**2. Results**

### *2.1. Mutations of OsITPK6 and Development of Homozygous Transgene-Free Mutant Lines*

In total, we obtained 23 hygromycin phosphotransferase (HPT)-positive T0 plants transformed with the CRISPR/Cas9 vector pH-itpk6. Among them, seven plants were found mutated at the target region, which represents an editing efficiency of 30.4%. T1 plants were tested for the presence of mutations and T-DNA, and transgene-free T1 plants with four types of mutation were identified. The mutations included a single-nucleotide (nt) insertion and three types of deletion (Figure 1A). Seeds

were harvested from T1 plants with different mutations and developed into homozygous mutant lines, which were designated *ositpk6\_1, \_2*, *\_3*, and *\_4*.

**Figure 1.** Schematic diagram of *OsITPK6* and sgRNA target site for CRISPR/Cas9-mediated mutagenesis of *OsITPK6*, and prediction of the related wild-type and mutant proteins. (**A**) Exons, introns, and UTRs are indicated by solid boxes, lines, and blank boxes, respectively. P6-F and P6-R are primers for mutation genotyping, and their positions are indicated by arrowheads. Mutation identified within the target site of *OsITPK6* generated through CRISPR/Cas9-mediated genome editing in rice. The PAM sequences (NGG) are boxed, and the 20-nt target sequences are underlined. Mutations are shown in lowercase letters (for insertions) or '–' (for deletions). (**B**) The amino acid sequences of mutant proteins were aligned to that of the wild-type protein using the Clustal Omega Multiple Sequence Alignment (https://www.ebi.ac.uk/Tools/msa/clustalo/). The numbers represent the total number of amino acids, and the amino acid V521 and P522 are highlighted in red box. (**C**) The three-dimensional structures of OsITPK6 and its mutants were analyzed on SWISS-MODEL (https://www.swissmodel.expasy.org/).

The *ositpk6\_1* (a 6-nt in-frame deletion) mutation would result in the loss of two amino acids at positions 91 to 92 (Figure 1B). In contrast, all the other three mutations, i.e., *ositpk6\_2* (a 1-nt insertion), *ositpk6\_3* (a 5-nt deletion), and *ositpk6\_4* (a 2-nt deletion), would generate a premature stop codon almost right after the mutation site and, hence, significantly shorten the encoded proteins (Figure 1B). Consequently, the *ositpk6\_2*, *\_3* and *\_4* mutant alleles were predicted to produce proteins of only 105, 103, and 104 amino acids, respectively (Figure 1C). Analysis of ITPK6 proteins of six organisms indicated

that the two amino acids missing in the *ositpk6\_1* mutant were located in a highly conserved segmen<sup>t</sup> (Figure S1), suggesting the mutation of *ositpk6\_1* could have a potential functional consequence.

### *2.2. Impact of ositpk6 Mutations on Plant Growth and Seed Germination*

Plant growth of *ositpk6\_2*, *\_3*, and *\_4* was significantly impaired. First, their panicles were significantly shorter (−30.1%, −28.8%, and −29.1%, respectively) than that of the WT parental cultivar Xidao 1 (Figure 2A). Second, the mutant panicles had a high percentage of empty grains with darkened glumes (Figure 2B). Third, the height of the mutant plants was significantly reduced (−37.5%, −36.9%, and −39.1%, respectively) compared with that of their parental cultivar Xidao 1 (Figure 2C). The impact of *ositpk6\_1* on plant growth and seed set was limited and not obvious (Figure 2A–C). No significant differences of tiller number per plant were observed between Xidao 1 and all four *ositpk6* mutant lines (Figure 2D). Compared with Xidao 1, the seed set and 1000-grain weight of *ositpk6\_1* were also significantly decreased (−11.7% and −10.8%, respectively) (Figure 2E,F). Due to the extremely low seed set, we were not able to harvest enough seeds from *ositpk6\_3* and *ositpk6\_4* for the evaluation of 1000-grain weight and other characteristics. The germination of *ositpk6\_1* was slower in the first three days (Figure 3A) but gradually caught up with that of the WT after five days and reached ~80% on the 7th day (Figure 3B). The germination rate of *ositpk6\_2* was far lower than that of the WT (Figure 3A), being only ~20% on the 7th day (Figure 3B).

