The plant ESCRT component FREE1 shuttles to the nucleus to attenuate abscisic acid signalling

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The endosomal sorting complex required for transport (ESCRT) machinery has been well documented for its function in endosomal sorting in eukaryotes. Here, we demonstrate an up-to-now unknown and non-endosomal function of the ESCRT component in plants. We show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body biogenesis, plays additional functions in the nucleus in transcriptional inhibition of abscisic acid (ABA) signalling. Following ABA treatment, SNF1-related protein kinase 2 (SnRK2) kinases phosphorylate FREE1, a step requisite for ABA-induced FREE1 nuclear import. In the nucleus, FREE1 interacts with the basic leucine zipper transcription factors ABA-RESPONSIVE ELEMENTS BINDING FACTOR4 and ABA-INSENSITIVE5 to reduce their binding to the cis-regulatory sequences of downstream genes. Collectively, our study demonstrates the crosstalk between endomembrane trafficking and ABA signalling at the transcriptional level and highlights the moonlighting properties of the plant ESCRT subunit FREE1, which has evolved unique non-endosomal functions in the nucleus besides its roles in membrane trafficking in the cytoplasm.

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Fig. 1: free1-ctmut is a newly identified free1 weak allele and is hypersensitive to ABA treatment.
Fig. 2: ABA treatment induces nuclear shuttling of FREE1.
Fig. 3: SnRK2 kinases interact with and phosphorylate the FREE1 protein.
Fig. 4: Identification of the phosphorylation sites located in the C terminus of FREE1.
Fig. 5: FREE1 interacts with and supresses the transcriptional activation activities of ABF4 and ABI5.
Fig. 6: Genetic interactions between FREE1 and ABF4 or ABI5.

Data availability

The data that support the findings of this study are available from the corresponding authors on reasonable request.


  1. 1.

    Henne, W. M., Buchkovich, N. J. & Emr, S. D. The ESCRT pathway. Dev. Cell 21, 77–91 (2011).

  2. 2.

    Liu, C., Shen, W., Yang, C., Zeng, L. & Gao, C. Knowns and unknowns of plasma membrane protein degradation in plants. Plant Sci. 272, 55–61 (2018).

  3. 3.

    Leung, K. F., Dacks, J. B. & Field, M. C. Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage. Traffic 9, 1698–1716 (2008).

  4. 4.

    Valencia, J. P., Goodman, K. & Otegui, M. S. Endocytosis and endosomal trafficking in plants. Annu. Rev. Plant Biol. 67, 309–335 (2016).

  5. 5.

    Gao, C., Zhuang, X., Shen, J. & Jiang, L. Plant ESCRT complexes: moving beyond endosomal sorting. Trends Plant Sci. 22, 986–998 (2017).

  6. 6.

    Otegui, M. S. ESCRT-mediated sorting and intralumenal vesicle concatenation in plants. Biochem. Soc. Trans. 46, 537–545 (2018).

  7. 7.

    Buono, R. A. et al. ESCRT-mediated vesicle concatenation in plant endosomes. J. Cell Biol. 216, 2167–2177 (2017).

  8. 8.

    Gao, C. et al. A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr. Biol. 24, 2556–2563 (2014).

  9. 9.

    Kolb, C. et al. FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol. 167, 1361–1373 (2015).

  10. 10.

    Shen, J. B. et al. AtBRO1 functions in ESCRT-I complex to regulate multivesicular body protein sorting. Mol. Plant 9, 760–763 (2016).

  11. 11.

    Zhuang, X. et al. A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell 25, 4596–4615 (2013).

  12. 12.

    Gao, C. et al. Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc. Natl Acad. Sci. USA 112, 1886–1891 (2015).

  13. 13.

    Barberon, M. et al. Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant–soil interface plays crucial role in metal homeostasis. Proc. Natl Acad. Sci. USA 111, 8293–8298 (2014).

  14. 14.

    Miyakawa, T., Fujita, Y., Yamaguchi-Shinozaki, K. & Tanokura, M. Structure and function of abscisic acid receptors. Trends Plant Sci. 18, 259–266 (2013).

  15. 15.

