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FERONIA coordinates plant growth and salt tolerance via the phosphorylation of phyB

Nature Plants volume 9, pages 645–660 (2023)Cite this article

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Abstract

Phosphorylation modification is required for the modulation of phytochrome B (phyB) thermal reversion, but the kinase(s) that phosphorylate(s) phyB and the biological significance of the phosphorylation are still unknown. Here we report that FERONIA (FER) phosphorylates phyB to regulate plant growth and salt tolerance, and the phosphorylation not only regulates dark-triggered photobody dissociation but also modulates phyB protein abundance in the nucleus. Further analysis indicates that phosphorylation of phyB by FER is sufficient to accelerate the conversion of phyB from the active form (Pfr) to the inactive form (Pr). Under salt stress, FER kinase activity is inhibited, leading to delayed photobody dissociation and increased phyB protein abundance in the nucleus. Our data also show that phyB mutation or overexpression of PIF5 attenuates growth inhibition and promotes plant survival under salt stress. Together, our study not only reveals a kinase that controls phyB turnover via a signature of phosphorylation, but also provides mechanistic insights into the role of the FER-phyB module in coordinating plant growth and stress tolerance.

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Fig. 1: Mutation of phyB suppresses the phenotypes of lrx345 and fer-4 mutants.
Fig. 2: FER interacts with and phosphorylates the N-terminal domain of phyB.
Fig. 3: FER-mediated phosphorylation controls photobody dissociation and phyB protein abundance in the nucleus.
Fig. 4: FER-mediated phosphorylation of phyB accelerates the dark-triggered Pfr-to-Pr conversion in vitro.
Fig. 5: Salt stress delays photobody dissociation and increases phyB protein abundance in the nucleus via the inactivation of FER.
Fig. 6: Overexpression of PIF5 suppresses the phenotypes of lrx345 and fer-4 mutants.
Fig. 7: A proposed working model.

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Data availability

All materials in this study are available from the corresponding author upon request. The raw data of BSA sequencing have been deposited in the NCBI BioProject database under accession number PRJNA914087. RNA-seq data have been deposited in the NCBI GEO under accession number GSE188335. Arabidopsis reference genome (TAIR10) was used in this study. Source data are provided with this paper.

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Acknowledgements

We thank H. Yang (Shanghai Normal University, China) for providing phyBpro::phyB-Myc plasmid, R. Fang (Institute of Microbiology, CAS, China) for providing OsphyB mutant, and J. Clark Lagarias (University of California, Davis, USA) for providing YHB seeds. This work was supported by National Natural Science Foundation of China Grant No. 32070295 (to C.Z.), Shanghai Pujiang Program Grant No. 20PJ1414800 (to C.Z.), Shanghai Agriculture Applied Technology Development Program Grant No. X20200101 (to C.Z.), and Strategic Priority Research Program of the Chinese Academy of Sciences Grant No. XDA27040104 (to J.-K.Z.).

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Authors and Affiliations

  1. Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China

    Xin Liu, Wei Jiang, Yali Li, Li Tan, Li Peng, Chao Li, Jinyan Luo, Ming Li, Pengcheng Wang, Jian-Kang Zhu & Chunzhao Zhao

  2. University of the Chinese Academy of Sciences, Beijing, China

    Xin Liu, Yali Li, Lina Cui & Bing Zhou

  3. Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, China

    Haozhen Nie, Hongxia Wang & Jun Yang

  4. State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Lina Cui & Bing Zhou

  5. Shanghai Bioprofile Technology Company Ltd, Shanghai, China

    Rongxia Li

  6. Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China

    Bing Zhou

  7. Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China

    Bing Zhou

  8. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Hongtao Liu

  9. Institute of Advanced Biotechnology and School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Jian-Kang Zhu

  10. Center for Advanced Bioindustry Technologies, Chinese Academy of Agricultural Sciences, Beijing, China

    Jian-Kang Zhu

  11. Hainan Yazhou Bay Seed Laboratory, Sanya, China

    Jian-Kang Zhu

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  1. Xin Liu
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Contributions

X.L. and C.Z. conceived and designed the experiments; X.L., W.J., Y.L., H.N., C.L., J.L. and M.L. performed experiments; X.L., L.C., L.P. and B.Z. conducted bioinformatic analysis; X.L. and R.L. performed proteomics analysis; L.T., H.W., J.Y., P.W., H.L., J.-K.Z. and C.Z. analysed data; X.L., J.-K.Z. and C.Z. wrote the paper.

