Centro de Investigação e de Tecnologias Agro-ambientais e Biológicas, CITAB-UMinho Pole, Departamento de Biologia, Escola de Ciências, Universidade do Minho, Braga, Portugal.
i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
Planta. 2017 Dec;246(6):1083-1096. doi: 10.1007/s00425-017-2754-0. Epub 2017 Aug 11.
The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca , Na and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca, demonstrating a role in Ca transport. The truncated VvCAX3 was further shown to be involved in the transport of Na, Li, Mn and Cu in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca, and Na in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.
葡萄蔓 VvCAX3 介导液泡中的钙转运，主要在绿色葡萄浆果中表达，并受 Ca 、 Na 和茉莉酸甲酯上调。钙是一种重要的植物营养物质，在葡萄（Vitis vinifera L.）浆果中具有重要的调节和结构作用。另一方面，质子-阳离子交换器 CAX 蛋白已被证明会影响 Ca 稳态，对果实完整性和对生物/非生物胁迫的抗性有重要影响。在这里，在转录组数据库中发现的 CAX 基因作为葡萄组织中表达最高的基因之一，VvCAX3 被克隆并进行了功能表征。在酵母中的异源表达表明，缺乏其 NNR 自动抑制域的 VvCAX3 截断版本（sCAX3）恢复了酵母菌株在 100-200 mM Ca 中生长的能力，证明了其在 Ca 转运中的作用。进一步表明，截断的 VvCAX3 还参与酵母细胞中 Na 、 Li 、 Mn 和 Cu 的转运。使用荧光标记蛋白进行的亚细胞定位研究证实 VvCAX3 是液泡膜转运蛋白。VvCAX3 在葡萄茎、叶、根和浆果中表达，尤其是在豌豆大小的阶段，在整个发育过程中逐渐减少，与果实中钙积累的模式平行。在葡萄细胞悬浮液中，VvCAX3 的转录丰度被证明受茉莉酸甲酯（MeJA）、Ca 和 Na 调节，并且 VvCAX3 启动子包含几个与发育和应激反应过程相关的预测顺式作用元件。总的来说，这些结果为葡萄钙稳态和细胞内区室化的机制提供了新的见解，并表明 VvCAX3 可能是开发增强葡萄浆果特性的策略的一个有趣目标。