    Yu, F. & Xie, Q. Non-26S proteasome endomembrane trafficking pathways in ABA signaling. Trends Plant Sci. 22, 976–985 (2017).

  16. 16.

    Yu, F., Wu, Y. R. & Xie, Q. Precise protein post-translational modifications modulate ABI5 activity. Trends Plant Sci. 20, 569–575 (2015).

  17. 17.

    Kong, L. et al. Degradation of the ABA co-receptor ABI1 by PUB12/13 U-box E3 ligases. Nat. Commun. 6, 8630 (2015).

  18. 18.

    Belda-Palazon, B. et al. FYVE1/FREE1 interacts with the PYL4 ABA receptor and mediates its delivery to the vacuolar degradation pathway. Plant Cell 28, 2291–2311 (2016).

  19. 19.

    Yu, F. F. et al. ESCRT-I component VPS23A affects ABA signaling by recognizing ABA receptors for endosomal degradation. Mol. Plant 9, 1570–1582 (2016).

  20. 20.

    Bueso, E. et al. The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. Plant J. 80, 1057–1071 (2014).

  21. 21.

    Rodriguez, L. et al. C2-domain abscisic acid-related proteins mediate the interaction of PYR/PYL/RCAR abscisic acid receptors with the plasma membrane and regulate abscisic acid sensitivity in Arabidopsis. Plant Cell 26, 4802–4820 (2014).

  22. 22.

    Zhao, Q. et al. Fast-suppressor screening for new components in protein trafficking, organelle biogenesis and silencing pathway in Arabidopsis thaliana using DEX-inducible FREE1-RNAi plants. J. Genet. Genomics 42, 319–330 (2015).

  23. 23.

    Shen, J. et al. A plant Bro1 domain protein BRAF regulates multivesicular body biogenesis and membrane protein homeostasis. Nat. Commun. 9, 3784 (2018).

  24. 24.

    Gao, X., Chen, J., Dai, X., Zhang, D. & Zhao, Y. An effective strategy for reliably isolating heritable and Cas9-free Arabidopsis mutants generated by CRISPR/Cas9-mediated genome editing. Plant Physiol. 171, 1794–1800 (2016).

  25. 25.

    Gao, Y. B. & Zhao, Y. D. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349 (2014).

  26. 26.

    Raghavendra, A. S., Gonugunta, V. K., Christmann, A. & Grill, E. ABA perception and signalling. Trends Plant Sci. 15, 395–401 (2010).

  27. 27.

    Planes, M. D. et al. A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions. J. Exp. Bot. 66, 813–825 (2015).

  28. 28.

    Ng, L. M. et al. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases. Proc. Natl Acad. Sci. USA 108, 21259–21264 (2011).

  29. 29.

    Gao, S. et al. ABF2, ABF3, and ABF4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis. Mol. Plant 9, 1272–1285 (2016).

  30. 30.

    Nardozzi, J. D., Lott, K. & Cingolani, G. Phosphorylation meets nuclear import: a review. Cell Commun. Signal. 8, 32 (2010).

  31. 31.

    Liu, Z. Y. et al. Plasma membrane CRPK1-mediated phosphorylation of 14-3-3 proteins induces their nuclear import to fine-tune CBF signaling during cold response. Mol. Cell 66, 117–128 (2017).

  32. 32.

    Nishino, T. G. et al. 14-3-3 regulates the nuclear import of class IIa histone deacetylases. Biochem. Biophys. Res. Commun. 377, 852–856 (2008).

  33. 33.

    Zhang, Z. et al. KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis. Proc. Natl Acad. Sci. USA 114, 4011–4016 (2017).

  34. 34.

    Takeo, K. & Ito, T. Subcellular localization of VIP1 is regulated by phosphorylation and 14-3-3 proteins. FEBS Lett. 591, 1972–1981 (2017).

  35. 35.

    Yoshida, T. et al. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress. Plant Cell Environ. 38, 35–49 (2015).

  36. 36.

    Skubacz, A., Daszkowska-Golec, A. & Szarejko, I. The role and regulation of ABI5 (ABA-Insensitive 5) in plant development, abiotic stress responses and phytohormone crosstalk. Front. Plant Sci. 7, 1884 (2016).

  37. 37.

