid Vitis vinifera L. varieties sweet sapphire and sweet surprise . Food Chem. 2022; 366: 130644.

[27] Bihmidine S, Hunter CT, Johns CE et al. Regulation of assimilate import into sink organs: update on molecular drivers of sink strength. Front Plant Sci. 2013; 4: 177.

[28] Beauvoit B, Colombié S, Monier A et al. Model-assisted analysis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion. Plant Cell. 2014; 26: 3224-42.

[29] Chen HY, Huh JH, Yu YC et al. The Arabidopsis vacuolar sugar transporter SWEET2 limits carbon sequestration from roots and restricts Pythium infection. Plant J. 2015; 83: 1046-58.

[30] Conde C, Agasse A, Glissant D et al. Pathways of glucose regulation of monosaccharide transport in grape cells. Plant Physiol. 2006; 141: 1563-77.

[31] Ren C, Liu Y, Guo Y et al. Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters. Hortic Res. 2021; 8: 52.

[32] Cai Y, Yin L, Tu W et al. Ectopic expression of VvSUC27 induces Stenospermocarpy and sugar accumulation in tomato fruits. Front Plant Sci. 2021; 12: 759047.

[33] Li CL, Fang KF, Lei H et al. Phloem unloading follows an extensive apoplastic pathway in developing strawberry fruit fruit. J Hortic Sci Biotechnol 2012; 87: 470-7.

[34] Jia H, Wang Y, Sun M et al. Sucrose functions as a signal involved in the regulation of strawberry fruit development and ripening. New Phytol. 2013; 198: 453-65.

[35] Gambetta GA, Matthews MA, Shaghasi TH et al. Sugar and abscisic acid signaling orthologs are activated at the onset of ripening in grape. Planta. 2010; 232: 219-34.

[36] Seymour GB, Østergaard L, Chapman NH et al. Fruit development and ripening. Annu Rev Plant Biol. 2013; 64: 219-41.

[37] Shiraishi M, Fujishima H, Chijiwa H . Evaluation of table grape genetic resources for sugar, organic acid, and amino acid composition of berries. Euphytica 2010; 174: 1-13.

[38] Yan N . Structural advances for the major facilitator superfamily (MFS) transporters. Trends Biochem Sci. 2013; 38: 151-9.

[39] Coombe BG . Adoption of a system for identifying grapevine growth stages. Aus J Grape Wine Res. 1995; 1: 104-10.

[40] Zha Q, Xi XJ, Jiang AL et al. Identification of the appropriate reference gene through using a real-time quantitative PCR in grapes. J Fruit Sci. 2016; 33: 268-74.

[41] Manning K, Davies C, Bowen HC, White PJ . Functional characterization of two ripening-related sucrose transporters from grape berries. Ann Bot. 2001; 87: 125-9.

[42] Ishimaru M, Kobayashi S . Expression of a xyloglucan endotransglycosylase gene is closely related to grape berry softening. Plant Sci. 2002; 162: 621-8.

[43] Dal Santo S, Vannozzi A, Tornielli GB et al. Genome-wide analysis of the expansin gene superfamily reveals grapevine-specific structural and functional characteristics. PLoS One. 2013; 8: e62206.

[44] Zhu X, Wang M, Li X et al. Genome-wide analysis of the sucrose synthase gene family in grape (Vitis vinifera): structure, evolution, and expression profiles . Genes. 2017; 8: 111.

[45] Sun T, Xu L, Sun H et al. VvVHP1;2 is transcriptionally activated by VvMYBA1 and promotes anthocyanin accumulation of grape berry skins via glucose signal . Front Plant Sci 2017; 8: 1811.

[46] Shahood R, Torregrosa L, Savoi S, Romieu C . First quantitative assessment of growth, sugar accumulation and malate breakdown in a single ripening berry. OENO One. 2020; 54: 1077-92.

[47] Didion T, Regenberg B, Jorgensen MU et al. The permease homologue Ssy1p controls the expression of amino acid and peptide transporter genes in Saccharomyces cerevisiae. Mol Microbiol. 1998; 27: 643-50.

[48] Popova Y, Thayumanavan P, Lonati E et al. Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor. Proc Natl Acad Sci U S A. 2010; 107: 2890-5.

[49] Ma QJ, Sun MH, Liu YJ et al. Molecular cloning and functional characterization of the apple sucrose transporter gene MdSUT2. Plant Physiol Biochem. 2016; 109: 442-51.

[50] Ren Y, Sun H, Zong M et al. Localization shift of a sugar transporter contributes to phloem unloading in sweet watermelons. New Phytol 2020; 227: 1858-71.

[51] Hou BZ, Xu C, Shen YY . A leu-rich repeat receptor-like protein kinase, FaRIPK1, interacts with the ABA receptor, FaABAR, to regulate fruit ripening in strawberry. J Exp Bot. 2018; 69: 1569-82.