CHPO coordinates chilling recovery and nitrogen use in rice

Data availability

The RNA-seq data, DAP-seq data and CUT&Tag-seq data have been deposited in the Genome Sequence Archive in the National Genomics Data Center (https://ngdc.cncb.ac.cn/, bioproject ID PRJCA031750). CHPO sequence data of Fig. 5g and Extended Data Fig. 10a–c are available from the Rice Annotation Project Database (RAP-DB) (https://rapdb.dna.naro.go.jp/), Pangenome Database of Wild and Cultivated Rice (RicePandb) (http://ricepandb.ncgr.ac.cn/) and the dataset associated with the published study ‘Multiple domestications of Asian rice’29 (https://doi.org/10.1038/s41477-023-01476-z). CHPO GCG allele data of Extended Data Fig. 10d–f are available from Rice Variation Map v.2.0 (https://ricevarmap.ncpgr.cn/). Geographical distribution information of varieties of Extended Data Fig. 10d–f is available from Rice Variation Map v.2.0 (https://ricevarmap.ncpgr.cn/) and Sandbox (https://sandbox.genesys-pgr.org). Soil temperature data of Extended Data Fig. 10d–f are available from the NOAA-CIRES-DOE 20th Century Reanalysis v.3 dataset provided by the NOAA Physical Sciences Laboratory, using the daily long-term mean soil temperature file tsoil.day.ltm.nc (https://psl.noaa.gov/data/gridded/data.20thC_ReanV3.html). Altitude data of Extended Data Fig. 10d–f are available from R package elevatr (https://cran.r-project.org/web/packages/elevatr/index.html). Soil nitrogen content data of Extended Data Fig. 10d–f are obtained from the Global Soil Dataset for Earth System Modeling (http://globalchange.bnu.edu.cn/research/soilw). Full version of all gels and blots are provided in Supplementary Fig. 1. Source data are provided with this paper.

Code availability

This paper does not report original code, custom software or custom algorithms. Data analyses and figure generation were performed using standard functions from R and publicly available R packages, as described in the Methods.

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Acknowledgements

We thank C. Chu for providing the genetic materials of OsTCP19 and K. Wu for providing the genetic materials of OsGRF4 and NGR5.

Funding

This work was supported by the Major Program of the National Natural Science Foundation of China (grant no. 32494772), and the Basic Science Center Project of the National Natural Science Foundation of China (grant no. 32388201), the National Natural Science Foundation of China (grant no. 32470291), and the Science and Technology Innovation 2030—Agricultural Biological Breeding Major Projects (grant no. 2022ZD040040104).

Author information

Authors and Affiliations

State Key Laboratory of Forage Breeding-by-Design and Utilization, Institute of Botany, Chinese Academy of Sciences, Beijing, China

Jie Cao, Wei Luo & Kang Chong

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China

Jie Cao, Yunyuan Xu, Zhitao Li, Wei Luo & Kang Chong

China National Botanical Garden, Beijing, China

Jie Cao, Yunyuan Xu, Zhitao Li, Jingdan Han, Song Ge, Wei Luo & Kang Chong

University of Chinese Academy of Sciences, Beijing, China

Jie Cao, Song Ge, Hong Wang & Kang Chong

State Key Laboratory of Plant Diversity and Specialty Crops, Institute of Botany, Chinese Academy of Sciences, Beijing, China

Jingdan Han & Song Ge

Yazhouwan National Laboratory, Sanya, China

Qian Qian

Authors

Jie Cao

Yunyuan Xu

Zhitao Li

Jingdan Han

Qian Qian

Song Ge

Hong Wang

Wei Luo

Kang Chong

Contributions

J.C., Y.X., H.W., W.L. and K.C. designed the experiments, analysed the data and wrote the paper. J.C. performed most of the experiments. W.L. performed experiments on the generation of the RIL genetic population, QTL analysis and genetic analysis. J.H. and S.G. provided the genetic variation data of wild rice and landrace, as well as performed evolutionary analysis. Z.L. performed GWAS analysis, and Q.Q. designed the genetic experiments and analysed the data. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to

