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CRISPR/Cas9‐mediated elimination of OsHHO3, a transcriptional repressor of three AMMONIUM TRANSPORTER1 genes, improves nitrogen use efficiency in rice

2023; Wiley; Volume: 21; Issue: 11 Linguagem: Inglês

10.1111/pbi.14167

1467-7652

Kexin Liu, Yasuhito Sakuraba, Namie Ohtsuki, Mailun Yang, Yoshiaki Ueda, Shuichi Yanagisawa,

Rice Cultivation and Yield Improvement

Plants require large amounts of nitrogen (N) to biosynthesize N-containing compounds indispensable for growth. Most land plants uptake N as nitrate ( NO 3 − $$ {\mathrm{NO}}_3^{-} $$ ), whereas plants growing under anaerobic conditions, such as rice, acquire N predominantly in the form of ammonium ( NH 4 + $$ {\mathrm{NH}}_4^{+} $$ ). Since neither NO 3 − $$ {\mathrm{NO}}_3^{-} $$ nor NH 4 + $$ {\mathrm{NH}}_4^{+} $$ levels in soils are sufficient to support extensive plant growth, massive amounts of N fertilizers are applied to the field annually, causing environmental pollution. Thus, developing crops with high N utilization efficiency (NUE) is one of the goals of plant biotechnology (Han et al., 2015). Unlike previous studies, which explored N uptake- or metabolism-related genes, several recent studies reported genetic improvement of NUE through the overexpression or heterologous expression of several transgenes (Liu et al., 2022; Sandhu et al., 2021). However, promising strategies for improving NUE that do not involve gene transfer and can be easily applied to commercial food production have yet to be established. A recent weighted gene coexpression network analysis (WGCNA) of rice cultivars displaying differential N deficiency responses revealed a gene network regulating N deficiency responses in roots and predicted OsHHO3 as a central regulator in this network (Ueda et al., 2020). OsHHO3 is a member of the NIGT1/HHO subfamily of the GARP/G2-like transcription factor family, which consists of a nitrate-inducible NIGT1 protein and four nitrate-noninducible HHO proteins in rice (Sawaki et al., 2013; Ueda et al., 2020). To study whether OsHHO3 regulates NUE in rice, we generated four independent OsHHO3 overexpression (OsHHO3-OX) lines and OsHHO3-edited knockout mutants (oshho3-KO) in Nipponbare (japonica rice) background and investigated their NUE (Figures S1 and S2). In contrast with OsHHO3-OX seedlings, the oshho3-KO seedlings showed enhanced growth and greater shoot and root dry weight, especially under N-deficient conditions (Figure 1a–c; Figures S3–S5). Additionally, chlorophyll content and the maximum quantum yield of photosystem II (Fv/Fm) were higher in oshho3-KO leaves than in OsHHO3-OX leaves under N-deficient conditions, suggesting that OsHHO3 negatively regulates the utilization efficiency of N, particularly NH 4 + $$ {\mathrm{NH}}_4^{+} $$ . The effects caused by these genetic manipulations were still prominent in 120-day-old plants grown in N-deficient soil (Figure 1d; Figure S6), and 150-day-old oshho3-KO and OsHHO3-OX plants showed superior and inferior agronomic traits, respectively, especially in N-deficient soil. Notably, OsHHO3 expression-level changes influenced tiller number, resulting in increased and decreased per-plant yield of oshho3-KO and OsHHO3-OX lines, respectively (Figure 1e–g; Figure S7). Transcriptome analysis of oshho3-KO rice revealed that OsHHO3 inactivation affected gene expression more profoundly in roots than in shoots (Figure 1h; Table S1–S4). Heatmap illustrated the upregulation of ammonium transporter (AMT) and nitrate transporter (NRT) genes and other N assimilation-related genes in oshho3-KO roots (Figure S8). Among these upregulations, the upregulation of OsAMT1;1, OsAMT1;2 and OsAMT1;3 was likely most critical because these three AMT1 genes are responsible for more than 95% of NH 4 + $$ {\mathrm{NH}}_4^{+} $$ uptake activity in rice (Konishi and Ma, 2021). In roots, other than four unknown genes and one HSP gene, OsAMT1;3 was the most highly upregulated gene (Table S1), and OsAMT1;1 and OsAMT1;2 were also