# A RALF-brassinosteroid signaling circuit regulates Arabidopsis hypocotyl cell shape

David Biermann, Michelle von Arx, Kristina S Munzert-Eberlein, Kaltra Xhelilaj, David Séré, Martin Stegmann, Grégory Vert, Sebastian Wolf, Timo Engelsdorf, Cyril Zipfel, et al.

► To cite this version:

David Biermann, Michelle von Arx, Kristina S Munzert-Eberlein, Kaltra Xhelilaj, David Séré, et al. A RALF-brassinosteroid signaling circuit regulates Arabidopsis hypocotyl cell shape. Current Biology, 2025, 35 (20), pp.5002 - 5017.e5. 10.1016/j.cub.2025.09.016. hal-05335727

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**Current Biology**

# A RALF-brassinosteroid signaling circuit regulates *Arabidopsis* hypocotyl cell shape

## Graphical abstract

Wildtype

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Loss of RALF-mediated cell wall signaling

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## Authors

David Biermann, Michelle von Arx,
Kristina S. Munzert-Eberlein, ...,
Timo Engelsdorf, Cyril Zipfel,
Julien Gronnier

## Correspondence

cyril.zipfel@botinst.uzh.ch (C.Z.),
julien.gronnier@tum.de (J.G.)

## In brief

Biermann, von Arx, et al. show that RALF peptides regulate Arabidopsis hypocotyl cell shape by linking cell wall status to brassinosteroid signaling. Loss of FERONIA-mediated RALF signaling affects cell shape and cell wall composition, while RALF23 promotes BRI1-BAK1 complex formation, establishing a feedback circuit that coordinates morphogenesis.

## Highlights

* The plasma membrane RALF receptor complex FER-LLG1 regulates cell anisotropic growth

* RALF23 promotes BRI1-BAK1 complex formation and signaling

* RALF23 functions as a cell-wall-informed and wall-modifying signaling cue

<figure><img src="image_6.png" /></figure>
## Article

# A RALF-brassinosteroid signaling circuit regulates *Arabidopsis* hypocotyl cell shape

David Biermann,<sup>1,2,7</sup> Michelle von Arx,<sup>1,2,3,7</sup> Kristina S. Munzert-Eberlein,<sup>4</sup> Kaltra Xhelilaj,<sup>2</sup> David Séré,<sup>5</sup>

Martin Stegmann,<sup>5,8</sup> Grégory Vert,<sup>6</sup> Sebastian Wolf,<sup>2</sup> Timo Engelsdorf,<sup>4</sup> Cyril Zipfel,<sup>3,5,*</sup> and Julien Gronnier<sup>1,2,3,9,*</sup>

## SUMMARY

Plant cells survey and modulate their cell walls to control their shape and anisotropic growth. Signaling mediated by the plant steroid hormones brassinosteroids (BR) plays a central role in coordinating cell wall status and cell growth, and alterations in the cell wall-BR feedback loop leads to life-threatening defects in tissue and cellular integrity. How the status of the cell wall is relayed to BR signaling remains largely unclear. Increasing evidence shows that RAPID ALKALANIZATION FACTORs (RALFs), a class of secreted peptides, play structural and signaling roles at the cell surface. Here, we show that loss of RALF signaling mediated by the plasma membrane-localized RALF receptor complex FERONIA (FER)-LORELEI-LIKE GPI-anchor protein 1 (LLG1) leads to defects in cell morphology, organization, and composition of the cell wall—including pectin methylation status—and to an increase in BR signaling. We observed that increase in BR signaling mitigates the cellular defects associated with the loss of FER-mediated RALF signaling. We show that perception of RALF23 promotes the formation and signaling activity of the main BR receptor complex formed by BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED KINASE 1 (BAK1) and that its bioactivity relies on pectin status. Our observations suggest a model in which RALF peptides operate at the nexus between cell wall status and BR signaling, capable of inducing changes in the cell wall as well as functioning as a cell-wall-informed signaling cues initiating a feedback loop that solicits BR signaling and coordinates cell morphogenesis.

## INTRODUCTION

Plant cells are encased by the cell wall, a dynamic polysaccharide-rich matrix providing tensile strength and controlling cell shape and integrity.<fnref n="1" /> During development, expanding cells face a life-or-death dilemma: they must precisely control the modifications of the cell wall to allow anisotropic growth while preserving cell wall integrity.<fnref n="2" /> Accumulating evidence indicates that this tight balance is controlled by cell-wall-derived cues that are perceived as informative signals at the cell wall-plasma membrane interface to initiate cell wall signaling (CWS).<fnref n="3" />⁻¹⁰ Among the recently identified CWS components, the RAPID ALKALINIZATION FACTOR (RALF) family of secreted peptides and its binding partners play a pivotal role in regulating growth,¹¹⁻¹⁵ reproduction,<fnref n="4" />,¹⁶⁻¹⁹ responses to abiotic stresses,²⁰⁻²² and immunity.²³⁻²⁷ Recent studies show that positively charged RALF peptides associate with cell wall

carbohydrates in the form of de-esterified pectin.²⁸⁻³² RALF-pectin oligomers associate with cell wall-bound LEUCINE-RICH REPEAT EXTENSIN (LRX) proteins<fnref n="4" />,¹³,²⁸⁻³⁰,³³ and plasma-membrane-localized complexes composed of *Catharanthus roseus* RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) and LORELEI-LIKE glycosylphosphatidylinositol-anchored (LLG) proteins.¹²,³¹,³⁴⁻³⁹ In the cell wall of tip-growing cells, Pectin-RALF-LRX complexes assemble into a reticulated network providing structural support for cellular expansion.²⁸,²⁹ At the cell surface, RALF perception has been linked to the initiation of several signaling events such as changes in apoplastic pH,⁴⁰⁻⁴² the production of reactive oxygen species,²³,⁴³ and an influx of calcium.³⁵,⁴⁴,⁴⁵ We previously reported that perception of RALF23 by the plasma membrane-localized receptors LLG1³<fnref n="6" /> and the CrRLK1L FERONIA⁴⁶ inhibits complex formation of the immune leucine-rich repeat (LRR) receptor kinases (RKs) FLAGELLIN SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR)

<footnote n="1"><sup>1</sup>Plant Cell Biology, TUM School of Life Sciences, Technical University of Munich, 85354 Freising, Germany</footnote>

<footnote n="2"><sup>2</sup>Center for Plant Molecular Biology (ZMBP), University of Tübingen, 72076 Tübingen, Germany</footnote>

<footnote n="3"><sup>3</sup>Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, 8092 Zürich, Switzerland</footnote>

<footnote n="4"><sup>4</sup>Molecular Plant Physiology, Department of Biology, Philipps-Universität Marburg, 35043 Marburg, Germany</footnote>

<footnote n="5"><sup>5</sup>The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, UK</footnote>

<footnote n="6"><sup>6</sup>Plant Science Research Laboratory (LRSV), UMR5546 CNRS/Université Toulouse 3, 24 chemin de Borde Rouge, 31320 Auzeville Tolosane, France</footnote>

<footnote n="7"><sup>7</sup>These authors contributed equally</footnote>

<footnote n="8"><sup>8</sup>Present address: Molecular Botany, Institute of Botany, Ulm University, Ulm, Germany</footnote>

<footnote n="9"><sup>9</sup>Lead contact</footnote>

<footnote>*Correspondence: cyril.zipfel@botinst.uzh.ch (C.Z.), julien.gronnier@tum.de (J.G.)</footnote>

<footnote>https://doi.org/10.1016/j.cub.2025.09.016</footnote>
A

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B

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C

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with their co-receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED KINASE 1 (BAK1, also known as SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3, SERK3)²,³,⁹⁹ and that such inhibitory function is linked to the regulation of receptor and co-receptor plasma membrane organization.²⁵ It has recently been shown that this effect is not limited to FLS2 and BAK1, and that perception of RALF23 induces global changes in membrane organization,³¹,⁴⁷⁻⁴⁹ notably impacting the organization of additional cell-surface receptors, such as the LRR-RK BRI1.³¹ While the effect of RALF perception on immune signaling is well characterized,²³,²⁵,²⁷,⁴⁸,⁵⁰,⁵¹ the functional consequences of RALF perception for other RK signaling pathways and potential link with CWS remain, however, unexplored. BRI1, the main receptor for the plant steroid hormones brassinosteroids (BR), forms a ligand-induced complex with SERKs such as BAK1/SERK3.⁵²⁻⁵⁶ BRI1-BAK1 complex formation triggers a signaling cascade that involves the de-phosphorylation and nuclear translocation of the transcription factors BRI1 EMS-SUPRESSOR 1 (BES1) and BRASSINAZOLE RESISTANT 1 (BZR1),⁵⁷,⁵⁸ that initiates a transcriptional reprogramming regulating growth.⁵⁹⁻⁶² It is becoming clear that BR signaling functions at the crossroad between CWS and growth.⁸ Indeed, alteration of pectin methylation status triggers BR signaling,⁶³,⁶⁴ BR signaling modulates the expression of a multitude of cell-wall-associated transcripts,⁶⁵,⁶⁶ as well as the activity of wall-modifying enzymes,⁶⁷⁻⁶⁹ and the inhibition of BR signaling in a mutant affecting pectin modification leads to life-threatening defects in tissue and cellular integrity.⁷⁰ Despite the apparent vital importance of the interplay between the cell wall and BR signaling, little is known about the underpinning molecular actors. In recent years, however, several lines of evidence point toward the predominant role of cell-wall-monitoring cell-surface receptors in this process. Indeed, the Arabidopsis-receptor-like protein RECEPTOR-LIKE PROTEIN 44 (RLP44) and the receptor kinase WALL-ASSOCIATED KINASE (WAK)-LIKE 22/RESISTANCE TO FUSARIUM OXYSPORUM 1 (WAKL22/RFO1) mediate the activation of BR signaling in response to pectin modification.⁶⁴,⁷¹ In rice (Oryza sativa, Os), changes in pectin alleviate the inhibition of OsBRI1 signaling by OsWAK11.⁷²

Here, we show that the loss of RALF-mediated CWS leads to defects in cell wall organization and composition, cell morphology, and to an increase in BR signaling. We show that increase in BR signaling mitigates the cell morphological defects induced by the loss of *FER*, placing RALF-mediated CWS at the crossroad between cell wall status and a BR signaling feedback loop. In addition, we demonstrate that RALF23 promotes BRI1-BAK1 complex formation and signaling, and that its bioactivity relies on pectin status. We show that RALF23 perception induces changes in pectin organization and in the activity of pectin-modifying enzymes, thus unveiling cell-type and RALF receptor specificities in these processes. Our observations suggest a model in which RALF peptides operate at the nexus between cell wall status and BR signaling, capable of inducing changes in the cell wall as well as functioning as a cell wall-informed signaling cue, initiating a feedback loop that solicitates BR signaling and coordinates cell morphogenesis.

