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Vacuolar metabolomic and proteomic profiling reveals vacuole composition of ripe juice sacs and functions of CsTST2 and CsERDL6 for sugar accumulation in citrus

1 Department of Pomology, College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, China

2 State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, College of Horticulture, Northwest A&F University, Yangling 712100, China

 Highlights 

1. An efficient protocol for isolating intact vacuoles from ripe juice sacs across four citrus varieties has been established.

2. Vacuole multi-omics identified 640 metabolites and 1,782 proteins, pummelo MJY shows unique sucrose accumulation and tonoplast transporter profiles.

3. Tonoplast-localized CsTST2 and CsERDL6 overexpression enhance tomato fruit sugar content.

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摘要  

液泡组成,尤其是代谢物与液泡膜蛋白的种类及丰度,对果实品质和风味具有决定性影响。然而,完全成熟时不同风味的果实之间的液泡组成差异,特别是糖分累积方面的具体特征,目前尚不明确。本研究通过优化液泡提取方法,克服了成熟柑橘汁胞液泡分离的技术难点,成功从四种新鲜成熟柑橘汁胞中分离出完整液泡。随后,基于类靶向代谢组学分析和4D-非标记蛋白质组学分析,分别在液泡中鉴定到640种代谢物和1782种蛋白质。液泡代谢组分析表明,氨基酸、黄酮类、脂质、碳水化合物和有机酸共同占液泡总代谢物种类的70%。其中,柚类品种MJY的汁胞液泡中蔗糖含量最高,而宽皮柑橘品种NFMJ的蔗糖含量最低。液泡蛋白质组功能分析表明,液泡蛋白主要参与蛋白质命运调控、代谢过程、囊泡运输、溶质转运及能量供应等生物学过程。品种间比较表明,MJY与NFMJ的液泡蛋白质丰度差异显著大于其他品种间的差异。共鉴定到158种转运蛋白,包括多个糖相关转运蛋白,其中多数在MJY液泡中丰度更高。与其他品种相比,CsTST2和CsERDL6在MJY液泡中的蛋白质丰度更高,其编码基因在果实成熟过程中的表达模式与糖累积变化趋势一致。亚细胞定位试验证实这两种蛋白均定位于液泡膜。在过表达番茄果实中,相应基因的表达水平及果实糖含量均显著提高。本研究系统揭示了不同柑橘品种成熟果实汁胞的液泡组成特性及其糖累积模式,为果实品质的遗传改良提供了重要的液泡膜蛋白靶点与理论依据。



Abstract  

Vacuolar composition, particularly the type and abundance of metabolites and tonoplast proteins, critically determines fruit quality and flavor. However, the specific vacuolar composition of fruits with different flavors at the fully ripe stage, especially regarding sugar accumulation, remains unclear. In this study, we established an optimized protocol to overcome technical barriers in isolating intact vacuoles from four types of fresh ripe citrus juice sacs. Subsequently, quasi-targeted metabolomics analysis and 4D-label-free proteomic analysis were conducted, identifying 640 metabolites and 1,782 proteins, respectively. Notably, amino acids, flavonoids, lipids, carbohydrates, and organic acids collectively represented 70% of the total vacuolar metabolites. Pummelo MJY vacuoles exhibited the highest sucrose accumulation, whereas tangerine NFMJ showed minimal sucrose content. Proteomic profiling revealed vacuolar proteins participating in protein fate determination, metabolism, vesicle trafficking, solute transport, and energy supply. Comparative analysis demonstrated significantly greater protein abundance variation between MJY and NFMJ than between other varieties. In total, 158 transport proteins, including sugar-related transporters, were identified, and most of them were more abundant in MJY vacuoles. The protein abundance of CsTST2 and CsERDL6 was greater in MJY vacuoles compared to other varieties, and the relative expression patterns of their encoding genes were consistent with sugar accumulation during fruit ripening. Subcellular localization analysis confirmed their tonoplast localization. Importantly, transgenic tomato fruits overexpressing these genes demonstrated both enhanced gene expression and increased sugar content. This study systematically revealed cultivar-specific vacuolar composition and sugar accumulation strategies in ripe citrus fruits and provided key tonoplast proteins responsible for fruit quality improvement.

 

Keywords:  vacuole isolation       vacuolar metabolome       vacuolar proteome       tonoplast sugar transporter        CsTST2       CsERDL6       Citrus juice sacs  
Online: 02 January 2026  
Fund: 

This work was supported by the National Natural Science Foundation of China (32060675), the Earmarked Fund for Jiangxi Agriculture Research System (Citrus Simplified and Efficient Cultivation Post, Grant No. JXARS-05), the Natural Science Foundation of Jiangxi Province (20244BAB28059) and the Education Department of Jiangxi Province (GJJ190231).