markedly activated (Figure 1h). NH 4 + $$ {\mathrm{NH}}_4^{+} $$ deficiency-induced expression of these AMT1 genes in roots (Kumar et al., 2003) was similarly enhanced in oshho3-KO seedlings but was mitigated in OsHHO3-OX seedlings (Figure 1i). Consistently, NH 4 + $$ {\mathrm{NH}}_4^{+} $$ uptake activity and accumulation were higher in oshho3-KO seedlings but lower in OsHHO3-OX seedlings compared with the wild type (WT), especially under N deficiency stress (Figure 1j–l). Chromatin immunoprecipitation (ChIP) and cotransfection assays revealed that OsHHO3 represses AMT1 gene promoters by binding to sequences identical to or resembling the NIGT1-binding motifs (Sawaki et al., 2013; Figure S9a,b). Another cotransfection assay revealed that the activities of these AMT1 promoters were higher in oshho3-KO protoplasts but lower in OsHHO3-OX protoplasts (Figure S9c), further confirming OsHHO3 as the transcriptional repressor responsible for the synchronous regulation of three critical AMT1 genes and NH 4 + $$ {\mathrm{NH}}_4^{+} $$ uptake activity. Although OsHHO3 inactivation upregulated NRT genes and increased NO 3 − $$ {\mathrm{NO}}_3^{-} $$ uptake slightly, its effects were much more potent on NH 4 + $$ {\mathrm{NH}}_4^{+} $$ uptake (Figures S8 and S10). OsHHO3 expression gradually declined during the N-free treatment and remained low even after this treatment (Figure 1m). Examining OsHHO3 levels in 13 rice cultivars used in the previous WGCNA (Ueda et al., 2020) revealed that the magnitude of the N deficiency-induced decrease in OsHHO3 expression differed among the 13 cultivars (Figure 1n,o). Therefore, negative correlations between OsHHO3 and AMT1 expression levels were investigated and indeed clarified by scatterplots (Figure S11a; Figure 1o). Furthermore, negative correlations between OsHHO3 expression levels and shoot or root biomass were also detected (Figure 1p). Interestingly, three cultivars (Kasalath, Shoni and Arc5955), displaying lower reduction in OsHHO3 expression and weaker activation of AMT1 genes under N deficiency stress, exhibited more serious growth impairment than the other 10 cultivars under N-deficient conditions (Figure S11b,c). Hence, natural variation in OsHHO3 expression level may be one of the critical genetic factors responsible for the differences in AMT1 expression and NUE among rice cultivars. Consistently, we found that there are SNPs uniquely conserved only in Kasalath, Shoni and Arc5955 but not in other 10 cultivars (Figure S12), suggesting that the polymorphisms in the OsHHO3 promoter sequence may be associated with reduced downregulation of OsHHO3 under N-deficient conditions and low NUEs in Kasalath, Shoni and Arc5955. The synchronous upregulation of essential AMT1 genes can explain why OsHHO3 was predicted as a central regulator in the N deficiency response network and why growth was reduced in OsHHO3-OX plants but improved in oshho3-KO plants. However, OsHHO3 may also simultaneously regulate genes not directly involved in N uptake to mediate N deficiency responses properly. In fact, expression levels of cytokinin biosynthesis genes OsIPT4, OsIPT5 and OsCYP735A were altered in OsHHO3-OX and oshho3-KO plants (Figure S13a). Cotransfection and ChIP assays consistently showed that OsHHO3 represses OsIPT4 and OsIPT5 promoters in planta (Figure S13b,c). Consistently, the cytokinin content of oshho3-KO and OsHHO3-OX seedlings was higher and lower than that the WT, respectively (Figure S13d). Since active cytokinins are necessary for axillary bud outgrowth (Ohashi et al., 2017), and the number of panicle-bearing active tillers is a critical determinant of grain yield in rice (Sakamoto and Matsuoka, 2008), the higher cytokinin content of oshho3-KO plants may be responsible for their greater tiller number and grain yield. These results suggest that OsHHO3 is critically involved in the multifaceted regulation of N deficiency responses. We demonstrated that the inactivation of OsHHO3 facilitates NH 4 + $$ {\mathrm{NH}}_4^{+} $$ uptake and improves