**3. Discussion**

ITPK6 is a unique gene in the ITPK gene family, and knowledge of its function has so far been very limited. The identification and characterization in rice of two *itpk6* mutant lines were reported in a study, which is the only study on the function of ITPK6 in all organisms [30]. Our present study demonstrated that the knockout of *OsITPK6* could severely impair plant growth and reproduction, implying that ITPK6 may play important roles in plant growth and development, in addition to the biosynthesis of inositol polyphosphates.

First, we observed that the *OsIPTK6* knockout mutants (*ositpk6\_2*, *\_3*, and *\_4*) generated in the present study grew poorly, e.g., their plant height was reduced to almost half of that of the WT, and their fertility was almost abolished (Figure 2). These results suggested that *ITPK6* plays an important role not only in the vegetative growth but also in the reproduction of rice. Because the reduction of phytic acid content in *ositpk6\_1* grains (−32.1%) was less than in the P522L mutant line (−46%) [30], the reduction of phytic acid alone could not explain the inferior performance of *ositpk6\_1*. Further studies are needed to uncover the biological basis leading to the discrepancy between our present study and that reported in reference [30] regarding the mutational effect on rice growth and reproduction.

In the present study, we identified an *ositpk6* mutant, i.e., *ositpk6\_1*, with a 6-bp deletion. Though only two amino acids are expected to be removed from the derived protein, *ositpk6\_1* did reduce IP6 content. This may be because these two amino acids are located in a conserved region (Figure S1). We previously also observed a similar case of *lpa* rice, where a 6-bp deletion (and, hence, a deletion of two amino acids) in *OsSultr3;3* significantly reduced grain phytic acid content [14]. Although this mutation only reduced phytic acid content by less than 20%, it did exert a negative effect on seed set, grain weight, seed germination, and tolerance to abiotic stresses. This was somehow unexpected, because the P522L mutant line with a 46% reduction of phytic acid content still had normal plant growth as its WT parent [30]. Further studies are needed to fully evaluate the mutational effect by examining more *ositpk6* mutants.

The usefulness of CRISPR/Cas9-based mutagenesis for improving a particular trait is strictly dependent on the performance of the generated mutants. There are often trade-offs for mutating a gene for a specific purpose, and the overall performance of the mutated plant could be affected as a consequence of pleiotropic effects. Enlightened by the findings of Kim and Tai [30], we hoped to establish a fast and effective method for breeding yield-competitive, *lpa* rice cultivars by using genome-editing techniques. However, our results sugges<sup>t</sup> that *OsITPK6* or its product plays an important role in multiple cellular processes, and simply knocking out *OsIPTK6* would impair rice growth and reproduction and, hence, would not work for our purpose.

Because [30] of the success in the production of *lpa* mutants without a significant negative impact on plant growth and seed development, it is still possible to generate *ositpk6* mutants without a significant effect on plant growth, if more appropriate vectors can be designed and more mutants are identified and assessed.

In summary, the present study demonstrates that the *OsITPK6* gene is essential for rice growth and reproduction.
