阿拉伯芥蔗糖轉化酶CINV1磷酸化與根部生長發育的功能性研究

Abstract

蔗糖是植物養分運輸的主要形式，而蔗糖轉化酶可以催化蔗糖水解成葡萄糖與果糖，提供細胞能量使用或形成訊號分子。在細胞質中的蔗糖轉化酶在非酸性環境有較高的活性，因此又被稱為中鹼性蔗糖轉化酶。在阿拉伯芥中共有九種中鹼性蔗糖轉化酶，其中CINV1與CINV2為最主要的中鹼性蔗糖轉化酶。過去研究發現這類蔗糖轉化酶參與植物根部生長發育、滲透壓逆境調節、開花及細胞壁生合成等途徑。除此之外，CINV1胺基酸序列第547的位置被認為是能與14-3-3蛋白交互作用的磷酸化位點。本研究發現在鹽分處理後，磷酸化蛋白質體分析發現CINV1具第547胺基酸位點的磷酸化胜肽片段比例上升。本研究欲探究CINV1是否影響植物生長發育或逆境反應。在鹽分處理後，CINV1的突變株其主根具有較大的生長偏角。而於鹽分處理情況下外加醣類 (葡萄糖、果糖、海藻糖)，其主根生長偏角則與野生型類似。另外，觀察互補株在鹽分處理後的主根生長偏角，發現性狀可以被回復，與野生型類似。此外，將Ser547點突變之後，蛋白質在細胞中的表現位置會改變。最後，利用雙分子螢光互補系統驗證CINV1可以和translationally controlled tumor protein (TCTP)有交互作用，且外加target of rapamycin (TOR)途徑的抑制劑之後，CINV1突變株主根長的縮短幅度較野生型來的小，且側根密度增加，暗示CINV1可能參與TOR訊號傳遞路徑。

Sucrose is the major form of nutrient in plant transport. Invertase catalyzes the hydrolysis of sucrose to glucose and fructose, which provides cell energy or as a signaling molecule. Invertases in the cytoplasm are highly active in a non-acidic environment and are therefore also referred to as alkaline/neutral invertases (A/N-Invs). There are nine alkaline/neutral invertases in Arabidopsis, of which CINV1 and CINV2 are the major ones. Previous studies have found that CINV1 is involved in root growth and development, regulation of osmotic stress, flowering and cell wall synthesis. In addition, Ser547 of CINV1 is considered to be a phosphorylation site that can interact with 14-3-3 proteins. This study found that after salt treatment, the phosphorylated peptides containing Ser547 increased abundance. The present study aims to study whether Ser547 phosphorylation is important for plant growth and stress responses. It was found that under salt stress condition, CINV1 mutants have larger primary root deviation angle from vertical orientation than wild type. The growth angle can be recover by addition of glucose, fructose, or trehalose, but not by sucrose. In complementation lines of CINV1, deviation angle of primary root was recovered. This study also found point mutation of Ser547 led to subcellular localization of CINV1 change. In addition, the bimolecular fluorescence complementation assay showed that CINV1 can interact with translationally controlled tumor protein (TCTP). After adding target of rapamycin (TOR) inhibitor, primary root length of CINV1 mutant was less affect than wild type, and the lateral root density was increased, which suggests that CINV1 is involved in TOR signaling pathway.