## RESULTS

### Loss of *FER* leads to an increase in basal BRI1-BAK1 complex formation and signaling

The *FER* loss-of-function mutant *fer-2* was reported to be hypersensitive to exogenous treatment with 24-epi-brassinolide (BL), a potent BR.⁷³ To confirm these observations, we used an additional knockout allele for *FER*, *fer-4*, and a corresponding complementation line expressing FER-GFP under the control of its native promotor.⁷⁴ In hypocotyl elongation assays, we observed that *fer-4* is hypersensitive to BL treatment and that expression of FER-GFP restored BL responsiveness to wild-type (WT) levels (Figure 1A). Since FER regulates ligand-induced complex formation between FLS2 and BAK1,²³ we asked whether the increased BL responsiveness observed in *fer-4* can be explained by changes in BRI1-BAK1 complex formation. In co-immunoprecipitation experiments, we observed an increase in the association between BRI1-GFP and BAK1 in *fer-4* compared with WT and that *fer-4* and WT showed similar relative increase in complex formation upon exogenous BL treatment (Figures 1B and 1C). The loss of *FER* was accompanied by

### Figure 1. Loss of *FERONIA* leads to an increase in basal BRI1-BAK1 complex formation and signaling

(A) Quantification of hypocotyl length of 5-day-old Arabidopsis seedlings grown on half MS medium containing 200 nM brassinolide (BL) or corresponding mock control solution (EtOH). Scattered data points indicate measurements of individual seedlings. Conditions that do not share a letter are significantly different in Dunn's multiple comparison test (*p* < 0.0001).

(B) Immunoprecipitation of BRI1-GFP in Arabidopsis seedlings after treatment with 1 µM BL or corresponding mock control solution (EtOH) for 90 min. Membranes were probed with anti-GFP or anti-BAK1 antibodies. Membrane stained with Coomassie brilliant blue (CBB) is presented to show equal loading. Similar observations were made in at least three independent experiments.

(C) Quantification of co-immunoprecipitated BAK1 in Col-0 or *fer-4* seedlings expressing BRI1-GFP treated with 1 µM BL or mock control solution (EtOH) for 90 min. Individual scatter data points indicate independent experiments. *p* value was determined using Students *t* test.

(D) Quantitative real-time PCR of *CPD* and *DWF4* transcripts from 12-day-old seedlings. Scattered data points indicate measurements of individual biological samples, each corresponding to 2–3 seedlings pooled, obtained from three independent experiments. *U-BOX* was used as a house keeping gene and values are expressed relative to Col-0. *p* values report Mann-Whitney statistical test.

(E) Representative *z* stack projections of hypocotyl confocal images of 5-day-old seedlings expressing pBZR1::BZR1-Ypet or pBES1::BES1-Ypet treated for 60 min with 1 µM BL or mock control solution (EtOH).

(F) Quantification of mean nuclear fluorescence intensity of BZR1-Ypet or BES1-Ypet in Col-0 or *fer-c*. Graphs are combined violin and box plots with *n* = 336–892 nuclei from 3–5 seedlings. Individual scatter points show outliers. Conditions that do not share a letter are significantly different according to one-way ANOVA with Tukey post-hoc HSD test (*p* < 0.05). The experiment was performed three times.

(G) Western blot analysis of BES1 in 12-day-old seedlings in presence and absence of 200 nM BL treatment. Note that the BL response in WT seedlings deviates from the canonical BL-induced accumulation of dephosphorylated BES1; therefore, these experiments are not informative regarding BL responsiveness. Membranes were probed with anti-BAK1 antibodies and stained with CBB as loading controls.

See also Figure S1.
an increase in BAK1 protein accumulation and a decrease in BRI1 and BRI1-GFP protein accumulation (Figure S1), which correlated with variation in the accumulation of the corresponding transcripts (Figures S1C and S1F).

We next monitored transcript accumulation of two marker genes negatively regulated by BRs, *CPD* and *DWF4*.⁷⁵ We observed a decrease in the transcript accumulation of *CPD* and *DWF4* in *fer*-4, indicative of an increase in BR signaling (Figure 1D). In agreement, in the hypocotyl of *FER* loss-of-function CRISPR mutants (*Figures S1K–S1N*), using confocal microscopy, we observed an increase in nuclear localization of two BR-responsive transcription factors BES1 and BZR1 tagged with the fluorescent protein Ypet and expressed under their corresponding native promoters (*Figures 1E and 1F*). Western blot analysis indicated an overall decrease in Ypet-BES1, Ypet-BZR1, and endogenous BES1 in *FER* loss-of-function mutants (*Figures 1G and S1O–S1T*). This decrease was not apparent for Ypet-BES1 and Ypet-BZR1 in hypocotyl epidermal cells (*Figures 1E and 1F*) and may be linked to changes in transcription, translation, or degradation of BES1 and BZR1 in other cell types. We observed that BL-induced dephosphorylation of BES1-Ypet and BZR1-Ypet was marginally higher in *fer* CRISPR mutants compared with WT (*Figures S1P and S1S*). Taken together, these results show that loss of *FER* leads to an increase in basal BRI1-BAK1 complex formation and BR signaling leading to an increase hypocotyl growth upon exogenous BL treatment. Co-immunoprecipitation experiments suggest that FER is not in close proximity to BRI1 (Figure S1U) implying that regulation of BRI1-BAK1 association by FER may be indirect.

FER regulates hypocotyl cell anisotropic growth and the activity of pectin-modifying enzymes

FER was shown to contribute to the mechanical integrity in the Arabidopsis root and shoot,⁷⁶,⁷⁷ and the loss of *FER* affects pavement cell anisotropic growth.²⁵,⁷⁸ We set to investigate the potential effect of loss of *FER* on cell growth and morphology in the hypocotyl of light-grown seedlings. Microscopic observation of *fer*-4 revealed defects in cell shape (Figures 2, S2A, and S2B). To gain quantitative information on these morphological defects, we performed automated high resolution 3D confocal imaging of optically cleared samples and computed 3D volumes of the hypocotyl epidermis using MorphographX.⁷⁹ Compared with WT, *fer*-4 showed a pronounced increase in cell volume and a decrease in cell anisotropy (Figures 2A–2C). Moreover, we repeatedly observed cellular outgrowths and loss of cellular integrity in *fer*-4, both in fixed and living samples (Figures S2A and S2B). We conclude that *FER* is genetically required to control anisotropic growth of hypocotyl epidermal cells.

In the cell wall, pectin forms a complex and dynamic meshwork that actively participates in shaping plant cells, although the exact contribution of pectin to morphogenesis remains enigmatic.⁸⁰⁻⁸³ Changes in pectin methylation degree correlate with cell wall biophysical properties⁸⁴,⁸⁵ and the overexpression of enzymes modifying pectin methylation, pectin methyl esterases (PMEs), or PME inhibitors (PMEIs) is sufficient to alter cell morphology.⁸⁶,⁸⁷ As loss of *FER* is associated with a decrease in de-esterified pectin at the filiform apparatus of synergids

