Root development progression involves RALF peptide-mediated ROS signaling and pectin dynamics in rice

Domain: neuroscience
Published: 2026-04-10
Editorial outcome: major_revision

Abstract
Root development involves the maintenance of longitudinal cell division, cell expansion, and maturation, which is coordinated by signaling networks composed of peptide ligand-receptor complexes and reactive oxygen species (ROS) homeostasis, and is coupled with dynamic cell-wall remodeling. However, these regulatory modules remain disconnected, and the systematic mechanisms that efficiently orchestrate root development remain elusive. Here, we uncover an intrinsic regulatory hub in rice that coordinates root development by integrating ROS transmembrane signaling with pectin structural organization. THESEUS3, a receptor-like kinase, interacts and phosphorylates RbohB to promote superoxide anion (O2−) generation, negatively regulating pectin demethylesterification in the meristem. Rice RAPID ALKALINIZATION FACTOR4 (OsRALF4), functioning as a ligand, balances O2− homeostasis through inhibiting THESEUS3-RbohB signaling in the meristem and modulates pectin demethylesterification. Demethylesterified pectin subsequently binds with OsRALF4 to mediate pectin bundling into a condensed structure, marking the onset of cell expansion. Our study establishes the integrated mechanistic framework underlying root development.

Introduction
Root development is a tightly regulated process that is essential for anchoring plants, absorbing water and nutrients, and facilitating interactions with soil environment1. Root growth arises from cell division in the root apical meristem (RAM), known as the meristematic zone (MZ) and stem cell niche2. Most cells begin to expand and differentiate once they leave the MZ, thereby entering the elongation zone (EZ) and mature zone. Root developmental progression is processed along the timeline in longitudinal axis, reflecting a dynamic interplay of genetic pathways, hormonal signals, and environmental responses3,4. Elucidating the mechanism that integrates major regulatory networks during root development will advance our knowledge and ultimately enable the rational design of root systems5.

Among the three developmental zones, MZ size makes the main contribution to root growth, which is highly associated with the maintenance of cell stemness. Root meristem growth factor 1-3 (RGF1-3) are crucial peptide signals controlling the size of MZ6. Five leucine-rich repeat RLKs (LRR-RLKs), including RGF1 INSENSITIVES (RGIs) and RGF1 RECEPTORS (RGFRs), have been found to act as receptors of RGF17–9. The RGF1-RGIs complexes regulate meristem homeostasis through stabilizing the transcription factor PLETHORA 1/2 (PLT1/2) that controls stem cell activity10,11, or activating PLT1/2 via a MAPK cascade12. Another notable signal hub of RGF1 pathway is reactive oxygen species (ROS) homeostasis along the root developmental zones, which is known controlled by a transcription factor RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1)11. However, how cell surface ligand-receptor complexes are directly connected to ROS homeostasis in roots is an unsolved issue.

ROS that encompass singlet oxygen (O2), superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH•) are ubiquitous second messengers involved in myriad basic processes of plant growth and stress adaptations13. ROS are the intrinsic signal for establishing MZ size11,14,15. O2− primarily accumulates in the MZ to maintain stemness, whereas H2O2 is abundant in the EZ to promote stem cell differentiation, which is coordinated by counteracting the activities of ROS-producing and scavenging enzymes (Supplementary Fig. 1a)16,17. The plasma membrane NADPH oxidases/respiratory burst oxidase homologs (Rbohs) are the well-studied proteins for O2− production18. The transcription factor UPBEAT1 (UPB1) balances the ROS signals in the MZ and EZ through modulating the expression of a set of peroxidase genes. Mutation in UPB1 alters this balance and delays the onset of cell expansion16. Disruption of ROOT MERISTEMLESS1, a gene encoding a glutathione biosynthesis enzyme, causes disability in the formation of root meristem19. Redox thus plays a crucial role in sustaining root meristem homeostasis. How this confined O2− distribution is maintained, as well as the implications, remains elusive.

The transition of meristematic cells from quiescence to expansion is usually accompanied by cell wall dynamics. Pectin, characterized by α-1,4-linked D-galacturonic acid, is the main cell wall component in growing roots. In general, newly synthesized pectin are highly methylesterified, and are demethylated by pectin methylesterases (PMEs) while inserting into cell walls20. Pectin demethylesterification, a key process that modulates the physicochemical properties of pectin, represents a major form of cell-wall remodeling during plant development21–23. Thus, modulating the degree and pattern of methyl esters on pectin can balance cell wall rigidity and elasticity required for plant cell growth24–27. However, whether pectin remodeling is incorporated into ROS-mediated root development, and how these two processes are integrated, are unknown.

RAPID ALKALINIZATION FACTORs (RALFs) are important peptide signals. The recognition of a RALF by the cell surface receptor FERONIA, a Catharanthus roseus receptor-like kinase1 like protein (CrRLK1L), triggers the phosphorylation of the plasma membrane H+–adenosine triphosphatase, regulating extracellular alkalinization and cell expansion28. Over the past decade, several pairs of RALFs and CrRLK1L members have been reported, identifying RALFs as crucial signals for plant growth and development29–35. Recently, Arabidopsis (Arabidopsis thaliana, At) RALF1 can bind extracellular pectin and serves as the cell surface sensor in response to environmental stressors36. Furthermore, AtRALF4 and AtRALF22 can interact with cell wall–anchored protein LEUCINE-RICH REPEAT EXTENSINs, facilitating wall assembly in pollen tube and root hair37,38. Thus, clarifying biological functions of RALFs is crucial to deciphering the mechanisms of plant growth and development.

Here, we identify a RALF-mediated intrinsic regulatory hub in rice (Oryza Sativa, Os) that maintains meristem homeostasis and drives the transition from division to expansion. THESEUS3 (THE3), a CrRLK1L member homologous to THE1, interacts and phosphorylates RbohB to promote O2− generation in the RAM. OsRALF4 controls O2− homeostasis in root meristem through inhibiting THE3-RbohB signaling. O2− level, in turn, negatively regulates the stability of PMEs, suppressing pectin demethylesterification. With the decrease of O2−, demethylesterified pectin are generated and bound directly to OsRALF4, facilitating pectin condensation and cell elongation. Hence, our study elucidates the systematic mechanism that governs the progression of root development from MZ maintenance to the onset of cell expansion, shedding lights in breeding crops with elite root architecture.

