Biochemical Properties and Physiological Roles of Plant Inositol Hexakisphosphate Kinases

肌醇焦磷酸盐已被证明在阿米巴、哺乳动物和真菌中发挥着重要的调节作用，尽管这些分子如何执行其调节功能的确切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，在植物提取物中可以很容易地检测到肌醇焦磷酸盐InsP7和InsP8，基于InsP8合成酶在所有植物门中的普遍存在，这些分子可能发挥重要的功能。我们的工作提供了强有力的证据表明，依赖于VIH2的InsP8是COI1-Jaz茉莉酸受体复合体的关键辅助因子，因此在植物抵御草食昆虫和真菌坏死菌方面发挥着重要作用。刺激诱导的产物和与细胞受体的结合，然后触发下游反应，使InsP8有资格成为参与茉莉酸信号转导的真正的第二信使。然而，肌醇焦磷酸生物学的许多方面仍然没有解决。例如，InsP7的功能以及植物InsP7(6个可能的异构体)和植物InsP8(15个可能的异构体)的异构体身份仍然未知。这是由于三大缺点：i)这些分子的数量太少，无法进行直接的核磁共振分析；ii)标准方法，如PAGE/甲苯胺分离和高效液相分析[~3H]-myo-inositol标记的植物提取物，无法分离各自的异构体；iii)负责植物InsP7合成的酶尚未被确定(直到我们最近的未发表发现)，因为植物基因组不编码存在于变形虫、哺乳动物和酵母中的IP6K/Kcs1家族的InsP6激酶。为了解决这些和其他悬而未决的问题，我们提出了以下三个具体目标：(I)研究两个新发现的拟南芥InsP6激酶(AtInsP6K1和AtInsP6K2)合成肌醇焦磷酸的机制，并确定植物InsP7和植物InsP8的异构体特性(Ii)研究缺乏InsP6激酶活性的植物InsP6同源物的作用，并与其中一个非常相关的同源物进行定向进化实验，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到的拟南芥中生长素感知强烈依赖InsP6K1功能(因此可能依赖InsP7)而不是全局InsP6的分子机制。

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isomers, and iii) enzymes responsible for InsP7 synthesis in plants have not been identified (until our recent unpublished discovery) since plant genomes do not encode InsP6 kinases of the IP6K/Kcs1 family present in amoebae, mammals and yeast.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.