Rapid production of novel beneficial alleles for improving rice appearance quality by targeting a regulatory element of <scp><i>SLG7</i></scp>

Rice, an important food crop consumed by more than half of the world's population, is also a tradeable commodity. The appearance quality of rice, characterized by grain shape, chalkiness, transparency and colour, greatly influences its commercial value. Generally, varieties with slender grains are associated with good appearance quality (Zhao et al., 2022). Several quantitative trait loci (QTL) for slender grains have been cloned and characterized (Liu et al., 2018; Wang et al., 2015a,b; Zhao et al., 2018). We previously characterized a major QTL for grain shape, slender grain on chromosome 7 (SLG7; Zhou et al., 2015), which is allelic to GL7 and GW7 (Wang et al., 2015a,b). Highly-expressed alleles of SLG7/GL7/GW7 produce slender grains with low chalkiness. SLG7 is valuable in breeding because it has few negative effects on grain yield-related traits. Although the beneficial SLG7 alleles might be introduced by traditional breeding to improve grain appearance, this process would typically take years. Here, we describe a rapid strategy to enhance rice appearance quality by editing the SLG7 promoter via CRISPR/Cas9. We predicted some cis-regulatory elements of the SLG7 promoter using Plant-CARE (Lescot et al., 2002; Figure 1a). To investigate which regulatory element is responsible for SLG7 expression, five target sites (T1–T5) near the predicted elements were selected for gene editing (Figure 1a). These five constructs were independently transformed into Wuyunjing30 (WYJ30), a high-yield japonica variety with round grains. In total, 12 homozygous T-DNA-free mutants were identified, all with one or two nucleotide variations at T1, T2, T4 and T5 target sites (Figure 1b). Interestingly, a 21/34-bp substitution and 14-bp and 36-bp deletions were found at T3 target site in mutants WYJ30-T3-1, WYJ30-T3-2, and WYJ30-T3-3, respectively (Figure 1b). Compared with WYJ30, SLG7 expression in WYJ30-T3-1, WYJ30-T3-2 and WYJ30-T3-3 increased by 34.6%, 47.2% and 66.2%, respectively (Figure 1c). In contrast, SLG7 transcription in the other mutants did not differ from that in WYJ30 (Figure 1c). We named the three novel alleles with increased expression SLG7P-s21 (21/34-bp substitution), SLG7P-d14 (14-bp deletion) and SLG7P-d36 (36-bp deletion; Figure 1b). Compared with WYJ30, the mutants WYJ30-T3-1, WYJ30-T3-2 and WYJ30-T3-3 produced more slender grains (Figure 1c, e). More importantly, appearance quality was greatly improved (Figure 1d). The percentage of chalky grains in WYJ30 was 12.1%, whereas that of WYJ30-T3-1, WYJ30-T3-2 and WYJ30-T3-3 was 10.0%, 7.8% and 4.5%, respectively (Figure 1f). The degree of chalkiness of the mutants was also significantly lower (Figure 1g). These results demonstrate that editing the T3 target site of the SLG7 promoter can produce novel alleles with higher expression levels and better appearance quality. We also measured cooking and eating quality. Compared with WYJ30, the apparent amylose contents of WYJ30-T3-2 and WYJ30-T3-3 were 3.5% and 8.8% higher, respectively (Figure 1h). In contrast, the gel consistencies of WYJ30-T3-2 and WYJ30-T3-3 decreased by 9.1% and 9.5%, respectively (Figure 1i). Viscosity indexes of WYJ30-T3-2 and WYJ30-T3-3 were also changed (Figure 1j). No significant difference was found in apparent amylose content, gel consistency, and viscosity index between WYJ30-T3-1 and WYJ30 (Figure 1h–j), possibly because SLG7 expression increased slightly and grain shape was only weakly modified in WYJ30-T3-1. The grain yield per plant of the mutants did not differ from that of WYJ30 (Figure 1k). The taste values of cooked rice declined slightly in the mutants, but the differences were not significant (Figure 1l). We conclude that editing the T3 target site of the SLG7 promoter via CRISPR/Cas9 can rapidly create beneficial alleles that improve appearance quality without diminishing yield and eating quality. We next analysed the molecular mechanism of T3 target-site regulation of SLG7 expression. A putative AC II element (ACCAATCC) was found near the T3 target site (Figure 1a,b). We used a yeast one-hybrid (Y1H) assay to examine possible interactions between the SLG7 promoter region containing the AC II element and 10 