Hong Wang, Wei Luo or Kang Chong.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Nicolaus von Wiren and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Identification of candidate gene of qCR2 locus.

a, Post-chilling tillering recovery in KY131 and ZF802 under equivalent survival rates.P = 0.931 (survival rate); P = 1.233 × 10−5 (tillering rate of surviving plants); statistical significance was determined using a two-sided Student’s t-test (n = 4 biologically independent experiments). Scale bars, 5 cm for plant and 5 mm for the shoot bases. Red arrows indicated tiller. b and c, Correlation analysis between survival rates and tillering rates of surviving plants in KY131, ZF802 (b) and recombinant inbred lines (RILs) (c). Pearson’s r was calculated to assess the linear correlation between the two variables, and R² was calculated as r². Pearson’s r value see and R² value is shown in the plot. In c, the blue line indicates the ordinary least-squares linear regression fit; grey shaded band represents the 95% confidence interval of the fitted regression line. d, Analysis of QTL associated with the chilling resilience in RILs. QTLs threshold, logarithm of odds (LOD) ≥ 3, P < 0.05. P-values were generated by genome-wide scanning in QTL.gCIMapping.GUI v2.039 using its random-QTL-effect mixed linear model framework, with likelihood-ratio tests used to evaluate QTL effects. No multiple-comparison adjustment was applied to P-values39. e, Fine-mapping of the CHPO gene (n = 3 biologically independent experiments). Yellow and white blocks represent introgressed segments from KY131 (japonica) and ZF802 (indica), respectively. f, Protein structural predictions for CHPOjap and CHPOind. The white square indicated the predicted secondary-structure differences between CHPOjap and CHPOind arising from variation in the alanine repeat. g, Chilling-induced expression patterns of candidate genes identified by qRT-PCR (n = 3 biologically independent experiments). Letters denote statistically significant differences (P.adjust < 0.05, one-way ANOVA followed by Tukey’s HSD test, P.adjust values see Source data). In a,e,g, data were presented as mean ± s.e.m.

Source data

Extended Data Fig. 2 Identification and representative phenotypic images of CHPO transgenic lines.

a, Identification of the loss-of-function of CHPO (chpo-1, chpo-2 and chpo-3) using sanger sequencing. chpo-1 carried a 1-bp deletion at +62 bp, resulting in a frameshift. chpo-2 contains a substitution spanning +453 to +1039 bp, replaced by a 12-bp insertion. chpo-3 harbored a 1-bp insertion between +205 and +206 bp, resulting in a frameshift. b,d,f, Representative phenotypes showing tillering of surviving plants in chpo-1, chpo-2, KY131, OEjap-1, OEjap-2, OEind-1, OEind-2 and ZH11 after 4 °C treatment and recovery growth for 40 days. Untreated seedlings served as controls for the 40 day post-chilling recovery group. Red arrows indicated tillers. Scale bars, 5 mm. c,e Relative expression of CHPO in CHPOjap overexpression lines and CHPOind overexpression lines in the ZH11 using qRT-PCR. Data were presented as mean ± s.e.m. n = 3 biologically independent experiments. Statistical significance was determined using a two-sided student’s t-test (**, P < 0.01). P = 1.217 × 10−8 (OEjap-1 vs ZH11) and P = 7.953 × 10−8 (OEjap-2 vs ZH11) in b; P = 2.414 × 10−4 (OEind-1 vs ZH11) and P = 1.750 × 10−4 (OEind-2 vs ZH11) in c.

