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| Vacuolar metabolomic and proteomic profiling reveals the vacuole composition of ripe juice sacs and functions of CsTST2 and CsERDL6 in sugar accumulation in citrus |
| Youfu Fan1, Wenxin Shangguan1, Rong Hu1, Yong Liu1, Li Yang1, Wei Hu1, Jie Song1, Jingheng Xie1, Yingjie Huang1, Mingjun Li2, Dechun Liu1#, Liuqing Kuang1# |
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
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Highlights
● An efficient protocol was established to isolate intact vacuoles from ripe juice sacs of four citrus varieties.
● Vacuole multi-omics analysis identified 640 metabolites and 1,782 proteins, and pummelo MJY showed unique sucrose accumulation and tonoplast transporter profiles.
● Overexpression of tonoplast-localized CsTST2 and CsERDL6 enhanced sugar content in tomato fruits.
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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 fresh
ripe citrus juice sacs of four citrus cultivars: NFMJ, Nanfeng tangerine (Citrus reticulata ‘97-2’); WZMG, Wenzhou satsuma (C. unshiu ‘Miyagawa’); NHER,
‘Newhall’ navel orange (C. sinensis ‘Newhall’); MJY, Majiayou
pomelo (C. grandis ‘Xipi Majia’).
Subsequently, quasi-targeted metabolomics analysis and 4D-label-free
proteomics 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.
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Received: 25 May 2025
Accepted: 05 November 2025
Online: 02 January 2026
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| Fund:
This work was supported by the National Natural Science Foundation of China (32060675), the earmarked fund for Jiangxi Agriculture Research System, China (JXARS-05), the Natural Science Foundation of Jiangxi Province, China (20244BAB28059) and the Education Department of Jiangxi Province, China (GJJ190231).
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| About author: Youfu Fan, E-mail: fyoufu@126.com; #Correspondence Liuqing Kuang, E-mail: kuangliuqing@jxau.edu.cn; Dechun Liu, E-mail: ldc873380800@163.com
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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 the vacuole composition of ripe juice sacs and functions of CsTST2 and CsERDL6 in sugar accumulation in citrus. Journal of Integrative Agriculture, 25(10): 4155-4174.
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