Cite this article: 

Youfu Fan, Wenxin Shangguan, Rong Hu, Yong Liu, Li Yang, Wei Hu, Jie Song, Jingheng Xie, Yingjie Huang, Mingjun Li, Dechun Liu, Liuqing Kuang. 2026. Vacuolar metabolomic and proteomic profiling reveals vacuole composition of ripe juice sacs and functions of CsTST2 and CsERDL6 for sugar accumulation in citrus. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.12.074

Antony E, Taybi T, Courbot M, Mugford S T, Smith J A, Borland A M. 2008. Cloning, localization and expression analysis of vacuolar sugar transporters in the CAM plant Ananas comosus (pineapple). Journal of Experimental Botany59, 1895–1908.

Breia R, Conde A, Conde C, Fortes A M, Granell A, Gerós H. 2020. VvERD6l13 is a grapevine sucrose transporter highly up-regulated in response to infection by Botrytis cinerea and Erysiphe necatorPlant Physiology and Biochemistry154, 508–516.

Çakir B, Giachino R R. 2012. VvTMT2 encodes a putative tonoplast monosaccharide transporter expressed during grape berry (Vitis vinifera cv. Sultanine) ripening. Plant Omics Journal, 5: 576–583.

Carter C, Pan S, Zouhar J, Avila E L, Girke T, Raikhel N V. 2004. The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell16, 3285–3303.

Cheng J, Wen S, Xiao S, Lu B, Ma M, Bie Z. 2018. Overexpression of the tonoplast sugar transporter CmTST2 in melon fruit increases sugar accumulation. Journal of Experimental Botany69, 511–523.

Endler A, Meyer S, Schelbert S, Schneider T, Weschke W, Peters S W, Keller F, Baginsky S, Martinoia E, Schmidt U G. 2006. Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach. Plant Physiology141, 196–207.

Farré E M, Tiessen A, Roessner U, Geigenberger P, Trethewey R N, Willmitzer L. 2001. Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method. Plant Physiology127, 685–700.

Feng G, Wu J, Xu Y, Lu L, Yi H. 2021. High-spatiotemporal-resolution transcriptomes provide insights into fruit development and ripening in Citrus sinensisPlant Biotechnology Journal19, 1337–1353.

Fontes N, Gerós H, Delrot S. 2010. Grape Berry Vacuole: A Complex and Heterogeneous Membrane System Specialized in the Accumulation of Solutes. American Journal of Enology and Viticulture‌63, 270–278.

Hu B, Huang W, Dong L, Liu S, Zhou Y. 2019. Molecular cloning and functional analysis of a sugar transporter gene (CsTST2) from cucumber (Cucumis sativus L.). Biotechnology & Biotechnological Equipment33, 118–127.

Jaquinod M, Villiers F, Kieffer-Jaquinod S, Hugouvieux V, Bruley C, Garin J, Bourguignon J. 2007. A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture. Molecular & Cellular Proteomics6, 394–412.

Jones P, Binns D, Chang H Y, Fraser M, Li W, McAnulla C, Maslen J, Mitchell A, Nuka G, Pesseat S, Quinn A F, Sangrador-Vegas A, Scheremetjew M, Yong S Y, Lopez R, Hunter S. 2014. InterProScan 5: genome-scale protein function classification. Bioinformatics30, 1236–1240.

Juan J X, Yu X H, Jiang X M, Gao Z, Zhang Y, Li W, Duan Y D, Yang G. 2015. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene. Genetics and Molecular Research14, 597–608.

Jung B, Ludewig F, Schulz A, Meißner G, Wöstefeld N, Flügge UI, Pommerrenig B, Wirsching P, Sauer N, Koch W, Sommer F, Mühlhaus T, Schroda M, Cuin TA, Graus D, Marten I, Hedrich R, Neuhaus HE. 2015. Identification of the transporter responsible for sucrose accumulation in sugar beet taproots. Nature Plants1, 14001.

Khan A, Cheng J, Kitashova A, Fürtauer L, Nägele T, Picco C, Scholz-Starke J, Keller I, Neuhaus H E, Pommerrenig, B. 2023. Vacuolar sugar transporter EARLY RESPONSE TO DEHYDRATION6-LIKE4 affects fructose signaling and plant growth. Plant Physiology193, 2141–2163.

Kim D S, Lee S, Park S M, Yun S H, Gab H S, Kim S S, Kim H J. 2021. Comparative metabolomics analysis of citrus varieties. Foods10, 2826.