NUE, growth and yield in the low N environment because OsHHO3 is a repressor of essential AMT1 genes. Since the effects of OsHHO3 inactivation were potent even in Nipponbare, which shows a relatively lower expression level of OsHHO3 than most other cultivars examined (Figure 1n), OsHHO3 inactivation will improve NUE in most commercially grown rice cultivars. Future studies on yield-related indexes, such as lodging and disease resistance, in the field will be needed to substantiate the positive effect of the OsHHO3 inactivation on rice yields. However, this study provides a strong foundation for reducing N fertilizer input in rice production and also suggests that identification of the negative regulators of N acquisition and utilization in other major crops will facilitate the establishment of sustainable agriculture. In Arabidopsis, reductions in the activity of NIGT1 repressors for NRT2 genes in low NO 3 − $$ {\mathrm{NO}}_3^{-} $$ environments enhance NO 3 − $$ {\mathrm{NO}}_3^{-} $$ uptake activity (Kiba et al., 2018), implicating that the release of the negative regulation of N uptake, which is necessary to avoid energy-wasting under N-sufficient environments, is critical for enhancing N uptake in N-deficient environments. Since rice prefers NH 4 + $$ {\mathrm{NH}}_4^{+} $$ rather than NO 3 − $$ {\mathrm{NO}}_3^{-} $$ as the N source, OsHHO3, a protein structurally very close to NIGT1s in Arabidopsis and rice but not encoded by any nitrate-inducible gene, may predominately act as a pivotal regulator to enhance N uptake in rice. Analyses of the NIGT1/HHO families may expand the possibility of genetically improving NUE in various crops. We thank the National Agriculture and Foot Research Organization (NARO) for providing the pZH_OsU6gRNA_PubiMMCas9 vector. This work was supported, in part, by the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (grant no. JPMJCR15O5) and the Japan Society for the Promotion of Science KAKENHI (grant no. 22H04977). The authors declare no competing interests. S.Y. initiated the project. K.Y., Y.S. and S.Y. designed the experiments and analysed the data. K.L., Y.S., N.O., Y.M. and Y.U. performed the experiments. K.Y. and S.Y. wrote the manuscript. Figure S1 OsHHO3 transcript levels in four independent OsHHO3-OX lines. Figure S2 Sequence analysis of the OsHHO3 locus in four independent oshho3-KO lines. Figure S3 WT, oshho3-KO, and OsHHO3-OX seedlings grown under different N conditions. Figure S4 Growth retardation phenotypes of four independent OsHHO3-OX lines. Figure S5 Enhanced growth phenotypes of four independent oshho3-KO lines under the low NH 4 + $$ {\mathrm{NH}}_4^{+} $$ condition. Figure S6. Effects of OsHHO3 inactivation and overexpression on growth at the late vegetative growth stage. Figure S7 Effects of OsHHO3 inactivation and overexpression on agronomic traits. Figure S8 Heatmaps presenting OsHHO3 inactivation-induced changes in the expression levels of N uptake-, transport-, or assimilation-related genes in shoots and roots. Figure S9 Direct suppression of OsAMT1;1, OsAMT1;2, and OsAMT1;3 by OsHHO3. Figure S10 NO 3 − $$ {\mathrm{NO}}_3^{-} $$ uptake in oshho3-KO and OsHHO3-OX seedlings. Figure S11 Variation in N deficiency-induced AMT1 and OsHHO3 expression levels and growth retardation among rice cultivars. Figure S12 Comparison of OsHHO3 promoter sequences from 13 rice cultivars. Figure S13 OsHHO3 regulates cytokinin biosynthesis. Table S1 Top 50 genes upregulated in the roots of oshho3-KO plants. Table S2 Top 50 genes downregulated in the roots of oshho3-KO plants. Table S3 Top 50 genes upregulated in the shoots of oshho3-KO plants. Table S4 Top 50 genes downregulated in the shoots of oshho3-KO plants. Table S5 List of oligonucleotides used for genome editing, cloning, and gene expression analysis. Table S6 Predicted off-target sites of the guide RNA at the OsHHO3 locus. Table S7 List of PCR primers for ChIP-qPCR. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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