Results
RbohB interacts with THE3 to sustain the profile of O2− in root meristem

Root meristem homeostasis depends on the O2− accumulation in the RAM17. To test this in rice roots, we examined O2− in the root apical of a japonica variety, Nipponbare (NP), using nitro blue tetrazolium (NBT), a reagent that specifically stains O2−. Meanwhile, we defined the RAM using 5-ethynyl-2′-deoxyuridine (EdU), a chemical that indicates de novo DNA synthesis. The signal profile of NBT almost overlapped with that of EdU (Fig. 1a,b), indicating that O2− accumulates in the MZ of rice roots. O2− is usually produced by Rbohs39, and rice has nine Rboh genes (RbohA-I) 40. To determine which Rbohs is responsible for O2− production in the MZ, we analyzed the expression level of Rbohs in the NP root tips. Quantitative PCR (qPCR) revealed that RbohB and RbohC are highly expressed among the nine Rbohs (Fig. 1c). We then generated a rbohb and rbohc mutants using CRISPR/Cas9 genome editing in a japonica variety Zhonghua11 (ZH11), and examined root growth and O2− level in these mutants (Supplementary Fig. 1b,c). Compared with rbohc, rbohb-2 exhibited more significant decline in the root growth and O2− content (Supplementary Fig. 1d–g), indicating RbohB as a dominant Rboh functioning in rice roots. To confirm this, we obtained the rbohb-1 mutant and RbohB overexpressing (RBox) lines in the NP background18 (Supplementary Fig. 1h). Consistent with their performance in root growth (Supplementary Fig. 1i,j), rbohb-1 also showed a reduction in O2−, whereas RBox lines had increased O2− in the root tips (Fig. 1d,e). Hence, RbohB serves as the major gene driving O2− production in the rice RAM.

To dissect the signaling pathway associated with RbohB, we performed TurboID-mediated proximity labeling coupled with mass spectrometry-based proteomic analysis in the rice protoplasts expressing RbohB-TurboID-cMyc fusion protein. Among the identified proteins (Supplementary Dataset 1), CrRLK1L family member THE3 was identified as an interacting protein (Supplementary Fig. 2a,b). To verify this, we used THE3 as prey to perform reciprocal TurboID labeling assay. In contrast to the controls, the specific antibody for RhobB probed RbohB in the streptavidin-pulled proteins extracted from the protoplasts expressing THE3-TurboID-cMyc (Fig. 1f and Supplementary Fig. 2c). RhobB interacts with THE3.

THE3 belongs to CrRLK1L subfamily and encodes an 860-aa protein harboring a malectin-like domain and a protein kinase domain (Supplementary Fig. 3a,b). Subcellular localization assay showed that THE3 is co-localized with mRFP-tagged HIR1, a plasma membrane marker (Supplementary Fig. 3c). The N terminus of Rbohs, critical for ROS production, often serves as the interaction site with protein kinases41. As revealed by split-luciferase complementary assay in Nicotiana benthamiana leaves, we found that the N-terminus of RbohB (RbohBN) mediates the interaction with THE3 (Fig. 1g and Supplementary Fig. 3d). This interaction was further confirmed by the Co-IP analysis in the rice protoplasts and the pull-down assay (Fig. 1h,i).

To determine whether THE3 affects O2− production, we generated a rice mutant in ZH11 using CRISPR/Cas9 genome editing. We obtained the the3 mutant, which carries an 1-bp deletion in the first exon, resulting in a frameshift and premature translational termination (Supplementary Fig. 4a,b). For genetic validation, we prepared the complementary transgenic plants by introducing 6,977 bp genomic fragment of THE3 in the3 (Comthe3) (Supplementary Fig. 4b,c). The transformation fully rescued the defects of the3 in root growth and O2− level (Fig. 1j,k and Supplementary Fig. 4e,f). We further overexpressed THE3 in ZH11 (THE3ox) (Supplementary Fig. 4c,d). The overexpression further elevated the root length and O2− level compared to the complementary transgene (Fig. 1j,k and Supplementary Fig. 4e,f), indicating that THE3 regulates root growth. To validate the genetic relationship between THE3 and RbohB, we overexpressed RbohB in the3 mutant (RBoxthe3) (Supplementary Fig. 4c,d). The O2− level in the RAM of RBoxthe3 was restored to the wild-type level; the comparable root growth suggests that RbohB functions downstream of THE3 (Fig. 1j,k and Supplementary Fig. 4e,f). THE3 is involved in RbohB-mediated O2− production in the RAM.

THE3-RbohB interaction is crucial for meristem homeostasis

The accumulation of O2− in the MZ maintains MZ stemness16. We thus detected the DNA synthesis activity in the RAM of THE3 and RbohB relevant genetic materials using EdU staining. The rbohb and the3 mutants exhibited shorter EdU-staining region than the wild type, whereas the complementary and overexpressing plants displayed slightly extended EdU staining (Fig. 2a,b), largely consistent with the alterations in MZ size (Fig. 2c and Supplementary Fig. 4g). The THE3-RbohB module orchestrates root growth through sustaining meristem homeostasis.

To explore the underlying mechanism, we performed in vitro phosphorylation assay using the purified THE3 proteins. The phosphorylation of THE3 and a universal substrate confirmed it as an authentic protein kinase (Supplementary Fig. 5a). By separating the reaction products of THE3 and RbohBN in the phos-tag gel, we detected the phosphorylated RbohBN, and this signal was abolished by the addition of calf intestinal alkaline phosphatase (CIP) (Fig. 2d). We further mutated RbohB at Ser140, Ser191 and Thr278 to alanine to mimic the non-phosphorylated forms according to the report41 (Supplementary Fig. 5b). In the presence of THE3, in vitro phosphorylation assay confirmed that these variants mediated lower phosphorylation levels than the wild-type RbohB (Supplementary Fig. 5c). To determine whether THE3 can phosphorylate RbohB in vivo, we co-expressed RbohBN-NanoLUC and THE3-GFP in the rice protoplasts. Phosphorylation of RbohB was detected in the protoplasts expressing THE3, while it was eliminated by treating the proteins with CIP (Fig. 2e). Thus, THE3 can phosphorylate RbohB.

As phosphorylation usually affects protein stability, we compared RbohB level in the wild-type, the3, and THE3ox plants. Indeed, RbohB level showed a decrease in the3 mutant and an increase in the THE3ox plants (Fig. 2f). We further performed a cell-free degradation assay by incubating the purified RbohB-His proteins with the total proteins extracted from the wild-type, the3, and THE3ox roots, separately. RbohB-His was degraded more rapidly while adding the the3 extracts than the wild-type and THE3ox extracts (Fig. 2g). The stability of RbohB showed a dependence on the level of THE3 (Fig. 2h). THE3-mediated phosphorylation augments RbohB stability.

