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Rab5 GTPases mediate the targeting of ROP signalling to establish polarity for pollen germination

Nature Plants volume 11, pages 2319–2331 (2025)Cite this article

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Abstract

The establishment of polarity is an evolutionarily recurring theme in all eukaryotes. Pollen germination in angiosperms establishes polarity, which is critical for reproduction and a barrier for inter-species crosses. Arabidopsis Rho of plants (ROPs) and their guanine nucleotide exchange factor 8 (RopGEF8) are essential for pollen germination through their polar distribution at the plasma membrane and asymmetric activation of downstream activities. Events leading to the polar plasma membrane association of RopGEF8 and ROPs during pollen germination are yet to be uncovered. We report here that canonical and plant-unique Rab5 GTPases, regulators of vesicular trafficking, synergistically regulate pollen germination. Intriguingly, the role of Rab5s in this process is independent of their own GEFs. Instead, Rab5 GTPases directly interact with RopGEF8 in an activity-independent way and mediate its polar plasma membrane targeting from endosomes. Our finding that Rab5 GTPases influence ROP signalling through RopGEF8 suggests an evolutionary innovation by plants. By using the combination of both canonical and plant-unique Rab5 GTPases, ROP signalling can be regulated in a more delicate manner. Consequently, various internal and external inputs are interpreted to ensure the proper timing of pollen germination.

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Fig. 1: Functional loss of both cRab5s and pRab5s causes no pollen germination.
Fig. 2: The promoting role of Rab5s in pollen germination does not rely on Vps9s.
Fig. 3: Functional loss of Rab5 GTPases compromises ROP signalling during pollen germination.
Fig. 4: Polar PM targeting of RopGEF8 during pollen germination relies on Rab5.
Fig. 5: Rab5 GTPases interact with RopGEF8.
Fig. 6: Rab5 GTPases interact with RopGEF8 at endosomes in pollen.
Fig. 7: The establishment of polarity, that is, pollen germination, requires Rab5-mediated targeting of ROP signaling.

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Data availability

All data generated or analysed during this study are included in this published article and its Supplementary Information files.

References

  1. Johnson, M. A., Harper, J. F. & Palanivelu, R. A fruitful journey: pollen tube navigation from germination to fertilization. Annu. Rev. Plant Biol. 70, 809–837 (2019).

    Article  PubMed  Google Scholar 

  2. Li, E. et al. Signaling network controlling ROP-mediated tip growth in Arabidopsis and beyond. Plant Commun. 4, 100451 (2023).

    Article  PubMed  Google Scholar 

  3. Ruan, H., Li, J., Wang, T. & Ren, H. Secretory vesicles targeted to plasma membrane during pollen germination and tube growth. Front. Cell Dev. Biol. 8, 615447 (2020).

    Article  PubMed  Google Scholar 

  4. Liu, C., Zhang, Y. & Ren, H. Actin polymerization mediated by AtFH5 directs the polarity establishment and vesicle trafficking for pollen germination in Arabidopsis. Mol. Plant 11, 1389–1399 (2018).

    Article  PubMed  Google Scholar 

  5. Cascallares, M. et al. A complex journey: cell wall remodeling, interactions, and integrity during pollen tube growth. Front Plant Sci. 11, 599247 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Zhang, Y., He, J., Lee, D. & McCormick, S. Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes. Plant Physiol. 152, 2200–2210 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Hao, G. J. et al. Vesicle trafficking in Arabidopsis pollen tubes. FEBS Lett. 596, 2231–2242 (2022).

    Article  PubMed  Google Scholar 

  8. Xiang, X. et al. RHO OF PLANT proteins are essential for pollen germination in Arabidopsis. Plant Physiol. 193, 140–155 (2023).

    Article  PubMed  Google Scholar 

  9. Bouatta, A. M., Anzenberger, F., Riederauer, L., Lepper, A. & Denninger, P. Polarized subcellular activation of Rho proteins by specific ROPGEFs drives pollen germination in Arabidopsis thaliana. PLoS Biol. 23, e3003139 (2025).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Ueda, T., Yamaguchi, M., Uchimiya, H. & Nakano, A. Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. EMBO J. 20, 4730–4741 (2001).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lee, G. J., Sohn, E. J., Lee, M. H. & Hwang, I. The Arabidopsis rab5 homologs rha1 and ara7 localize to the prevacuolar compartment. Plant Cell Physiol. 45, 1211–1220 (2004).

