**1. Introduction**

The inositol phosphate (InsP) signaling pathway has been implicated in a diverse array of cellular processes across eukaryotic organisms (for reviews, see [1–3]). Inositol phosphates are intracellular signaling molecules built around a simple 6-carbon *myo*-inositol (inositol) scaffold used in signal transduction (Figure 1). Differing phosphorylation patterns on the inositol ring convey specific cellular information, and several combinations of phosphorylation events are possible. Inositol hexakisphosphate (InsP6), also called phytate when complexed with cations, is a molecule where all hydroxyl (OH) groups on the inositol ring are phosphorylated, that serves as both a storage pool of phosphate as well as a signaling molecule in plants [4–6].

InsP6 is the major phosphorus storage sink within the plant seed, comprising up to ~1% of an Arabidopsis seed's dry weight, and roughly 65–85% of total seed phosphorus in cereal crops [6]. InsP6 is one of the most highly electronegative molecules present in the cell, resulting in a greatly limited ability to cross cell membranes. It can be transported into the vacuole by MRP5, an ABC-transporter localized in the vacuolar membrane that has been shown to specifically transport InsP6 [4]. During seed development, the electronegative properties of InsP6 result in the chelation of positively charged metal ions such as Mg<sup>2</sup>+, Fe2+, Zn2+, Mn2+, Ca2+, and these InsP6 metal complexes accumulate within the protein storage vacuole (PSV) (Figure 2) [7,8].

**Figure 1.** Simplified structures of *myo*-inositol, InsP6, phytate, and PtdInsP. Inorganic phosphate (P*i*) groups, covalently bound to the *myo*-inositol ring by an oxygen molecule, are represented in red. For simplification purposes, this figure does not take into account the axial and equatorial positions of the moieties.

While InsP6 is important for plant survivorship and yield, it is also, unfortunately, an anti-nutrient, as it can prevent useful cations and minerals from being absorbed by the animal gu<sup>t</sup> [9–11]. Additionally, non-ruminant animals cannot digest InsP6, and that which is excreted from livestock is a major environmental concern as it leads to phosphorus pollution, eutrophication, and toxified watersheds [12,13]. Given this, plant breeders and biotechnologists have sought to limit the production of InsP6 in plants, resulting in the so-called low-phytate crops [14]. However, reducing InsP6 synthesis and accumulation in plants can come with consequences, such as a significant decline in vital plant signaling molecules derived from InsP6.

**Figure 2.** Diagram illustrating phytate storage in wheat granules. Phytate and other cations are stored in the protein storage vacuole (PSV) within small, crystalline storage bodies, known as globoids [9,15]. While the location of globoids varies across species, a majority of these are found within the aleurone layer of cereal crops, such as wheat and barley, as well as oilseed crops including peanuts, hemp, sunflower, and cotton [6,9,16]. Maize globoids are concentrated within the germ, whereas soybean phytate is distributed throughout the entire seed [6,17,18]. PSVs also contain crystalloid bodies, containing stored proteins and lipids [9].

Our primary expertise and focus are on an emerging class of InsPs derived from InsP6 that contain one or two diphosphate or pyrophosphate groups (PP) attached to the inositol ring (Figure 1) [5,19]. These inositol pyrophosphates (PP-InsPs) are gaining significant attention due to their newly discovered roles in energetic metabolism as well as hormone signaling and P*i* sensing (for a review, see [20]). The presence of pyrophosphate bonds in PP-InsPs has resulted in their consideration as "energetic

signaling" molecules, and we note that PP-InsPs are similar in structure to ADP or ATP. PP-InsP biosynthesis is well described in non-plant eukaryotes, such as yeas<sup>t</sup> and mammals, and many physiological roles have been linked to these molecules [1–3]. Elucidating the route of PP-InsP biosynthesis in plants has recently been accomplished and is fundamentally critical to our understanding of these molecules, which is described in the following sections. We will start with an overview of how plants synthesize the precursor to PP-InsPs, InsP6, as several of the enzymes in this pathway are key to understanding the PP-InsP pathway.
