Finessing, Extending and Developing an Overview of the Regulation of Ascorbate in plants (FEDORA)

抗坏血酸是从真核藻类到被子植物的所有植物的关键代谢物。它是最丰富的水溶性抗氧化剂，在光合作用、跨膜电子传递、细胞分裂、生长以及生物和非生物胁迫的耐受性中发挥重要作用。它通过充当作为光合作用副产物产生的活性氧的主要清除剂，在通过水-水循环耗散过量光子能量中以及作为叶黄素循环中的辅助因子来执行这些不同的角色。它作为双加氧酶的辅因子，所述双加氧酶在植物激素、乙烯、脱落酸和赤霉素的合成中以及在羟脯氨酸的产生中是活性的，所述羟脯氨酸对于装饰小信号肽如CLAVATA 3和阿拉伯半乳聚糖蛋白（AGP）是重要的，所述小信号肽是普遍存在的细胞表面蛋白聚糖，被认为在植物生长和发育中起重要作用，包括细胞扩增、细胞分裂、生殖发育和体细胞胚胎发生。因此，抗坏血酸水平的调节是所有植物生长和发育中大量生理过程的关键，有大量的文献描述了影响抗坏血酸水平的过程，但很少有文献描述调节机制，特别是在分子水平上。不用说，这种对机制的缺乏理解限制了我们通过生物技术或植物育种来改造依赖于抗坏血酸盐的过程的能力。这种局限性甚至延伸到提高新鲜水果和蔬菜中抗坏血酸水平的努力，尽管最近有令人震惊的报道称，由于抗坏血酸/维生素C缺乏导致坏血病的发病率增加。例如，在英国，2009年至2014年期间，由于营养不良和过度消费垃圾食品饮食导致的肥胖，与坏血病相关的住院人数增加了27%。这一切都被两篇里程碑式的论文改变了，一篇是Laing等人的，在2015年的The Plant Cell中，证明了控制GDP-L-半乳糖磷酸化酶（GGP）的翻译的独特负反馈调节机制，GGP是一种在抗坏血酸合成的Smirnoff-Wheeler途径中具有活性的酶，另一种由Fenech等人，在Plant Physiology（2021）中显示GGP活性是决定植物组织中抗坏血酸水平的唯一重要控制步骤，并且显示通过Smirnoff-Wheeler途径几乎线性控制通量。我们的目标是确定GGP翻译的负反馈调节的分子机制，并了解GGP的翻译控制是如何设置在转录控制所定义的边界内。我们建议了解GGP活性的控制如何影响抗坏血酸的通量。我们建议通过主要研究两种植物物种，拟南芥和番茄，整合我们的调查机制的调节，以发展通用的理解机制，可以广泛适用的植物。我们建议通过合作将我们对GGP负反馈调节机制的理解扩展到绿色藻类。为了了解抗坏血酸在不同植物物种中的生理作用，我们将研究一系列GGP突变体的表型结果与误调节抗坏血酸水平，并确定是否在拟南芥中观察到相似或不同的表型效应。

Ascorbate is a key metabolite for all plants, from eukaryotic algae to angiosperms. It is the most abundant water-soluble antioxidant with major roles in photosynthesis, transmembrane electron transport, cell division, growth and tolerance of biotic and abiotic stress. It performs these diverse roles by serving as a major scavenger of active oxygen species generated as by-products of photosynthesis, in the dissipation of excess photonic energy through the water-water cycle and as a co-factor in the xanthophyll cycle. It serves as a cofactor for dioxygenases, which are active in the synthesis of the phytohormones, ethylene, abscisic acid and gibberellins and in the generation of hydroxyproline, important for decorating small signalling peptides like CLAVATA3 and arabinogalactan-proteins (AGPs), which are ubiquitous cell surface proteoglycans proposed to play essential roles in plant growth and development, including cell expansion, cell division, reproductive development and somatic embryogenesis. Consequently the regulation of ascorbate levels is key to a large number of physiological processes in the growth and development of all plants.There is a vast body of literature describing the processes that influence ascorbate levels, but remarkably few papers describe the mechanisms of regulation, particularly at the molecular level. Needless to say, this lack of understanding of mechanism has limited our ability to engineer processes dependent on ascorbate through biotechnology or plant breeding for crop improvement. This limitation extends even to efforts to enhance ascorbate levels in fresh fruit and vegetables, despite the alarming reports of recent increases in the incidence of scurvy resulting from ascorbate/vitamin C deficiency. For example, in the UK between 2009 and 2014, hospital admissions related to scurvy went up by 27% due to malnutrition and obesity related to over-consumption of junk food diets. All that was changed by the publication of two landmark papers, one by Laing et al., in The Plant Cell in 2015 demonstrating a unique negative feedback regulatory mechanism controlling the translation of GDP-L-galactose phosphorylase (GGP) an enzyme active in the Smirnoff-Wheeler pathway for ascorbate synthesis, and the other by Fenech et al., in Plant Physiology (2021) showing that GGP activity is the only significant controlling step determining ascorbate levels in plant tissues and shows almost linear control of flux through the Smirnoff-Wheeler pathway.The aim of this application is to integrate our understanding of the regulation of ascorbate in plant cells. We aim to define the molecular mechanism for the negative feedback regulation of GGP translation and to understand how translational control of GGP is set within the boundaries defined by transcriptional control. We propose to understand how the control of GGP activity impacts flux to ascorbate. We propose to integrate our investigations of the mechanisms of regulation by studying primarily two plant species, Arabidopsis and tomato, to develop generic understanding of mechanisms for plants that could be broadly applicable. We propose to extend our understanding of the mechanism of negative feedback regulation of GGP to green algae through collaboration. To understand the physiological roles of ascorbate in different plant species we will investigate the phenotypic consequences of a series of GGP mutants with mis-regulated ascorbate levels, and determine whether similar or different phenotypic effects are observed in Arabidopsis.