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Liquid–liquid phase separation of TZP promotes PPK-mediated phosphorylation of the phytochrome A photoreceptor

Nature Plants (2024)Cite this article

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Abstract

Phytochrome A (phyA) is the plant far-red (FR) light photoreceptor and plays an essential role in regulating photomorphogenic development in FR-rich conditions, such as canopy shade. It has long been observed that phyA is a phosphoprotein in vivo; however, the protein kinases that could phosphorylate phyA remain largely unknown. Here we show that a small protein kinase family, consisting of four members named PHOTOREGULATORY PROTEIN KINASES (PPKs) (also known as MUT9-LIKE KINASES), directly phosphorylate phyA in vitro and in vivo. In addition, TANDEM ZINC-FINGER/PLUS3 (TZP), a recently characterized phyA-interacting protein required for in vivo phosphorylation of phyA, is also directly phosphorylated by PPKs. We reveal that TZP contains two intrinsically disordered regions in its amino-terminal domain that undergo liquid–liquid phase separation (LLPS) upon light exposure. The LLPS of TZP promotes colocalization and interaction between PPKs and phyA, thus facilitating PPK-mediated phosphorylation of phyA in FR light. Our study identifies PPKs as a class of protein kinases mediating the phosphorylation of phyA and demonstrates that the LLPS of TZP contributes significantly to more production of the phosphorylated phyA form in FR light.

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Fig. 1: PPKs interact with phyA and regulate the phosphorylation of phyA in vivo.
Fig. 2: PPKs catalyse the phosphorylation of phyA in vitro and in vivo.
Fig. 3: TZP facilitates PPK-mediated phosphorylation of phyA.
Fig. 4: TZP undergoes LLPS in vivo and in vitro.
Fig. 5: The TZP IDRs are indispensable for TZP function in FR light.
Fig. 6: PPKs play an important role in regulating FR light signalling.
Fig. 7: The LLPS of TZP promotes PPK-mediated phosphorylation of phyA in FR light.

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Data availability

All data supporting the findings of this study are available in the main text or the supplementary information. The biological materials used in this study are available from J.L. on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Q. Wang for the ppk123 and ppk124 mutants and the ACT2p::GFP–PPK1 rdr6-11 transgenic line. This work was supported by grants from the National Natural Science Foundation of China (no. 32225006 to J.L. and no. 32200245 to L.Q.) and the Beijing Natural Science Foundation (no. 5232011 to J.L.).

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Author notes

  1. These authors contributed equally: Ziyi Feng, Meijiao Wang.

Authors and Affiliations

  1. State Key Laboratory of Plant Environmental Resilience, College of Biological Sciences, Frontiers Science Center for Molecular Design Breeding (MOE), China Agricultural University, Beijing, China

    Ziyi Feng, Meijiao Wang, Yan Liu, Cong Li, Shaoman Zhang, Jie Duan, Jiaqi Chen, Lijuan Qi, Yanru Liu, Hong Li, Yan Guo & Jigang Li

  2. State Key Laboratory of Protein and Plant Gene Research, School of Advanced Agricultural Sciences and School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Lijuan Qi, Weiqiang Qian & Xing Wang Deng

  3. Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Jie Wu & Xiaofeng Fang

  4. College of Life Sciences, Nanjing Normal University, Nanjing, China

    Yannan Liu & Bojian Zhong

  5. Department of Biology, Wilkes University, Wilkes-Barre, PA, USA

    William Terzaghi

  6. State Key Laboratory of Plant Environmental Resilience, Frontiers Science Center for Molecular Design Breeding (MOE), China Agricultural University, Beijing, China

    Feng Tian

  7. State Key Laboratory of Wheat Improvement, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences at Weifang, Weifang, China

    Weiqiang Qian & Xing Wang Deng

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  1. Ziyi Feng
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Contributions

J.L., Z.F. and M.W. designed the research. Z.F., M.W., Yan Liu, C.L., S.Z., J.D., J.C., L.Q., Yanru Liu, H.L., J.W. and Yannan Liu performed the research. J.L., Z.F., M.W., F.T., B.Z., X.F., W.Q., Y.G. and X.W.D. discussed and interpreted the data. J.L., Z.F., M.W. and W.T. wrote the paper.

