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RLCKs phosphorylate RopGEFs to control auxin-dependent Arabidopsis development

Nature Plants (2025)Cite this article

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Abstract

Auxin plays a critical role throughout plant development. We have established earlier that auxin activates the ROP (plant RHO GTPase) molecular switch, and that guanine nucleotide exchange factors ROPGEFs, which activate ROPs, are important regulators for myriad auxin-regulated processes. Here we show that auxin induces phosphorylation of RopGEF1 and that four receptor-like cytoplasmic kinases (RLCKs), named RopGEF1-activating kinases 1/2/3/4 (RAK1/2/3/4), mediate this process. We show that RAKs interact with RopGEFs and specifically phosphorylate S488 on RopGEF1, promoting its stability and recruitment to the cell membrane, and enhance ROP activation. Knockout of these RLCKs blocked auxin-stimulated RopGEF1 phosphorylation, reduced ROP activation and led to defects in PIN-FORMED (PIN)-mediated auxin distribution and multiple developmental processes. Phospho-mimic RopGEF1 (S488D) shows enhanced guanine nucleotide exchange activity in vitro, and its expression in rak quadruple mutants reverses their phenotypes. The RLCK–RopGEF linkage represents an important functional node and elucidates a critical missing link in ROP-meditated auxin signaling.

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Fig. 1: RAK1/2/3/4 affects auxin-related growth and development in Arabidopsis.
Fig. 2: RAKs are important for the maintenance of auxin maximum, PIN1 polarity and abundance of PINs.
Fig. 3: RAK kinases interact with RopGEFs and phosphorylate RopGEF1.
Fig. 4: RAKs are indispensable for the maintenance of RopGEF1 protein PM localization and stability.
Fig. 5: Overexpression of the phosphomimic RopGEF1S488D restores the development of rak mutants, and the phosphorylation status of S488 affects its activity and the PM targeting of RopGEF1.
Fig. 6: Auxin promotes RAK-dependent RopGEF phosphorylation to regulate auxin-related growth and development.

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Data availability

All data that support the findings of this study are available in the Extended data and Supplementary Information. The biological materials used in this study are available from the corresponding author upon request. Sequence data from this paper can be found in the Arabidopsis Genome Initiative databases under the following accession numbers: RAK1 (At3g62220), RAK2 (At2g47060), RAK3 (At2g30740), RAK4 (At1g06700), RAK5 (At3g17410), RopGEF1 (At4g38430), RopGEF6 (At3g55660), RopGEF7 (At5g02010), ACTIN2 (At3g18780), PIN1 (At1g73590), PIN2 (At5g57090), PIN3 (At1g70940), PIN7 (At1g23080), VSR2 (At2g30290), VAMP711 (At4g32150) and RabF2a (At5g45130). Source data are provided with this paper.

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Acknowledgements

We thank F. Li for providing the CRISPR/Cas9-related vectors; H. Wang and C. Gao for providing the PVC and vacuole reporter lines and plasmids; J. Huang and C. Liu for TEM sample preparation and imaging; X. A. Zhu for assistance in drawing the diagram for Fig. 6d. This work was supported by grants from the National Natural Science Foundation of China (32370327 and 32070320) to L.-Z.T.

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  1. Wanqing Zhang

    Present address: CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory for the Development Biology and Environmental Adaptation of Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China

    Xiaojing Zhang, Hao Jiang, Guangqi Zhu, Fuhai Chen, Weichao Yuan, Yaqin Li, Wanqing Zhang, Dian Wu, Huili Liu & Li-Zhen Tao

  2. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China

    Xiaojing Zhang, Hao Jiang, Guangqi Zhu, Fuhai Chen, Weichao Yuan, Yaqin Li, Wanqing Zhang, Dian Wu, Huili Liu & Li-Zhen Tao

  3. Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA

    Hen-Ming Wu & Alice Y. Cheung

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Contributions

L.-Z.T. and X.Z. designed the research. X.Z., H.J., G.Z., F.C., W.Y., Y.L., W.Z., D.W. and H.L. performed the experiments. L.-Z.T., H.-M.W. and A.Y.C. conferred on overall visioning of the project, and together with X.Z. analysed the data. X.Z. and L.-Z.T. wrote the manuscript. L.-Z.T. and A.Y.C. revised the manuscript.