Klemens P A W, Patzke K, Trentmann O, Poschet G, Büttner M, Schulz A, Marten I, Hedrich R, Neuhaus H E. 2014. Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination. The New Phytologist202, 188–197.

Ku Y S. 2023. How sweet! Transcription factor CitZAT5 regulates CitSUS5 and CitSWEET6 to promote sugar accumulation in citrus fruit. Plant Physiology192, 1669–1670.

Kuang L, Chen S, Guo Y, Ma H. 2019. Quantitative proteome analysis reveals changes in the protein landscape during grape berry development with a focus on vacuolar transport proteins. Frontiers in Plant Science10, 641.

Kuang L, Chen S, Guo Y, Scheuring D, Flaishman M A, Ma H. 2022. Proteome analysis of vacuoles isolated from fig (Ficus carica L.) flesh during fruit development. Plant & Cell Physiology63, 785–801.

Lado J, Gambetta G, Zacarias L. 2018. Key determinants of citrus fruit quality: Metabolites and main changes during maturation. Scientia Horticulturae233, 238–248.

Li J M, Zheng D M, Li L T, Qiao X, Wei S W, Bai B, Zhang S L, Wu J. 2015. Genome-wide function, evolutionary characterization and expression analysis of sugar transporter family genes in pear (Pyrus bretschneideri Rehd). Plant & Cell Physiology56, 1721–1737.

Li M, Mao Z, Zhao Z, Gao S, Luo Y, Liu Z, Sheng, X, Zhai X, Liu J H, Li C 2024. CBL1/CIPK23 phosphorylates tonoplast sugar transporter TST2 to enhance sugar accumulation in sweet orange (Citrus sinensis). Journal of Integrative Plant Biology, 67, 327–344.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods25, 402–408.

Lu X, Zhao C, Shi H, Liao Y, Xu F, Du H, Xiao H, Zheng J. 2023. Nutrients and bioactives in citrus fruits: Different citrus varieties, fruit parts, and growth stages. Critical Reviews in Food Science and Nutrition63, 2018–2041.

Mao Z, Wang Y, Li M, Zhang S, Zhao Z, Xu Q, Liu J H, Li C. 2023. Vacuolar proteomic analysis reveals tonoplast transporters for accumulation of citric acid and sugar in citrus fruit. Horticulture Research11, uhad249.

Martinoia E, Meyer S, De Angeli A, Nagy R. 2012. Vacuolar transporters in their physiological context. Annual Review of Plant Biology63, 183–213.

Martinoia E, Mimura T, Hara-Nishimura I, Shiratake K. 2018. The multifaceted roles of plant vacuoles. Plant & Cell Physiology59, 1285–1287.

Moskowitz A H, Hrazdina G. 1981. Vacuolar contents of fruit subepidermal cells from Vitis species. Plant Physiology68, 686–692.

Ohnishi M, Anegawa A, Sugiyama Y, Harada K, Oikawa A, Nakayama Y, Sasaki R, Shichijo C, Hatcher P G, Fukaki H, Kanaya S, Aoki K, Yamazaki M, Fukusaki, E, Saito K, Mimura T. 2018. Molecular components of Arabidopsis intact vacuoles clarified with metabolomic and proteomic analyses. Plant & Cell Physiology59, 1353–1362.

Oikawa A, Matsuda F, Kikuyama M, Mimura T, Saito K. 2011. Metabolomics of a single vacuole reveals metabolic dynamism in an alga Chara australisPlant Physiology157, 544–551.

Poschet G, Hannich B, Raab S, Jungkunz I, Klemens P A, Krueger S, Wic S, Neuhaus H E, Büttner M. 2011. A novel Arabidopsis vacuolar glucose exporter is involved in cellular sugar homeostasis and affects the composition of seed storage compounds. Plant Physiology157, 1664–1676.

Ranganna S, Govindarajan V S, Ramana K V. 1983. Citrus fruits - varieties, chemistry, technology, and quality evaluation. Part II. Chemistry, technology, and quality evaluation. A. Chemistry. Critical Reviews in Food Science and Nutrition18, 313–386.

Ren Y, Guo S, Zhang J, He H, Sun H, Tian S, Gong G, Zhang H, Levi A, Tadmor Y, Xu Y. 2018. A tonoplast sugar transporter underlies a sugar accumulation QTL in watermelon. Plant Physiology176, 836–850.

Reuscher S, Akiyama M, Mori C, Aoki K, Shibata D, Shiratake K. 2013. Genome-wide identification and expression analysis of aquaporins in tomato. PLoS One8, e79052.