Corresponding author

Correspondence to Jigang Li.

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Nature Plants thanks Enamul Huq, Jeong-Il Kim and Cornelia Klose for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 PPKs interact with TZP in vitro and in vivo.

a, Schematic diagram of prey proteins (TZP, TZP-N, TZP-N1, TZP-C, TZP-C1 and TZP-C2 fused with AD domains). ZF, zinc finger. b, Yeast two-hybrid assays showing that PPKs interact with the C-terminal domain of TZP. c–f, Pull-down assays showing that both the zinc-finger and PLUS3 domains are required for interactions between TZP and PPK1 (c), PPK2 (d), PPK3 (e) and PPK4 (f) in vitro. g, BiFC assays showing that TZP and PPK1 interact in the nuclei of N. benthamiana plants expressing red fluorescent protein (RFP)-H2B fusion proteins89. An unrelated protein (GUS) fused with the C-terminal domain of YFP77 was used as the negative control. Super:CFP was used as the transformation control. Scale bar = 20 μm (the whole image) and 10 μm (the enlarged nuclei). h,i, LCI assays showing that TZP interacts with all four PPKs in N. benthamiana leaf cells. In (h), scale bars = 1 cm. In (i), the values are mean ± s.d. (n = 3 independent assays). P values are from two-tailed Student’s t-test. j, Co-IP assays showing the associations of TZP with phyA and PPK1 in vivo. TZP-GFP, phyA-FLAG and MYC-PPK1 were coexpressed in Col protoplasts, then total proteins were extracted and incubated with anti-GFP beads (AlpaLife). The total and precipitated proteins were subjected to immunoblotting with antibodies against TZP, phyA, MYC, HSP and GFP, respectively. The asterisk and arrowhead represent the phosphorylated and unphosphorylated phyA forms, respectively.

Source data

Extended Data Fig. 2 PPK2 phosphorylates apo- and holo-phyA in vitro.

In vitro kinase assays showing that PPK2-catalyzed phosphorylation of holo- and apo-phyA with different concentrations of phyA (a,c) or for different times (b,d). For each panel, CBB staining (top), and autoradiographs (bottom) are shown, with zinc fluorescence (Zinc) shown in the middle of a and b. PhyA-GST proteins were expressed in and purified from Saccharomyces cerevisiae BJ2168, and phycocyanobilin (PCB) extracted from Spirulina was added to the supernatant at a final concentration of 10 µM in the dark to allow the assembly of holo-phyA proteins in vitro. The holo-phyA proteins were exposed to 15 min of FR light alone, or 15 min of FR light immediately followed by 15 min of R light to allow holo-phyA to form Pr or Pfr forms in vitro, respectively.

Extended Data Fig. 3 PPKs phosphorylate TZP in vitro and in vivo.

a, Identical amounts of Super:TZP-GFP and 35 S:MYC-PPK1 plasmids were transiently cotransfected into Col protoplasts. After transfection, the protoplasts were incubated in the dark for 16 h, and then treated with FR light (80 μmol m−2 s−1) for 2 h. The total proteins were extracted and treated with active (+) or inactive (boiled) or without (−) λ-PPase for 30 min, and then analyzed by immunoblots using anti-TZP, anti-MYC or anti-HSP antibodies, respectively. b, In vitro kinase assays showing that PPKs directly phosphorylate the full-length TZP proteins. CBB staining (top) and autoradiographs (bottom) are shown. c, Immunoblot assays showing the patterns of TZP proteins in Col and ppk mutant seedlings grown under FR light. Numbers below the immunoblots indicate the relative band intensities of TZP proteins normalized to those of HSP, respectively. The ratio was set to 100 for the first TZP band.

