Abstract
Pressurized cells with strong walls make up the hydrostatic skeleton of plants. Assembly and expansion of such stressed walls depend on a family of secreted RAPID ALKALINIZATION FACTOR (RALF) peptides, which bind both a membrane receptor complex and wall-localized LEUCINE-RICH REPEAT EXTENSIN (LRXs) in a mutually exclusive way. Here we show that, in root hairs, the RALF22 peptide has a dual structural and signalling role in cell expansion. Together with LRX1, it directs the compaction of charged pectin polymers at the root hair tip into periodic circumferential rings. Free RALF22 induces the formation of a complex with LORELEI-LIKE-GPI-ANCHORED PROTEIN 1 and FERONIA, triggering adaptive cellular responses. These findings show how a peptide simultaneously functions as a structural component organizing cell wall architecture and as a feedback signalling molecule that regulates this process depending on its interaction partners. This mechanism may also underlie wall assembly and expansion in other plant cell types.
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Data availability
Source data have been made available for this paper. Raw imaging data and accompanying information will be provided upon request due to the size and complexity of the datasets. Root-specific transcription profiles were retrieved from The Arabidpsis eFP Browser 2.0 (https://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html). Single-cell transcriptome data were collected from the Root scRNA-Seq Atlas database (https://www.zmbp-resources.uni-tuebingen.de/timmermans/plant-single-cell-browser-root-atlas/). The crystal structures of RALF4 (PDB: 6TME), LRX2 (PDB: 6QXP) and LLG2 (PDB: 6A5E) are available via the PDB. Source data are provided with this paper.
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Acknowledgements
This work was funded by the Research Foundation Flanders (Fonds Wetenschappelijk Onderzoek (FWO); grants 1225120N and G013023N), the University of Antwerp (UA; BOF-KP; DOCPRO4) and LASERLAB-EUROPE (grant 654148) to S.S. and K.V.; the Agence Nationale pour la Recherche (ANR), project ‘HOMEOWALL’ to H.H.; and the University of Lausanne, the European Research Council grant agreement no. 716358 and the Swiss National Science Foundation grant 310030_204526 to J.S. We thank A. Peaucelle and K. Haas for the fruitful discussions and J. Mravec, C. Ringli and S. Gilroy for sharing OG7-13647, lrx1-1/2-1 and lrx1-1 × pLRX1::cmyc-LRX1 seeds and Col-0 × 35S::GCaMP3 seeds, respectively. Our gratitude also goes to CPKelco (Denmark) for donating industrial low and high methoxyl pectin samples. The Institut Jean-Pierre Bourgin benefits from the support of Saclay Plant Sciences-SPS (ANR-17-EUR-0007). This work has benefited from the support of Institut Jean-Pierre Bourgin’s Plant Observatory technological platforms. Access to fluorescence microscopy infrastructure was provided by the Antwerp Centre for Advanced Microscopy. The purchase of the PerkinElmer UltraVIEW Vox and Leica SP8 confocal microscopes was supported by FWO-HERCULES infrastructure grants AUHA-09-001, AUHA-15-12. The Nikon Sora microscope was purchased with the support of an FWO mid-size infrastructure (I003420N) and FWO International Research Infrastructure grant (I000123N).
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S.S., K.V., H.H. and J.S. conceived the project. S.M. aided in conceptualizing the research based on his work on LRX8–RALF4. H.K.L. and J.S. designed, produced and characterized all recombinant proteins by SEC and SDS analysis. C.B. provided technical assistance for protein production. H.K.L. performed the TSAs. S.S. generated all crosses and performed the cloning of the RALF22-related constructs. E.A. generated and characterized the ralf22-1 line. E.A. and S.S. identified and characterized the ralf22-2 line. S.S. and E.F. optimized microfluidics for imaging. S.S. performed the phenotyping, staining and imaging of live cell and fixed-tissue samples. S.S. performed image analysis, bioinformatics and statistics. N.C. assisted with live cell imaging, cloning and sample preparations. D.B. assisted with cloning, phenotyping and sample preparation. S.S. and M.G. performed MST analysis. H.H. and B.C. conceived the QCM-D analysis. T.L. and H.H. performed the QCM-D analysis. T.L. characterized the pectin solutions. E.B. and C.M. provided technical and conceptual assistance with the in vitro QCM-D and pectin work. H.A. aided in conceptualizing the work and provided assistance with sample preparation for phenotyping. A.B., A.C., D.S.C.D and J.A.F. aided in the visualization and analysis of oscillatory parameters in live cell imaging. J.S. generated 3D protein structures and performed peptide-docking modelling. S.S., H.H. and K.V. wrote the paper.
