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

液泡中的糖积累对于植物的胁迫耐受性非常重要，特别是对于冷或渗透胁迫刺激。我们确定了第一个液泡糖转运蛋白（TMT，液泡膜单糖转运蛋白），并显示在最后一个资助期间，TMT作为质子偶联液泡糖反向转运蛋白。此外，我们能够揭示，TMT活性是细胞内糖区室化的关键，参与细胞糖传感和控制拟南芥种子产量。此外，TMT活性调节的一种新的模式控制的有丝分裂原激活的三重激酶（MAP 3 K，以前命名为VIK，能够结合VH 1/BRL 2受体样激酶在血管发育中发挥作用的蛋白激酶）被确定。VIK属于拟南芥中预测的MAP 3 K大家族的一个小的亚支（6个成员）。值得注意的是，VIK敲除株系紧密表型复制缺乏液泡TMT活性的拟南芥突变体，VIK磷酸化TMT 1环蛋白，它与真正的TMT 1蛋白相互作用并刺激葡萄糖输入到分离的叶肉液泡中。在拟议的资助期内，我们将破译相应的MAP 3 K亚支的其他5个成员的功能，我们将确定VIK的上游和下游伴侣蛋白，我们将寻找与TMT 1相互作用的其他蛋白，我们将研究TMT 1突变体的特性，无论是缺乏拟议的磷酸化位点，还是模仿组成性磷酸化。此外，它似乎可能是独特的天冬氨酸簇位于中央TMT 1环有助于亚细胞靶向TMT 1。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，将分析所有未来的突变体在分子，代谢和生理变化引起的非生物胁迫刺激。

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.