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A receptor–kinase cascade confers cold-induced root growth inhibition in Arabidopsis

Abstract

Cold stress restricts root growth by disrupting stem cell activity in plants. C-REPEAT BINDING FACTORs (CBFs) are central regulators of cold signalling and also modulate root stem cell activity. While the receptor-like cytoplasmic kinase CRPK1 promotes CBF destabilization under cold stress, its regulatory partner remains unclear. Here we identify KINASE ON THE INSIDE (KOIN), a plasma-membrane-localized receptor-like kinase, as a crucial interactor of CRPK1. The loss of either KOIN or CRPK1 results in cold-insensitive root phenotypes, characterized by sustained primary root elongation and enhanced cortex cell proliferation via the 14-3-3–CBF3–SHR pathway. Under cold stress, KOIN undergoes endocytosis and is recycled back to the plasma membrane in a CRPK1-dependent manner. Although catalytically inactive, KOIN modulates CRPK1 protein levels and phosphorylation through a non-catalytic mechanism. These findings uncover a membrane-to-nucleus signalling module that integrates receptor trafficking with intracellular kinase activity to mediate cold-induced root growth inhibition in Arabidopsis.

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Fig. 1: CRPK1 promotes cold-induced inhibition of root growth.
Fig. 2: KOIN interacts with CRPK1 and mitigates cold-induced inhibition of root growth.
Fig. 3: CRPK1 is required for KOIN recycling to the PM.
Fig. 4: Phosphorylation-dependent interaction between CRPK1 and KOIN.
Fig. 5: CRPK1-mediated KOIN phosphorylation regulates cold-induced root growth inhibition.
Fig. 6: The KOIN–CRPK1–14-3-3 module acts upstream of CBFs to regulate cold-induced root growth inhibition.
Fig. 7: Proposed model of the KOIN–CRPK1 module in cold-induced inhibition of root growth.

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Data availability

All data supporting the findings of this study are available in the article, the extended data figures or the Supplementary information. The biological materials used in this study are available from the corresponding author upon reasonable request. The Arabidopsis gene sequences referenced in this study can be accessed through the TAIR database (https://www.arabidopsis.org/) under the following accession numbers: KOIN (AT5G58300), CRPK1 (AT1G16670), 14-3-3λ (AT5G10450), 14-3-3κ (AT5G65430), CBF1 (AT4G25490), CBF2 (AT4G25470), CBF3 (AT4G25480) and SHR (AT4G37650). Source data are provided with this paper.

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Acknowledgements

We thank Y. Guo for providing seeds of marker lines and helpful discussion. We thank Y. Liu and S. Wang for help in the imaging. This work was supported by grants from the National Natural Science Foundation of China (no. 32230005) and Pinduoduo-China Agricultural University Research Fund (no. PC2023B01001) to S.Y.

Author information

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Authors

Contributions

S.Y. designed the study. Xiuyue Zhang performed the experiments with the help of M.L. and Xiaoyan Zhang. Xiuyue Zhang, R.Z., Y.P., Y.S., X.W., W.Z. and Z.G. analysed the data. Xiuyue Zhang and S.Y. wrote the paper.

Corresponding author

Correspondence to Shuhua Yang.

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Extended data

Extended Data Fig. 1 Characterization of CRPK1 and KOIN.

a, Representative diagrams showing root growth measurement of 5-d-old seedlings grown at 22 °C and then either maintained at 22 °C or transferred to 4 °C for an additional 10 days. b, Root length of 5-d-old Col-0, crpk1-1 and crpk1-1 CRPK1 complementation seedlings grown at 22 °C and then kept at 22 °C for an additional 10 days. Data represent mean ± s.d. from one biological replicate (n = 20), representative of three independent experiments. c, Dual-membrane yeast two-hybrid assays showing negative controls. d, Schematic diagram of KOIN ___domain structure. SP, signal peptide; LRR, leucine-rich repeat; TM, transmembrane ___domain, CD, cytoplasmic ___domain. Amino acid positions at ___domain boundaries are indicated. e, In vitro pull-down assay showing the interaction between KOIN-CD with CRPK1. His-KOIN-CD and MBP-His-CRPK1 were immunoprecipitated with MBP beads and detected by anti-His immunoblotting. f, Co-IP assays in Nicotiana benthamiana showing interaction between KOIN–GFP and CRPK1–Myc. g, RT–qPCR analysis of KOIN transcript levels in Col-0 and koin-2 mutants. h, Immunoblot analysis of KOIN protein levels in Col-0 and koin-2 mutants using anti-KOIN antibody. Actin serves as a loading control in (g) and (h).