Saini R K, Ranjit A, Sharma K, Prasad P, Shang X, Gowda K G M, Keum Y S. 2022. Bioactive compounds of citrus fruits: A review of composition and health benefits of carotenoids, flavonoids, limonoids, and terpenes. Antioxidants11, 239.

Salvino R A, Colella M F, Luca G D. 2021. NMR-based metabolomics analysis of Calabrian citrus fruit juices and its application to industrial process quality control. Food Control121, 107619.

Schmidt U G, Endler A, Schelbert S, Brunner A, Schnell M, Neuhaus H E, Marty-Mazars D, Marty F, Baginsky S, Martinoia E. 2007. Novel tonoplast transporters identified using a proteomic approach with vacuoles isolated from cauliflower buds. Plant Physiology145, 216–229.

Schulz A, Beyhl D, Marten I, Wormit A, Neuhaus E, Poschet G, Büttner M, Schneider S, Sauer N, Hedrich R. 2011. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2. The Plant Journal68, 129–136.

Schulze W X, Schneider T, Starck S, Martinoia E, Trentmann O. 2012. Cold acclimation induces changes in Arabidopsis tonoplast protein abundance and activity and alters phosphorylation of tonoplast monosaccharide transporters. The Plant Journal69, 529–541.

Shiratake K, Martinoia E. 2007. Transporters in fruit vacuoles. Plant Biotechnology24, 127–133.

Shitan N, Yazaki K. 2013. New insights into the transport mechanisms in plant vacuoles. International Review of Cell and Molecular Biology305, 383–433.

Tadeo F R, Cercos M, Colmenero-Flores J M, Iglesias D J, Naranjo M A, Rios G, Carrera E, Ruiz-rivero O, Lliso I, Morillon R, Ollitrault P, Talon M. 2008. Molecular physiology of development and quality of citrus. Advances in Botanical Research47, 147–223.

Timm S, Eisenhut M. 2023. Four plus one: vacuoles serve in photorespiration. Trends in Plant Science28, 1340–1343.

Tohge T, Ramos M S, Nunes-Nesi A, Mutwil M, Giavalisco P, Steinhauser D, Schellenberg M, Willmitzer L, Persson S, Martinoia E, Fernie A R. 2011. Toward the storage metabolome: profiling the barley vacuole. Plant Physiology157, 1469–1482.

Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America76, 4350–4354.

Wang S, Shen S, Wang C, Wang X, Yang C, Zhou S, Zhang R, Zhou Q, Yu H, Guo H, Zheng W, Liu X, Xu J, Deng X, Xu Q, Luo J. 2023. A metabolomics study in citrus provides insight into bioactive phenylpropanoid metabolism. Horticulture Research11, uhad267.

Wen B, Mei Z, Zeng C, Liu S. 2017. metaX: a flexible and comprehensive software for processing metabolomics data. BMC Bioinformatics18, 183.

Wiśniewski J R, Zougman A, Nagaraj N, Mann M. 2009. Universal sample preparation method for proteome analysis. Nature Methods6, 359–362.

Wormit A, Trentmann O, Feifer I, Lohr C, Tjaden J, Meyer S, Schmidt U, Martinoia E, Neuhaus H E. 2006. Molecular identification and physiological characterization of a novel monosaccharide transporter from Arabidopsis involved in vacuolar sugar transport. Plant Cell18, 3476–3490.

Wu G A, Sugimoto C, Kinjo H, Azama C, Mitsube F, Talon M, Gmitter F G J, Rokhsar D S. 2021. Diversification of mandarin citrus by hybrid speciation and apomixis. Nature Communications12, 4377.

Yamaki S. 2010. Metabolism and accumulation of sugars translocated to fruit and their regulation. Journal of the Japanese Society for Horticultural Science79, 1–15.

Yoshida K, Ohnishi M, Fukao Y, Okazaki Y, Fujiwara M, Song C, Nakanishi Y, Saito K, Shimmen T, Suzaki T, Hayashi F, Fukaki H, Maeshima M, Mimura T. 2013. Studies on vacuolar membrane microdomains isolated from Arabidopsis suspension-cultured cells: local distribution of vacuolar membrane proteins. Plant & Cell Physiology54: 1571–1584.

Zhu L, Li B, Wu L, Li H, Wang Z, Wei X, Ma B, Zhang Y, Ma F, Ruan Y L, Li, M. 2021. MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato. Proceedings of the National Academy of Sciences of the United States of America118, e2022788118.

Zhu L, Lan J, Zhao T, Li M, Ruan Y L. 2025. How vacuolar sugar transporters evolve and control cellular sugar homeostasis, organ development and crop yield. Nature plants, 11, 1102–1115.

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