**4. PP-InsP Synthetic Pathway**

While a grea<sup>t</sup> majority of InsP6 is stored as phytate, a small pool can be further phosphorylated to form PP-InsPs. Our group and others have examined a class of enzymes involved in PP-InsP synthesis named the diphosphoinositol-pentakisphosphate kinases (PPIP5Ks), known as VIP or VIH in plants, and Vip1 in *Chlamydomonas reinhardtii* (algae) [5,34,47]. Mammalian and yeas<sup>t</sup> PPIP5K enzymes phosphorylate the 1-position on InsP6 and 5PP-InsP5 to generate an InsP7 molecule, 1PP-InsP5, and an InsP8 molecule, 1,5(PP)2-InsP4, respectively [48–52]. Two Arabidopsis genes, *AtVIP1* and *AtVIP2*, are orthologous to the mammalian *PPIP5K* genes [5,34]. 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-InsP5 in vitro [38]. Additionally, the Hothorn group used NMR and showed that the product of the AtVIP enzymes is indeed 1,5-PP-InsP4 [37].

A second class of enzymes, known as the inositol hexakisphosphate kinases (IP6Ks), function in non-plant organisms by phosphorylating the 5-position of InsP5 and InsP6 to generate InsP7 [53]. While the genes coding for IP6Ks are present in humans and yeast, there is no identifiable *IP6K* gene in the plant genome [5]. This prompted us and other groups to speculate the AtVIPs might be bifunctional enzymes, phosphorylating both InsP6 and InsP7. While our biochemical analyses of the AtVIP KDs

did not rule out the possibility that these enzymes can phosphorylate InsP6, 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 InsP6 in vitro [36,38]. We also demonstrated that the AtITPK1 product could be further phosphorylated by the AtVIP1-KD, resulting in InsP8 [38]. Based on our findings, as well as recent work by Laha et al., we conclude that the AtITPKs are the missing enzyme in the pathway [36,38].

### **5. How Do PP-InsPs Function in Plants?**

Williams et al. suggested, in a review published in 2015, that a major function of PP-InsPs in plants was as a "glue" to bring together various protein binding partners [20]. At the time of the review, it was known that InsP6 could bind to the transport inhibitor response 1 (TIR1) auxin receptor [54]. Additionally, InsP7 was hypothesized to bind to the jasmonate (JA) receptor based on structural modeling experiments [34]. Exciting data, of importance to crop breeders, details how InsP6 and InsP7 are able to complex with key proteins involved in P*i*-sensing in plants [55]. Soils depleted in phosphorus lead plants to induce a suite of molecular and physiological mechanisms to enhance P*i* scavenging, known as the P*i* starvation response (PSR) [56,57]. The PSR is facilitated by an increase in transcription of a group of PSR genes, leading to increases in P*i* transport and uptake. Upregulation of PSR gene expression is regulated by Phosphate Starvation Response Regulator 1 (PHR1), a transcription factor, along with its homologs [56,57]. PHR1 and homologous transcription factors have a high binding affinity for promoters containing PHR1 binding sequences (P1BS), which allows for the binding and up-regulation of PSR genes under low P*i* conditions [56,57].

InsP6 and PP-InsPs regulate P*i* sensing via facilitating complex formations between the PHR1 transcription factor and the SPX domain-containing proteins (Figure 4) [58]. PHR and SPX proteins isolated from *Oryza sativa* (rice), known as OsPHR2 and OsSPX4, respectively, can complex with InsP6 or InsP7 in vitro [55]. InsP8 has an even lower dissociation constant than InsP7 in the OsPHR2–OsSPX4 complex formation [59]. Together, these data support the idea that InsP8 is the main mediator of the PHR1:SPX complex formation in plants.

**Figure 4.** Model depicting how PP-InsPs regulate plant P*i* sensing and the P*i* starvation response (PSR). (**a**) PSR gene regulation under deplete (top) and replete P*i* conditions (bottom). SPX1 binds to PHR1 under replete P*i*, preventing SPX1 from binding to promoters containing P1BS. Under deplete P*i*, PHR1 is uninhibited from binding to P1BS promoters. Adapted from [60]. (**b**) Model depicting a complex formation between SPX1, PHR1, and InsP8. This model represents interactions between other SPX proteins and PSR regulators, such as PHR1 and its homolog, PHT1, along with others.

