Biochemical Properties and Physiological Roles of Plant Inositol Hexakisphosphate Kinases

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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isomers, and iii) enzymes responsible for InsP7 synthesis in plants have not been identified (until our recent unpublished discovery) since plant genomes do not encode InsP6 kinases of the IP6K/Kcs1 family present in amoebae, mammals and yeast.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the molecular mechanism of plant InsP6-kinase function (III) To investigate the molecular mechanism underlying our unpublished observation that auxin-perception in Arabidopsis strongly depends on AtInsP6K1 function (and thus likely on InsP7) and not on global InsP6.