Suppressors of Cytokinin Receptor Mutant pal1/ohk4 Confer Favorable Alleles of Grain Number 1a (Gn1a) for Improving Grain Yield in japonica Rice

doi:10.1016/j.rsci.2025.10.008

Abstract

Abstract:

Panicle number per plant, grain number per panicle, and grain weight are three key factors influencing rice grain yield. Gn1a, a major QTL for grain number per panicle, encodes the cytokinin oxidase/dehydrogenase (CKX) OsCKX2. While the use of elite Gn1a alleles has been well documented in indica rice cultivars, their potential in japonica rice remains largely unexplored. In this study, we characterized three suppressor mutants of the rice cytokinin receptor mutant pal1/ohk4 and found that all causal genes were novel alleles of Gn1a identified through the MutMap approach. These three suppressor mutants caused single amino acid substitutions in the FAD-binding domain (G556D and G156D) and the cytokinin-binding domain (Y357C), resulting in significantly reduced enzymatic activity of OsCKX2 and elevated cytokinin levels in the panicle. Haplotype analysis of Gn1a using a natural population from the 3K Rice Genomes Project showed that G556D, G156D, and Y357C were novel alleles of Gn1a. G556, G156, and Y357 were highly conserved, whereas four natural variants G54A, A105V, H116R, and N535K identified in different haplotypes of Gn1a showed extremely low conservation. By backcrossing the suppressor mutants with their original wild-type Huaidao 5, an elite japonica rice variety, we developed improved lines carrying only the gn1a mutation. The improved lines showed a significant increase in grain number per panicle, grain weight, panicle number per plant, plant height, and stem thickness, leading to a 25.7%-28.7% increase in grain yield per plot compared with Huaidao 5. This study provides valuable Gn1a alleles ‌for synergistic improvement of the three key yield factors and offers germplasm resources for high-yielding breeding in japonica rice.

Key words: grain number, panicle size, cytokinin, Gn1a, OsCKX2, OHK4/OsHK4, suppressor, Oryza sativa

Jia Huichao, Chun Yan, Ashmit Kumar, Mo Tianyu, Wang Haifeng, Guo Shengyuan, Fang Jingjing, Zhao Jinfeng, Sun Wei, Zhang Shiyong, Yuan Shoujiang, Li Xueyong. Suppressors of Cytokinin Receptor Mutant pal1/ohk4 Confer Favorable Alleles of Grain Number 1a (Gn1a) for Improving Grain Yield in japonica Rice[J]. Rice Science, 2026, 33(1): 81-98.

Figures/Tables 7

Fig. 1. Phenotypic analysis of wild type (WT, Huaidao 5), pal1, and suppressor mutants pal1s35, pal1s205, and pal1s242. A, Morphology of mature plants of WT, pal1, and suppressor mutants. Scale bar, 15 cm. B and C, Morphology of closed (B) and spread (C) panicles generated on the primary tillers of WT, pal1, and suppressor mutants. Scale bars, 3 cm. D-I, Comparison of plant height (D), primary tiller number (E), panicle length (F), primary branch number (G), secondary branch number (H), and grain number per panicle (I) among WT, pal1, and suppressor mutants. Panicles in (F) to (I) include only those generated on primary tillers. J and K, Cross‐sections (J) and front views (K) of the first (I), second (II), and third (III) internodes of WT, pal1, and suppressor mutants. Scale bars, 1 mm in (J) and 1 cm in (K), respectively. L-N, Comparison of the diameters of the first (internode I, L), second (internode II, M), and third (internode III, N) internodes among WT, pal1, and suppressor mutants. In D-I and L-N, data are mean ± SD (n = 10). Different lowercase letters above bars indicate significant differences at the 0.05 level by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig. 2. Cloning of causal genes of three pal1 suppressor mutants through MutMap. A, Identification of single nucleotide polymorphisms (SNPs) and candidate genes tightly linked with three pal1 suppressor mutants pal1s35, pal1s205, and pal1s242 through MutMap. The most likely candidate gene LOC_Os01g10110 (OsCKX2) is highlighted in red. Mut, Mutation; Chr, Chromosome; WT, Wild type; AA, Amino acid. B, Schematic representation of Gn1a/OsCKX2 gene structure and base and amino acid variations at the three sites in the suppressor mutants. The boxes and lines represent exons and introns, respectively. The start codon (ATG) and stop codon (TGA) are indicated. C, Alignment of partial sequences surrounding the mutation site indicated by a triangle in cytokinin oxidase/dehydrogenase (CKX) homologs from different plant species, including Oryza sativa OsCKX2 (NP_001393243), OsCKX1 (NP_001388401), OsCKX5 (NP_001388663), OsCKX6 (XP_015624918), OsCKX7 (XP_015624361), and OsCKX10 (XP_025881924); Zea mays ZmCKX1 (NP_001105591), ZmCKX8 (AQK59741), and ZmCKX10 (XP_008660924); Arabidopsis thaliana AtCKX1 (NP_181682) and AtCKX6 (NP_191903); Hordeum vulgare HvCKX1 (KAE8806376) and HvCKX7 (XP_044954246); and Triticum aestivum TaCKX1 (AEK84310), TaCKX2 (AEV76971), and TaCKX7 (XP_044409379).