We further found that the phosphorylation level of RbohB was reduced in the3 root tips, but was enhanced in THE3ox root tips (Fig. 2i). To explore the effect of RbohB phosphorylation on enzyme activity, we analyzed NADPH oxidase activity using the proteins extracted from the root tips of these plants. By applying equal amount of isolated RbohB, we detected lower oxidase activity in the3 and higher activity in THE3ox (Fig. 2j), consistent with their alterations in O2− level (Fig. 1k). Thus, THE3-mediated phosphorylation enhances the activity of RbohB. To obtain the evidence in O2− generation, we transfected N. benthamiana leaves with THE3, RbohB and the combinations of THE3 with either wild-type RbohB or its variant with a mutation at the phosphorylation site (Supplementary Fig. 5b,c). After ensuring that all the transgenes were expressed in N. benthamiana leaf discs (Supplementary Fig. 5d), NBT staining revealed that co-infiltration of THE3 with RbohB variants generated significantly less O2− level than that cotransfecting THE3 with wild-type RbohB (Fig. 2k,l). Despite the comparable protein abundance, the changed RbohB phosphorylation levels led us conclude that phosphorylation is crucial for driving O2− production (Fig. 2m and Supplementary Fig. 5c).

Therefore, THE3-RbohB mediated phosphorylation regulates the activity and stability of RbohB, influencing O2− production and meristem homeostasis in RAM.

Methylesterified pectin is rich in the RAM accumulating O2−

Pectin demethylesterification is a crucial step in cell growth22. To investigate the methylesterification state of pectin in the RAM, we immunolabeled the NP root tips using LM20 and 2F4, the antibodies preferentially recognizing methylesterified pectin (mPectin) and demethylesterified pectin (dmPectin), respectively. LM20 generated obvious signal for mPectin in the MZ (Fig. 3a), whereas dmPectin recognized by 2F4 was mainly present in the EZ (Supplementary Fig. 6a). Furthermore, the distribution of the LM20 signal coincides with the MZ in the root tips (Fig. 3b), suggesting that the RAM is rich in mPectin.

As root meristem also accumulates O2− (Fig. 1a), to ascertain the relationship between O2− and pectin demethylesterification, we treated the wild-type roots with Tiron, a scavenger of O2−, and analyzed the pectin in the root tips using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) (Supplementary Fig. 6b). Tiron treatment significantly reduced the levels of methyl esters on pectin (Fig. 3c and Supplementary Fig. 6c), in agreement with the effects of Tiron on root growth, MZ size, and O2− content (Supplementary Fig. 6d–h). O2− accumulation may sustain pectin in a methylesterified state in root tips.

To test this, we examined the methylesterification degree of pectin in the root tips of the3 and rbohb mutants and the relevant transgenic plants. By immunolabeling their RAMs using LM20, the fluorescent signal for mPectin almost disappeared in the rbohb and the3 mutants, but was restored to the wild-type level in the complementary plants (Fig. 3d,e). The RbohB overexpression plants exhibited the extended mPectin region along the longitudinal axis (Fig. 3d,e). We further chemically analyzed the methylesterified degree of pectin in the root tips using MALDI–TOF MS (Supplementary Fig. 7). Consistently, the methyl esters on pectin were dramatically reduced in the rbohb and the3 mutant root tips, whereas those in the complementary and overexpression plants were comparable to the wild-type plants (Fig. 3f). Therefore, O2− level maintained by the THE3-RbohB module locally affects pectin methylesterification degree in the RAM.

The activity of PMEs is suppressed by O2−

To dissect how the accumulated O2− regulates pectin demethylesterification, we extracted total proteins from NP root tips treated with Tiron and analyzed the PME activity. The pectoplate assay revealed that the PME activity of the Tiron treated root tips was higher than the control (Supplementary Fig.8a,b). Thus, O2− accumulation may suppress PME activity. To test this, we incubated a commercial PME with an increasing gradient of O2−, which was released from the xanthine (XON)-xanthine oxidase (XOD) reaction in the pectoplate (Supplementary Fig.8c). The PME activity decreased with the increase of O2− release (Supplementary Fig.8c,d). Consistently, the total PME activity was elevated in rbohb and the3 root-tip extracts compared to the wild-type, whereas it was significantly reduced in the overexpression lines (Fig. 4a). Thus, O2− can block PME activity. Since O2− usually affects protein stability, we measured the protein level of the commercial PME in the presence of O2−. The continuous drop of protein abundance with the increase of O2− indicates that O2− impairs PME stability (Supplementary Fig.8e).

This finding prompted us to identify the endogenous PMEs in rice RAM. LC-MS analysis revealed that PME30 abundance, as indicated by Peptide Spectrum Match (PSM) value, increased by 75 folds after Tiron treatment (Supplementary Fig. 8f and Supplementary Dateset 2), suggesting it as a major PME functioning in rice roots. Cell-free protein degradation assay and in vitro enzyme activity analysis showed that the stability and the activity of the recombinant PME30 proteins were reduced with the release of O2− (Fig. 4b, and Supplementary Fig.8g,h). We further transfected the N. benthamiana leaves with PME30-Myc and performed protein blotting. Tiron treatment stabilized PME30 (Fig. 4c). All those demonstrate that O2− disables PME30 through disrupting the protein stability.

Rice PME30 was grouped into a subclade containing Arabidopsis PME30 (Supplementary Fig. 9). To obtain the genetic support, we isolated two allelic mutants, both containing frameshifts that introduce premature stop codons (Supplementary Fig. 10a,b). The proteins extracted from the root tips of the ospme30 mutants displayed significantly lower PME activity than those extracted from the wild type (Fig. 4d). Similarly, immunolabeling using LM20 revealed the extended mPectin area in ospme30 roots (Fig. 4e), consistent with the augmented level of methyl esters on the pectin (Fig. 4f and Supplementary Fig. 10c). Notably, the promoted root growth in ospme30 mutants was accompanied by the increased pectin methylesterification, enhanced cell division, and an enlarged MZ (Fig. 4g–j and Supplementary Fig. 10d,e). Hence, OsPME30, as a major PME in rice RAM, is disabled by O2− produced by the THE3-RbohB module.