cells,⁸⁸ we wondered whether defects in cell shape of the hypocotyl epidermis observed in *fer*-4 are linked to changes in pectin status in vegetative tissue. To this end, we used a fluorescently labeled chitosan oligosaccharide probe (COS-488), which binds to de-esterified pectin oligomers,⁸⁹ to monitor pectin status. We observed an increase in COS-488 staining in *fer*-4 compared with WT, indicating an increase in de-esterified pectin (Figures 2C and 2D). In dot-blot assays, probing water-extracted pectin with antibodies that present distinct affinity toward highly esterified, partially esterified, and mostly de-esterified pectin,⁹⁰ we observed a decrease in signal of all anti-pectin antibodies used to probe *fer*-4 cell wall fractions (Figure S2C). The analysis of cell wall monosaccharide composition of the pectin extracts by high-performance anion-exchange chromatography with pulsed amperometry detection (HPAEC-PAD) indicated that this reduced signal reflects a decrease in the relative proportion of pectin in *fer*-4 extracts but not a decrease in the total amount of galacturonic acid (GalA, forming homogalacturonan) (Figures 2E, S2D, and S2E; Table S1). HPAEC-PAD analyses of cell walls showed complex alterations in the cell wall of *fer*-4, in line with FER's proposed function as a cell wall sensor⁹,²⁰ (Figure 2E; Table S1), and a decrease in pectin methylation degree in *fer*-4 compared with WT, further supporting COS-488 staining (Figure 2F). To corroborate these observations, we examined the activity of PMEs, the enzymes responsible for pectin de-esterification.⁹¹ We observed an increase in PME activity in *fer*-4 proteinaceous cell wall extracts (Figure 2G). In good agreement, the pharmacological inhibition of PMEs using epigallocatechin-3-gallate (EGCG)⁹² mitigated the increase in COS-488 staining in *fer*-4 (Figure 2D). Altogether, these observations indicate that *FER* is genetically required to control cell wall composition, pectin modifications, and cell anisotropy. Since BR is a major determinant promoting cell growth and expansion,⁹³,⁹⁴ we asked whether increased BR signaling causes the increase in cell volume and the loss of anisotropy observed in *fer*-4. We crossed *fer*-4 with *bak1-4*, a *BAK1* knockout allele⁹⁵ and *bri1-301*, an hypomorphic allele of *BRI1*⁹⁶ (Figure 3A). As expected, *bri1-301* and *bak1-4* were hyporesponsive to BL treatment (Figure 3B). We observed that introducing *bri1-301* or *bak1-4* alleles in *fer*-4 led to cumulative growth defects (Figure 3A) and alleviated *fer*-4 hypersensitivity to exogenous BL treatment (Figure 3B), indicating that they effectively mitigate the increase in BR signaling observed in *fer*-4. However, quantitative analysis of hypocotyl epidermal cell 3D morphology showed that introducing *bak1-4* or *bri1-301* in *fer*-4 does not alleviate cell volume and cell shape defects of *fer*-4 (Figures 3C and 3D). In contrast, cell anisotropy appears more severely affected in *fer*-4/*bak1-4* and *fer*-4/*bri1-301* than in *fer*-4 (Figures 3C and 3D). These results indicate that increased BR signaling is not causal for the *fer*-4 morphological defects. On the contrary, these observations suggest that the increase in BR signaling attenuates the defects caused by the loss of *FER*. Corroborating this hypothesis, we observed that BL treatment alleviated *fer*-4 defects in cell anisotropy (Figures S2F–S2K). Altogether, these observations indicate that the increase in BR signaling compensates for the morphological defects associated with the loss of FER-mediated cell-wall-monitoring and place FER-mediated CWS at the crossroad between cell wall status and BR signaling.
<figure><img src="image_14.png" /><figcaption>**Figure 2. FERONIA regulates cell anisotropic growth and pectin methylation status**</figcaption></figure>

(A) 3D segmentation of the hypocotyl epidermal cells of fixed 5-days-old seedlings stained with SR2200. Cells are colored according to their volume.

(B) Quantification of cell volume and anisotropy in Col-0 and *fer-4*. Graphs are combined violin and boxplots, *n* = 647–760 cells from 5–6 seedlings. Individual scatter points show outliers. Conditions that do not share a letter are significantly different according to one-way ANOVA with Tukey's post-hoc HSD test (*p* < 0.05).

(C) Representative z stack projections of hypocotyl confocal images stained with COS-488 labeling de-esterified pectin in Col-0 and *fer-4*.

(D) Quantification of COS-488 fluorescence intensity in Col-0 or *fer-4*. Plants were either treated with 50 µM epigallocatechin-3-gallate (EGCG) for 2.5 h or corresponding mock solution (DMSO). Individual scatter points show *n* = 50 hypocotyl epidermis cells from 5 seedlings. Conditions not sharing a letter are significantly different according to Dunn's multiple comparison test (*p* < 0.05). The experiment was performed three times.

(E) Heatmap showing cell wall composition of 2-week-old Col-0 and *fer-4* seedlings. Boxes are colored according to the mean amount of µg.mg⁻¹ alcohol-insoluble residue (AIR) from 5 biological replicates. GalA, galacturonic acid; GlcA, glucuronic acid. The symbols +, −, or = indicate significant increase, decrease or no significant change in sugar abundance as determined by Student's *t* test, respectively.

(F) Pectin methylation degree (mol %) in Col-0 and *fer-4*. Scatter points show 5 independent biological replicates, *p* value was determined by Student's *t* test.

(G) Relative PME activity determined by gel diffusion assay. Scatter points show replicates from 3 experiments, *p* value was determined by pairwise Mann-Whitney test.

See also Figure S2 and Table S1.

## RALF peptides regulate hypocotyl epidermal cell morphology and pectin methylation status

We sought to further investigate the molecular basis for FER function in regulating cell anisotropy. FER has been shown to bind to RALF peptides and pectin.<sup>20,35,39,78,97</sup> The FER ectodomain contains two malectin-like domains, MalA and MalB.<sup>98</sup> FER MalA domain was shown to bind pectin *in vitro*<sup>20,78,97</sup> and is required for FER function in regulating pavement cell shape and root hair growth.<sup>25,78</sup> On the other hand, FER MalB is sufficient for RALF23 responsiveness.<sup>25,39</sup> To test whether FER function in regulating cell anisotropy and BR signaling involves pectin sensing and/or RALF perception, we analyzed *fer-4* mutant complemented with a truncated version of FER lacking its MalA domain (FER<sup>ΔMalA</sup>).<sup>25</sup> We noticed that FER<sup>ΔMalA</sup> complements the BL hypersensitivity, hypocotyl cell volume and anisotropy defects observed in *fer-4* to a level that is comparable to the

complementation line expressing full-length FER (Figures 1 and S3A–S3E). These observations suggest that FER's function in regulating hypocotyl cell morphology and BR signaling is primarily achieved by RALF perception and that MalA-mediated pectin sensing has a minor role in this context. To confirm these observations, we analyzed *llg1-2*, a loss-of-function mutant of *LLG1*, which encodes the main RALF co-receptor in vegetative tissues.<sup>37,39,99</sup> We observed that loss of *LLG1* leads to defects in hypocotyl epidermal cell morphology that are reminiscent of *fer-4* (Figures S3F–S3H). Further, similar to *fer-4*, we observed that *llg1-2* is hypersensitive to exogenous BL treatment (Figure S3I), indicating that BR signaling is hyperactive in *llg1-2*. In agreement, we observed a decrease in *CPD* and *DWF4* transcript levels in *llg1-2* (Figure S3J). Altogether, these observations indicate that hypocotyl epidermal cell morphology and BR signaling are regulated by RALF signaling.
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 3.** Increase in BR signaling does not underlie morphological defects of *fer-4*</figcaption></figure>

(A) Rosettes of 4-week-old Arabidopsis plants grown in a 16-h light regime.

(B) Quantification of hypocotyl lengths of 5-day-old seedlings grown on half Murashige and Skoog (MS) medium containing 200 nM brassinolide (BL) or corresponding mock control solution (EtOH). Graphs are boxplots, scattered data points show individual measurements pooled from three independent experiments. Conditions that do not share a letter are significantly different in Dunn's multiple comparison test (p < 0.0001).

(C) 3D segmentation of the hypocotyl epidermal cells of fixed 5-day-old seedlings stained with SR2200. Cells are colored according to their volume.

(D) Quantification of cell volume and anisotropy. Graphs are combined boxplots and violin plots, *n* = 401–630 cells from 4–5 seedlings. Individual scatter points show outliers. Conditions not sharing a letter are significantly different according to one-way ANOVA with Tukey's post-hoc HSD test (p < 0.05).
See also Figure S2.

RALF peptides constitute a multigenic family with over 30 members in Arabidopsis Col-0.⁴³ RALF1, RALF22, RALF23, and RALF33 are among the most abundant RALF transcripts in the hypocotyl (Figure S4A). We observed that the quadruple loss-of-function mutant for RALF1, RALF22, RALF23, and

RALF33 (ralf1/22/23/33, previously referred to as *ralf quad*¹⁸) was affected in overall plant growth, phenocopying loss of *FER* and *LLG1* (Figure 4A). The analysis of the effect of BL treatment on hypocotyl growth, and measurement of *CPD* and *DWF4* transcript accumulation (Figures 4B–4D) suggested that loss of
<figure><img src="image_17.png" /><figcaption>Figure 4. RALFs regulate cell morphology and pectin methylation status</figcaption></figure>

(A) Rosettes of 4-week-old Arabidopsis plants grown in a 16-h light regime.

(B) Quantification of hypocotyl length of 5-day-old seedlings growth on half MS medium containing 200 nM brassinolide (BL) or corresponding mock control solution (EtOH). Graphs are boxplots, scattered data points show individual measurements from three independent experiments. Conditions that do not share a latter are significantly different according to Dunn's multiple comparison test (*p* < 0.0001).

(C) Quantitative real-time PCR of *CPD* and *DWF4* transcripts from 12-day-old seedlings. Scattered data points indicate measurements of individual biological samples, each corresponding to 2-3 seedlings pooled, obtained from three independent experiment. *UBQ* was used as a house keeping gene, and values are expressed relative to Col-0. Conditions that do not share a latter are significantly different according to Dunn's multiple comparison test (*p* < 0.05).

(D) 3D segmentation of hypocotyl epidermal cells of fixed 5-day-old seedlings stained with SR2200. Cells are colored according to their volume.

