Impact of vacuolar monosaccharide accumulation on cellular stress response of Arabidopsis. Function of AtTMT type carriers

糖在液泡中的积累对植物的抗寒性，特别是对冷胁迫和渗透胁迫的耐受性具有重要意义。我们发现了第一个液泡糖转运蛋白（TMT， tono质体单糖转运蛋白），并在最后的资助期内证明了TMT作为质子偶联的液泡糖反转运蛋白。此外，我们能够揭示TMT活性对细胞内糖区隔至关重要，参与细胞糖感知并控制拟南芥的种子产量。此外，研究人员还发现了一种新的TMT活性调节模式，该模式由一种预测的丝裂原激活的三激酶（MAP3K，以前称为VIK，一种能够结合在血管发育中起作用的VH1/BRL2受体样激酶的蛋白激酶）控制。VIK属于拟南芥中预测的MAP3K大家族的一个小亚枝（6个成员）。值得注意的是，VIK敲除了缺乏液泡TMT活性的拟南芥表型突变体，VIK磷酸化了TMT1环蛋白，它与真正的TMT1蛋白相互作用，刺激葡萄糖进入分离的叶肉液泡。在计划的资助期内，我们将破译MAP3K亚分支的其他5个成员的功能，我们将识别VIK的上游和下游伙伴蛋白，我们将寻找与TMT1相互作用的其他蛋白，我们将研究TMT1突变体缺乏所提出的磷酸化位点或模仿组成磷酸化的特性。此外，位于TMT1中央环的独特天冬氨酸簇可能有助于TMT1的亚细胞靶向。因此，详细分析天冬氨酸簇的靶向功能也是我们项目的一部分。与过去一样，所有未来的突变体都将在非生物应激刺激引起的分子、代谢和生理变化方面进行分析。

Sugar accumulation in vacuoles is of high importance for plant stress tolerance, especially in respect to cold or osmotic stress stimuli. We identified a first vacuolar sugar transporter (TMT, tonoplast monosaccharide transporter) and showed in the last funding period that TMT acts as a proton-coupled vacuolar sugar antiporter. Moreover, we were able to reveal that TMT activity is critical for intracellular sugar compartmentation, is involved in cellular sugar sensing and governs seed yield in Arabidopsis. In addition, a novel mode of TMT activity regulation controlled by a predicted mitogen-activated triple kinase (MAP3K, previously named VIK, a protein kinase able to bind a VH1/BRL2 receptor-like kinase functioning in vascular development) was identified. VIK belongs to a small subclade (6 members) of the large family of predicted MAP3K in Arabidopsis. Remarkably, VIK knock out lines closely phenocopy Arabidopsis mutants lacking vacuolar TMT activity, VIK phosphorylates the TMT1 loop protein, it interacts with the authentic TMT1 protein and stimulates glucose import into isolated mesophyll vacuoles. In the proposed funding period we will decipher the function of the other 5 members of the corresponding MAP3K subclade, we will identify upstream- and further down-stream partner proteins of VIK, we will search for further proteins interacting with TMT1, and we will study the properties of TMT1 mutants either lacking proposed phosphorylation sites, or mimicking constitutive phosphorylation. Moreover, it appears likely that the unique aspartate cluster located in the central TMT1 loop contributes to the subcellular targeting of TMT1. Thus, detailed analysis of the targeting function of the aspartate cluster is also part of our project. As in the past, all future mutants will be analysed in respect to molecular-, metabolic- and physiological changes induced by abiotic stress stimuli.