**Figure 2.** Agronomic traits of mutant *OsITPK6* line and wild-type plant. (**A**) Panicle phenotype of mutant *OsITPK6* and wild-type plants. (**B**–**D**) Twenty replicates were performed for four *OsITPK6* mutant lines and the wild-type plant. Error bars represent the standard error. The different letters show significant differences at a probability of *p* < 0.05. (**E**,**F**) Twenty replicates were performed for four *OsITPK6* mutant lines and the wild type. Error bars represent the standard error. Data with an asterisk(s) are significantly different from those of the wild type (\* *p* < 0.05, \*\* *p* < 0.01).

**Figure 3.** Germination rate of mutant seeds. (**A**) The pictures were taken on the 5th day after soaking. (**B**) The germination rate was recorded from 1 to 7 days after soaking, and three replicates were examined in each group.

### *2.3. E*ff*ect of ositpk6 Mutations on Inorganic Phosphorus (Pi), Phytic Acid Phosphorus (PA-P), and Total Phosphorus (TP) in Brown Rice*

A colorimetric assay showed that *ositpk6\_1* and *ositpk6\_2* had significantly higher Pi levels than the control (Figure 4A). To quantify the mutational effect of *ositpk6\_1* and *ositpk6\_2,* the Pi, PA-P, and TP contents were assessed in seeds of these two mutant lines, together with Xidao 1 as the WT control. All mutant lines had significantly lower levels of PA-P and higher levels of Pi compared with the control, while TP was not significantly different from that of the control (Figure 4B–D). *ositpk6\_1* and *ositpk6\_2* had Pi levels of 1.13 mg g<sup>−</sup><sup>1</sup> and 1.43 mg g<sup>−</sup>1, respectively, which were 4.12- and 5.18-fold higher than those of the control (0.28 mg g<sup>−</sup>1), respectively (Figure 4B). Xidao 1 seeds had a PA-P content of 2.30 mg g<sup>−</sup>1, which was significantly greater than those of the two mutant lines; the reduction of PA-P levels was 10.1% and 32.1% in *ositpk6\_1* and *ositpk6\_2*, respectively (Figure 4C). Xidao 1 seeds had a TP content of 3.9 mg g<sup>−</sup>1, which was not significantly different from those of the two mutant lines (Figure 4D).

**Figure 4.** *Cont.*

**Figure 4.** Inorganic P (Pi), phytic acid P (PA-P), and total phosphorus (TP) contents of the mutant *OsITPK6* lines and the wild type. (**A**) Qualitative assay of inorganic phosphorus (Pi) in mutant seeds. The concentration of the Pi standard samples is shown above. Five replicates were performed for two *OsITPK6* mutant lines and the wild type. (**B**–**D**) Six replicates were performed for two *OsITPK6* mutant lines and the wild type. Error bars represent the standard error. Data with an asterisk(s) are significantly different with respect to the wild-type data (\* *p* < 0.05, \*\* *p* < 0.01).

### *2.4. E*ff*ect of ositpk6 Mutation on Stress Tolerance*

To further test whether the mutation also had any impact on stress tolerance, we subjected the *ositpk6\_1* and Xidao 1 plants to osmotic stress treatment (because of the limited number of seeds harvested and the low germination rate, *ositpk6\_2*, was not further analyzed). The growth of *ositpk6\_1* seedlings appeared to be inferior to that of the WT control grown either under normal or stressed conditions (Figure 5A). The shoot length of *ositpk6\_1* was shorter than that of the WT with or without stress treatment (100 mM NaCl or 20 mM mannitol), while the root length of *ositpk6\_1* was shorter than that of the WT only under stress (Figure 5A,B). There was no significant difference in the number of leaves and roots with or without stress treatment between *ositpk6\_1* and WT (Figure 5C).

**Figure 5.** The phenotypes of the mutant *OsITPK6\_1* and wild type under salt stress (100 mM NaCl) and drought stress (20 mM mannitol). (**A**) The picture was taken on the 7th day after treatment. (**B**,**C**) Six replicates were performed for the *OsITPK6* mutant line and the wild type. Error bars represent the standard error. The different letters show significant differences at a probability of *p* < 0.05.