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Correspondence to Jian-Kang Zhu or Chunzhao Zhao.

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Nature Plants thanks Ferenc Nagy, Eunkyoo Oh, Christa Testerink and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 slrx620 mutation suppresses the phenotypes of lrx345 mutant.

a, Phenotype of wild type, lrx345, and slrx620 mutant grown on 1/2 MS medium with or without NaCl (120 mM) under white light conditions (16 h light/8 h dark cycle). b, Quantification of the survival rate of seedlings grown on NaCl medium. Values are the means ± SD of three biological replicates. c, Representative images of wild type, lrx345, and slrx620 mutant grown on soils for 22 days under LD conditions. Scale bar, 1 cm. d, Quantification of the petiole length of plants shown in c. Values are the means ± SD (n = 11 seedlings). e, RT–qPCR analysis of the transcript levels of PDF1.2 and PDF1.3 genes in each genotype. Values are the means ± SD of three biological replicates. f, Identification of mutations in slrx620 mutant by bulked segregant analysis (BSA). Red asterisk indicates the location of the mutated gene that is associated with the slrx620 mutant phenotypes. g, Diagram illustrating the genomic structure of phyB and the mutation site in the slrx620 mutant. h. Hypocotyl growth of seedlings grown under white light conditions for 4 days. Scale bar, 2 mm. i, Quantification of the hypocotyl length of the seedlings shown in h. Values are the means ± SD (n = 49-63 seedlings). Asterisks in b, d and e indicate statistically significant differences (**P < 0.01, Student’s t test, two-sided). Different letters in i indicate statistically significant differences (P < 0.01, one-way ANOVA).

Source data

Extended Data Fig. 2 phyB is involved in FER-mediated regulation of plant growth and salt tolerance.

a, Representative images of wild type, fer-4, phyB-9, and phyB-9 fer-4 siliques. Scale bar, 0.5 mm. b, Representative images of wild type, fer-4, phyB-9 fer-4, and 35 S::phyB-GFP/phyB-9 fer-4 grown on soils for 22 days under white light conditions (16 h light/8 h dark cycle). Scale bar, 1 cm. c, Quantification of the petiole length of plants shown in b. Values are the means ± SD (n = 21 plants). d, Phenotype of wild type, fer-4, phyB-9 fer-4, and 35 S::phyB-GFP/phyB-9 fer-4 grown on 1/2 MS media supplemented with or without NaCl (120 mM) under white light conditions. e, Quantification of the survival rate of seedlings grown on NaCl medium. Values are the means ± SD of three biological replicates. f, Phenotype of wild type, phyB-9, and 35 S::phyB-GFP/phyB-9 transgenic seedlings grown on 1/2 MS medium with or without NaCl (120 mM) under white light conditions. g, Quantification of the survival rate of seedlings grown on NaCl medium. Values are the means ± SD of three biological replicates. h, Representative images of wild type, fer-4, phyB-9, phyB-9 fer-4, 35 S::phyB-GFP/phyB-9, and 35 S::phyB-GFP/phyB-9 fer-4 seedlings grown on soils for 35 days under white light conditions. Scale bar, 5 cm. i. Quantification of the plant height as shown in h. Values are the means ± SD (n = 18-25 plants). Different letters in c, e, g and i indicate statistically significant differences (P < 0.01, one-way ANOVA).

Source data

Extended Data Fig. 3 phyB mutation suppresses the salt-hypersensitivity of the lrx345 and fer-4 mutants under monochromatic red light illumination.

a, Phenotype of each genotype grown on 1/2 MS media supplemented with or without NaCl (120 mM) under continuous red light. b, Quantification of the survival rate of seedlings grown on NaCl medium as shown in a. Values are the means ± SD of three biological replicates. c, Phenotype of wild type, lrx345, and fer-4 seedlings grown on 1/2 MS and 1/2 MS + NaCl (120 mM) media under continuous high red-light intensity (~15 μmol m-2 sec-1) or continuous low red-light intensity (~1 μmol m-2 sec-1). d, Quantification of the survival rate of seedlings grown on NaCl medium as shown in c. Values are the means ± SD of three biological replicates. e, Fluence rate response curves to analyze hypocotyl elongation inhibition under different red light fluence rates at 22 °C. Wild type and fer-4 mutant were grown on 1/2 MS medium without sucrose in dark or continuous red light for 4 days. Relative hypocotyl length of the seedings grown under different red light fluence rates was calculated by comparing with that under dark conditions. Values are the means ± SD (n = 32-41 seedlings). Asterisks in b and d indicate statistically significant differences (**P < 0.01, Student’s t test, two-sided).

