Biochemical Properties and Physiological Roles of Plant Inositol Hexakisphosphate Kinases

植物肌醇六磷酸激酶的生化特性和生理作用

• 批准号: 361089095
• 负责人: Professor Dr. Gabriel Schaaf
• 金额: --
• 依托单位: Bereich Pflanzenernährung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/361089095?language=en
• 关键词: Biochemical; Properties; Physiological; Roles; Plant

Inositol pyrophosphates have been shown to play important regulatory roles in amoebae, mammals and fungi even though the precise mechanism of how these molecules execute their regulatory function is in most cases still under debate. Our work in Arabidopsis demonstrates that the inositol pyrophosphates InsP7 and InsP8 can be readily detected in plant extracts and that these molecules are likely to play important functions based on the ubiquitous presence of InsP8 synthetases in all plant phyla. Our work provides strong evidence that VIH2-dependent InsP8 is a critical cofactor of the COI1-JAZ jasmonate receptor complex and thus plays an important role in plant defenses against herbivorous insects and fungal necrotrophs. Stimulus-induced production and binding to a cellular receptor which then triggers a downstream response qualifies InsP8 as a bona fide second messenger involved in jasmonate signaling. However, many aspects of inositol pyrophosphate biology remain unresolved. For instance, the function of InsP7 and the isomer identities of both plant InsP7 (6 possible isomers) and plant InsP8 (15 possible isomers) remain unknown. This is due to three major shortcomings: i) the amount of these molecules is too low for direct NMR analyses, ii) standard methods such as separation by PAGE/toluidine and HPLC analyses of [3H]-myo-inositol-labelled plant extracts fail to allow separation of the respective isomers, and iii) enzymes responsible for InsP7 synthesis in plants have not been identified (until our recent unpublished discovery) since plant genomes do not encode InsP6 kinases of the IP6K/Kcs1 family present in amoebae, mammals and yeast. To address these and other open questions we propose the following 3 Specific Aims:(I) To investigate the mechanism of inositol pyrophosphate synthesis by two newly discovered Arabidopsis InsP6 kinases (AtInsP6K1 and AtInsP6K2) and to identify the isomer identity of plant InsP7 and plant InsP8 (II) To investigate the role of plant InsP6 kinase homologs that lack InsP6 kinase activity and to perform directed evolution experiments with one of the very closely related homologs to better understand the molecular mechanism of plant InsP6-kinase function (III) To investigate the molecular mechanism underlying our unpublished observation that auxin-perception in Arabidopsis strongly depends on AtInsP6K1 function (and thus likely on InsP7) and not on global InsP6.

肌醇焦磷酸已被证明在变形虫、哺乳动物和真菌中发挥重要的调节作用，尽管这些分子如何执行其调节功能的精确机制在大多数情况下仍存在争议。我们在拟南芥中的工作表明，可以在植物提取物中轻松检测到肌醇焦磷酸 InsP7 和 InsP8，并且基于所有植物门中普遍存在的 InsP8 合成酶，这些分子可能发挥重要功能。我们的工作提供了强有力的证据，证明 VIH2 依赖性 InsP8 是 COI1-JAZ 茉莉酸受体复合物的关键辅助因子，因此在植物防御草食性昆虫和真菌坏死生物中发挥重要作用。刺激诱导产生并与细胞受体结合，然后触发下游反应，使 InsP8 成为真正参与茉莉酸信号转导的第二信使。然而，肌醇焦磷酸生物学的许多方面仍未解决。例如，InsP7 的功能以及植物 InsP7（6 种可能的异构体）和植物 InsP8（15 种可能的异构体）的异构体身份仍然未知。这是由于三个主要缺点：i) 这些分子的量太低，无法进行直接 NMR 分析，ii) 标准方法，例如通过 PAGE/甲苯胺分离和 [3H]-肌醇标记植物提取物的 HPLC 分析无法分离各自的异构体，以及 iii) 植物中负责 InsP7 合成的酶尚未确定（直到我们最近未发表） 发现），因为植物基因组不编码变形虫、哺乳动物和酵母中存在的 IP6K/Kcs1 家族的 InsP6 激酶。为了解决这些和其他悬而未决的问题，我们提出以下3个具体目标：（I）研究两种新发现的拟南芥InsP6激酶（AtInsP6K1和AtInsP6K2）合成肌醇焦磷酸的机制，并鉴定植物InsP7和植物InsP8的异构体身份（II）研究缺乏InsP6激酶活性的植物InsP6激酶同源物的作用并进行定向进化 使用非常密切相关的同源物之一进行实验，以更好地了解植物 InsP6 激酶功能的分子机制 (III) 研究我们未发表的观察结果的分子机制，即拟南芥中的生长素感知强烈依赖于 AtInsP6K1 功能（因此可能依赖于 InsP7）而不是全局 InsP6。