    Article  PubMed  Google Scholar 

  12. Sohn, E. J. et al. Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins. Plant Cell 15, 1057–1070 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Rutherford, S. & Moore, I. The Arabidopsis Rab GTPase family: another enigma variation. Curr. Opin. Plant Biol. 5, 518–528 (2002).

    Article  PubMed  Google Scholar 

  14. Cui, Y. et al. Activation of the Rab7 GTPase by the MON1–CCZ1 complex Is essential for PVC-to-vacuole trafficking and plant growth in Arabidopsis. Plant Cell 26, 2080–2097 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ebine, K. et al. Plant vacuolar trafficking occurs through distinctly regulated pathways. Curr. Biol. 24, 1375–1382 (2014).

    Article  PubMed  Google Scholar 

  16. Singh, M. K. et al. Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB–vacuole fusion. Curr. Biol. 24, 1383–1389 (2014).

    Article  PubMed  Google Scholar 

  17. Uemura, T. & Ueda, T. Plant vacuolar trafficking driven by RAB and SNARE proteins. Curr. Opin. Plant Biol. 22, 116–121 (2014).

    Article  PubMed  Google Scholar 

  18. Bottanelli, F., Foresti, O., Hanton, S. & Denecke, J. Vacuolar transport in tobacco leaf epidermis cells involves a single route for soluble cargo and multiple routes for membrane cargo. Plant Cell 23, 3007–3025 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Wolfenstetter, S., Wirsching, P., Dotzauer, D., Schneider, S. & Sauer, N. Routes to the tonoplast: the sorting of tonoplast transporters in Arabidopsis mesophyll protoplasts. Plant Cell 24, 215–232 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Ebine, K. et al. A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6. Nat. Cell Biol. 13, 853–859 (2011).

    Article  PubMed  Google Scholar 

  21. Ito, E. et al. Integration of two RAB5 groups during endosomal transport in plants. eLife 7, e34064 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Goh, T. et al. VPS9a, the common activator for two distinct types of Rab5 GTPases, is essential for the development of Arabidopsis thaliana. Plant Cell 19, 3504–3515 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Nielsen, M. E., Jürgens, G. & Thordal-Christensen, H. VPS9a activates the Rab5 GTPase ARA7 to confer distinct pre- and postinvasive plant innate immunity. Plant Cell 29, 1927–1937 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Hao, G. J. et al. Two functionally interchangeable Vps9 isoforms mediate pollen tube penetration of style. N. Phytol. 244, 840–854 (2024).

    Article  Google Scholar 

  25. Hao, G. J. et al. Canonical Rab5 GTPases are essential for pollen tube growth through style in Arabidopsis. N. Phytol. 239, 1740–1753 (2023).

    Article  Google Scholar 

  26. Wang, Y. et al. Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis. Plant Physiol. 148, 1201–1211 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Francis, K. E., Lam, S. Y. & Copenhaver, G. P. Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene. Plant Physiol. 142, 1004–1013 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Ebine, K. et al. Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants. Small GTPases 3, 23–27 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Rosa, P., Barr, F. A., Stinchcombe, J. C., Binacchi, C. & Huttner, W. B. Brefeldin A inhibits the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. Eur. J. Cell Biol. 59, 265–274 (1992).

    PubMed  Google Scholar 

  30. Zhu, J. et al. Annexin5 plays a vital role in Arabidopsis pollen development via Ca2+-dependent membrane trafficking. PLoS ONE 9, e102407 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Takáč, T. et al. Wortmannin treatment induces changes in Arabidopsis root proteome and post-Golgi compartments. J. Proteome Res. 11, 3127–3142 (2012).