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Correspondence to Li-Zhen Tao.

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Extended data

Extended Data Fig. 1 Expression pattern of RAK1/2/3/4.

GUS staining of RAKspro:GUS lines indicated that RAK1, RAK2, RAK3 and RAK4 were expressed in embryos at various developmental stages, including globular (a1, e1, i1, m1), transition (a2, i2), and heart stages (a3 – a4, e2 – e4, i3 – i4, m2 – m4). RAK1, RAK2, RAK3, RAK4 were expressed in 4-d-old seedlings (b, f, j, n), root apical meristems (c, g, k, o) and flowers of 40-d-old plants (d, h, l, p).GUS staining durations were 16-24 h for embryos, seedlings and flowers of RAK1pro:GUS plants, 1.5-4 h for embryos, seedlings and flowers of RAK2 pro:GUS and RAK4pro:GUS plants; 1.5 h for seedlings and flower organs, 24-36 h for embryos of RAK3pro:GUS plants. The experiments in a-p are repeated independently three times with similar results. Scale bars = 25 μm in (a1) to (a4); (e1) to (e4); (i1) to (i4); (m1) to (m4) and (c), (g), (k), (o); 1 mm in (b), (d), (f), (h), (j), (l), (n) and (p).

Extended Data Fig. 2 Subcellular localization of RAK1/2/3/4 – GFP fusion proteins.

a, Expression of 35Spro:RAK1/2/3/4-GFP in rice hypocotyl protoplasts. b–e, GFP – tagged RAK1 (b), RAK2 (c), RAK3 (d), RAK4 € localization at the root epidermal cells of apical meristem and elongation zones of 4-d-old stable transformed Arabidopsis plants. Scale bars, 25 μm.

Extended Data Fig. 3 The expression of RPS5apro: RAKs and 35Spro: RAKs-GFP have complemented the embryo and root phenotypes of the rak4m mutants.

a, Expression of RAK1, RAK2, RAK3 or RAK4 under the control of embryonic promoter (RPS5a pro:RAKs) rescued the embryonic defects of rak4m mutants. Representative images of indicated stage (globular and heart) embryos are shown. b and c, Expression of RAK1, RAK2, RAK3 or RAK4 (35Spro: RAKs-GFP) rescued root phenotypes of rak4m mutants. 9-d-old seedlings were used for the analysis and representative images are shown. Vertical lines indicate meristem length. d and e, Quantification of the primary root length (d) and meristem cell numbers (e) of 9-day-old seedlings of wild type, rak4m mutants and representative complementation lines. Data are means ± SD (n = 38). Asterisks indicate significant differences from the wild type as tested by one-way ANOVA, followed by Dunnett’s post hoc test (*P < 0.05; ****P < 0.0001; ns, not significant). The experiments in a–e were performed at least three times with similar results. Scale bars, 25 μm in (a) and (c); 1 cm in (b).

Source Data

Extended Data Fig. 4 Deficiency of RAKs affect PIN1 polarity in cotyledon primordium cells of embryos.

Quantification of the PIN1 polarity were shown by the ratio of GFP fluorescence intensity for lateral to apical plasma membrane of PIN1-GFP in the cotyledon primordium cells of wild type (WT) and rak4m heart embryos. Data are presented as mean ± SD. The median (horizontal lines), interquartile ranges (boxes), whiskers (±1.5×interquartile range) and outliners of the data are shown (n = 16 individual cells from five embryos). The P value was calculated using two-tailed Student’s t-test (****P < 0.0001). The experiments were performed at least three times with similar results.

Source Data

Extended Data Fig. 5 Quantitative comparison of fluorescence intensity of PINs-GFP in the root cells of wild type and rak4m mutants.

Quantification of fluorescent intensity of PIN1-GFP, PIN2-GFP, PIN3-GFP and PIN7-GFP in wild type and rak4m root tips of 3-day-old seedlings. Data are means ± SD (n = 10 individual seedlings). The median (horizontal lines), interquartile ranges (boxes), whiskers (±1.5×interquartile range) and outliners of the data are shown. Asterisks indicate significant differences from the wild type as tested by one-way ANOVA, followed by Dunnett’s post hoc test (****P < 0.0001;).