Source data

Extended Data Fig. 3 RNA-seq analysis of CHPOjap transgenic line.

a, Subcellular localization of CHPOjap and CHPOind in rice protoplasts at 25 °C and 4 °C. Box plots show the median as the centre line, the first and third quartiles as the lower and upper bounds of the box, and whiskers extending to the smallest and largest observations within 1.5 × the interquartile range from the lower and upper quartiles. n = 3 cells of CHPOjap and n = 6 cells of CHPOind. P = 0.002 (CHPOjap); P = 0.806 (CHPOind). b, Diagram of transcriptome in the OEjap-2 and ZH11. Arrows indicated the time points at which the samples were harvested. n = 3 biologically independent experiments. c, PCA analysis of transcriptome profiles across all samples. Grey: 4 °C, 0 h; blue: 4 °C, 48 h; yellow: Re 24 h after 4 °C 48 h. d, Clustered heatmap of transcriptome profiles across all groups. e, Volcano plot of differentially expressed genes (DEGs). Blue dots indicated downregulated DEGs (log2 fold change ≤ –1, P.adjust < 0.05), while red dots indicated upregulated DEGs (log2 fold change ≥ 1, P.adjust < 0.05). P values were calculated by two-sided Wald tests based on negative binomial generalized linear models and adjusted using the Benjamini–Hochberg method40. f, KEGG pathway enrichment analysis of DEGs between OEjap-2 and ZH11 at recovery stage, performed using clusterProfiler. Pathways with significant enrichment (q-value < 0.05) are shown. g, Log2 fold changes of genes within enriched KEGG pathways in OEjap-2 relative to ZH11. h, Log2 fold changes of known chilling tolerance genes and CHPOjap target genes in OEjap-2 relative to ZH11. i, Changes in expression of chilling tolerance and nitrogen metabolism genes in the cog4 mutant and its wild type NIP before and after exposure to 4 °C. Heatmap colors indicated log2(expression ratio cog4-1/NIP); n = 3 biologically independent experiments. Exact P-values see Source Data. In a and i, statistical significance was determined using a two-sided student’s t-test (NS, P ≥ 0.05; *, P < 0.05; **, P < 0.01).

Source data

Extended Data Fig. 4 RT-qPCR validation of CHPOjap-mediated genes.

a, Regulation of nitrogen metabolism-related genes by CHPOjap in different tissues at various stages of chilling treatment. At recovery stage, three concentrations of NH4NO3 (0.1, 1.0, and 5.0 mM) were applied. b, RT-qPCR validation of CHPOjap-mediated regulation of genes related to tillering, chilling tolerance, and nitrogen metabolism in chpo-1, chpo-2, OEjap-1, OEjap-2 and their wild types. In this figure, log2-transformed expression levels ratios of measured by RT-qPCR; heatmap colors indicate log2 (expression ratio). n = 3 biologically independent experiments. Statistical significance was determined using a two-sided student’s t-test (*, P < 0.05; **, P < 0.01), exact P values see Source Data.

Source data

Extended Data Fig. 5 CHPOjap is associated with nitrogen availability at recovery stage.

a, Survival rates and tillering rates of surviving plants in chpo-2 and KY131 under varying nitrogen concentration at recovery stage. b, Survival rates and tillering rates of surviving plants in chpo-3 and ZH11 under varying nitrogen concentration at recovery stage. c, Survival rates and tillering rates of surviving plants in OEjap-1 and ZH11 at different nitrogen concentration post-chilling. d, Survival rates and tillering rates of surviving plants in cog4-1 and NIP at different nitrogen concentration under varying nitrogen concentration at recovery stage. e, f, Survival rates and tillering rates of surviving plants in osgrf4, OsGRF4-OE, ngr5, NGR5-OE and WYJ7. In this figure, n = 3 biologically independent experiments; data were presented as mean ± s.e.m; statistical significance was determined using a two-sided student’s t-test (NS, P ≥ 0.05; *, P < 0.05; **, P < 0.01), exact P values see Source Data. Interaction effects between genotype and nitrogen concentration were assessed by two-way ANOVA. P(G × N) denoted the significance of the interaction. Scale bars, 5 cm.