Source data

Extended Data Fig. 2 KOIN negatively regulates cold-induced root growth inhibition.

a,b, Root length of 5-d-old Col-0, koin-2 and koin-2 KOIN complementation seedlings grown at 22 °C and then maintained at 22 °C (a) or transferred to 4 °C (b) for 10 days. Data represent mean ± s.d. from one biological replicate (n = 20), representative of three independent experiments. c-h, Freezing tolerance of Col-0, koin mutants (c-e) and koin-2 KOIN complementation seedlings (f-h) under cold-acclimated conditions. Shown are representative seedling phenotypes (c,f), survival rates (d,g), and ion leakage (e,h). In (d-e) and (g-h), data are mean ± s.d. (n = 3 biological replicates). In (b, d-e) and (g-h), different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s post hoc test). Exact P values are provided in the Supplementary Table 2.

Source data

Extended Data Fig. 3 Expression pattern and subcellular localization of KOIN.

a, Tissue specific expression of KOIN in Col-0 based on Genevestigator data. Expression potential (%) indicates average expression across tissues relative to the highest expression sample (set as 100%). b, GUS staining of pKOIN::GUS transgenic plants. c, RT–qPCR analysis of KOIN expression in Col-0 under cold treatment. Data represent mean ± s.d. (n = 3), representative of three independent experiments. d, Genevestigator-based expression patterns of KOIN under cold stress. e, Subcellular localization of KOIN-GFP in various tissues of koin-2 KOIN complementation plants. GFP, GFP fluorescence; PI, propidium iodide staining (red); merged images show overlays with chlorophyll, PI, or FM4-64. Scale bars: 10 μm (root tip, meristem zone, hypocotyl, epidermal cells); 5 μm (elongation zone, root hairs). f, KOIN-GFP localization in root tip. Scale bar, 20 μm. g, KOIN protein levels in Col-0 under cold stress. Anti-KOIN and anti-Actin antibodies were used. h, Identification of Super::KOIN-GFP (KOIN-OE) transgenic plants in Col-0 and crpk1-1 background. i-j, KOIN protein levels in Col-0 (i) or crpk1-1 (j) backgrounds under cold. In (h-j), anti-GFP and anti-Actin antibodies were used.

Source data

Extended Data Fig. 4 Re-localization of KOIN-GFP from BFA compartments to the plasma membrane is impaired in crpk1-1 seedlings.

a, Quantification of KOIN-GFP fluorescence intensity in Col-0 and crpk1-1 roots under cold treatment, corresponding to Fig. 3a–d. b, Relative fluorescence intensity of KOIN-GFP during recycling assays, corresponding to Fig. 3g, h. c, Number of BFA bodies per cell after sequential CHX, BFA + CHX, and BFA washout treatments as described in Fig. 3g, h. d, Quantitation of plasma membrane and cytosol fluorescence intensities of KOIN-GFP in Col-0 and crpk1-1 roots under the same treatments. Data are mean ± s.d. (n = 3 biological replicates). Different letters indicate significant differences (P < 0.05, one-way ANOVA with Tukey’s test). Exact P values are provided in the Supplementary Table 2.

Source data

Extended Data Fig. 5 Sequence analysis of KOIN.

a, Alignment of subdomains VIb and VII within the kinase catalytic cores of active RLKs (BRI1, FLS2, EFR, CRK7) and inactive RLKs (PRK4, PRK5, BIR2, SUB). b, Phylogenetic analysis of kinase catalytic core sequences in Arabidopsis. The tree was constructed using MEGA7 based on amino acid sequences of the kinase domains. c, Repeated in vitro phosphorylation assay showing CRPK1-mediated phosphorylation of KOIN, corresponding to Fig. 4d. Autoradiograph (top) and CBB staining (bottom) are shown. Phosphorylation intensities are normalized to wild-type KOIN (set as 1.0).

Source data

Extended Data Fig. 6 Identification of KOIN complemented transgenic plants.

a, Immunoblot analysis of KOIN, KOINS314A and koin-2 KOINS314E expression in koin background under the native promoter. KOIN was detected with anti-GFP antibodies; Actin serves as a loading control. b, In planta phosphorylation of wild-type and S314A mutant KOIN after cold treatment. Immunoprecipitated KOIN-GFP and KOINS314A-GFP were analyzed by immunoblotting using anti-GFP and anti-phosphoserine antibodies. c,d, Root length of 5-d-old Col-0, koin-2, koin-2 KOINS314A and koin-2 KOINS314E seedlings grown at 22 °C and then maintained at 22 °C (c) or transferred to 4 °C (d) for 10 days. Data represent mean ± s.d. (n = 20, one biological replicate), representative of three independent experiments. Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s post hoc test). Exact P values are provided in the Supplementary Table 2.