While InsP7 has a ~7-fold stronger binding a ffinity than InsP6 to OsSPX4, recent genetic analyses greatly support the idea that InsP8 is the major signaling molecule, or proxy, that conveys information on the P*i* status within the plant cell [37,61]. First, *atipk1*, *atitpk1*, *atitpk4* and *atvip1*/*vip2* mutants commonly show defects in P*i* sensing, such that the PSR is turned on even when grown under <sup>P</sup>*i*-replete conditions [30,33,37,61]. Additionally, all of these particular mutants have decreased InsP8 levels (Table 1). In the case of *atvip1*/*vip2* double mutants, the PSR is likely so highly upregulated that growth becomes stunted and lethality occurs in double homozygotes [37,61]. In contrast, *atipk1* and *atitpk1* mutants can grow fairly normally under certain conditions, and can be stimulated to further increase the PSR under <sup>P</sup>*i*-replete conditions [33,62]. All of this information suggests that InsP8 functions to turn o ff the PSR.

We now know in greater detail that PP-InsPs likely function in binding to plant hormone receptors and transcription factor complexes involved in P*i* sensing. One example where PP-InsPs potentially function as cofactors is in the case of auxin signaling [54]. Auxin is a phytohormone which regulates numerous plant developmental processes and responses to environmental stress [63,64]. Auxin modulates gene expression by binding to the auxin receptor TIR1, an F-box protein, and mediates the SCF ubiquitin–ligase-catalyzed proteolysis of AUX/IAA transcriptional repressors [65,66]. The crystallized Arabidopsis TIR1 protein complex has a tightly bound InsP6 in the leucine-rich repeat (LRR) domain of TIR1, suggesting that InsP6 is a cofactor for the auxin receptor [54].

A later study identified Ins(1,2,4,5,6)P5 in the crystallized structure of a homologous plant hormone-JA co-receptor [67]. JA is a phytohormone critical for environmental and pathogen defense signaling as well as plant physiology [68]. Similar to auxin signaling with TIR1, JA is also perceived by interactions between F-box protein, coronatine-insensitive 1 (COI1), and the JA zim domain (JAZ) transcriptional repressors [69–71]. JA gene regulation is modulated by JA-hormone binding to COI1 and the degradation of JAZ repressors, freeing repressed transcription factors to upregulate JA genes [67]. Sheard et al. showed that Ins(1,2,4,5,6)P5 binds to and stabilizes COI, suggesting that Ins(1,2,4,5,6)P5 is a cofactor for the JA receptor [67,72]. A study using structural modeling predicted that the PP-InsPs have a much stronger binding a ffinity for COI1-JAZ1 than Ins(1,2,4,5,6)P5 and InsP6, suggesting that the PP-InsPs might be the true cofactor for the JA receptor [34].

### **6. Consequences for Targeting InsP6 for Reduction**

With our current understanding of PP-InsP synthesis, and analyses of genetic mutants in the pathway, it seems reasonable to question whether reducing InsP6 in plants will also result in reduction in PP-InsPs. Most characterized Arabidopsis mutants showing alterations in InsP6 also have impacted the intracellular levels of InsP7 and InsP8 (Table 1). Mutants with reduced PP-InsPs, such as *atipk1*, *atitpk1*, and *atvip1*/*vip2* mutants, show an upregulation of the PSR, which could impact engineered crop performance in the field [33,37,61]. Specifically, altered P*i* sensing could negatively impact plant growth and development, reduce viability, and alter root architecture [56,57]. Alterations in P*i* sensing can affect other signaling systems, such as the sensing and accumulation of nitrate and other micronutrients, along with the ABA signaling pathway, often linked to stress pathways [73,74].

Given the link between InsP8 and JA signaling, decreasing PP-InsPs might result in crops that are more susceptible to pathogens and insects [34]. This impact is seen in Arabidopsis *atvip1* mutants, which have reduced InsP8, and show increased susceptibility to insect herbivory as compared to WT plants [34]. Additionally, both transgenic potato plants and Arabidopsis mutants with reduced *myo*-*inositol phosphate synthase* (MIPS), the first committed step in inositol synthesis, have decreased InsP6 and are more susceptible to pathogenic viruses [75]. While the PP-InsPs were not quantified in these mutants, it is possible that the PP-InsPs were also reduced and are a causative factor in the increased susceptibility to pathogens.