Source data

Extended Data Fig. 6 CHPOjap enhances NUE at recovery stage.

a, Root length, plant height and first tiller length of chpo-1 and KY131 under varying nitrogen concentration at recovery stage (n = 30 plants). b, Dry biomass per plant, root length, plant height and first tiller length of chpo-2 and KY131 under varying nitrogen concentration at recovery stage (n = 10 plants). c, Dry biomass per plant, Root length, plant height and first tiller length of chpo-3 and ZH11 under varying nitrogen concentration at recovery stage (n = 10 plants). d, Dry biomass per plant, root length, plant height and first tiller length of OEjap-1 and ZH11 under varying nitrogen concentration at recovery stage (n = 10 plants). e, Root length, plant height and first tiller length of OEjap-2 and ZH11 under varying nitrogen concentration at recovery stage (n = 30 plants). f, Dry biomass per plant, root length, plant height of chpo-1, chpo-2, KY131, OEjap-1, OEjap-2 and ZH11 before chilling treatment (n = 10 plants). g, Dry biomass per plant, root length, plant height and first tiller length of cog4-1 and ZH11 under varying nitrogen concentration at recovery stage (n = 10 plants). In this figure, box plots show the median as the centre line, the first and third quartiles as the lower and upper bounds of the box, and whiskers extending to the smallest and largest observations within 1.5 × the interquartile range from the lower and upper quartiles; first tiller length was recorded as NA for surviving plants without tillers. Statistical significance was determined using a two-sided student’s t-test (NS, P ≥ 0.05; *, P < 0.05; **, P < 0.01), exact P values see Source Data. Interaction effects between genotype and nitrogen concentration were assessed by two-way ANOVA. P(G × N) denoted the significance of the interaction. Scale bars, 5 mm.

Source data

Extended Data Fig. 7 CHPOjap contributes to post-chilling rice yield.

a, Tiller number of field-transplanted KY131, chpo-1, ZH11 and OEjap−2 (surviving plants with tillering) under varying nitrogen concentration at recovery stage. n see Source Data. Scale bars, 10 cm. Plants were transplanted to the field in Beijing. Box plots show the median as the centre line, the first and third quartiles as the lower and upper bounds of the box, and whiskers extending to the smallest and largest observations within 1.5 × the interquartile range from the lower and upper quartiles. b, Agronomic traits of field-transplanted ZH11 and chpo-3 (surviving plants with tillering) under varying nitrogen concentration at recovery stage. n = 15 plants. n. Scale bars, 10 cm. Plants were transplanted to the field in Lingshui, Hainan province. c, CHPOjap transgenic lines did not alter the tiller number without chilling stress. n, see Source Data; Scale bars, 10 cm. d, Nitrogen distribution of field-transplanted ZH11 and chpo-3 (surviving plants with tillering) under varying nitrogen concentration at recovery stage (n = 5 independent biological experiments). In this figure, plants subjected to the same field management practices, with all plants grown under the same moderate external nitrogen application; data were presented as mean ± s.e.m in b–d; statistical significance was determined using a two-sided student’s t-test (NS, P ≥ 0.05; *, P < 0.05; **, P < 0.01), exact P values see Source Data. Interaction effects between genotype and nitrogen concentration were assessed by two-way ANOVA. P(G × N) denoted the significance of the interaction; S, seed; O, Other (d).

Source data

Extended Data Fig. 8 CHPOjap targets OsTCP19 and OsNRT2.4 for tillering recovery.

a, Genome-wide distribution of CHPOjap-binding peaks identified by DAP-seq. b, Putative CHPOjap-binding motifs detected by DAP-seq assays. c, DAP-seq showing that CHPOjap bound the promoters of OsTCP19 and OsNRT2.4. Peak regions indicated by red lines were identified using MACS3. Black arrows indicated gene regions, with the direction of the arrows showing the transcription direction. d, OsTCP19 negatively regulated chilling resilience in rice. e, Identification of osnrt2.4 mutant by sanger sequencing. f, OsNRT2.4 positively regulated chilling tolerance and resilience. In d,f, n = 3 biological independent experiments; scale bars, 5 cm of plants and 5 mm of sheet bases; red arrows indicated tillers; data were presented as mean ± s.e.m.; In d, letters denote statistically significant differences (P.adjust <0.05, one-way ANOVA followed by Tukey’s HSD test, exact P.adjust values see Source Data). In f, P-values were determined using two-sided student’s t-test; *, P < 0.05; **, P < 0.01, P = 0.002 (survival rate); P = 0.013 (tillering rate of surviving plants).