    Kagale, S. & Rozwadowski, K. EAR motif-mediated transcriptional repression in plants: an underlying mechanism for epigenetic regulation of gene expression. Epigenetics 6, 141–146 (2011).

  38. 38.

    Reynolds, N., O’Shaughnessy, A. & Hendrich, B. Transcriptional repressors: multifaceted regulators of gene expression. Development 140, 505–512 (2013).

  39. 39.

    Yoshida, H. et al. DELLA protein functions as a transcriptional activator through the DNA binding of the INDETERMINATE DOMAIN family proteins. Proc. Natl Acad. Sci. USA 111, 7861–7866 (2014).

  40. 40.

    Pauwels, L. & Goossens, A. The JAZ proteins: a crucial interface in the jasmonate signaling cascade. Plant Cell 23, 3089–3100 (2011).

  41. 41.

    Pauwels, L. et al. NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature 464, 788–791 (2010).

  42. 42.

    Bunney, T. D. et al. Association of phosphatidylinositol 3-kinase with nuclear transcription sites in higher plants. Plant Cell 12, 1679–1688 (2000).

  43. 43.

    Drobak, B. K. & Heras, B. Nuclear phosphoinositides could bring FYVE alive. Trends Plant Sci. 7, 132–138 (2002).

  44. 44.

    Zhou, X. N. et al. SOS2-LIKE PROTEIN KINASE5, an SNF1-RELATED PROTEIN KINASE3-type protein kinase, is important for abscisic acid responses in Arabidopsis through phosphorylation of ABSCISIC ACID-INSENSITIVE5. Plant Physiol. 168, 659–676 (2015).

  45. 45.

    Nakashima, K. et al. Three Arabidopsis SnRK2 protein kinases, SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1 and SRK2I/SnRK2.3, involved in ABA signaling are essential for the control of seed development and dormancy. Plant Cell Physiol. 50, 1345–1363 (2009).

  46. 46.

    Benedetti, C. E. & Arruda, P. Altering the expression of the chlorophyllase gene ATHCOR1 in transgenic Arabidopsis caused changes in the chlorophyll-to-chlorophyllide ratio. Plant Physiol. 128, 1255–1263 (2002).

  47. 47.

    Song, S. et al. Interaction between MYC2 and ETHYLENE INSENSITIVE3 modulates antagonism between jasmonate and ethylene signaling in Arabidopsis. Plant Cell 26, 263–279 (2014).

  48. 48.

    Gao, C. et al. The Golgi-localized Arabidopsis endomembrane protein 12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus. Plant Cell 24, 2086–2104 (2012).

  49. 49.

    Yamaguchi, N. et al. Protocols: chromatin immunoprecipitation from Arabidopsis tissues. Arabidopsis Book 12, e0170 (2014).

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This work was supported by grants from the National Natural Science Foundation of China (31671467 and 31870171), the China 1000-Talents Plan for young researchers (C83025) to C.G., the National Natural Science Foundation of China (31701246) to W.S., the China Postdoctoral Science Foundation (2018M630963) and the National Science Foundation of Guangdong Province (2018A030310505) to C.Y., the National Natural Science Foundation of China (91854201) and the Research Grants Council of Hong Kong (C4011-14R, C4012-16E, C4002-17G and AoE/M-05/12) to L.J. We thank H. Deng for her assistance on the EMSA experiments.

Author information

H.L., Y.L., Q.Z. and C.G. designed the project. H.L., Y.L., Q.Z., T.L., J.W., B.L. and Y.Z. performed the experiments. H.L., Y.L., Q.Z., W.S., C.Y., P.L.R., Y.Z., L.J., X.W. and C.G. analysed the results. H.L., Q.Z., R.L.R., L.J., X.W. and C.G. wrote the manuscript.

Correspondence to Liwen Jiang or Xiaojing Wang or Caiji Gao.

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Journal peer review information: Nature Plants thanks Masa Sato and other anonymous reviewers for their contribution to the peer review of this work.

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Li, H., Li, Y., Zhao, Q. et al. The plant ESCRT component FREE1 shuttles to the nucleus to attenuate abscisic acid signalling. Nat. Plants 5, 512–524 (2019) doi:10.1038/s41477-019-0400-5

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