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Supplementary Figs. 1–6.
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Supplementary Tables 1 and 2 (overview of statistics, product overview).
Supplementary Video 1
Time lapse video of Col-0 and ralf22-2 roots showing the short and bursting ralf22-2 RH phenotype. RALF22 loss-of-function RHs display aberrant growth and frequent bursting. Representative 2 h time lapse acquisitions of growing Col-0 and ralf22-2 (−/−) RHs.
Supplementary Video 2
Live cell imaging of changes in the growth rate, [Ca2+]cyt, pHex and cell wall physico-chemistry of Col-0 and ralf22-2 RHs being treated with 5 µM RALF22. Exogenous RALF22 supplementation induces a FER-dependent growth arrest/signalling response and a FER-independent change in cell wall physico-chemistry. Representative time lapse acquisitions (10 min) of longitudinal optical sections of growing Col-0 and fer-4 RHs expressing the [Ca2+]cyt sensor GCaMP3, in the presence of the dextran-coupled dyes FITC (110 kDa dextran, pH sensitive) and TRITC (20 kDa dextran, pH insensitive). RHs were imaged in a microfluidics chip for 4 min before the addition of 5 µM RALF22. The RH’s response was followed for another 6 min (scale bar, 5 µm).
Supplementary Video 3
Live cell imaging of ralf22-2 × pRALF22::mCherry-RALF22mature RHs showing RALF22 microdomain formation in the RH CW. RALF22 is secreted to the RH CW where it forms immobile periodic microdomains. a, Representative time lapse acquisition (10 min) of a longitudinal optical section of a growing ralf22-2 × pRALF22::mCherry-RALF22mature RH showing mCherry–RALF22mature localization to the entire RH CW. Corresponding kymograph showing the immobility of secreted mCherry–RALF22mature microdomains (shown as vertical red lines) throughout the acquisition (scale bar, 5 µm). b, RALF22 microdomains form in the growing RH dome. Consecutive time lapse frames of the growing tip have been aligned to allow visual tracking of mCherry–RALF22mature microdomains as they move from tip to shank while the tip grows forward. The concave shape of the growing dome was straightened to generate a kymograph. Red lines in the kymograph depict mCherry–RALF22mature microdomains, which originate at the very apex and remain immobile in the cell wall as they move towards the shank (scale bar, 5 µm).
Supplementary Video 4
FRAP of ralf22-2 × pRALF22::mCherry-RALF22mature RHs showing that RALF22 is secreted at the growing tip. RALF22 is secreted at the growing RH tip. Representative time lapse acquisition (10 min) of a longitudinal optical section of a growing ralf22-2 × pRALF22::mCherry-RALF22mature RH. After 4 min of growth, mCherry–RALF22mature fluorescence was bleached in a ROI in the tip and shank. FRAP was followed for an additional 6 min. Rapid mCherry–RALF22mature fluorescence recovery was observed in the tip, but not in subapical regions (scale bar, 5 µm).
Supplementary Video 5
Time lapse video of Col-0, ralf22-2 and lrx1-1/2-1 roots showing that the lrx1-1/2-1 phenotype is indistinguishable from ralf22-2. The lrx1-1/2-1 RH phenotype is indistinguishable from ralf22-2. Representative 2 h time lapse acquisitions of growing Col-0, ralf22-2 and lrx1-1/2-1 RHs.
Supplementary Video 6
Live cell imaging of ralf22-2 and lrx1-1/2-1 RHs expressing mCherry-RALF22mature, showing that LRX1/2 is required for secretion of RALF22 to the CW. LRX1/2 is required for RALF22 secretion. Representative time lapse acquisitions (10 min) of a longitudinal optical section of growing ralf22-2 × pRALF22::mCherry-RALF22mature and lrx1-1/2-1 × pRALF22::mCherry-RALF22mature RHs illustrating the accumulation of mCherry–RALF22mature in intracellular compartments in lrx1-1/2-1 RHs.
Supplementary Data 1
Statistical source data for Supplementary Fig. 5.
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Source Data Fig. 5e
Unprocessed gels.
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Schoenaers, S., Lee, H.K., Gonneau, M. et al. Rapid alkalinization factor 22 has a structural and signalling role in root hair cell wall assembly. Nat. Plants 10, 494–511 (2024). https://doi.org/10.1038/s41477-024-01637-8
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DOI: https://doi.org/10.1038/s41477-024-01637-8
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