OsRALF4 is a ligand of THE3, suppressing THE3-RbohB signaling

THE3 homologs often serve as the receptors of RALF peptides28. To determine the ligand of THE3, we analyzed the transcriptomes of rice root developmental zones, and found four RALF peptides highly expressed in the rice RAM (Supplementary Fig. 11a and Table 1). We chemically synthesized these RALF peptides, and measured their binding affinity with the ectodomain of THE3 (THE3ECD) using micro-scale thermophoresis (MST). OsRALF4 specifically bound THE3ECD with a dissociation constant (Kd) of 6.1 µM (Fig. 5a), whereas the other OsRALFs exhibited higher Kd value or did not bind to THE3ECD (Supplementary Fig. 11b). Split-luciferase complementation assay verified the interaction between OsRALF4 and THE3 (Fig. 5b). OsRALF4, clustered in the subclade with AtRALF4 and AtRASLF2237,38 (Supplementary Fig. 11c), encodes a potential ligand of THE3.

Next, we explored the impact of this binding on the THE3-RbohB signaling pathway. In the presence of OsRALF4, split-luciferase complementation assay detected the obvious attenuation in the interaction between THE3 and RbohBN (Fig. 5c). This finding is strengthened by in vitro phosphorylation analysis, in which the addition of OsRALF4 significantly decreased the level of phosphorylated RbohB (Fig. 5d). To determine its impact on O2− production, we applied different concentrations of OsRALF4 onto N. benthamiana leaves (Supplementary Fig. 12a). NBT staining of the treated N. benthamiana leaf discs revealed that OsRALF4 treatment resulted in a dose-dependent inhibition of O2− levels (Fig. 5e). Furthermore, by transiently transfecting the N. benthamiana leaves with the constructs containing THE3 or/and RbohB, the addition of OsRALF4 weakened the NBT signal enhanced by coexpressing THE3 and RbohB (Fig. 5f,g). OsRALF4 exerts an inhibitory effect on the THE3-RbohB pathway.

Next, we generated OsRALF4 mutants by CRISPR/Cas9 genome editing. Two allelic mutants resulted from 1 bp deletion and insertion are loss-of-function mutations and displayed increased root length and MZ size as revealed by EdU labeling (Fig. 5h–k and Supplementary Fig. 12b). OsRALF4 thus restrains root growth. To verify its inhibitory role in the THE3-RbohB signaling pathway, we treated the relevant genetic materials using the synthesized OsRALF4 polypeptides. Consistent with the compromised root growth (Supplementary Fig. 12c,d), OsRALF4 treatment attenuated NBT intensity in the root tips except the3 (Fig. 5l,m); meanwhile, the MZ size of the wild-type and the overexpression plants was shortened, but was almost unchanged in the3 and rbohb mutants (Fig. 5n and Supplementary Fig. 12e). Moreover, the polypeptide treatment restored the promoted root growth of osralf4 to the wild-type level, as indicated by NBT staining and MZ size measurement (Fig. 5l–n and Supplementary Fig. 12c–e). Hence, OsRALF4 attenuates the THE3-RbohB signaling pathway, reducing O2− production.

OsRALF4 binds dmPectin, facilitating pectin condensation and cell expansion

RALF peptides can play structural roles in the expandable cell wall of pollen tubes and root hairs37,38. To determine whether OsRALF4 has structural role in root development, we prepared a fluorescent tag Alexa Fluor 647-labeled OsRALF4 and Flag-tagged OsRALF4. MST assay and physiological treatment demonstrated that the addition of these tags did not affect OsRALF4 function (Supplementary Fig. 13a–e). After incubating the root longitudinal sections with LM20 and 2F4 antibodies, super-resolution 3D-stimulated emission depletion (STED) microscopy detected that OsRALF4 is colocalized with the 2F4-labeled dmPectin in the walls of the EZ cells (Fig. 6a,b), but failed to overlap with the LM20-targeted mPectin in the MZ cell walls (Supplementary Fig. 13f). Taken together with the cell-wall localization of OsRALF4-GFP in the N. benthamiana cells (Supplementary Fig. 13g), OsRALF4 can integrate into the expanding cell walls. We further prepared mPectin and dmPectin (Supplementary Fig. 13h), and examined their binding capacity to OsRALF4 by MST assay. OsRALF4 bound to dmPectin with a Kd of 6.7 µM and mPectin with a Kd of 201.5 µM (Fig. 6c and Supplementary Fig. 13i). Since a high Kd value indicates weak binding, OsRALF4 preferentially binds to dmPectin.

To determine its role in cell wall organization, we analyzed pectin structure in the EZ cell walls of wild-type and osralf4-1 roots using 3D direct stochastic optical reconstruction microscopy (3D-dSTORM). By imaging the 2F4-labeled walls of macerated cortex cells at XZ plane, we observed a bimodal distribution of dmPectin (Supplementary Fig. 14a,b and Supplementary Movie 1). In opposite to the condensed dmPectin in the wild-type walls, we observed more fine particles in that of osralf4-1 (Fig. 6d,e and Supplementary Fig. 14c). Notably, dmPectin formed random bundles in wild-type cell walls, whereas in the mutant, it appeared less aggregated (Fig. 6d). OsRALF4 facilitates pectin condensation in the EZ.

Given its dual roles in signaling and structure, a key question arises: how do this functional duality modulates root development? We thus explored the effect of dmPectin on the formation of OsRALF4-THE3 complex. Co-IP assay using the rice protoplasts coexpressing OsRALF4-Flag and THE3ECD showed that applying dmPectin weakened the interaction between OsRALF4 and THE3ECD; however, adding mPectin did not (Fig. 6f). The presence of dmPectin likely disturbs the perception of OsRALF4 by THE3. qPCR analysis further revealed an ascending expression gradient of OsRALF4 along the root developmental axis, spatially opposed to O2− distribution (Fig. 6g and Fig. 1b). Notably, immunolabeling of the root tips using LM20 exhibited the extended mPectin pattern in osralf4 mutants (Fig. 6h). OsRALF4 peptide may facilitate pectin demethylesterification. To verify this, we treated the osralf4, the3, rbohb and the overexpression plants with exogenous OsRALF4 polypeptides. The treatment significantly repressed mPectin pattern in the RAM, as probed by LM20 (Fig. 6i), demonstrating that OsRALF4 promotes dmPectin generation, boosting OsRALF4-dmPectin interaction and cell expansion.

Therefore, we propose that the increase of OsRALF4 promotes the production of dmPectin, which anchor free OsRALF4 into the cell wall. Thus, the location of pectin demethylesterification would mark the acquisition of a structural role for OsRALF4, in addition to its signaling function, thereby promoting the onset of cell elongation during root development.

