**2. InsP6 Synthesis: The Lipid-Dependent Pathway**

InsP6 can be synthesized by two interconnected pathways in plants. The pathways are named for their starting material: the Lipid-Dependent pathway and the Lipid-Independent pathway (Figure 3). The Lipid-Dependent pathway is present in all eukaryotic organisms [21–25]. The Lipid-Independent pathway for synthesizing InsP6 was originally discovered in *Dictyostelium* in a landmark paper by Stephens and Irvine in 1990, and followed up on in *Spirodela polyrhiza* [26,27]. This pathway was thought be unique to these organisms, along with land plants. However, a very recent publication by Desfougères et al. shows that the Lipid-Independent pathway is also present in mammals [28]. This work is the first to report evidence of the Lipid-Independent pathway in mammals and will be crucial for exploring the evolution of enzymes across organisms.

**Figure 3.** A simplified view of the InsP synthesis and degradation pathway. InsP synthesis starts in the Loewus Pathway (tan), where InsP is synthesized from Glucose-6-Phosphate (G6P). InsPs are synthesized through the Lipid-Dependent (pink) or Lipid-Independent (yellow) pathways. PP-InsPs in plants are synthesized from InsP6 (purple). The enzymes involved in the pathway are discussed throughout the review.

The lipid component in the Lipid-Dependent pathway is phosphatidylinositol phosphate (PtdInsP), a molecule containing inositol as the head group (Figure 1). While plants synthesize a myriad of lipid-soluble PtdInsPs, phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2), is important for the Lipid-Dependent pathway as it is acted on by the enzyme phospholipase C (PLC) [29]. Phospholipases, by definition, hydrolyze phospholipids. The hydrolysis of PtdIns(4,5)P2 by PLC produces Ins(1,4,5)P3 and diacylglycerol (DAG), which essentially converts a phosphorylated lipid-signaling molecule (PtdIns(4,5)P2) into a water-soluble, InsP-signaling molecule (Ins(1,4,5)P3) (Figure 3) [29].

Ins(1,4,5)P3 can be subsequently phosphorylated into InsP4, and then InsP5, by a dual specific inositol polyphosphate multikinase (IPMK). The IPMK enzyme is encoded by two genes in Arabidopsis, *AtIPK2*α and *AtIPK2*β (Table 1) [30]. Both genes encode enzymes with a 6/3-kinase activity, catalyzing the conversion of Ins(1,4,5)P3 to Ins(1,4,5,6)P4 and to a final Ins(1,3,4,5,6)P5 product [31]. 5-kinase activity towards Ins(1,3,4,6)P4 and Ins(1,2,3,4,6)P5 was also reported by Stevenson-Paulik et al. Notably, these genetic studies show that the *AtIPK2*β gene can complement a yeas<sup>t</sup> *ipk2* mutant, and that Arabidopsis T-DNA loss-of-function *atipk2*β mutants have a 35% reduction in seed InsP6 (Table 1) [30]. *atipk2*α mutants are not easily studied as those that are recovered are lethal. This, along with the generally ubiquitous expression of *AtIPK2*<sup>α</sup>, supports the idea that *AtIPK2*α supplies the major IPK2 or IPMK activity in the plant cell.

**Table 1.** Loss-of-function Arabidopsis mutants and impacts on InsP6 and PP-InsP levels. Arabidopsis is a simple model system that can be used to gauge the impacts of genetic changes on InsPs. The table shows the impact on InsP6 and PP-InsPs in Arabidopsis mutants for enzymes involved in InsP synthesis. Mutants for enzymes important in both the Lipid-Dependent and Lipid-Independent pathways are indicated (\*).


The last step in the Lipid-Dependent pathway of InsP6 biosynthesis is the phosphorylation of Ins(1,3,4,5,6)P5 to InsP6. 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 InsP5 [30]. While there are seven genes in Arabidopsis that are predicted to encode IPK1 enzymes, the *At5g42810* gene (*AtIPK1*) is the only one actively expressed in plants [30]. Complementation assays reveal that *AtIPK1* is able to complement a yeas<sup>t</sup> *ipk1* mutant, restore InsP6 levels, and rescue the mutant's temperature-sensitive growth phenotype [39]. As loss of IPK1 function results in an 83% reduction in InsP6 in seeds (Table 1), this shows that IPK1 plays a major role in maintaining seed InsP6 levels [30].