Source data

Extended Data Fig. 4 Yeast two-hybrid assays showing that the T418D and C323A mutations abolished phyA interaction with TZP.

a, In vivo plate assays showing that TZP interacts with phyA(WT) and phyA(D422R), but not with phyA(T418D) in yeast cells. b, Liquid culture assays showing the interactions between TZP and phyA(WT), phyA(D422R), phyA(T418D), phyA(Y242H) and phyA(C323A) in yeast cells. Yeast cultures incubated in darkness (D) were treated with 5 min of R light alone, or 5 min of R light immediately followed by 5 min of FR light, and then incubated in D for additional 3 h. β-Galactosidase activities were measured using ONPG as the substrate (the values are mean ± s.d., n = 4 independent assays). Different letters represent significant differences indicated by one-way ANOVA with Bonferroni’s multiple comparisons test (P < 0.05). The exact P values are provided in Supplementary Table 2.

Extended Data Fig. 5 TZP promotes PPK1-mediated phyA phosphorylation in FR light.

Immunoblots showing that TZP promotion of PPK1-mediated phyA phosphorylation in Col protoplasts became more evident after longer FR light exposure (a,c,e) and with higher intensities of FR light (b,d,f). Representative pictures are shown in (a) and (b), the percentages of phosphorylated phyA are shown in (c) and (d), and the total amounts of phyA are shown in (e) and (f). In (c-f), the values are mean ± s.d. (n = 3 independent assays). P values are from two-tailed Student’s t-test. In (e) and (f), the total amounts of phyA were not significantly changed under different times of FR light or under different intensities of FR light treatments.

Source data

Extended Data Fig. 6 FRAP of TZP NBs in N. benthamiana leaf cells and in vitro.

a, FRAP assays of the whole TZP-GFP NBs in N. benthamiana leaf cells. Time 0 indicates the time of the photobleaching pulse. Scale bar = 4 μm. b, Plot showing the time course of the recovery after photobleaching the whole TZP nuclear condensates. Data are representative of ten independent experiments. Data are presented as mean ± SD (n = 10 independent experiments). c, FRAP assays of the half of TZP nuclear condensates in N. benthamiana leaf cells. The dashed lines demarcate the area of bleaching. Time 0 indicates the time of the photobleaching pulse. Scale bar = 5 μm. d, Plot showing the time course of the recovery after photobleaching the half of TZP nuclear condensates. Data are presented as the mean ± SD (n = 10 independent experiments). e, FRAP assays of the whole TZP-IDR-GFP droplets. Time 0 s indicates the time of the photobleaching pulse. Scale bar = 3 μm. f, Plot showing the time course of the recovery after photobleaching the whole TZP-IDR-GFP droplets. Data are representative of ten independent experiments. Data are presented as the mean ± SD (n = 10 independent experiments). g, FRAP assays of the half of TZP-IDR-GFP droplets. The dashed lines demarcate the area of bleaching. Time 0 s indicates the time of the photobleaching pulse. Scale bar = 5 μm. h, Plot showing the time course of the recovery after photobleaching the half of TZP-IDR-GFP droplets. Data are representative of seven independent experiments. Data are presented as the mean ± SD (n = 7 independent experiments). i, FRAP assays of FUS-IDR-NLS-TZPC-GFP NBs in N. benthamiana leaf cells. Time 0 indicates the time of the photobleaching pulse. Scale bar = 4 μm. j, Plot showing the time course of the recovery after photobleaching FUS-IDR-NLS-TZPC-GFP nuclear condensates. Data are representative of ten independent experiments. Data are presented as mean ± SD (n = 10 independent experiments).