Methods
Plant materials and growth conditions
All the rice plants used in this study were planted during the growing seasons in the experimental fields at the Institute of Genetics and Developmental Biology in Beijing (China) and Lingshui (Hainan Province, China). The rice seedlings were cultivated in hydroponic medium under the long-day condition (16 h of light and 8 h of dark).

The the3 and osralf4 mutants were generated by CRISPR/Cas9 genome editing. The ospme30 mutants was purchased from BIOGLE GeneTech (China). To generate the THE3 overexpression lines, the coding sequence (CDS) of THE3 was cloned into pCAMBIA1300 vector between the 35S promoter and NOS terminator. For generation of RbohB overexpression lines, the CDS of RbohB was cloned into pJLI1460 vector between ubiquitin promoter and NOS terminator. These resulting constructs were introduced into japonica variety ZH11 or the3 mutant via the transformation of Agrobacterium tumefaciens (strain EHA105). For genetic complementation assays, the genomic fragment containing a 2,158 bp promoter, the full coding region, and a 1,239 bp terminator was introduced into the the3 mutant. After confirming the genotyping by sequencing, the homozygous T plants were used for the experiments. The primers and gRNAs used in this study were listed in Supplementary Dataset 3.

qPCR analysis
For gene expression analysis, total RNAs were extracted from rice root tips using Plant RNA Reagent (Invitrogen, 12322012) according to the manufacturer’s instructions. Approximately 2 μg of the total RNA were treated with DNase I (Takara, 2270A) and used to generate cDNA using the PrimeScript RT Reagent Kit (Takara, RR037A). qPCR was performed using a real-time PCR system (CFX96, Bio-Rad) with FastStart Universal SYBR Green Master (Roche, 4913914001). Three biological replicates were included. The primers were listed in Supplementary Dataset 3.

NBT staining
For detecting O2−, 5-day-old rice seedlings and infiltrated N. benthamiana leaves were immersed in the NBT solution (0.05%) freshly prepared in 50 mM sodium phosphate (pH 7.0) and 10 mM NaN3. After infiltrating under vacuum at 0.8 psi for 2 min (root tips) or 30 min (N. benthamiana leaves), the stained samples were bleached and stored in 95% (v/v) ethanol for photographing. For quantifying NBT length, we measured the distance from the root tip to the onset of diminished NBT staining (indicated by a white triangle). For quantifying formazan, approximately 20 mg of stained tissues was employed to extract the resultant formazan using 1 mL of 2 M potassium hydroxide: chloroform (1:1, v/v) at 4°C. The resulting extracts were dried under N2 at 4°C, and resuspended by dissolving in 350 μL of DMSO and 300 μL of 2 M potassium hydroxide at room temperature. Formazan amount was measured according to absorbance at 630 nm using a spectrophotometer (Enspire, PerkinElmer). Three biological replicates were included.

EdU staining
For measuring cell proliferation using EdU (5-ethynyl-2′-deoxyuridine) staining, three to seven 5-day-old rice seedlings were incubated in PBS solution containing 20 μM EdU (Beyotime, C0085S) for 4 h. The resulting seedlings were fixed in freshly prepared fixative solution (3.7% [v/v] paraformaldehyde and 1% [v/v] Triton-X 100 in 1 × PBS) for 1 h. After rinsing with 3% (w/v) bovine serum albumin (BSA) in 1 × PBS twice, the seedlings were treated with 100 μL Click-iT reaction mix for 30 min to label the incorporated EdU with Azide Alexa Fluor 488, and then applied to visualize DNA synthesis using a confocal microscope (LSM980, Zeiss). We recorded the length of the root axis exhibiting EdU fluorescence as the EdU length.

TurboID proximity labeling
To identify the interacting proteins of RbohB and THE3, the CDS of RbohB and THE3 were fused in frame with the CDS of TurboID between Ubiquitin promoter and NOS terminator in Raichu vector. The resulting plasmids were transformed into rice protoplasts prepared according to the previous report49. After incubating for 12 h, the protoplasts were treated with 50 µM biotin for 3 h and then subjected to total protein extraction using the extraction buffer (100 mM Tris-HCl, pH 7.5, 100 mM KCl, 10% glycerol, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, and 1 × protease inhibitor cocktail). After centrifuging at 12,000 × g and 4°C for 20 min, the protein extract supernatants were incubated with 30 µL of Dynabeads C1 Streptavidin (Invitrogen) at 4°C for 4 h. The biotinylated proteins bound to streptavidin beads were separated by SDS-PAGE. After destaining, reduction with dithiothreitol (DTT), and alkylation with iodoacetamide, Coomassie-stained gel bands were digested overnight with trypsin, followed by stepwise extraction of peptides with acetonitrile at different concentrations. The peptide mixture obtained from enzymatic digestion was subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and then used for database search and protein identification via the SEQUEST HT search engine in Thermo Proteome Discoverer (version 1.4.0.288) against the ZLJ_RB database. Search parameters include trypsin digestion with 2 allowed missed cleavage sites, precursor ion mass tolerance < 10 ppm, and fragment ion mass tolerance < 0.02 mDa. Spectrum filtering was performed using Percolator with Delta Cn < 0.1, false discovery rate (FDR) set to 1%, and peptide filtering parameter selected as "High" peptide confidence.

To detect RbohB in the TurboID-labelled samples, we prepared polyclonal antibodies against RbohB using a synthetic peptide (amino acids 30–43, CPNSGNLGSSNRSTK) in rabbit (HUABIO, China). For protein blotting analysis, the biotinylated proteins were extracted from the rice protoplasts expressing THE3-TurboID-cMyc fusion protein, and separated on 10% (w/v) SDS-PAGE gel. By transferring to nitrocellulose membrane (Bio-Rad), the blot was blocked in TBST buffer containing 5% (w/v) milk for at least 2 h, and incubated with anti-RbohB antibody at 1:1000 dilution, followed by incubating with HRP-conjugated secondary antibody at 1:10,000 dilution (Dingguo). After developing with chemiluminescent HRP substrate (Cytiva, RPN2235), the blot was imaged by Tanon 5200 Imaging System (Tanon). The relative amount of proteins was quantified using Image J software. The experiments were repeated three times with similar results.

Protein interaction assays
For split-luciferase assay, the CDSs of THE3, RbohB and OsRALF4 were cloned into the pCMABIA1300-cLUC vector and the pCAMBIA1300-nLUC vector, respectively. The resulting constructs were pairwise infiltrated into N. benthamiana leaves. After cultivation for 48 h, the infiltrated leaves were sprayed with 1 mM D-luciferin and then kept in dark for 5 min. The luciferase signal was captured using IndiGO software (Berthold). GFP-nLUC and cLUC-GFP were served as negative controls.

