The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis

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 Ca2+ 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 100200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ 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), Ca2+, 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.The authors thank Professor Kyle W. Cunningham (Department of Biology, Johns Hopkins University, USA) for providing the yeast strain K667. The authors also acknowledge Professor Manuela Côrte-Real and Dr. Nuria Genicio Bouza (Department of Biology, University of Minho, Portugal) for technical support in flow cytometry and critical analysis of the results. This work is supported by European investment funds by FEDER/COMPETE/POCI-Operacional Competitiveness and Internationalization Programme, under the Projects INTERACT-NORTE-01-0145FEDER-000017-Linha VitalityWine-ON 0013 and POCI-01-0145FEDER-006958, and by National Funds by FCT–Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013, and CherryCrackLess (PTDC/AGR-PRO/7028/2014). VM was supported by a FCT postdoctoral Grant (SFRH/BPD/107905/ 2015).