Studies of vacuolar transporters controlling nutrient storage and ion detoxification in Arabidopsis thaliana

液泡和TGN/EE（反式高尔基网络/早期内体）是细胞内的隔室，通过分隔不同的细胞成分，对营养物质积累和胞质离子解毒至关重要。这些隔室的多种作用与存在于其膜中的转运蛋白的活性密切相关。假设转运体利用质子泵如v型H+-腺苷三磷酸酶（V-ATPase）产生的电化学梯度的能量。对V-ATPase的研究表明了其作用的特异性：vha-a2 vha-a3敲除拟南芥液泡V-ATPase活性的突变体特异性地降低了硝酸盐的液泡储存，但不降低氯化物的固存，而vha-a1敲除突变体对NaCl盐胁迫的敏感性增加，该突变体在TGN/EE处降低了V-ATPase活性。本项目旨在通过分析拟南芥中V-ATPase与CLC阴离子转运体之间的相互作用，了解这一功能规范的分子基础。后一种蛋白质家族具有有趣的特征：(1)它们可以是通道（被动转运体）或阴离子/质子交换体（主动转运体），尽管所有成员在结构上非常接近；(2)它们对氯化物或硝酸盐的选择性更高，这取决于它们的选择性过滤器中丝氨酸/脯氨酸的存在；(3)成员既存在于TGN/EE膜上，也存在于液泡膜上。该项目将分为三个任务：任务1将调查液泡和TGN/EE中atclc的特异性/冗余性。另外两个任务将分析V-ATPase和atclc在液泡（任务2）和TGN/EE膜（任务3）中的共同调控。该项目将利用拟南芥初级和次级运输领域两个专家组的专门知识。法国合作伙伴将提供分子生理学和电生理学方面的知识，德国合作伙伴的细胞生物学和组学方法将予以补充。两个实验室之间的合作将在考虑不同环境波动（硝酸盐浓度、干旱和NaCl胁迫）的情况下，在分子、细胞和整个植物水平上深入分析V-ATPase和AtCLC之间的相互作用。总的来说，该项目将对叶绿体和TGN/EE的初级和次级运输过程的协调获得新的见解，并具有开发未来策略的潜力，以改善作物的营养同化和对非生物胁迫的耐受性。

The vacuole and TGN/EE (trans-Golgi network/early endosome) are intracellular compartments essential for nutrient accumulation and cytosolic ion detoxification by compartmentalizing the different cellular components. The multiple roles of these compartments are tightly linked to the activities of transporters present in their membranes. It is assumed that transporters use the energy of the electrochemical gradient generated by proton pumps such as the V-type H+-Adenosintriphosphatases (V-ATPase). The studies on V-ATPase suggest a specificity of action: a vha-a2 vha-a3 knock-out Arabidopsis mutant affected in vacuolar V-ATPase activity specifically reduces the vacuolar storage of nitrate but not the sequestration of chloride whereas the sensitivity to NaCl salt stress increases in a knock-down mutant of vha-a1, a mutant with reduced V-ATPase activity at the TGN/EE. The project aims at understanding the molecular basis of this functional specification through the analysis of interaction between V-ATPase and CLC anion transporters in Arabidopsis. This latter protein family is characterized by interesting features: (1) they can be channel (passive transporter) or anion/proton exchangers (active transporter) although all the members are structurally very close, (2) they can be either more selective for chloride or nitrate depending of a presence of a serine/proline in their selectivity filter and, (3) members are present on both TGN/EE and vacuolar membranes. The project will be divided in three Tasks: Task 1 will investigate the specificities/redundancies of AtCLCs localised in the vacuole and the TGN/EE. The two other Tasks will analyze the co-regulation between V-ATPase and AtCLCs in the vacuolar (Task 2) and TGN/EE membrane (Task 3). The project will take advantage of the expertise of two expert groups in the field of primary and secondary transport in Arabidopsis thaliana. The French partner will provide knowledge in molecular physiology and electrophysiology which will be complemented by cell biological and omics approaches of the German partner. The cooperation between the two laboratories will allow an in-depth analyses of the interaction between V-ATPase and AtCLC at the molecular, cellular and whole-plant level, taking different environmental fluctuations (nitrate concentrations, drought and NaCl stress) into account. Overall, this project will gain new insights into the coordination of primary and secondary transport processes at the tonoplast and TGN/EE and holds the potential for developing future strategies to improve crops for nutrient assimilation and tolerance to abiotic stress.