For Co-IP assay, the CDS of THE3 and RbohB was fused with cMyc in the pCAMBIA1300 vector or fused with GFP in the pBI221 vector to generate 35S:THE3KD-cMyc/35S:GFP or THE3KD-cMyc/35S:RbohBN-GFP constructs. The resulting constructs were transformed into rice protoplasts. After overnight cultivation, these protoplasts were applied to total protein extraction as described above. The protein samples were incubated with 20 µL of GFP-trap agarose at 4°C for 4 h. The co-precipitated proteins were eluted and separated by SDS–PAGE. Protein blotting was performed using anti-GFP antibody (Abcam) and anti-cMyc antibody (Abclonal) as described above.

For pull-down assay, the coding sequence of THE3 kinase domain was fused with GST (Glutathione-S-Transferase) in pGEX-6p-1 vector. The N terminal RbohB was cloned in pET28a vector. The resulting constructs were transformed into E. coli Rosetta (DE3), separately. The recombinant proteins of THE3KD-GST and RbohBN-His were purified following the manufacturer’s instructions. Approximately 3 µg purified RbohBN-His were incubated with 9 µg immobilized GST or GST-THE3KD at 4°C for 2 h. The eluants were separated by SDS-PAGE and then subjected to protein blotting using anti-GST (Abclonal) or anti-His (Biodragon) antibody. The above experiments were repeated three times respectively, and the results were similar.

Subcellular localization
To determine the subcellular localization of THE3, the CDS of THE3 was amplified and fused to GFP in the vector pBI221 (HonorGene) using the primers (Supplementary Dataset 3). The THE3-GFP construct, together with the construct harboring an mRFP-HIR1 (a plasma membrane marker), was transformed into the protoplasts prepared from 2-week-old NP seedlings as describes above. By incubating in the dark overnight, the transfected rice protoplasts were photographed under a confocal microscope (Zeiss, LSM980). 35S:GFP was used as a control.

For the subcellular localization of OsRALF4, the CDS of OsRALF4 was cloned into the pCAMBIA1300-GFP vector using the primers (Supplementary Dataset 3). OsRALF4-GFP was transformed into the 3-week-old Nicotiana tabacum leaves. The N. benthamiana leaves were soaked in 1 M mannitol for 10 minutes for plasmolysis. Fluorescence was oberved using a confocal laser-scanning microscope (Zeiss, LSM710). The above experiments were repeated three times with similar results.

Protein phosphorylation assay
For in vitro phosphorylation assay, the purified THE3KD-GST was incubated with a universal substrate, myelin basic protein (Sigma) or MIBP in reaction buffer (50 mM Tris–HCl pH 6.8, 10 mM MgCl2, 1 mM dithiothreitol [DTT], 0.1 mM ATP, and 5 μCi [γ-32P]ATP) for 1 h, and then terminated by the addition of stop buffer (0.25 M Tris–HCl pH 6.8, 10% [w/v] SDS, 50% [v/v] glycerol, 0.5% [w/v] bromophenol blue, 5% [v/v] β-mercaptoethanol). The labeled proteins were separated in 10% SDS–PAGE gel and subjected to autoradiography using a Typhoon 9410 imager (GE Healthcare). The proteins in parallel reactions without [γ-32P]ATP were separated by SDS-PAGE and stained with coomassie brilliant blue.

For the phosphoprotein mobility-shift assay, the purified RbohBN-His were incubated with GST-THE3KD in a kinase buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 10 mM ATP) at 30°C for 30 min. Additional parallel samples were incubated with CIP (NEB) at 37°C for 30 min. By separating in a 12% (w/v) SDS-PAGE gel containing 50 μM Phos-tag and 100 μM MnCl2, the phosphorylated proteins were detected using anti-His antibody (Biodragon) as described50.

To validate the in vivo phosphorylation of RbohB, the CDS of RbohB was fused to NanoLUC in Raichu vector. The resulting construct was transfected into the rice protoplasts together with THE3-GFP or GFP. After overnight incubation, the total proteins were extracted. The aliquots of the samples were treated with 1 U/μL CIP (NEB) or 2 μM OsRALF4 peptide for 30 min. After trichloroacetic acid precipitation, the samples were separated on 12% SDS-PAGE gel containing 50 μM Phos-tag. By washing the gel in an SDS-removing buffer (50 mM Tris [pH 7.5], 1 mM DTT, 0.1 mM EDTA) for 1 h, the gel was developed using Nano-Glo Luciferase Assay Substrate (Promega, N1110) and was imaged using a Tanon 5200 chemiluminescent imager. The relative protein level was determined by normalized densitometry. The experiments were repeated three times with similar results.

NADPH oxidase activity analysis
RbohB proteins were immunoprecipitated using anti-RbohB antibody, and the NADPH oxidase activity was detected following the manufacturer’s instructions (AIDISHENG, ADS-F-NA001). NADPH oxidase activity was measured as Δ450 per mg protein per min. Three biological replicates were included.

Protein degradation assays
Cell-free protein degradation was performed as described previously with some modifications50. In brief, the total proteins were extracted from 2-week-old wild-type, the3, and THE3ox seedlings using extraction buffer (50 mM Tris pH 8.0, 2 mM EDTA, 20% glycerol, 1 mM DTT, 0.1% Triton X-100, and 1 × inhibitor cocktail). The purified RbohB-His proteins (10 μg) were incubated with equal amount of the total extract proteins with 10 mM ATP. The protein mixtures were separated by SDS-PAGE and probed using anti-His monoclonal antibody. The loading control was indicated by the proteins probed by anti-actin (EASYBIO). The relative protein level was determined by normalized densitometry.

To examine the stability of OsPME30 protein, the purified MBP-OsPME30 or MBP proteins were treated with O2− generated from the reaction containing 3 mM xanthine (XON) and 0.5 U or 1 U xanthine oxidase (XOD). The residues were subjected to protein blotting using the anti-MBP monoclonal antibody (NEB). To obtain in vivo support, the cMyc-OsPME30 construct was infected into the 4-week-old N. benthamiana leaves along with the RNAi inhibitor vector P19 (OD = 1). After cultivation for 48 h, the leaves were treated with 200 µM Tiron for 1 h. Total proteins were extracted from the infected leaves and subjected to western blotting. The content of PME30 was determine by probing the blot with anti-cMyc antibody. The experiments were repeated three times with similar results.