Source data

Extended Data Fig. 9 Differential DNA-binding motifs and regulatory pattern of CHPOjap and CHPOind.

a, Differentially bound DNA motifs analysis between CHPOjap and CHPOind based on CUT&Tag-seq data. b, EMSA assays showing binding affinity of CHPOjap and CHPOind to probes 3×ATATATTATC and 3×CCAATCG. c, EMSA assays showing binding affinity of CHPOjap and CHPOind to the probes of OsTCP19 and OsNRT2.4 promoters. The probe sequences see Supplementary Table 8. The numbers (EMSA assays) represented the shift probe concentration measured by ImageJ, and the values for each band were normalized by dividing by the value of the first shift probe band. The numbers (western blot assays) represented the protein concentration measured by ImageJ, and the values for each band were normalized by dividing by the value of the first CHPO protein band. d, RT-qPCR analysis of the differential regulation of genes related to tillering, chilling tolerance, and nitrogen metabolism by CHPOjap and CHPOind. n = 3 biological independent experiments; green stars, potential CHPOjap target genes; yellow stars, potential CHPOind target genes, identified by CUT&Tag-seq, see Supplementary Table 5. Heatmap colors indicate log2 (expression ratio). P-values were determined using two-sided student’s t-test; *, P < 0.05; **, P < 0.01, exact P values see Source Data. For gel source data, see Supplementary Fig. 1c–h.

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Extended Data Fig. 10 Evolution and environmental adaption analysis of CHPO gene.

a, Whole-genome variation data from 684 accessions was used, including 137 Ruf, 77 Niv, 63 TEJ, 189 TRJ, and 218 indica, for phylogenetic analysis. A neighbor-joining tree was constructed to infer evolutionary relationships among these accessions. Accession names were color-coded based on species or subspecies. b, Phylogenetic analysis of the CHPO. The green dashed line indicates the temperate japonica genotype of CHPO. c, Haplotype network of CHPO. b and c, a total of 178 accessions (41 Ruf, 33 Niv, 39 TEJ, 30 TRJ and 35 indica, as high-depth dataset) and one O.barthii were used to construct the neighbor-joining tree. O.rufipogon (W1943, W3029, W3096, W3092) and O. nivara W1839 were collected in China. d, Association analysis ana between CHPO allele and annual accumulated temperature. e, Association analysis between CHPO allele and total soil nitrogen concentration. P values in d and e were calculated using a linear model including the first six principal components as covariates to correct for population structure. Two-sided Wald tests were used to test the association between genotype and phenotype. P values were not adjusted for multiple comparisons. f, The distribution of the CHPO allele across regions with varying levels of total soil nitrogen content and annual accumulated temperatures. In d–f, 3,335 georeferenced accession–location records with CHPO GCG-repeat genotype information (n = 1,780 of 6×GCG records, n = 1,555 of 2×GCG records) and matched environmental variables, detailed information is provided in the Source Data; box plots show the median as the centre line, the first and third quartiles as the lower and upper bounds of the box, and whiskers extending to the smallest and largest observations within 1.5 × the interquartile range from the lower and upper quartiles.

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Cao, J., Xu, Y., Li, Z. et al. CHPO coordinates chilling recovery and nitrogen use in rice.

Nature (2026). https://doi.org/10.1038/s41586-026-10682-6

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Received: 29 September 2025

Accepted: 20 May 2026

Published: 17 June 2026

Version of record: 17 June 2026

DOI: https://doi.org/10.1038/s41586-026-10682-6