Source data

Extended Data Fig. 4 Transcriptomic profiling reveals that FER regulates a subset of gene expression via phyB.

a, Venn diagrams showing the overlapped genes that were up-regulated in fer-4 mutant compared with wild type, but down-regulated in phyB-9 fer-4 compared with fer-4 mutant. b, Heat map of the 272 overlapped genes shown in a. c, GO enrichment analysis for the overlapped genes shown in a. Categories that were significantly enriched for these overlapped genes were selected and shown. Significance was determined using Student’s t test (two-sided). d, Venn diagrams showing the overlapped genes that were down-regulated in fer-4 mutant compared with wild type, but up-regulated in phyB-9 fer-4 compared with fer-4 mutant. e, Heat map of the 135 overlapped genes shown in d. f, GO enrichment analysis for the overlapped genes shown in d. Categories that were significantly enriched for these overlapped genes were selected and shown. Significance was determined using Student’s t test (two-sided).

Extended Data Fig. 5 Identification of the phosphorylation sites of phyB by FER.

a, Co-localization analysis of FER and phyB in tobacco leaves under dark and white light conditions. Scale bar, 10 μm. b, Split luciferase complementation assay showing the interaction of THE1 with phyB. Fluorescence was detected at 48 h after infiltration of the indicated constructs. c, Mass spectrometry analysis of the phosphorylation of phyBN and phyBNAA after incubation with FERCD. In the left panel, the two peaks represent nonphosphorylated phyBN and the biotin-tagged nonphosphorylated phyBN. In the middle and right panels, the newly developed peaks represent phosphorylated phyBN. d, The phosphorylation sites of phyB after incubation with FERCD were identified by LC–MS analysis. PH indicates the phosphorylated residues. e, In vitro kinase assay showing the phosphorylation of phyBN and phyBNS86A by FERCD. It should be noted that, due to the addition of a small amount of FERCD in the reaction buffer, FERCD band was not clearly detected in CBB. The experiments in a and e were repeated independently for at least twice with similar results.

Source data

Extended Data Fig. 6 Identification of the phosphorylation sites of phyB by FER in Arabidopsis.

a,b, In vivo phosphorylations of S106 (a) and S227 (b) residues in phyB were identified by using LC–MS assay. PH indicates the phosphorylated residues. c,d, Relative phosphopeptide signals of the peptides containing phosphorylated S106 (c) or phosphorylated S227S234 (d) residues in wild type and fer-4 seedlings based on LC–MS data. e, Analysis of the phosphorylation of phyB in wild type and fer-4 seedlings grown on 1/2 MS medium with or without NaCl (120 mM). Total proteins were extracted from seven-day-old etiolated seedlings. phyB-GFP protein was immunoprecipitated with anti-GFP magarose beads, and immunoblotting assays were performed using anti-phosphoserine and anti-GFP antibodies. The experiment was repeated independently for twice with similar results. f, Relative phosphopeptide signals of the phyB peptides containing phosphorylated S94S106 residues in Arabidopsis seedlings treated with or without NaCl (150 mM). Values in c, d and f are the means ± SD of two biological replicates. Asterisks in c and f indicate statistically significant differences (*P < 0.05, Student’s t test, two-sided).