(E) Quantification of cell volume and anisotropy. Graphs are combined boxplots and violin plots, *n* = 399–549 cells from 4–5 seedlings. Individual scatter points show outliers. Conditions not sharing a letter are significantly different according to one-way ANOVA with Tukey's post-hoc HSD test (*p* < 0.05).

(F) Confocal z stack projections of 5-day-old hypocotyl epidermis cells stained with COS-488 labeling de-esterified homogalacturonan.

(G) Quantification of COS-488 fluorescence intensity in Col-0 and *ralf1/22/23/33*. Plants were either treated with 50 µM EGCG for 2.5 h or corresponding mock solution (DMSO). Individual scatter points show *n* = 50 hypocotyl epidermis cells from 5 seedlings. Conditions not sharing a letter are significantly different according to Dunn's multiple comparison test (*p* < 0.05). The experiment was performed 3 times.

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RALF1, RALF22, RALF23, and RALF33 led to an increase in BR signaling. The analysis of hypocotyl epidermal cells showed that ralf1/22/23/33 was affected in cellular morphology with an increase in cell volume and in cell anisotropy (Figures 4D and 4E). Live cell imaging using COS-488 staining and HPAEC-PAD analysis of cell wall monosaccharides indicated that loss of RALF1, RALF22, RALF23, and RALF33 led to changes in cell wall composition comparable to fer-4 with a decrease in pectin methylation (Figures 4F-4I; Table S2). Consistently, we observed an increase in PME activity in proteaceous wall extract of ralf1/22/23/33 (Figure 4J) and inhibiting PMEs using EGCG alleviated the increase in COS-488 signal (Figure 4G). Altogether, these observations further support a tight functional link between RALF signaling, cell wall status, and BR signaling.

## RALF23 promotes BRI1-BAK1 complex formation

Emerging evidence shows that RALFs play structural and signaling roles at the cell surface.²⁸,²⁹ Positively charged RALFs associate with de-esterified pectin and mutations in RALF4, RALF22, or RALF1 basic residues affect their function.²⁸,²⁹,³¹,³² In the context of tip-growing cells, RALF4-pectin and RALF22-pectin oligomers associate with LRX8 and LRX1 proteins, respectively.²⁸,²⁹ In leaves, the association of RALF1 to pectin has been proposed to form condensates nucleating FER-LLG1 complex formation.³¹ RALF23 is a bona fide ligand of the FER-LLG1 complex.²³,³⁹ In addition, RALF23 associates with LRX3, LRX4, and LRX5 in planta, and the loss of LRX3, LRX4, and LRX5 affects RALF23 responsiveness,²¹,²⁵ suggesting that RALF23 binds to both LRXs in the cell wall and FER-LLG1 at the plasma membrane. In agreement, AlphaFold-multi-mer predicts binding of RALF23 to LRX3, LRX4, and LRX5 (Figure S4B). Analogous to LRX8-bound RALF4, RALF23 exposes a polycationic surface compatible with pectin binding in the predicted LRX3-RALF23, LRX4-RALF23, LRX5-RALF23, and FER-LLG1-RALF23 complexes (Figures S4B and S4C). Mutating surface-exposed positively charged residues of RALF23, and pharmacological inhibition of PME abolished RALF23 bioactivity (Figures S4D, S4E, and S4H), suggesting that, as for RALF1 and RALF4,²⁸,²⁹,³¹,³² pectin binding is required for RALF23 function. We next asked whether RALF23 regulates BR signaling. In co-immunoprecipitation experiments, we observed that RALF23 treatment promotes BRI1-BAK1 association (Figures 5A and 5B). In good agreement, we observed that plants overexpressing RALF23-GFP showed a decrease in CPD and DWF4 transcripts accumulation as well as a decrease in the accumulation of BES1 (Figures 5C and 5D). In addition, we observed that RALF23 treatment promoted the nuclear localization of BES1-Ypet and BZR1-Ypet (Figures 5E, 5F, and S4F). Furthermore, the macroscopic analysis of hypocotyl length and microscopy observation of hypocotyl cells showed that

overexpression of RALF23-GFP increases BL responsiveness, which was suppressed by introducing the *bri1-301* allele (Figures S5A-S5C). Finally, we observed that the inhibition of PME alleviated the effect of RALF23 on BR signaling (Figures S4H and S4I). Taken together, these results indicate that RALF23 perception promotes BRI1-BAK1 complex formation and BR signaling.

## RALF23 modulates the organization of the cell wall in a context and receptor-specific manner

Since an increase in BR signaling is linked to defects in cell morphology in *fer-4*, we asked whether the overexpression of RALF23 affects hypocotyl cell shape. The analysis of 3D segmentation of OxRALF23-GFP hypocotyl epidermal cells showed that overexpression of *RALF23* does not affect cellular morphology (Figures S5D and S5E). We thus hypothesized that the promotion of BR signaling by RALF23 is not caused by defects in cell morphology but is linked to its signaling function.

We next asked whether the effect of RALF23 on BR signaling is linked to modification of the cell wall as observed in *fer-4* and *ralf1/22/23/33*. The overexpression of RALF23-GFP did not affect PME activity nor pectin esterification status (Figures S5G, S5F, and S5H; Table S3), suggesting that the promotion of BR signaling by RALF23 is not linked to measurable changes in pectin. Corroborating these observations, RALF23 treatment did not lead to significant changes in cell wall composition, PME activity, or pectin esterification status in whole seedlings or in hypocotyl epidermal cells (Figures 5G-5K, S5I, S6A, and S6B; Table S4). We conclude that the effect of RALF23 on BR signaling is not linked to measurable changes in the cell wall composition. However, we observed that RALF23 treatment induced the formation of COS-488-stained punctuates in hypocotyl epidermal cells (Figure 5I). These spheric structures differed morphologically from RALF1-induced cell wall invaginations observed in root epidermis,³² which we also observed upon RALF23 treatment in root epidermis, and which occurred predominantly in the late meristematic and transition zones of the root (Figures S6C-S6F). Furthermore, we observed that both RALF23-induced cell invaginations in the root and RALF23-induced punctuates in the hypocotyl epidermis occurred in a *FER*-dependent but *LRX3/4/5*-independent manner (Figures 5, S6G, and S6H). This indicates that RALF23 modulates cell wall organization in a cell-type and receptor-specific manner. In addition, we observed that RALF23 treatment inhibits PME activity in *fer-4* (Figure 5K). The effects of RALF23 treatment on PME activity were abolished in the *fer-4/lrx3/4/5* quadruple mutant (Figure 5K), suggesting that RALF23 binding to LRXs could inhibit PME activity in *fer-4*. Interestingly, RALF23 treatment led to no discernable effect on pectin esterification status and on cell wall monosaccharide composition in *fer-4*, as measured

(H) Heatmap showing cell wall composition of 2-week-old seedlings. Boxes are colored according to the mean amount of µg.mg⁻¹ alcohol-insoluble residue (AIR) from 6 independent biological replicates, each corresponding to 3-4 seedlings pooled. GalA, galacturonic acid; GlcA, Glucuronic acid. The symbols +, -, or = indicate significant increase, decrease, or no significant change in sugar abundance as determined by Student's *t* test.

(I) Pectin methylation degree (mol %) in Col-0 and *ralf1/22/23/33*. Scatter points show 6 independent biological replicates, *p* value was determined by Student's *t* test.

(J) Relative PME activity determined by gel diffusion assay. Scatter points show individual biological replicates from 3 experiments. Conditions that do not share a letter are significantly different according to Dunn's multiple comparison test.
See also Figures S3 and S4 and Table S2.
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>Figure 5. RALF23 promotes BRI1-BAK1 complex formation and modulates pectin organization</figcaption></figure>

(A) Immunoprecipitation of BRI1-GFP in Arabidopsis seedlings after treatment with 1 µM BL, 1 µM RALF23, or corresponding mock control solution (EtOH) for 90 min. Membranes were probed with anti-GFP or anti-BAK1 antibodies. Membrane stained with Coomassie brilliant blue (CBB) is presented to show equal loading. Similar observations were made in at least three independent experiments.

(B) Quantification of co-immunoprecipitated BAK1 in Col-0 or *fer-4* seedlings expressing BRI1-GFP treated with 1 µM RALF23 or mock control solution (H₂O) for 90 min. Individual scatter points indicate independent experiments, *p* value was determined by Student's *t* test.

(legend continued on next page)
by HPAEC-PAD analyses, dot-blot assays and COS-488 staining experiments (Figures 5G–5J, S6B, S6G, and S6H; Table S4). This indicates that apoplastic PME activity is regulated by additional factors that are not represented in PME activity assays. Collectively, these observations unveil context dependency and receptor specificity in RALF23-induced modification of the cell wall status.

DISCUSSION

Our observations indicate that RALF-mediated CWS functions at the nexus between cell wall status and BR signaling (Figure 6). The genetic analysis of *FER*, *LLG1*, and *RALF1/22/23/33* mutants showed that the loss of RALF-mediated CWS leads to defects in the composition of the cell wall, cell morphology and to an increase in basal BR signaling. The genetic analysis of *fer-4/bri1-301* and *fer-4/bak1-4* and exogenous BL treatment indicate that an increase in BR signaling mitigates the morphological defects observed in *fer-4*. We hypothesize that increased BR signaling in *RALF1/22/23/33* serves a similar purpose. We observed that loss of *FER* and *RALF1/22/23/33* leads to an increase in PME activity and to a decrease in pectin methylation (Figures 2 and 4). These modifications could be sensed by other wall-monitoring pathways promoting BR signaling by regulating *BRI1-BAK1* association, as well as by regulating *BRI1*-like receptors¹⁰⁰ and may be mediated by changes in BR synthesis, accumulation, or transport. Interestingly, overexpression of RALF23 and treatment with exogenous RALF23 led to an increase in BR signaling (Figure 5), thus phenocopying the loss of *FER* and the loss of *RALF1/22/23/33*. Neither overexpression of RALF23 nor RALF23 treatment resulted in measurable changes in pectin chemistry (Figures 5, S5, and S6), indicating that this effect is mediated by RALF23 signaling function. More research is required to unravel whether RALF signaling also involves the previously identified BR-regulating cell-wall-monitoring cell-surface receptors RLP44⁶⁴ and RFO1/WAKL22,⁷¹ or whether

they correspond to parallel signaling pathways converging toward BR signaling.