Pectin analysis
By germinating wild type and mutant seeds in wet petri dish for 3 d, we further treated the wild-type seedlings in 200 μM Tiron for 12 h. The root tips from 20 individual plants of each sample were harvested and ball-milled into powder. The resulting samples were extracted with alcohol and treated with 1% α-amylase (w/v) to obtain destarched alcohol insoluble residues (AIRs). The AIRs were treated with endo-polygalacturonanase M2 (Megazyme) in 50 mM sodium acetate solution at 30°C for 16 h. The released oligogalacturonides were derivatized in 100 mM procainamide (Sigma) containing 250 mM sodium cyanoborohydride at 37°C overnight. For MALDI-TOF MS assay, the derivates were mixed with a matrix solution (25 mg/mL 2,5-dihydroxbenzoic acid) and spotted on the target plate with 0.5 μL of 5-chloro-2-mercaptobenothiazole (10 mg/mL). The mass spectra were collected within m/z range of 200–2,000 in the reflection positive ion mode (laser intensity 95%, ion source 1 voltage 20.00 kV, ion source 2 voltage 17.75 kV, lens voltage 7.60 kV, detector voltage 2,127 V, and pulsed ion extraction 180 ns). Each spectrum corresponded to an ion accumulation of 2,000–5,000 laser shots randomly distributed on the spot. The spectra were analyzed using FlexAnalysis v.3.4 software (Bruker Daltonics). The relative intensity of methyl esters on oligogalacturonides was determined by dividing the methyl-esterified signal by the total detected oligmers. At least three replicates were included, and the replicates without Tiron treatment were set as controls.

PME activity assay
Total PME activity was determined according to previous study52. Five-day-old root tips were ground in liquid nitrogen, and subjected to extracting total proteins using 200 μL extraction buffer (0.1 M citrate acid, 0.2 M Na2HPO4, and 1 M NaCl, pH 5.0). The samples were incubated on ice for 1 h and centrifuged at 4°C with 13,000 g for 15 min. Protein extracts were added in the reaction containing 0.5% citrus pectin (0.2 M NaCl, and 0.002% methyl red, pH 6.8) at 37°C for 1 h. The absorbance of the solution was measured at 525 nm using EnSpire Multimode Plate Reader (PerkinElmer). Calibration curve was obtained using a gradient of 0 to 200 nM HCl solution. Total PME activity was calculated according to the established standard curve.

PME activity was also measured using pectoplate method52. In brief, pectoplate was prepared as 1% (w/v) low-temperature agarose (USB) medium containing 0.1% (w/v) citrus pectin (Sigma), 12.5 mM citric acid, and 50 mM Na2HPO4, pH 6.5. Equal amounts (30 μL) of the eluent fractions or protein extracts were loaded into each punched well and incubated at 30°C for 16 h. The plates were stained with 0.02% (w/v) ruthenium red (Sigma) for 30 min. By rinsing with distilled water, the size of stained circle was measured to indicate PME activity. To examine PME activity with the increase of O2−, a gradient amount (0.5, 1, 2, 4, 10 U) of xanthine oxidase (XOD) were added in the pectoplate containing 3 mM xanthine (XON). PME activity was measured as described above.

The CDS of PME30 was fused with His-tag to its C-terminus in pPICZα-C vector (Invitrogen) and introduced into Pichia pastoris strain X-33 cells by electroporation following the manufacturer’s instruction. PME30-His recombinant proteins were purified using affinity chromatography53. The activity of PME30 was measured using pectoplate as described above. The experiments were repeated three times with similar results.

RNA-seq analysis
The total RNA was extracted from ZH11 roots using Trizol reagent. The samples were subjected to deep sequencing using Illumina HiSeq 2000 platform (Illumina) after library construction (Novogene). The obtained paired-end clean reads were aligned to the rice reference genome, Release 7 of the MSU Rice Genome Annotation Project (http://rice.uga.edu/), using HISAT2 (http://daehwankimlab.github.io/hisat2/). Read counting and normalization (FPKM) were performed using StringTie version 2.1.7 (https://ccb.jhu.edu/software/stringtie/) and the package Ballgown (https://github.com/alyssafrazee/ballgown). Three biological replicates were included in this experiment for each sample.

MST assay
The mature peptides of OsRALF1, OsRALF4, OsRALF6 and OsRALF15 were synthesized with high purity (> 95%) using the services of Sangon and HUABIO. To test their binding affinity to THE3 or pectin, OsRALF4 peptides, BSA or the purified GST-THE3ECD recombinant proteins were labeled using RED-NHS (Monolith, MOL011) in the buffer (50 mM sodium phosphate, pH 6.8, 150 mM NaCl, 0.05% Tween 20) for 30 min, separately. The resulting samples were purified by affinity chromatography using the B-Column to wash off free fluorescent chemicals. Followed by collecting the conjugated peptides, a gradient of the labeled RALFs was incubated with GST-THE3 pectin or peptides in 1 mg/mL BSA, respectively. The reactions were loaded into the silica capillaries (Monolith NT.115 Standard Treated Capillaries, MO-K002; Monolith NT.115 MST Premium Coated Capillaries, MOK005) and measured by using Monolith NT.115 (NanoTemper) at 20% or 40% LED power. Dissociation constant (Kd) value was calculated using Mo Affinity Analysis (V2.2.4) based on the fit curve. To examine the binding affnity between OsRALF4 and dmPectin or mPectin, the operation was same as decrisbed above, using BSA as a negative control. Three biological replicates were included in this experiment.

Confocal microscopy
For pectin immunolabeling, the rice seedlings were geminated in petri dish for 2 d, and then transferred to ½ MS medium containing 200 µM Tiron for additional 1 d. The roots were harvested and fixed in 4% paraformaldehyde and embedded in 4% agarose. Longitudinal sections with 15-µm thickness were prepared using a vibratome (Leica, VT1200) and probed with the LM20 and 2F4 antibody (Kerafast) at a 1:200 dilution. Alexa Fluor 488-coupled anti-rat IgG (Abcam) and Alexa Fluor 568-coupled anti-mouse IgG (Abcam) were used as the secondary antibody at a 1:500 dilution. Fluorescent signals of 10 images from three individual plants were recorded using a confocal microscope (Zeiss, LSM980). For OsRALF4 treatment, the germinated rice seeds were cultivated on ½ MS medium containing 2 µM OsRALF4. The immunolabeling assay was performed as described above. The replicates without Tiron and OsRALF4 treatment were set as controls.