reported transcription factors controlling rice grain size or shape. AH2, a MYB protein, that functions in grain and hull development (Ren et al., 2019), was found to bind to the SLG7 promoter of WYJ30 (Figure 1m,n). Moreover, the interaction between AH2 and the mutated promoters of WYJ30-T3 mutants was significantly weaker than with the wild type (WT) promoter (Figure 1m,n). In Y1H and transcriptional activity analysis, Ren et al. (2019) previously found that AH2 can bind to the GL7/SLG7 promoter and repress its expression. We further confirmed the interaction between AH2 and the SLG7 promoter in an electrophoresis mobility shift assay (EMSA). A shifted band was detected when GST–AH2 fusion protein was incubated with the AC II element-containing promoter segment (Figure 1o). The shifted band was gradually abolished upon addition of 20-, 50- and 100-fold unlabelled competitor oligonucleotides containing the AC II motif. In contrast, competitor oligonucleotides containing a mutated AC II motif had no effect on this band (Figure 1o). Binding capabilities of the mutated promoters of WYJ30-T3-2 and WYJ30-T3-3 were also markedly decreased (Figure 1p). Dual luciferase (LUC) assays further indicated that the inhibitory effect of the AH2 protein on WYJ30-T3 mutated-promoter expression was significantly reduced (Figure 1q). SLG7 expression was significantly increased in the ah2 mutant (Figure 1r), consistent with a previous report (Ren et al., 2019). Taken together, these results indicate that AH2 directly binds to the SLG7 promoter and represses its expression. In addition, the AC II element is an essential target site of AH2. We noticed that the mutated promoters of WYJ30-T3-1 and WYJ30-T3-2 still contained the AC II element (Figure 1b), which might be because the flanking sequence of AC II also affects the binding capability of AH2. Previously, GW8/OsSPL16 protein was found to bind to the GTAC motif of the GW7/SLG7 promoter and repress its expression (Wang et al., 2015a). Here, our results suggest that AH2 acts as a novel negative player that regulates SLG7 expression. The construct for targeting the T3 site was also introduced into Yandao8 (YD8), a japonica variety cultivated around the lower reaches of the Yangtze River in China. We obtained two independent homozygous mutants, YD8-T3-1 and YD8-T3-2, having 20- and 44-bp deletions in the SLG7 promoter, respectively (Figure 1s). The SLG7 expression of these two mutants was significantly increased compared with the WT (Figure 1t). As expected, both YD8-T3-1 and YD8-T3-2 produced longer, more slender grains (Figure 1t). Moreover, the chalkiness of milled rice was significantly improved (Figure 1u,v). The apparent amylose contents of YD8-T3 mutants were slightly increased, while the gel consistencies of YD8-T3 mutants were significantly decreased (Figure 1v). Although not statistically significant, the taste values of YD8-T3 mutants tended to be slightly lower (Figure 1v). In summary, we developed a rapid strategy for generating novel beneficial alleles for improving rice appearance quality by targeting the AC II element-containing region of the SLG7 promoter via CRISPR/Cas9 gene editing (Figure 1w). We thank Dr. Kejian Wang (China National Rice Research Institute) for providing the CRISPR/Cas9 plasmid. We thank Dr. Deyong Ren (China National Rice Research Institute) for kindly providing the ah2 mutant. This work was supported by the National Key Research and Development Program of China (2022YFD1200104), the National Natural Science Foundation of China (31971917), the Program of Jiangsu Province Government (JBGS[2021]001), the Project of Zhongshan Biological Breeding Laboratory (BM2022008-02), the Natural Science Foundation of Jiangsu Province (BK20200947), the Project of Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding (PLR202101) and the PAPD Program from Jiangsu Government. The authors have declared no conflict of interest. Y.Z., G.L. and S.W. contributed to the original concept of the project. W.T., J.M., B.X., C.Z., Y.W., X.G., S.L., B.W., C.C., J.Z., S.Z., Z.G. and A.Y. performed the research. Z.Y. helped to analyse the data. W.T. and Y.Z. wrote the manuscript. G.L., J.M. and S.W. contributed to the writing. All authors read and approved the final version of the manuscript.

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