    Article  PubMed  Google Scholar 

  32. Wang, J., Cai, Y., Miao, Y., Lam, S. K. & Jiang, L. Wortmannin induces homotypic fusion of plant prevacuolar compartments. J. Exp. Bot. 60, 3075–3083 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Guilluy, C., Garcia-Mata, R. & Burridge, K. Rho protein crosstalk: another social network? Trends Cell Biol. 21, 718–726 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Vernoud, V., Horton, A. C., Yang, Z. & Nielsen, E. Analysis of the small GTPase gene superfamily of Arabidopsis. Plant Physiol. 131, 1191–1208 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Zhang, Y. & McCormick, S. A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 104, 18830–18835 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Miao, Y., Yan, P. K., Kim, H., Hwang, I. & Jiang, L. Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells. Plant Physiol. 142, 945–962 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Oliviusson, P. et al. Plant retromer, localized to the prevacuolar compartment and microvesicles in Arabidopsis, may interact with vacuolar sorting receptors. Plant Cell 18, 1239–1252 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Foresti, O. & Denecke, J. Intermediate organelles of the plant secretory pathway: identity and function. Traffic 9, 1599–1612 (2008).

    Article  PubMed  Google Scholar 

  39. Zhou, X. et al. SPA, a stigma–style-transmitting tract physical microenvironment assay for investigating mechano-signaling in pollen tubes. Proc. Natl Acad. Sci. USA 120, e2314325120 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Zhu, D., Zhang, M., Gao, C. & Shen, J. Protein trafficking in plant cells: tools and markers. Sci. China Life Sci. 63, 343–363 (2020).

    Article  PubMed  Google Scholar 

  41. Hirano, T. et al. The SYP123–VAMP727 SNARE complex delivers secondary cell wall components for root hair shank hardening in Arabidopsis. Plant Cell 35, 4347–4365 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Bement, W. M., Goryachev, A. B., Miller, A. L. & von Dassow, G. Publisher correction: patterning of the cell cortex by Rho GTPases. Nat. Rev. Mol. Cell Biol. 25, 333 (2024).

    Article  PubMed  Google Scholar 

  43. Takeuchi, H. & Higashiyama, T. Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis. Nature 531, 245–248 (2016).

    Article  PubMed  Google Scholar 

  44. Chang, F., Gu, Y., Ma, H. & Yang, Z. AtPRK2 promotes ROP1 activation via RopGEFs in the control of polarized pollen tube growth. Mol. Plant 6, 1187–1201 (2013).

    Article  PubMed  Google Scholar 

  45. Lee, Y. J., Szumlanski, A., Nielsen, E. & Yang, Z. Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth. J. Cell Biol. 181, 1155–1168 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Yang, Z. Small GTPases: versatile signaling switches in plants. Plant Cell 14, S375–S388 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Winge, P., Brembu, T., Kristensen, R. & Bones, A. M. Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana. Genetics 156, 1959–1971 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Zhou, L. Z. et al. Protein S-ACYL Transferase10 is critical for development and salt tolerance in Arabidopsis. Plant Cell 25, 1093–1107 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Hwang, J. U., Vernoud, V., Szumlanski, A., Nielsen, E. & Yang, Z. A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex. Curr. Biol. 18, 1907–1916 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Qu, X. et al. Arabidopsis villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars. Plant Cell 25, 1803–1817 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Diao, M., Qu, X. & Huang, S. Calcium imaging in Arabidopsis pollen cells using G-CaMP5. J. Integr. Plant Biol. 60, 897–906 (2018).

    Article  PubMed  Google Scholar 

  52. Wang, Z. P. et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol. 16, 144 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Xiang, X. et al. Arabidopsis class A S-acyl transferases modify the pollen receptors LIP1 and PRK1 to regulate pollen tube guidance. Plant Cell 36, 3419–3434 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Wan, Z. Y. et al. Arabidopsis PROTEIN S-ACYL TRANSFERASE4 mediates root hair growth. Plant J. 90, 249–260 (2017).

    Article  PubMed  Google Scholar 

  55. Ma, T., Li, E., Li, L. S., Li, S. & Zhang, Y. The Arabidopsis R-SNARE protein YKT61 is essential for gametophyte development. J. Integr. Plant Biol. 63, 676–694 (2021).

    Article  PubMed  Google Scholar 

  56. Xing, H. L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Grefen, C. & Blatt, M. R. A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC). Biotechniques 53, 311–314 (2012).

