Kuang Liuqing, Chen Shangwu, Guo Yan, Ma Huiqin
Department of Fruit Tree Sciences, College of Horticulture, China Agricultural University, Beijing, China.
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Front Plant Sci. 2019 May 15;10:641. doi: 10.3389/fpls.2019.00641. eCollection 2019.
The vacuole plays a central role in fruit growth and quality formation, yet its proteomic landscape is largely unknown. In the present study, a protocol for isolating intact vacuoles from grape flesh tissue was successfully established. Quantitative proteome analysis identified 2533 proteins from five sampling dates along Cabernet Sauvignon berry development from stage I to III; among them, 1443 proteins were identified on all five sampling dates in at least two biological replicates per sample and were designated core proteome, and 1820 were recruited as differentially abundant proteins (DAPs) by sequential pairwise comparisons using arbitrary fold change of >1.5 and < 0.05. Metabolism consistently constituted the largest category of identified proteins for both core proteome and DAPs, together with a consistently high proportion of protein-fate category proteins, indicating that the classic lytic functions of vegetative cell vacuoles are maintained throughout berry development; accumulation of metabolites involved in high sugar and other berry qualities in the late developmental stage added to the conventional lytic role of the flesh cell vacuoles. Overall increases in abundance of the DAPs were seen in the transporter proteins, membrane fusion/vesicle trafficking, and protein-fate categories, and decreased abundance was seen for DAPs in the stress, energy and cytoskeleton categories as berry development progressed. A very pronounced proteomic change was revealed between late stage I and mid stage II, with 915 increased and 114 decreased DAPs, demonstrating a significant surge of the vacuolar proteome underlying the rather static phenotypical and physiological phase. We identified 161 transport proteins with differential abundance, including proton pumps, aquaporins, sugar transporters, ATP-binding cassette transporters and ion transport proteins, together with organic compound transport proteins, the highest number and variety of berry tonoplast transporters found in grape proteome efforts to date. We further found a pre-positive increment of 96 transport proteins from the middle of stage II, before the berry undergoes its dramatic physiological changes at and following véraison. Our results are the first to describe the proteome of a vacuole-enriched preparation, toward understanding the functions of the largest compartment in berry cells during grape growth and ripening.
液泡在果实生长和品质形成中起着核心作用，但其蛋白质组学概况在很大程度上尚不清楚。在本研究中，成功建立了一种从葡萄果肉组织中分离完整液泡的方法。定量蛋白质组分析从赤霞珠葡萄浆果发育的I期到III期的五个采样日期中鉴定出2533种蛋白质；其中，1443种蛋白质在所有五个采样日期的每个样品至少两个生物学重复中被鉴定出来，并被指定为核心蛋白质组，通过使用大于1.5和小于0.05的任意倍数变化进行顺序成对比较，有1820种蛋白质被招募为差异丰度蛋白质（DAPs）。代谢始终是核心蛋白质组和DAPs中鉴定出的最大蛋白质类别，同时蛋白质命运类别蛋白质的比例一直很高，这表明营养细胞液泡的经典裂解功能在整个浆果发育过程中得以维持；发育后期参与高糖和其他浆果品质的代谢物积累增加了果肉细胞液泡的传统裂解作用。随着浆果发育的进行，DAPs在转运蛋白、膜融合/囊泡运输和蛋白质命运类别中的丰度总体增加，而在应激、能量和细胞骨架类别中的DAPs丰度降低。在I期后期和II期中期之间揭示了非常明显的蛋白质组变化，有915种DAPs增加，114种减少，这表明在相当静态的表型和生理阶段下，液泡蛋白质组有显著增加。我们鉴定出161种丰度有差异的转运蛋白，包括质子泵、水通道蛋白、糖转运蛋白、ATP结合盒转运蛋白和离子转运蛋白，以及有机化合物转运蛋白，这是迄今为止在葡萄蛋白质组研究中发现的浆果液泡膜转运蛋白数量最多、种类最丰富的一次。我们进一步发现，在浆果在转色期及之后经历剧烈生理变化之前，从II期中期开始有96种转运蛋白出现预阳性增加。我们的研究结果首次描述了富含液泡制剂的蛋白质组，有助于了解葡萄生长和成熟过程中浆果细胞中最大区室的功能。
