**1. Introduction**

Myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6), also known as phytic acid (PA), is the main storage form of phosphorous (P) (65–80%) in cereal and legume seeds, accounting for ~1.5% of the dry weigh [1]. In most cereal grains, PA exists as mixed salts (phytates) in protein storage bodies and can chelate several mineral cations, including Zn2+, Fe2+, Ca2+, and Mg<sup>2</sup>+ [2]. During seed germination, endogenous grain phytase is activated to degrade phytate, releasing myo-inositol, phosphorus, and bound mineral cations [3], which are utilized by the developing seedlings. The PA biosynthetic pathway is still not well defined, but a number of genes involved in its biosynthesis or transport have already been cloned in several plants. Mutations of these genes could result in low-phytic-acid (*lpa*) grains in rice [4–14] and other plants, e.g., wheat [15] and maize [3,16,17]. In rice, 12 genes have been identified that catalyze the production of intermediate inositol polyphosphates in seeds [18].

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK) plays a pivotal role in phytic acid biosynthesis, whereby the inositol triphosphate (IP3) molecule is further phosphorylated at the 5th or 6th position [19,20]. ITPK belongs to the ATP-grasp fold proteins group [21] and is conserved from plants to humans with diverse functions. ITPK has even been found in the anaerobic protozoan *Entamoeba histolytica* [22], where

its transcription is slightly induced by heat shock, demonstrating its role in the cellular response to stress [21]. The first plant ITPK, AtITPK1, was identified in *Arabidopsis* [20]. AtITPK1 is involved in photomorphogenesis possibly by interacting with the constitutive photomorphogenic (COP) signalosome under red light [23]. The kinase activity of AtITPK1 is indispensable for maintaining inorganic phosphorus (Pi) homeostasis under Pi-replete conditions, and *itpk1* mutants exhibited decreased levels of IP6 and diphosphoinositolpentakisphosphate (IP7). Disruption of another ITPK family enzyme, ITPK4, also caused depletion of IP6 and IP7 but did not display similar Pi-related phenotypes as *itpk1* [24]. AtITPK4 is an outlier to its family and does not display inositol 3,4,5,6 tetrakisphosphate 1-kinase activity; rather, it displays inositol 1,4,5,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate isomerase activity [21]. AtITPK2 was required for seed coat development and lipid polyester barrier formation [25], and ABA or phosphorus deficiency could induce *AtITPK2* expression. In maize (*Zea mays* L.), ZmITPK1 exhibits multiple inositol phosphate kinase activities and is involved in phytic acid biosynthesis in developing seeds [17]. In soybean (*Glycine max* L.), GmITPK1 is a potential candidate for developing low-phytate soybean [26], and GmITPK2 may play a role as a dehydration and salinity stress regulator [27].

In rice (*Oryza sativa* L.), the *OsITPK* genes can be divided into three sub-families [18]. *OsITPK1*, *OsITPK2*, and *OsITPK3* belong to subgroup I, each with 10 exons and 9 introns; *OsITPK4* and *OsITPK5* belong to subgroup II, which has no intron; and *OsITPK6* belongs to subgroup III, with 12 exons and 11 introns. OsITPK2 is a negative regulator of osmotic stress signaling [28], and its disruption could affect the expression of some of its homologous genes, *OsITPK1* and *OsITPK4* [29]. The expression of *OsITPK4*, but not of *OsITPK1, 2, 3*, and *5* can be strongly induced by cold and heat stresses [29]. The IP3 level was not affected by the *ositpk2* mutation, probably owing to redundant functions of other homologs [29].The expression of *OsITPK6* could also be induced by heat [29], and mutations of *OsITPK6* were already demonstrated to result in significant reduction of IP6 in rice grains [30], i.e., mutant lines with the amino acid substitution P522L had IP6 content about half that of the wild-type (WT) line. Among the *ositpk6* mutants, one line with a P522L amino acid substitution had agronomic performance (seed weight, germination, and seedling growth) similar to that of its WT parent, suggesting *OsITPK6* could be a desirable target of mutagenesis for breeding yield-competitive *lpa* rice [30]. Since the binding site for nucleotide or ATP is between 200 and 500 amino acids in OsITPK6, the substitution mutation (P522L) is localized outside of this binding region. The effect of the P522L mutation in OsITPK6 on IP6 biosynthesis could be related to the interaction of the enzyme with another substrate inositol polyphosphate. On the other hand, a splicing mutant of *OsITPK6* at the 9th intron showed a more severe *lpa* phenotype: lower phytic acid content with reduced seed set [30]. Hence, it would be worthwhile to examine the function of OsITPK6 by generating more and different mutants, particularly by disruption of the ATP-binding region.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system, CRISPR/Cas9, is an efficient and precise genome-editing technique and has the potential to be used for crop improvement [31–33], including rice [34–36]. In the present study, we explored the possibility of establishing a genome-editing-based method for the fast breeding of yield-competitive *lpa* rice by evaluating *OsITPK6* mutants generated by CRISPR/Cas9-mediated mutagenesis. Our results showed that mutation of *OsITPK6* not only significantly reduced the accumulation of IP6 in rice grains but also impaired plant growth and tolerance to abiotic stress.
