Inositol Trisphosphate Kinase and Diphosphoinositol Pentakisphosphate Kinase Enzymes Constitute the Inositol Pyrophosphate Synthesis Pathway in Plants

Abstract

Inositol pyrophosphates (PP-InsPs) are an emerging class of "high-energy" intracellular signaling molecules containing one or two diphosphate groups attached to an inositol ring, with suggested roles in bioenergetic homeostasis and inorganic phosphate (Pi) sensing. Information regarding the biosynthesis of these unique class of signaling molecules in plants is scarce, however the enzymes responsible for their biosynthesis in other eukaryotes have been well described. Here we report the characterization of the two Arabidopsis VIP kinase domains, a newly discovered activity of the Arabidopsis ITPK1 and ITPK2 enzymes, and the subcellular localization of the enzymes involved in the synthesis of InsP6 and PP-InsPs. Our data indicate that AtVIP1-KD and AtVIP2-KD act primarily as 1PP-specific Diphosphoinositol Pentakisphosphate Kinases (PPIP5) Kinases. The AtITPK enzymes, in contrast, can function as InsP6 kinases, and thus are the missing enzyme in the plant PP-InsP synthesis pathway.Together, these enzyme classes can function in plants to produce PP-InsPs, which have been implicated in signal transduction and Pi sensing pathways. We measured a higher InsP7 level (increased InsP7/InsP8 ratio) in vip1/vip2 double loss-of-function mutants, and an accumulation of InsP8 (decreased InsP7/InsP8 ratio) in the 35S:VIP2 overexpression line relative to wild-type plants. We also report that enzymes involved in the synthesis of InsPs and PP-InsPs accumulate within the nucleus and cytoplasm of plant cells. Our work defines a molecular basis for understanding how plants synthesize PP-InsPs which is crucial for determining the roles of these signaling molecules in processes such as Pi sensing. 3 SIGNIFICANCE STATEMENTInositol pyrophosphate signaling molecules are of agronomic importance as they can control complex responses to the limited nutrient, phosphate. This work fills in the missing steps in the inositol pyrophosphate synthesis pathway and points to a role for these molecules in the plant cell nucleus. This is an important advance that can help us design future strategies to increase phosphate efficiency in plants.

Can Inositol Pyrophosphates Inform Strategies for Developing Low Phytate Crops?

“…While our biochemical analyses of the AtVIP KDs did not rule out the possibility that these enzymes can phosphorylate InsP 6 , they suggested that other enzymes likely had to exist in plants to drive this reaction [38]. As the ITPKs phosphorylate the 5-position of lower InsPs, we decided to target this class of enzymes, and found that both AtITPK1 and AtITPK2 are able to phosphorylate InsP 6 in vitro [36,38]. We also demonstrated that the AtITPK1 product could be further phosphorylated by the AtVIP1-KD, resulting in InsP 8 [38].…”

“…It is interesting to note that only Arabidopsis atitpk1 and atitpk4 mutants have reduced seed InsP 6 levels (Table 1) [33,35]. Our own work with AtITPK1 and AtITPK2 enzymes shows that these proteins are also efficient at converting InsP 6 to InsP 7 [38]. Laha et al additionally used NMR to show that 5PP-InsP 5 is the isoform synthesized by AtITPK1 and AtITPK2 [36].…”

“…This prompted us and other groups to speculate the AtVIPs might be bifunctional enzymes, phosphorylating both InsP 6 and InsP 7 . While our biochemical analyses of the AtVIP KDs did not rule out the possibility that these enzymes can phosphorylate InsP 6 , they suggested that other enzymes likely had to exist in plants to drive this reaction [38]. As the ITPKs phosphorylate the 5-position of lower InsPs, we decided to target this class of enzymes, and found that both AtITPK1 and AtITPK2 are able to phosphorylate InsP 6 in vitro [36,38].…”

“…This, along with the generally ubiquitous expression of AtIPK2α, supports the idea that AtIPK2α supplies the major IPK2 or IPMK activity in the plant cell. [34,37] vip1/vip2 No change reported [37] Increased InsP 7 and Decreased InsP 8 [37,38] The last step in the Lipid-Dependent pathway of InsP 6 biosynthesis is the phosphorylation of Ins(1,3,4,5,6)P 5 to InsP 6. This step is catalyzed by only one type of enzyme in nature, the inositol pentakisphosphate 2-kinase (IPK1), so named because all known IPK1 enzymes phosphorylate the 2-position of InsP 5 [30].…”

“…AtVIP1 (also referred to as AtVIH2) and AtVIP2 (AtVIH1) are dual-domain enzymes, consisting of an ATP-grasp N-terminal kinase domain (KD) and a C-terminal histidine phosphatase domain (PD) [5]. We recently found that the KD of both AtVIP enzymes can phosphorylate 5PP-InsP 5 in vitro [38]. Additionally, the Hothorn group used NMR and showed that the product of the AtVIP enzymes is indeed 1,5-PP-InsP 4 [37].…”