Our observations suggest a model in which RALF peptides function as cell-wall-informed signaling cues, initiating a feedback loop solicitating BR signaling coordinating cell morphogenesis (Figure 6). Our data and previous studies²¹,²³,²⁵,³⁹ indicate that RALF23 binds to both cell-wall-located LRXs and the plasma membrane-localized receptor complex FER-LLG1. As observed with other positively charged RALF peptides, RALF23 activity relies on pectin; presumably through direct binding and the formation of RALF23-pectin oligomers. Since RALF's binding affinity to LRXs is an order of magnitude higher than to FER-LLG1,³⁰,³⁹ we hypothesize that the availability of cell-wall-located epitopes defines whether RALF23 is perceived by FER-LLG1. Thereby, RALF23 perception by FER-LLG1 is informative of cell wall status and initiates adaptive responses, which involved the promotion of *BRI1-BAK1* complex formation and signaling. It is conceivable that in case of cell wall perturbations, such as upon salt²⁰ or mechanical stress,⁷⁷ or manipulation by microbes,¹⁰² RALF peptides are released from LRXs so that they can be perceived by FER-LLG1. The FER-dependent effect of RALF23 on BR signaling appears not to be caused by overall changes in cell wall composition. However, we cannot exclude the possibility that RALF23 induces changes in the nano-organization of the cell wall, as observed for RALF1 and RALF4 in tip-growing cells,²⁸,²⁹ or local modification of the cell wall composition that may not be detectable with the methods employed in this study. Interestingly, we observed that RALF23 treatment induces the formation of COS-488 punctuate structures (Figures 5 and S6) that were reminiscent of RALF1-pectin condensates observed *in vitro*.³¹ This might be linked to the regulation of plasma membrane organization and signaling of cell-surface receptors²⁵,³¹ and may be induced by local BR-induced modifications of the cell wall. The origin and potential functional implications of these punctuates await further investigations. Further, it remains possible that the effect of RALF23 on BR signaling may involve *BRI1*-like receptors and

(C) Western blot analysis of BES1 in 12-day-old seedlings treated with 200 nM of BL or corresponding control EtOH for 30 min. Note that the BL response in WT seedlings deviates from the canonical BL-induced accumulation of dephosphorylated BES1; therefore, these experiments are not informative regarding BL responsiveness. Membranes were probed with anti-BAK1 antibodies to show equal protein loading.

(D) Quantitative real-time PCR of *CPD* and *DWF4* transcripts from 12-day-old seedlings. Scattered data points indicate measurements of individual biological samples, each corresponding to 2–3 seedlings pooled, obtained from three independent experiment. *U-BOX* was used as a house keeping gene and values are expressed relative to *Col-0*. Conditions not sharing a letter show significant differences according to Dunn’s multiple comparison test.

(E) Representative z stack confocal images of the hypocotyl of 5-day old seedlings expressing pBES1::BES1-Ypet treated for 60 min with 1 µM BL, 90 min with 1 µM RALF23 or corresponding mock control solution (EtOH).

(F) Quantification of mean nuclear fluorescence intensity of *BZR1-Ypet* or *BES1-Ypet* when treated with 1 µM BL for 60 min, 1 µM RALF23 for 90 min or mock solution (EtOH). Graphs are combined violin and boxplots with *n* = 964–1,124 nuclei from 5 seedlings. Individual scatter points show outliers. Conditions not sharing a letter are significantly different according to one-way ANOVA with Tukey post-hoc HSD test (*p* < 0.05).

(G) Pectin methylation degree (mol %) in *Col-0* and *fer-4* treated with 1 µM RALF23 or H₂O for 90 min. Scatter points show 6 independent replicates, each corresponding to 3–4 seedlings pooled, conditions that do not share a letter are significantly different according to pairwise Mann-Whitney test.

(H) Heatmap showing cell wall composition of 2-week-old seedlings. Boxes are colored according to amount of µg·mg⁻¹ alcohol-insoluble residue (AIR). Each corresponds to the mean from 6 independent replicates, each corresponding to 3–4 seedlings pooled. GalA, galacturonic acid; GlcA, glucuronic acid. +, −, or = indicate significant increase, decrease, or no significant change in sugar abundance as determined by Student’s *t* test, respectively.

(I) Confocal z stacks of 5-day old hypocotyl epidermis cells stained with COS-488 to label de-esterified homogalacturonan, treated 90 min with 1 µM RALF23 or corresponding mock condition (H₂O).

(J) Quantification of COS-488 stained punctate structures in hypocotyl epidermal cells. Scatter points show independent biological replicates from 4 experiments. Conditions that do not share a letter are significantly different according to Dunn’s multiple comparison test (*p* < 0.05).

(K) Relative PME activity determined by gel diffusion assay. Scatter points show 3 independent biological replicates from 3 experiments. Conditions that do not share a letter are significantly different according to pairwise Mann-Whitney test (*p* < 0.05).

See also Figures S4, S5, and S6 and Tables S3 and S4.
<figure><img src="image_28.png" /><figcaption>Figure 6. A proposed working model for a RALF-brassinosteroid signaling circuit</figcaption></figure>

RALF23 binds to wall-located LRXs and/or plasma-membrane-localized receptor complex FER-LLG1. We hypothesize that the availability of wall-located epitopes defines whether RALF23 is perceived by FER-LLG1 or LRXs. Thereby, RALF23 perception by FER-LLG1 is informative of cell wall status and initiates adaptative responses, which involve the promotion of BRI1-BAK1 complex formation and signaling to regulate morphogenesis. RALF23 modulates pectin patterning in a FER-dependent and the activity of PME in a LRX3/4/5-dependent manner. In the case of the regulation of PME activity, RALF23 effects may be induced locally upon binding to LRXs. BR-triggered and BIN2-mediated FER phosphorylation modulates FER plasma membrane localization, thereby promoting cell wall integrity signaling and an appropriate cell elongation rate.¹⁰ The definition of the exact molecular ramification connecting RALF23 signaling to BR signaling, and to cell wall status awaits further experimental investigation.

additional plasma membrane-localized receptors, for instance, other CrRLKL1s, while cell-type-specific alterations in pectin organization require FER.

Our study shows that FER regulates BRI1-BAK1 complex formation and signaling, adding to the growing list of cell-wall-monitoring cell-surface receptors regulating BR signaling and providing the first example of a peptide ligand-receptor complex module in this context. Interestingly, a recent report showed that BR signaling, through BIN2 phosphorylation of FER, modulates FER plasma membrane localization, thereby promoting cell wall integrity signaling and maintaining an appropriate cell elongation rate.¹⁰ Together, the study from

Chaudhary and co-authors, and this study indicate that RALF and BR signaling regulate each other, thus providing a molecular circuit coordinating morphogenesis. It will also be particularly interesting to test whether the regulation of BRI1-BAK1 association and cell anisotropy by FER involves antagonist RALF peptides, as proposed in the context of immune signaling²³ and recently demonstrated in the context of reproduction.¹⁸ RALF23 perception modifies the plasma membrane organization of several cell-surface receptors such as FLS2, BAK1, and BRI1.²⁵,³¹ While these observations are associated with an inhibition of FLS2-BAK1 complex formation, the functional consequences of RALF23 for other LRR-RK signaling pathways remained unknown. Here, we show that RALF23 promotes BRI1-BAK1 association and BR signaling and may function as a tipping point in the trade-off between immunity and growth by rewiring cell-surface signaling. A mechanistic understanding of the molecular links between wall rheology, RALF peptides signaling,

be mediated by changes in BR synthesis, accumulation, or transport.

We observed that RALF23 modulates pectin organization and the activity of PME in a context- and receptor-specific manner (Figures 5 and S6). RALF-mediated CWS likely involves multiple RALF peptide-receptor modules providing an elaborate system to sense, relay and modify the cell wall in a context-dependent manner. RALFs may regulate the amount of cell-wall-located RALF binding epitopes and, consequently, potentially modulate cell wall properties and signaling from CrRLK1Ls including FER. In this scenario, RALFs could thus finely balance their own structural and signaling function acting within a self-regulating signaling pathway. Interestingly, we observed that modulation of pectin organization and PME activity by RALF23 occurs in a FER-dependent and -independent manner. In the case of FER-independent changes in PME activity, RALF23 effects could be induced upon binding to LRXs, or mediated by
and the organization of the plasma membrane organization
awaits further investigation.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Julien Gronnier (julien.gronnier@tum.de).

### Materials availability

Plasmids and *Arabidopsis* transgenic lines generated in this study are available upon request.