Extended Data Fig. 7 TZP NBs are reversible by light-to-dark transition in Arabidopsis.

a,b, Partition coefficients and numbers of TZP-GFP NBs per nucleus in response to FR light (a) or dark treatment (b) shown in Fig. 4g. Data are presented as means ± SD (n = 10 nuclei in [a] and n = 8 nuclei in [b]). c, Time-lapse images showing the behaviors of TZP NBs in one nucleus of TZPp:TZP-GFP tzp-2 seedlings in response to white light or dark treatment. White slash indicates the treatment of white light. Data are representative of ten independent experiments. Scale bar = 15 μm.

Extended Data Fig. 8 TZP phase separates into droplets in vitro.

a, Coomassie staining of MBP-His-TZP-IDR-GFP protein samples before and after TEV cleavage to remove the MBP tag. The TEV protease-treated solution was then flowed through Superdex 200 increase 10/300 column (SD200) (GE healthcare), and then the fraction containing the main peak of TZP-IDR-GFP was collected for in vitro phase separation assays shown in (b) and (c). b, In vitro phase separation assay of TZP-IDR-GFP proteins (10 μM) in the presence of 100 mM NaCl and PEG8000 (n = 3 independent assays, scale bar = 10 μm). c, Images showing formation of droplets at different concentrations of TZP-IDR-GFP and NaCl in the presence of PEG8000. Data are representative of three independent experiments. Scale bar = 5 μm.

Extended Data Fig. 9 Representative images for the subcellular localizations of FUS-IDR-NLS-TZPC-GFP, phyA-CFP and RFP-PPK1.

a,b, Confocal microscopy assays showing the subcellular localizations of GFP-, CFP- and RFP-tagged proteins coexpressed in Col protoplasts (a) and N. benthamiana leaf cells (b). The representative confocal images for each channel were subsequently merged to show colocalization. The arrow heads indicate the locations of nuclear bodies. Scale bars = 10 μm. c,d, Fluorescence profiles of FUS-IDR-NLS-TZPC-GFP/phyA-CFP/RFP-PPK1, phyA-CFP/RFP-PPK1, FUS-IDR-NLS-TZPC-GFP/phyA-CFP, FUS-IDR-NLS-TZPC-GFP/RFP-PPK1 and FUS-IDR-NLS-TZPC-GFP in Col protoplasts (c) and N. benthamiana leaf cells (d) over the white lines shown in (a) and (b), respectively. The arrow heads indicate the locations of nuclear bodies.

Extended Data Fig. 10 Evolutionary analyses of phytochromes, PPKs and TZP.

a, Evolution of the core light signaling components of Archaeplastida. Cryptochrome (CRY) was the first type of photoreceptor to originate in red algae66. PPKs first originated in the chlorophytes, and PPK1/4 and PPK2/3 separated from PPKs in lycophytes and ferns, respectively. Both phytochrome and TZP originated in the charophytes, while Arabidopsis phyA and phyB separated after the divergence of seed plants66. Figure created with BioRender.com. b, Alignment of the PLUS3 domain in multiple species from charophytes to angiosperms. The sequences were aligned using ClustalX and modified using Adobe Illustrator.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12, Methods and uncropped scans of blots and gels for the supplementary figures.

Reporting Summary

Supplementary Table 1

Primers.

Supplementary Table 2

Exact P values.

Supplementary Data 1

Statistical source data for the supplementary figures.

Source data

Source Data Fig. 1

Unprocessed western blots.

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 1

Unprocessed western blots.

Source Data Extended Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

Source Data Figs. 1–6 and Extended Data Figs. 1, 4–7 and 9

Summary of all statistical source data.

Source Data Figs. 1 and 3–5 and Extended Data Figs. 1 and 6–9

Summary of all confocal microscopy pictures.

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Feng, Z., Wang, M., Liu, Y. et al. Liquid–liquid phase separation of TZP promotes PPK-mediated phosphorylation of the phytochrome A photoreceptor. Nat. Plants (2024). https://doi.org/10.1038/s41477-024-01679-y

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