Source data

Extended Data Fig. 7 Phosphorylation of Ser106 and Ser227 is required for the regulation of photobody dissociation under dark conditions.

a, Protein abundance of phyB in wild type (Col-0), 35 S::phyB-GFP/phyB-9 (WT), and 35 S::phyBS106AS227A-GFP/phyB-9 (AA) transgenic plants was analyzed using anti-phyB antibody. b, Hypocotyl phenotype of four-day-old seedlings grown on 1/2 MS medium under white light conditions (16 h light/8 h dark cycle). Scale bar, 2 mm. c, Quantification of the hypocotyl length of the seedlings shown in b. Values are the means ± SD (n = 50-55 seedlings). d, Time-course analysis of the hypocotyl length of seedlings before and after dark treatment (n = 33 seedlings). e, Protein abundance of phyB in each genotype was analyzed by using anti-phyB antibody. DD represents 35 S::phyBS106DS227D-GFP/phyB-9 transgenic plants. f, Photobody dissociation under dark conditions. Five-day-old seedlings were exposed to white light for 5 h (ZT5) after dawn, and then the seedlings were treated in darkness for 1 h and 3 h before observation of photobodies. Scale bar, 4 μm. g, Quantification of the diameter of phyB photobodies shown in f. Values are the means ± SD (n > 100 bodies from 30-40 cells). h, Hypocotyl length of four-day-old seedlings grown on 1/2 MS medium under white light conditions. Values are the means ± SD (n = 54 seedlings). i, Protein abundance of phyB in each genotype was analyzed using anti-phyB antibody. j, Hypocotyl phenotype of four-day-old seedlings grown on 1/2 MS medium under white light conditions. Scale bar, 2 mm. k, Quantification of the hypocotyl length of the seedlings shown in j. Values are the means ± SD (n = 38-53 seedlings). Asterisks in g indicate statistically significant differences (**P < 0.01, Student’s t test, two-sided). Different letters in c, h, and k indicate statistically significant differences (P < 0.01, one-way ANOVA). The experiments in a, e and i were repeated independently for at least three times with similar results.

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Extended Data Fig. 8 Phosphorylation of phyB is not required for far-red light-triggered photoconversion.

a, Spectral analysis of the photoswitching of wild type phyBN, phyBNS106AS227A, phyBNS86A, and phyBNS86AS106AS227A after irradiance with far-red light. Proteins were exposed to red light illumination (~17 μmol m-2 sec-1) for 5 min and then exposed to far-red light illumination (~5 μmol m-2 sec-1) for 5 min. Red lines indicate absorbance spectra after red light illumination and blue lines indicate absorbance spectra after far-red light illumination. The absorption spectra of phyBN were measured using Nanodrop 2000C spectrophotometer. b, Quantification of the absorption spectra of the wild type phyBN and point-mutated phyB under red light and far-red light illumination. Y axis represents Pfr/Pr ratio. Values are the means ± SD of three independent replicates. Different letters indicate statistically significant differences (p < 0.01, one-way ANOVA). c, Recombinant His-FERCD and His-FERCDK565R were detected by Coomassie Brilliant Blue (CBB) staining. His-FERCD was treated with or without λPPase. The experiments were repeated independently for three times with similar results.

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Extended Data Fig. 9 phyB-PIFs module is required for the regulation of plant survival under salt stress.

a, The survival rate of wild type, 35 S::phyB-GFP/phyB-9 and 35 S::phyBS106AS227A-GFP/phyB-9 (AA) seedlings grown on 1/2 MS medium supplemented with NaCl (120 mM) under white light conditions (16 h light/8 h dark cycle). Values are the means ± SD of three independent replicates. b, RT–qPCR analysis of the transcript level of PIF5 gene in wild type and 35 S::PIF5-YFP transgenic plants. ACTIN8 was used as an internal control. c, RT–qPCR analysis of the transcript level of PIF3 gene in wild type and PIF3OE/fer-4 seedlings. d, Phenotypes of wild type, fer-4, PIF3OE/WT, and PIF3OE/fer-4 seedlings grown on 1/2 MS medium with or without NaCl (120 mM) under white light conditions. e, Quantification of the survival rate of seedlings grown on NaCl medium. Values in a, b, c and e are the means ± SD of three independent replicates. Different letters in a and e indicate statistically significant differences (p < 0.01, one-way ANOVA). Asterisks in c indicate statistically significant differences (**P < 0.01, Student’s t test, two-sided).

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Supplementary information

Supplementary Information

Supplementary Tables 2 and 3.

Reporting Summary

Supplementary Table 1

List of differentially expressed genes.

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Liu, X., Jiang, W., Li, Y. et al. FERONIA coordinates plant growth and salt tolerance via the phosphorylation of phyB. Nat. Plants 9, 645–660 (2023). https://doi.org/10.1038/s41477-023-01390-4

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