### Data and code availability

* All data reported in this paper will be shared by the lead contact upon request.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We thank the members of the Zipfel, Grossniklaus, Ringli, Sanchez-Rodriguez, and Keller laboratories for sharing results and comments during our stimulating CCWI meetings. We thank Elke Barbez for fruitful discussions and for sharing detailed protocols for COS-488 synthesis and staining. We thank Isabel Monte and Kyle W. Bender for discussion and comments on the manuscript. We thank Steven Huber, Li-Jia Qu, and Jürgen Kleine-Vehn for providing materials. Confocal imaging was performed at the ZMBP microscopy facility of the University of Tübingen and at the Center for Advanced Light Microscopy (CALM) of the TUM School of Life Sciences. This research was funded by the Gatsby Charitable Foundation (to C.Z.), the University of Zürich (to C.Z.), the University of Tübingen (to J.G.), the European Research Council under the grant agreements 309858 and 773153 (grants PHOSPHinnATE and immuno-PEPTALK to C.Z.), the European Molecular Biology Organization (postdoctoral fellowship EMBO LTF 438-2018, to J.G.), the Deutsche Forschungsgemeinschaft (DFG) (postdoctoral fellowship STE 2448/1, to M.S.), and DFG grants A08-SFB1101 and B01-TRR356 to J.G.

## AUTHOR CONTRIBUTIONS

Conceptualization and project administration, J.G. and C.Z.; investigation, D.B., M.v.A., J.G., M.S., K.S.M.-E., K.X., D.S., T.E., and M.S.; funding acquisition, J.G., C.Z., and M.S.; resources, G.V.; supervision, J.G., C.Z., M.S., S.W., and T.E.; writing – original draft, D.B. and J.G.; writing – review and editing, D.B., M.v.A., M.S., G.V., S.W., T.E., J.G., and C.Z.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Plant materials and growth

* METHOD DETAILS

* Generation of BZR1-Ypet, BES1-Ypet and corresponding FERONIA CRISPR lines

* Confocal microscopy and image analysis of BZR1-Ypet/BES1-Ypet

* RNA isolation and quantitative RT-PCR

* Brassinosteroid-sensitivity assay

* Mobility shift-based estimation of BES1 phosphorylation status

* Co-immunoprecipitation

* Immunoblotting

* Structural modelling and analysis

* Confocal laser scanning microscopy of propidium iodide-stained samples

* Synthesis, imaging and quantification of COS-488 staining

* 3D Segmentation and analysis of Arabidopsis hypocotyl epidermis cells

* Pectin isolation and dot-blot assay

* Gel diffusion PME activity assay

* Analysis of cell wall composition by high-performance anion-exchange chromatography with pulsed amperometry detection

* Analysis of pectin methyl-esterification

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cub.2025.09.016.

Received: March 29, 2024

Revised: May 12, 2025

Accepted: September 4, 2025

Published: September 26, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Rabbit polyclonal anti-BAK1</td><td>Agrisera</td><td>Cat#AS121858</td></tr><tr><td>Anti-BES1</td><td>Steven Hubermann, Perraki et al.<sup>103</sup></td><td>N/A</td></tr><tr><td>Mouse monoclonal anti-GFP-HRP</td><td>Santa Cruz</td><td>Cat#sc-9996-HRP</td></tr><tr><td>Mouse monoclonal anti-GFP</td><td>Santa Cruz</td><td>Cat#sc-9996</td></tr><tr><td>Goat polyclonal anti-Rabbit IgG-HRP</td><td>Sigma</td><td>Cat#A0545</td></tr><tr><td>Rat monoclonal LM20</td><td>Kerafast</td><td>Cat#ELD004</td></tr><tr><td>Rat monoclonal JIM5</td><td>Kerafast</td><td>Cat#ELD003</td></tr><tr><td>Rabbit monoclonal LM19</td><td>Absolute Antibody</td><td>Cat#AB02545-23.0</td></tr><tr><td>Goat polyclonal anti-Rat-HRP</td><td>Thermo Scientific</td><td>Cat#31470</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>RALF23 (ATRRYISYGALRRNTIPCSRRGASYYNCRR GAQANPYSRGCSAITRCRRS)</td><td>Synpeptide</td><td>N/A</td></tr><tr><td>RALF23n (ATRRYISYGALRRNTIPCSAAGASYYNCAAG AQANPYSAGCSAITACAAS)</td><td>Synpeptide</td><td>N/A</td></tr><tr><td>Pectin methylesterase from orange peel</td><td>Sigma</td><td>Cat#P5400</td></tr><tr><td>epi-Brassinolide</td><td>Sigma</td><td>Cat#E1641</td></tr><tr><td>Pierce Fast Western Blot Kits, SuperSignal West Femto</td><td>Thermo Scientific</td><td>Cat#35080</td></tr><tr><td>Chitosan oligosaccharides</td><td>Carbosynth</td><td>Cat#OC09272</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>A. thaliana: Columbia-0</td><td>N/A</td><td>N/A</td></tr><tr><td>A. thaliana: fer-4</td><td>Duan et al.<sup>74</sup></td><td>N/A</td></tr><tr><td>A. thaliana: fer-4/pFER::FER-GFP</td><td>Duan et al.<sup>74</sup></td><td>N/A</td></tr><tr><td>A. thaliana: fer-4/p35S::FER<sup>ΔMALA</sup>-YFP</td><td>Gronnier et al.<sup>25</sup></td><td>N/A</td></tr><tr><td>A. thaliana: Col-0/p35S::RALF23-GFP</td><td>Stegmann et al.<sup>23</sup></td><td>N/A</td></tr><tr><td>A. thaliana: Ilg1-2</td><td>Li et al.<sup>37</sup></td><td>N/A</td></tr><tr><td>A. thaliana: bak1-4</td><td>Chinchilla et al.<sup>95</sup></td><td>N/A</td></tr><tr><td>A. thaliana: bri1-301</td><td>Xu et al.<sup>96</sup></td><td>N/A</td></tr><tr><td>A. thaliana: fer-4/bak1-4</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: fer-4/bri1-301</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: bri1-301/p35S::RALF23-GFP</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: Col-0/pBRI1::BRI1-GFP</td><td>Geldner et al.<sup>104</sup></td><td>N/A</td></tr><tr><td>A. thaliana: fer-4/pBRI1::BRI1-GFP</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: lrx3/4/5</td><td>Dünser et al.<sup>13</sup></td><td>N/A</td></tr><tr><td>A. thaliana: fer-4/lrx3/4/5</td><td>Dünser et al.<sup>13</sup></td><td>N/A</td></tr><tr><td>A. thaliana: ralf1/22/23/33</td><td>Lan et al.<sup>18</sup></td><td>N/A</td></tr><tr><td>A. thaliana: Col-0/pBES1::BES1-Ypet</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: Col-0/pBZR1::BZR1-Ypet</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: fer-c/pBES1::BES1-Ypet</td><td>This paper</td><td>N/A</td></tr><tr><td>A. thaliana: fer-c/pBZR1::BZR1-Ypet</td><td>This paper</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td colspan="3">See Table S5 for oligonucleotides used</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>Fiji</td><td>Schindelin et al.<sup>105</sup></td><td>https://imagej.net/software/fiji/downloads</td></tr></tbody></table>

(Continued on next page)
**Continued**

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>AlphaFold Multimer</td><td>Evans et al.<sup>106</sup></td><td>https://alphafoldserver.com/</td></tr><tr><td>ChimeraX</td><td>Meng et al.<sup>107</sup></td><td>https://www.cgl.ucsf.edu/chimerax/download.html</td></tr><tr><td>MorphographX</td><td>Barbier de Reuille et al.<sup>79</sup></td><td>https://www.morphographx.org</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Plant materials and growth

Arabidopsis thaliana ecotype Columbia (Col-0) was used as WT control. The *fer-4*, *fer-4/FER-GFP*,<sup>74</sup> *fer-4/FER<sup>ΔMalA</sup>-YFP*,<sup>25</sup> *lrx3/4/5*,<sup>13</sup> *fer-4/lrx3/4/5*,<sup>13</sup> Col-0/p35S::RALF23-GFP,<sup>23</sup> *lgl1-2*,<sup>37</sup> *ralf1/22/23/33*, previously reported as *ralf quad*,<sup>18</sup> lines were previously reported. *fer-4/bak1-4* and *fer-4/bri1-301* were obtained by crossing *fer-4*<sup>74</sup> with *bak1-4*<sup>95</sup> and *bri1-301*<sup>96</sup> respectively. Col-0/ *pBRI1::BRI1-GFP*<sup>104</sup> was crossed *fer-4*<sup>74</sup> to obtain *fer-4/pBRI1::BRI1-GFP*. Seeds were surface sterilized using chlorine gas for 5 hours or by incubating them in 0.1 % Tween20 in 70 % EtOH for 10 minutes, following 70 % EtOH for 10 minutes and 100% EtOH for 1 min. Seeds were stratified for 2 days in the dark at 4 °C and grown on half Murashige and Skoog (MS) media supplemented with vitamins, 1 % sucrose and 0.8 % agar at 22 °C and a 16-hour light photoperiod.

## METHOD DETAILS

### Generation of BZR1-Ypet, BES1-Ypet and corresponding FERONIA CRISPR lines

Fusions of BES1 and BZR1 to the YFP variant Ypet protein were constructed by recombineering the Ypet-encoding DNA sequence in frame with the 3' of *BES1* and *BZR1* genes using a bacterial recombineering approach and transformed into wild-type plants by floral dipping. Mono-insertional homozygous transgenic plants were selected for further analyses. For the generation of *FER* CRISPR mutants, CRISPR-Cas9 constructs carrying a gRNA targeting *FER* (Figure S4) were transformed into mono-insertional homozygous transgenic plants expression *pBZR1::BZR1-Ypet* and *pBES1::BES1-Ypet*. Transformants were selected and alteration of *FER* coding sequence confirmed by Sanger sequencing. Homozygous *fer-c/pBZR1::BZR1-Ypet* and *fer-c/pBES1::BES1-Ypet* not carrying CRISPR-Cas9 constructs were used for further analyses.