Source Data

Extended Data Fig. 6 The interactions between RAKs and RopGEF6/7.

a and b, Luciferase complementation assays displayed the interactions of RAK1/2/3/4 with RopGEF6 (a) and RopGEF7 (b). Circles indicate areas that were infiltrated with Agrobacterium containing the indicated constructs. c, MBP-tagged RAKs pull down GFP-RopGEF6 from 7-day-old transgenic plants harboring RopGEF6p:GFP-RopGEF6. Lower panel shows total plant proteins stained with CBB. MBP was used as a control. d and e, Both GST-tagged RAK1/3 (d) and MBP-tagged RAK2/4 (e) pull down YFP-RopGEF7 from 9-day-old transgenic plants harboring 35Spro:YFP-RopGEF7. Lower panel shows total plant proteins stained with CBB. GST in (d) or MBP in (e) was used as a control. The experiments in c–e were performed independently three times with similar results.

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Extended Data Fig. 7 The endomembrane integrity of rak4m is not different from that of wild type in the root cells.

a–c, Transmission electron microscope (TEM) analysis of root meristem cells displayed similar endomembrane structure of 4-day-old wild type (WT) and ram4m mutants. However, ram4m mutants have enlarged vacuoles in the root meristem cells in contrast to wild type. Representative TEM images of root meristem cells in wild type (a), rak4m-1 (b) and rak4m-2 (c). d–f, Magnified view of the boxed area in wild type (a), rak4m-1 (b) and rak4m-2 (c), with multiple ER and Golgi bodies. PM denotes plasma membrane, ER denotes Endoplasmic reticulum, Golgi indicates Golgi body. Scale bars = 5 μm in a to c, and 500 nm in d to f.

Extended Data Fig. 8 Phosphatase treatment of immunoprecipitated RopGEF1-GFP.

Phosphatase treatment of immunoprecipitated RopGEF1-GFP extracted from 7-d old 35S:RopGEF1-GFP transgenic plants caused migration shift in gradient SDS-PAGE gel. Immunoprecipitated RopGEF1-GFP protein was treated with lambda (λ) phosphatase for 1 h. Anti-GFP was used to detect RopGEF1-GFP. The experiments were performed at least three times with similar results.

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Extended Data Fig. 9 Wortmannin treatment causes RopGEF1-GFP proteins sequestered to large intracellular aggregates in the root cells of rak4m seedlings.

a, Confocal images showed the subcellular distribution of GFP-labeled RopGEF1 in the root epidermis cells of meristem and elongation zones of 4-day-old wild type (upper panel) and rak4m (lower panel) treated with 0.1% (v/v) DMSO (wortmannin solvent) or 20 μM wortmannin for 3 h. Scale bars=20 μm. b, Immunoblot analysis of RopGEF1-GFP protein levels in wortmannin-treated and untreated rak4m seedlings. Total protein was isolated from 7-day-old rak4m transgenic seedlings carrying RopGEF1pro:RopGEF1-GFP treated without and with 20 μM wortmannin for 3 h. RopGEF1-GFP proteins were probed with GFP antibody and ponceau S staining was used as a loading control. The experiments in a and b were performed at least three times with similar results.

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Extended Data Fig. 10 Guanine nucleotide exchange activity of ROP6 were enhanced by RopGEF1 in a dosage-dependent manner.

a, The activity of the GEF domain of Dbs for a human Cdc42 protein were detected as a positive control by a fluorescence spectroscopy-based assay. Results are representative of three independent assays that gave similar results. b, Data showed dosage-dependent guanine nucleotide exchange activity of RopGEF1 for ROP6. Various concentrations (0, 0.5, 1, 2 μM) of RopGEF1 were tested toward ROP6. The experiments in a and b were performed independently at least three times with similar results.

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Supplementary Figs. 1–7, Tables 1–10, and Materials and Methods.

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Supplementary Data 1

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Unprocessed western blots and gels.

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Source Data Extended Data Figs. 3–5 and 10

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Unprocessed western blots.

Source Data Extended Data Fig. 9

Unprocessed western blots and gels.

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Zhang, X., Jiang, H., Zhu, G. et al. RLCKs phosphorylate RopGEFs to control auxin-dependent Arabidopsis development. Nat. Plants (2025). https://doi.org/10.1038/s41477-025-02111-9

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