**3. InsP6 Synthesis: The Lipid-Independent Pathway**

Given the importance of P*i* storage in plants, it is not surprising that plants evolved a separate way to synthesize InsP6, apart from the PtdInsP pathway. In the Lipid-Independent pathway, "free" *myo-*inositol is acted on by a series of inositol kinases. The first is the *myo-*inositol kinase (MIK), which was first identified in maize as a product of the *Low Phytic Acid* gene [40]. Loss-of-function maize *lpa3* mutants have reduced InsP6 and elevated inositol levels in the seeds [40]. Arabidopsis *atmik* mutants have a large reduction in total seed mass InsP6 levels (Table 1) [35]. The second step in the Lipid-Independent pathway is likely catalyzed by a gene/protein named LPA1 in rice [41]. This gene product was originally categorized as a potential 2-phosphoglycerate kinase that impacts InsP6 accumulation [41]. However, recent structural modeling indicates that InsP3 can be accommodated in the active site of this kinase, supporting a role for it as an InsP kinase [42].

The next step in this pathway should involve an inositol kinase capable of phosphorylating an InsP2 substrate. As no such kinase has been identified, this prompted speculation that InsP2's conversion to InsP3 is catalyzed by a moonlighting function of another inositol phosphate kinase, which has ye<sup>t</sup> to be identified. The last novel component of the Lipid-Independent pathway is the inositol triphosphate kinase (ITPK), which can phosphorylate specific InsP3 and InsP4 molecules [39,43]. A gene encoding ITPK1 was also identified in a maize mutant named *lpa2* [44]. There are four ITPK enzymes (AtITPK1–4) in Arabidopsis [45]. It is interesting to note that only Arabidopsis *atitpk1* and *atitpk4* mutants have reduced seed InsP6 levels (Table 1) [33,35]. Our own work with AtITPK1 and AtITPK2 enzymes shows that these proteins are also e fficient at converting InsP6 to InsP7 [38]. Laha et al. additionally used NMR to show that 5PP-InsP5 is the isoform synthesized by AtITPK1 and AtITPK2 [36]. These are key findings that highlight the catalytic flexibility of the ITPKs, as well as indicating that the Lipid-Independent pathway may have an important relationship with, and impact on, PP-InsP synthesis.

The ITPKs are thought to act in concert with the IPK2 enzymes in producing Ins(1,3,4,5,6)P5 in the Lipid-Independent pathway [27,31,43]. The final step is the conversion of Ins(1,3,4,5,6)P5 to InsP6. Both pathways utilize IPK1 to synthesize InsP6 and converge at this last step in InsP synthesis. This further highlights the importance of IPK1 in InsP6 synthesis. A very recent publication on non-plant organisms suggests that there might be some conserved functions of the Lipid-Independent pathway in other eukaryotes. Humans, for example, contain ITPK genes, and the expression of these enzymes appears to complement mutants defective in PLC, which cannot synthesize InsP6 [28,46]. Although more work needs to be done, this suggests that animals may also utilize ITPKs in a Lipid-Independent InsP6 pathway. In the same work, these authors also found that the plant ITPK1 could complement the yeas<sup>t</sup> PLC mutant, suggesting that the plant ITPK may also have a very flexible substrate preference, and may act at several di fferent steps in the Lipid-Independent pathway [28].