    Article  PubMed  Google Scholar 

  58. Zhong, S. et al. RALF peptide signaling controls the polytubey block in Arabidopsis. Science 375, 290–296 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Johnson-Brousseau, S. A. & McCormick, S. A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J. 39, 761–775 (2004).

    Article  PubMed  Google Scholar 

  60. Wu, F. H. et al. Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Methods 5, 16 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  PubMed  Google Scholar 

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Acknowledgements

This work is funded by the National Natural Science Foundation of China (grant nos 32270805 and 32470807), by collaborative funds from the National Natural Science Foundation of China and RGC (grant nos 32461160286 and N_CUHK482/24) and by a grant from the China Post-doctoral Science Foundation (grant no. 2024M751520).

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Author notes

  1. These authors contributed equally: Guang-Jiu Hao, Fei Yu.

Authors and Affiliations

  1. Frontiers Science Center for Cell Responses, College of Life Sciences, Nankai University, Tian’jin, China

    Guang-Jiu Hao, Gui-Min Yin & Yan Zhang

  2. State Key Laboratory of Wheat Improvement, College of Life Sciences, Shandong Agricultural University, Tai’an, China

    Guang-Jiu Hao, Fei Yu, Le-Yan Su, Xin-Ying Zhao & Sha Li

  3. School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong, Hong Kong, China

    Zi-zhen Liang

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  1. Guang-Jiu Hao
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Contributions

G.-J.H. and F.Y. performed the research with the assistance of Z.-z.L. and L.-Y.S. X.-Y.Z. contributed new reagents/analytic tools. G.-J.H., S.L., G.-M.Y. and Y.Z. analysed the data. G.-J.H., X.-Y.Z., S.L. and Z.-z.L. provided funding. Y.Z. and G.-J.H. wrote the paper with input from all other authors.

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Correspondence to Sha Li or Yan Zhang.

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Nature Plants thanks Peishan Yi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and Table 1.

Reporting Summary

Supplementary Video 1

GFP-ROP1 highly accumulates at the PM of the germination site in a qrt1 pollen grain.

Supplementary Video 2

GFP-ROP1 is enriched at the tip of a growing qrt1 pollen tube.

Supplementary Video 3

GFP-ROP1 distributes in cytoplasmic vesicles in a qrt1 rha1 ara7 ara6 pollen grain.

Supplementary Video 4

Active ROPs as indicated by CRIBRIC4-GFP are highly enriched at the PM of the germination site in a qrt1 pollen grain.

Supplementary Video 5

Active ROPs as indicated by CRIBRIC4-GFP are enriched at the apical PM of a growing qrt1 pollen tube.

Supplementary Video 6

Active ROPs as indicated by CRIBRIC4-GFP are associated with vesicular structures in a qrt1 rha1 ara7 ara6 pollen grain.

Supplementary Video 7

Cortical actin bundles as indicated by LifeAct-GFP in a qrt1 pollen grain.

Supplementary Video 8

Actin cables in the shank and dynamic actin bundles as indicated by LifeAct-GFP at the subapical region of a qrt1 pollen tube.

Supplementary Video 9

Ring-like actin structures indicated by LifeAct-GFP in a qrt1 rha1 ara7 ara6 pollen grain.

Supplementary Video 10

Post-Golgi vesicles labeled by mRFP-RabA4b distribute to the germination site in a qrt1 pollen grain.

Supplementary Video 11

Post-Golgi vesicles labeled by mRFP-RabA4b form in inverted cone at the tip of a qrt1 pollen tube.

Supplementary Video 12

Post-Golgi vesicles labeled by mRFP-RabA4b remain randomly distributed in a qrt1 rha1 ara7 ara6 pollen grain.

Supplementary Video 13

A growing pollen tube co-expressing nYFP-ARA7 and cYFP-RopGEF8.

Supplementary Video 14

A growing pollen tube co-expressing ARA6-nYFP and cYFP-RopGEF8.

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Hao, GJ., Yu, F., Liang, Zz. et al. Rab5 GTPases mediate the targeting of ROP signalling to establish polarity for pollen germination. Nat. Plants 11, 2319–2331 (2025). https://doi.org/10.1038/s41477-025-02130-6

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