### Confocal microscopy and image analysis of BZR1-Ypet/BES1-Ypet

Five-day old Arabidopsis seedlings were treated for 60 minutes with 1 µM eBL, 90 minutes 1 µM RALF23 or corresponding mock solution (EtOH) in ½ MS + 1 % Suc 50 mM MES. Seedlings were mounted in ½ MS + 1 % sucrose and z-focal planes imaged using a Zeiss LSM880 confocal laser scanning microscope equipped with a Plan-Apochromat 10x/0.45 M27 objective. Ypet was excited with a 514 nm argon laser and fluorescence collected between 520-570 nm. Z-stacks maximum projections were reconstructed in Fiji<sup>105</sup> and used to automatically segment nuclei and measure their fluorescence intensity using a custom-made pipeline.

### RNA isolation and quantitative RT-PCR

RNA isolation, cDNA synthesis and quantitative real-time PCR (qRT-PCR) was performed as previously described<sup>75</sup> with minor modifications. Five-day-old seedlings grown on half MS 1 % sucrose pH 5.8 were transferred to liquid half MS 1 % sucrose pH 5.8 and grown for seven days in 24 well plates. Each sample, constituted of three individual seedlings, were blot-dried, transferred to 2 mL tubes containing 2 mm glass beads and flash-frozen in liquid nitrogen. Samples were grinded while frozen for 1.5 minute at 1.500 rpm using BioRad TissueLyser. RNA was extracted using TRIzol™ Reagent (ThermoFischer scientific). First-strand cDNA synthesis was performed using RevertAid first strand cDNA synthesis kit (ThermoFischer scientific) and oligo(dT)18 according to the manufacturer's instructions. cDNA was amplified in triplicate by quantitative PCR by using PowerUp™ SYBR™ Green Master Mix (ThermoFischer scientific) and a 7500 Fast real-time PCR detection system. The relative transcript accumulation values were determined by using U-box as reference and the comparative Ct method (2-ΔΔCt). The following primers were used for cDNA amplification, CPD (AT5G05690): forward: 5'-CCCAAACCACTTCAAAGATGCT-3' and reverse 5'-GGGCCTGTCGTTACCGAGTT-3', DWF4 (AT3G50660): forward '5-CATAAAGCTCTTCAGTCACGA-3' and reverse '5-CGTCTGTTCTTTGTTTCCTAA-3' and U-BOX (AT5G15400): forward '5-TGCGCTGCCAGATAATACACTATT-3' and reverse '5-TGCTGCCCAACATCAGGTT-3'.

### Brassinosteroid-sensitivity assay

Seeds were surface-sterilized and individually placed in line on square Petri dishes containing half MS 1 % sucrose, 0.8 % phytoagar, supplemented with 200 nM epi-brassinolide or corresponding control solution (EtOH). The plates were placed at 4 °C for 2 days and then placed vertically in a growth chamber for 5 days. Pictures of the plates were then taken to measure root hypocotyl length which were measured using Fiji.<sup>105</sup>
Mobility shift-based estimation of BES1 phosphorylation status

Brassinolide-induced BES1 dephosphorylation assays were performed as previously described,¹⁰³ seedlings were germinated on half MS-agar supplemented with 1 % sucrose for five days before transplanting to 24-well plates (two seedlings per well) containing liquid half MS supplemented with 1 % sucrose. One day before the assays, the medium was replaced with fresh medium. Twelve-day-old seedlings were treated with epi-brassinolide or ethanol (mock) at the indicated concentrations for 30 min. Samples were blot-dried, transferred to 2 mL tubes containing 2 mm glass beads and flash-frozen in liquid nitrogen and store at -80 °C before protein extraction and immunoblotting.

Co-immunoprecipitation

Co-immunoprecipitations were performed as previously described.¹⁰⁸ Twenty to thirty seedlings per plate were grown in wells of a 6-well plate for 2 weeks, transferred to 2 mM MES-KOH, pH 5.8, and incubated overnight. The next day, BL (final concentration 1 µM) and/or RALF23 (final concentration 1 µM) were added and incubated for 90 min. Seedlings were then frozen in liquid nitrogen and subjected to protein extraction. To analyze BRI1-GFP/BAK1 association, proteins were isolated in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol, 5 mM dithiothreitol, 1 % protease inhibitor cocktail (Sigma-Aldrich), 2 mM Na2MoO4, 2.5 mM NaF, 1.5 mM activated Na3VO4, 1 mM phenylmethanesulfonyl fluoride, and 0.5% IGEPAL. For immunoprecipitations, GFP-Trap agarose beads (ChromoTek) were used and incubated with the crude extract for 3 to 4 hours at 4°C. Subsequently, beads were washed three times with wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 0.1 % IGEPAL) before adding Laemmli sample buffer and incubating for 10 minutes at 95°C. Analysis was carried out by SDS-PAGE and immunoblotting.

Immunoblotting

Protein samples were separated in 8-10 % bisacrylamide gels at 150 V for approximately 2 hours and transferred into activated PVDF membranes at 100 V for 90 min. Immunoblotting was performed with antibodies diluted in blocking solution (5 % fat-free milk in TBS with 0.1 % [v/v] Tween-20). Antibodies used in this study were α-BAK1 (1:5000,¹⁰⁹ or Agrisera AS12 1858), α-BES1 (1:1000,¹⁰⁵), α-GFP-HRP (1:5000, sc-9996-HRP, Santa Cruz), α-GFP (1:5000, sc-9996, Santa Cruz). Blots were developed with Pierce ECL/ECL Femto Western Blotting Substrate (Thermo Scientific). The following secondary antibody was used: anti-rabbit IgG (whole molecule)-HRP (A0545, Sigma, dilution 1:10,000).

Structural modelling and analysis

RALF23-LRXs and RALF23-FER-LLG1 complexes were predicted using AlphaFold Multimer.¹⁰⁶,¹¹⁰ Predicted complexes were analyzed using ChimeraX.¹⁰⁷

Confocal laser scanning microscopy of propidium iodide-stained samples

Arabidopsis seedlings were stained with 50 µg/µl propidium iodide (PI) in water for 20 min, then washed with water 3 times to remove unbound PI. Samples were mounted in H₂O on coverslips an z-stacks imaged using a Zeiss LSM880 confocal laser scanning microscope, equipped with a Plan-Apochromat 10x/0.45 M27 objective. PI was excited with a DPS 561-nm diode laser and fluorescence collected between 570-642 nm using 600-800 V gain. Z-projections of the images were created in Fiji.¹⁰⁵

Synthesis, imaging and quantification of COS-488 staining

COS-488 was synthesized as described before.⁸⁹ Briefly, COS was generated by dissolving chitosan oligosaccharides (Carbosynth OC09272) in 100 mM sodium acetate buffer pH 4.9 to a final concentration of 1 mg/ml. Then, 16 µl of 10 mg/ml AlexaFlour 488 hydroxylamine in DMSO was added to 0.5 ml of COS solution and incubated in dark at 37 °C for two days under shaking. For staining of hypocotyls, five-day-old seedlings were stained as described before⁸⁹ with minor modifications. For treatments, seedlings were incubated in 50 µM EGCG or 1 µM RALF23 or corresponding mock solution in ½ MS + 1% Sucrose for indicated time. For staining of hypocotyls, COS-488 was added at 1:500 dilution for 2.5 h. Afterwards, seedlings were washed in ½ MS + 1% Sucrose 3 times. Roots were stained with a 1:1000 COS-488 dilution for 10 minutes and washed in ½ MS + 1% sucrose three times. Samples were mounted in ½ MS + 1 % Sucrose and z-stacks acquired using a Zeiss LSM880 confocal laser scanning microscope, equipped with a Plant-Apochromat 10x/0.45 M27 objective. COS-488 was excited with 488-nm argon laser and fluorescence collected between 490-560 nm using 600-800V gain. Z-projections were created and grey values measured using Fiji.¹⁰⁵

3D Segmentation and analysis of Arabidopsis hypocotyl epidermis cells

Prior to analysis, seedlings were fixed, cleared and stained as previously described¹¹¹ with minor modifications. Briefly, five-day-old seedlings grown on half MS-agar (0.8 % w/v) supplemented with 1 % sucrose, were fixed using 4 % Paraformaldehyde in PBS pH 6.9 while applying mild vacuum (100 mBar). Fixed samples were washed 3 times with PBS and then incubated in ClearSee solution (25 % Urea (w/v), 15 % Sodium deoxycholate (w/v), 10 % Xylitol (w/v)). Clearing was performed for at least 2 weeks while changing ClearSee every second day. Seedlings were stained using 0.1 % (v/v) SR2200 stain in ClearSee for 1 h, then washed with fresh ClearSee for at least 30 minutes. For imaging, samples were mounted in ClearSee and imaged on a Zeiss LSM880 confocal laser scanning microscope, equipped with a C-Apochromat 40x/1.2W Autocorr M27 water objective. SR2200 was excited with a 405 nm diode laser and emission detected between 413 nm-472 nm. Z-stacks acquisition was performed with an interval size of 0.3 µm to obtain cubic voxels (pixel size 0.3 µm x 0.3 µm). To analyze whole hypocotyls, z-stacks from one seedling were combined
in Fiji¹⁰⁴ using the stitching plug-in.¹¹² 3D segmentation and analysis were carried out in MorphoGraphX.⁷⁹ Hypocotyl stacks were blurred using Gaussian Blur Stack with values between 1 µm-1.5 µm. Automated 3D segmentation was carried out using the Water-shed Auto Seeded process with a threshold ranging from 800 – 1000. Segmentation was manually corrected, and cells not fully rep-resented in the stack deleted. The 3D mesh was created using Marching Cubes 3D process with a cube size of 2 and 3 smoothing steps. To achieve optimal cell axis, a Bezier cord was formed according to hypocotyl shape and custom cell axis created with the process Create Bezier Grid Directions. Measurements were exported as CSV files and index of anisotropy calculated as follows: Cell Length/Cell Width = Index anisotropy.