Source data

Extended Data Fig. 7 Phenotypic analysis of KOIN-OE and koin crpk1 transgenic lines expressing KOIN variants.

a, Subcellular localization of KOIN-GFP in root tips. Scale bar, 20 μm. b, Confocal images of median longitudinal sections of root tips; asterisks indicate the meristematic cortex. Scale bar, 20 μm. c, Quantification of cortical cell numbers shown in (b) at 0, 24, and 48 h of cold treatment. Data represent mean ± s.d. (n = 3 biological replicates). d, Root phenotypes of 5-d-old Col-0, koin-2, and KOIN-OE seedlings grown at 22 °C and then maintained at 22 °C or transferred to 4 °C for 10 days. Scale bar, 0.5 cm. e, Immunoblot analysis of KOIN protein levels in koin crpk1 mutants expressing KOIN, KOINS314A, or KOINS314E. Anti-GFP and anti-Actin antibodies were used for detection. f, Root length of 5-d-old Col-0, crpk1-1, koin-2 crpk1-1, and koin crpk1 mutants expressing KOIN variants. Seedlings were grown at 22 °C, and then maintained at 22 °C (top) or transferred to 4 °C (bottom) for 10 days. Data represent mean ± s.d. (n = 20, one biological replicate), representative of three independent experiments. Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s post hoc test). Exact P values are provided in the Supplementary Table 2. g,h, FM4-64 labeling of koin crpk1 double mutants expressing KOIN variants after cold treatment, showing KOIN-GFP recycling defects. Seedlings were incubated with FM4-64 for 5 min before imaging. Scale bar, 10 μm.

Source data

Extended Data Fig. 8 KOIN interacts with 14-3-3λ and modulates of CBF signaling under cold stress.

a, Co-IP assays in Arabidopsis protoplasts showing interaction between KOIN-GFP and Flag-HA-14-3-3λ. Proteins were detected using anti-GFP and anti-HA antibodies. b, In vitro pull-down assay showing the interaction between KOIN and 14-3-3λ. Anti-His antibodies were used for detection. c, BiFC assay in Arabidopsis protoplasts showing KOIN-14-3-3λ interaction at the plasma membrane. FM4-64 staining was used to visualize endosomes. BRI1–YFPN+14-3-3λ–YFPC served as a negative control. Negative control (BRI1-YFPN+14-3-3λ-YFPC) is shown. Scale bar: 5 μm. d,e, Transcript levels of CBFs (d) and their target genes (e) in Col-0, koin-2, koin-2 KOIN complementation lines under cold treatment. Expression is shown relative to untreated Col-0 (set as 1.0), with ACTIN2/8 as internal reference. Data represent mean ± s.d. (n = 3, one biological replicate), representative of three independent experiments. **P < 0.01 (one-way ANOVA with Tukey’s test). Exact P values are provided in the Supplementary Table 2.

Source data

Extended Data Fig. 9 KOIN, CRPK1 and 14-3-3s act upstream of CBFs to regulate cold-induced root growth inhibition.

a, Root length of 5-d-old Col-0, 14-3-3kλ, 14-3-3k/14-3-3λ, 14-3-3λ-OE, crpk1-1, koin-2, crpk1 14-3-3λ-OE and koin 14-3-3λ-OE seedlings grown at 22 °C, then either maintained at 22 °C or transferred to 4 °C for 10 days. b, Root length of 5-d-old Col-0, 14-3-3kλ, cbfs, 14-3-3kλ cbfs, crpk1-1, koin-2, crpk1 cbfs and koin cbfs mutants under the same conditions. Data represent mean ± s.d. (n = 20, one biological replicate), representative of three independent experiments. Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s post hoc test). Exact P values are provided in the Supplementary Table 2.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

List of primer sequences used in this study.

Supplementary Table 2

P values for Figs. 1, 2 and 4–6 and Extended Data Figs. 1, 2, 4 and 6–9.

Source data

Source Data Fig. 2

Unprocessed western blots for Fig. 2.

Source Data Fig. 4

Unprocessed western blots for Fig. 4.

Source Data Fig. 6

Unprocessed western blots for Fig. 6.

Source Data Extended Data Fig. 1

Unprocessed western blots and gels for Extended Data Fig. 1.

Source Data Extended Data Fig. 3

Unprocessed western blots for Extended Data Fig. 3.

Source Data Extended Data Fig. 5

Unprocessed western blots for Extended Data Fig. 5.

Source Data Extended Data Fig. 6

Unprocessed western blots for Extended Data Fig. 6.

Source Data Extended Data Fig. 7

Unprocessed western blots for Extended Data Fig. 7.

Source Data Extended Data Fig. 8

Unprocessed western blots for Extended Data Fig. 8.

Source Data Figs. 1, 2 and 4–6 and Extended Data Figs. 1–4 and 6–9

Statistical source data for Figs. 1, 2 and 4–6 and Extended Data Figs. 1–4 and 6–9.

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Zhang, X., Li, M., Zhang, X. et al. A receptor–kinase cascade confers cold-induced root growth inhibition in Arabidopsis. Nat. Plants (2025). https://doi.org/10.1038/s41477-025-02034-5

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