Fig. 3. Knocking out Gn1a in pal1 mutant (pal1 gn1a-ko1 and pal1 gn1a-ko2) using CRISPR/Cas9 system. A, Schematic representation of Gn1a target site and target sequence alignment of wild type (WT, Huaidao 5) and knockout (KO) lines (Gnla-KO1 and Gnla-KO2). The guide RNA target sequence is shown in blue, and the protospacer adjacent motif (PAM) sequence is underlined. Two mutations generated by CRISPR/Cas9 are shown in the lower panel. The start codon (ATG) and stop codon (TGA) are indicated. B, Morphology of mature plants of WT, pal1, and two homozygous KO lines. Scale bar, 15 cm. C and D, Morphology of closed (C) and spread (D) panicles generated on the primary tillers of WT, pal1, and two homozygous KO lines. Scale bars, 3 cm. E-J, Comparison of plant height (E), primary tiller number (F), panicle length (G), primary branch number (H), secondary branch number (I), and grain number per panicle (J) among WT, pal1, and two homozygous KO lines. Panicles in (G) to (J) only include those generated on primary tillers. Data are mean ± SD (n = 10). Different lowercase letters above histograms indicate significant differences at the 0.05 level by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig. 4. Increased endogenous cytokinin levels and responsiveness in pal1 suppressor mutants pal1s35, pal1s205, and pal1s242. A, Seedling roots of wild type (WT), pal1, and pal1 suppressor mutants treated with various concentrations of trans-zeatin (tZ). Scale bars, 2 cm. B, Comparison of root length in WT, pal1, and pal1 suppressor mutants after 10 d of treatment with various concentrations of tZ. C, Effect of different concentrations of tZ on dark-induced leaf senescence in WT, pal1, and pal1 suppressor mutants. D, Quantification of chlorophyll content in leaves of WT, pal1, and pal1 suppressor mutants on 10 d of dark treatment. E, Comparison of cytokinin (tZ and isopentenyladenine) contents in young panicles (~0.5 cm) of WT, pal1, and pal1 suppressor mutants. F, Relative expression levels of type-A OsRRs genes in young panicles (~0.5 cm) of WT, pal1, and pal1 suppressor mutants. The rice Ubiquitin gene (LOC_Os03g13170) served as an internal control. G and H, Relative expression levels of genes related to meristem maintenance (G) and panicle development (H) in young panicles (~0.5 cm) of WT, pal1, and pal1 suppressor mutants. The rice Ubiquitin gene (LOC_Os03g13170) served as an internal control. In E-H, data are mean ± SD (n = 3). In E, ns, *, and ** indicate P > 0.05, P < 0.05, and P < 0.01, respectively, by Student’s t-test. In F-H, different lowercase letters above the bars indicate significant differences at the 0.05 level by one-way ANOVA followed by Tukey’s multiple comparison test.

Fig. 5. Effects of three variants G556D, G156D, and Y357C on OsCKX2 protein structure and enzymatic activity. A, Ribbon diagram of the three-dimensional structure of OsCKX2 predicted by AlphaFold3. The Flavin adenine dinucleotide (FAD)-binding domain and substrate-binding domain are shown in blue and yellow, respectively. The FAD molecule is represented as a stick model in green. B-G, Diagrams showing mutation sites and their surrounding structure: G556 (B), G556D (C), G156 (D), G156D (E), Y357 (F), and Y357C (G). Mutation sites are shown in red. The FAD molecule is represented as a stick model in green and broken lines indicate potential H-bonds with FAD. H, In vitro OsCKX2 activity assay of recombinant maltose-binding protein (MBP)-OsCKX2 (WT) and three variants containing G556D, G156D, and Y357C. Data are mean ± SD (n = 3). **, P < 0.01 by Student’s t-test.

Fig. 6. Improvement of agronomic traits of Huaidao 5 by pal1 suppressor mutation. A, Morphology of mature plants of Huaidao 5 (HD5) and gn1aG556D. Scale bar, 15 cm. B and C, Morphologies of spread (B) and closed (C) panicles from the primary tillers of HD5 and gn1aG556D lines. Scale bars, 3 cm. D-I, Comparison of plant height (D), panicle number per plant (E), panicle length (F), primary branch number (G), secondary branch number (H), and grain number per panicle (I) between WT and gn1aG556D lines. Panicles in (D) to (I) include only those from primary tillers. J and K, Front views (J) and cross-sections (K) of the first (I), second (II), and third (III) internodes of HD5 and gn1aG556D. Scale bars, 1 cm in (J) and 1 mm in (K). L-N, Comparison of the diameters of first (internode I, L), second (internode II, M), and third (internode III, N) internodes between HD5 and gn1aG556D. O, Grain yield per plot (1.5 m2) of HD5 and gn1aG556D lines in the field. In D-I and L-O, data are mean ± SD (n = 10). **, P < 0.01 by Student’s t-test.

Fig. 7. Haplotype analysis of Gn1a. A, Structure and natural variations of Gn1a gene in the 3K Rice Genomes dataset. AA, Amino acid; UTR, Untranslated region. Blue, green, and yellow represent single nucleotide polymorphisms or insert/deletions in the promoter region, 5′-UTR, and coding region, respectively. B, Sequence alignment around the variation sites among each haplotype in the coding region (▼, Difference variation sites). Os, Oryza sativa; Zm, Zea mays; At, Arabidopsis thaliana; Hv, Hordeum vulgare. C, Composition of Hap1, Hap2, Hap3, and Hap4 in rice subspecies. D, Comparison of panicle length among Hap1, Hap2, Hap3, and Hap4. Different lowercase letters indicate significant differences (P < 0.05) by one-way ANOVA followed by Tukey’s multiple comparison test. E, Statistical analysis of panicle length for Hap1, Hap2, Hap3, and Hap4. F, Comparison of transcriptional activity of promoters from different Gnla haplotypes. The luciferase gene driven by the Gn1a promoter was transiently expressed in Nicotoana benthamiana leaves.

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