## Pectin isolation and dot-blot assay

Pectin dot-blot assay was performed as previously described with slight modifications.¹¹³ Five-day-old Arabidopsis seedlings were transferred to liquid half MS 1 % sucrose pH 5.8 with 2 mM MES-KOH pH 5.8 and grown for six days. Three seedlings per biological replicate were harvested by freezing in liquid nitrogen. Samples were incubated with 500µl protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 % glycerol, 5 mM dithiothreitol, 1 % protease inhibitor cocktail (Sigma-Aldrich), 2 mM Na₂MoO₄, 2.5 mM NaF, 1.5 mM activated Na₃VO₄, 1 mM phenylmethanesulfonyl fluoride, and 0.5 % IGEPAL) at 8 °C for 30 min. Cell wall debris was collected by centrifugation at 16.000 g 4 °C for 30min. The supernatant was collected and directly used for subsequent analysis or stored at -80 °C. Cell wall pellet was incubated in 1 ml 70 % pre-warmed (60°C) ethanol for 30 minutes and recovered by centrifu-gation at 16.000 g for 15 min. To remove residual proteins and lipids, the pellet was washed twice with 1 ml Chlorform:Methanol (1:1) for 30 min. Subsequently, cell wall pellet was washed twice with 80 % acetone. After removal of acetone, pellet was dried overnight at room temperature. To extract pectins, the dry pellet was boiled in 200 µl H₂O for 1 h. The cell debris were collected by centrifugation (13.000 g, 15 min) and the supernatants were either directly used for western blotting or stored at -20 °C. For western blotting, total sugar content was quantified using the phenol/H₂SO₄ method¹¹⁴ as described in Nielsen¹¹⁵ with minor changes. 200 µl of 1:10 diluted pectin solutions were incubated with 200 µl of 5% phenol solution and 1 ml 95 % sulfuric acid. After 1 h incubation, absorbance at 490 nm was measured and total sugar content calculated using galacturonic acid as a standard. Sugar concentration was adjusted to 0.5 µg/µl and 2 µl with a total of 1 µg sugars spotted on nitrocellulose membrane. Spots were dried overnight. Then, membranes were blocked with 5 % milk in PBS for 1 h. Membranes were incubated for 1 h with JIM5 (1:250 in 5 % milk in PBS), LM20 (1:250 in 5 % milk in 20 mM Tris-HCL pH 8.2, 0.5 mM CaCl₂ and 150 mM NaCl) rat primary antibody or LM19 (1:5000 in 5 % milk in 20 mM Tris-HCL pH 8.2, 0.5 mM CaCl₂ and 150 mM NaCl) rabbit primary antibody. Following washing with PBS + 0.1 % Tween20, membranes were incubated with anti-rat HRP-conjugated secondary antibody (1:6000 in 5 % milk in PBS, for JIM5 and LM20) and anti-rabbit HRP-conjugated secondary antibody (1:10000 in 5 % milk in PBS, for LM19) for 1 hour. Once again washing was performed with PBS + 0.1 % Tween20. Blots were revealed with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).

## Gel diffusion PME activity assay

Estimation of PME activity by gel diffusion assay was adapted from Bethke et al.¹¹⁶ Proteins were extracted as described above and concentration determined using Bio-Rad protein assay kit according to the manufacturer's instructions. Sample protein concentra-tion was equally diluted to 0.33 µg/µl. 20 ml of a gel containing 1.2 % agarose and 0.1 % esterified pectin from apple (Sigma, 93854) and 12.5 mM citric acid, 50 mM Na2HPO4 pH 7 was prepared in 120 mm square plates. Three millimetres holes were filled with 15 µl of protein extract. Plates were incubated for 16 h at 37 °C. Afterwards, the plates were briefly washed and stained with 0.05 % Ruthe-nium Red (Sigma, R2751) for 30 min. Residual dye was removed by washing. Plates were captured using a Cannon EOS 750D cam-era. Areas with higher staining intensity corresponding to de-esterified pectin were quantified using Fiji.¹⁰⁵ Active PME units per µg of protein were calculated by using a standard curve generated with known amounts of commercial PME (Sigma, P5400).

## Analysis of cell wall composition by high-performance anion-exchange chromatography with pulsed amperometry detection

Arabidopsis seedlings were lyophilized and ball-milled in a Retsch mixer mill. All samples were extracted three times with 70 % ethanol and three times with 1:1 (v:v) chloroform:methanol in the Retsch mill, washed with acetone and dried in a vacuum concen-trator. Starch was removed from the resulting alcohol-insoluble residue (AIR) as described by Pettolino et al. ¹¹⁷ with minor modifi-cations. Approximately 6 mg AIR were soaked with 100 µl 10 mM Tris-maleate buffer and incubated for 30 min at room temperature. Samples were boiled for 5 min to gelatinize starch granules. After equilibration to 40 °C, 100 µl of α-amylase solution containing 2 U α-amylase from porcine pancreas (Sigma-Aldrich, A3176) per mg AIR were added and samples incubated for 1 h. Another 50 µl of the same α-amylase solution was added and samples were incubated for 30 min at 40°C. To precipitate polysaccharides, 1 ml of cold absolute ethanol was added, and samples were incubated at -20°C overnight. After centrifugation for 5 min at 1500 g, de-starched AIR was washed three times with 0.5 ml of cold absolute ethanol and dried in a vacuum concentrator.

For analysis of the cell wall monosaccharide composition, 1–2 mg de-starched AIR was weighed in 2-mL tubes with screw caps and used for cell wall hydrolysis according to the ‘one-step two-step’ protocol described by Yeats et al.¹¹⁸ Briefly, neutral cell wall sugars and uronic acids from non-crystalline cell wall matrix polymers were hydrolyzed in 4% (w/v) sulfuric acid by autoclaving at 121°C for 60 min. A second set of samples was pretreated with 72 % (w/w) sulfuric acid for 1 h to induce cellulose swelling. Samples were than diluted to 4% (w/v) sulfuric acid and hydrolyzed as described above to obtain monosaccharides from matrix polymers and cellulose. After cooling to room temperature, all samples were centrifuged to remove any insoluble material and diluted 30-fold in ultrapure water while adding 50 µM ribose as an internal standard. Monosaccharide quantification was performed via
high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a biocompatible Knauer Azura HPLC system and an Antec Decade Elite SenCell detector heated to 40 °C. Monosaccharides were separated on a Thermo Fisher Dionex CarboPac PA20 BioLC analytical column (3 x 150 mm) equipped with a CarboPac PA20 BioLC guard column (3 x 30 mm) in a solvent gradient of (A) water, (B) 10 mM NaOH and (C) 700 mM NaOH at 0.4 mL/min flow rate. The gradient program was as follows (solvent % in A): 0 to 25 min: 20 % B; 25 to 28 min: 20 % to 0 % B, 0 % to 70 % C; 28 to 33 min: 70 % C; 33 to 35 min: 70 % to 100 % C; 35 to 38 min: 100 % C; 38 to 42 min: 0 % to 20 % B, 100 % to 0 % C; 42 to 60 min: 20 % B. To calculate cellulose amounts, glucose quantified in matrix polymer hydrolysates was subtracted from glucose in matrix + cellulose hydrolysates as detailed in Yeats et al.¹¹⁸

## Analysis of pectin methyl-esterification

The degree of pectin methyl-esterification was analyzed as described by Lionetti et al.¹¹⁹ with minor modifications. One mg de-starched AIR was saponified in 0.2 ml 250 mM NaOH for 1 h at room temperature and neutralized with 1 M HCl. After centrifugation for 2 minutes at 16000 g, the supernatant was diluted 1:10 with ultrapure water to 50 µl and incubated with an equal volume of 0.1 M sodium phosphate buffer (pH 7.5) containing 0.03 units alcohol oxidase from *Pichia pastoris* (Sigma-Aldrich, A2404) for 15 minutes at room temperature with shaking. After addition of 100 µl 0.02 M 2,4-pentanedione in 2 M ammonium acetate and 0.5 M acetic acid, samples were incubated at 68 °C for 10 min, briefly cooled on ice and then transferred to 96-well microtiter plates. Absorbance was measured at 412 nm in a Tecan infinite 200Pro microplate reader and calibrated against a 0–350 µM formaldehyde standard curve. All samples and standards were measured in technical duplicates. The degree of pectin methyl-esterification was calculated as the molar ratio of methanol to uronic acid determined via HPAEC-PAD analysis.

## QUANTIFICATION AND STATISTICAL ANALYSIS

The number of independent experiments and the number of individual cells analyzed per condition and collected across these experiments are indicated in each figure legends. The statical tests used are reported in the figure legends and have been performed using R or GraphPad Prism.