To determine the meristem length, root tips of at least eight 3-d old seedlings were used to prepare root longitudinal sections as described above. By staining with Calcofluor White (CFW), the images were taken by a confocal microscope (Zeiss, LSM980) and analyzed using ZEN software. In these images, the distance from the root tip to the onset of cell expansion was determined as MZ size. At least 10 images from at least eight individual plants were analyzed.

Super-resolution microscopy
For STED microscopy, OsRALF4 peptides were labeled with Alexa Fluor 647 (Invitrogen) according to the manufacturer’s instructions. The root longitudinal sections were prepared and incubated in the T/Ca/S buffer (20 mM Tris-HCl, 1 mM CaCl2, 150 mM NaCl, pH 8.2) containing 0.2 μM labeled OsRALF4 for 1 h. The sections were further probed with LM20 and 2F4 antibodies, followed by incubating with Alexa Fluor 488-coupled anti-rat IgG and STAR ORANGE conjugated anti-mouse IgG (Aberior, NC1933863) at a 1:500 dilution. The images were acquired using STEDYCON (Aberrior microscope system) with excitation wavelengths of 640 nm for Alexa Fluor 647, 550–610 nm for STAR ORANGE and 495 nm for Alexa Fluor 488. At least 10 images from three individual plants were analyzed.

For dSTORM microscopy, at least three 3-day-old rice roots were pretreated in 10% peracetic acid solution (v/v) for 30 min. The cells in root elongation zone were isolated and transferred onto the 35-mm glass bottom dishes (Cellvis, D35-14-1.5-N) coated with poly-L-lysine (Merck, P4707). By incubating with primary and secondary antibodies sequentially, root cells were post-fixed for 15 min in 3.5% paraformaldehyde to prevent thermal drift of antibodies. For imaging, T/Ca/S buffer was replaced with the STORM imaging buffer (Nano-Microimaging, NWIB#1). Images were acquired by a commercial dSTORM system (Nano-Microimaging, STORM Ultra300) based on an inverted fluorescence microscope (Nikon, Ti2-E) equipped with a sCMOS camera (TUCSEN, Dhyana 95V2) and a 100 × oil-immersion objective (Nikon CFI Plan Apochromat λ, NA 1.49). The 642 nm laser (~2 kW/cm2) was used to excite the sample to photoswitch with most of the labeled Alexa Fluor 647 dye into a dark state. The 405 nm laser (typically 0~1 W/cm2) was applied to stochastically reactivate fluorophores into the activation state for detection of single-molecule fluorescence. The images were collected at ~100 frames/sec. Typically, dSTORM images were reconstructed from ~50,000 frames based on single-molecule localization algorithm as described previously54,55. For data analysis, peak fitting was performed using a Gaussian function in OriginPro 2021. A custom MATLAB program was used for cluster analysis of the primary cell wall. Clustering was performed using the DBSCAN function with the arguments (epsilon, minpts). The polyarea function was used to calculate the area of each cluster, which was used for distribution statistics. At least 5 images from three individual plants were analyzed.

Statistical analysis
Two-tailed Student’s t-test was performed using Excel, and one-way analysis of variance (ANOVA) was conducted with Holm-Sidak method using GraphPad Prism (v9.1.1).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The overall concept of a RALF-CrRLKL-ROS signaling axis and RALF-pectin interaction providing a dual structural and functional role are not novel as they have been demonstrated in several previous studies. This work here, carried out in rice, still could represent an important entry. The combinations of experiments are not often reported in studies of the CrRLKL signaling.\nWhile I find the data presented are consistent with prevalent models and the authors conclusion could advance the current knowledge in both of the linkages referred to, they are not as robust as needed to draw exclusive conclusions on which components are specific or representative of a class of components. Either of the scenarios are fine, but “specific” gives some satisfaction of finality and without the necessity of much interpretation; “representative of…” might in fact be the biological reality, thus the subtlety in the data, will require some discussions, which is relatively weak.\nThis study, being in rice, it is difficult to place the various gene #, whether they bear any relationship to homologs in Arabidopsis. Is RALF4 here most similar to A thaliana RALF4 (pollen high), or it is just #4 in the rice family nomenclature. ExtD Fig. 12 has some RALF#s missing, suggesting most like the rice genes are named in their closeness to A. thaliana counterpart. This by itself is not important, but just distracting. I suggest just name them OsRALF4 etc. –it does not really matter whether and in which way it is related to the A. thaliana peptides.\nI do not understand why the authors only focused on RBOHB, and ignored RBOHC, when the two are quite comparably expressed Fig. 1c (even if the qRT-PCR was perfect, B represents 3/5, C represent 2/5 of the two together.). I think the experiments should at least test how much contribution is from RBOHC, and also test the double double to get to a lower baseline for root ROS in these mutants. As it is, All the ROS comparisons are quite subtle, e.g., Fig. 1d and others. With a lower baseline, I believe the ensuing data will follow suit and together make a stronger study.\nAnother example of more robust dataset will be more persuasive is the in vivo RBOHB phosphorylation in WT, the3 and THE3ox lines (Fig. 2). Also, most of the phospho-study panels (e.g. ghij) correlate THE or lack of to ROS levels, the only ones that address the phospho-state of RBOHB to ROS are Fig. 2k,l , where the data in k (images) were considerably less discernable in differences than the quantified data in l. These data, being among the most crucial of the study and not deducible from existing knowledge, need to be more robust.\nFig. 5 is an important data set, but much of the comparative data have saturated exposure making comparative quantification difficult. Also in Fig. 5j, the author should include loss of raf4 mutant, this would inform on the importance of RALF4 to the signaling axis, does it play a prominent or almost exclusive role, or a contributing role etc. These lines exist and were used in later figures. They could easily be tested and results added to the study.\nFig. 6, authors report RALF4 binds to dmPectin, facilitate pectin condensation into “Bands” vs. in ralf4 mutant, dmpectin occurs as particles. The patterns shown are clearly different. The perspective shown for the WT pattern does not quite persuade a “banding” pattern. There are some longer forms separated by unstained regions.\nThe Discussion is somewhat weak. I think it is because the data need substantive interpretation, and gaps from one set of experiments to another need to be explained.\nI see considerable potential for this to be a much stronger and comprehensive study as the authors would like to deliver (Discussion). I hope the authors will further consider the rationale behind each set of inquiry and aim for more depth in experimental strategies and data.", "reviewer_2": "This study provides a systematic elucidation of a sophisticated mechanism governing rice root tip development, mediated by the RALF4–THE3–RbohB signaling module, which coordinates cell division and expansion through the regulation of reactive O₂⁻ homeostasis and pectin dynamics. The work demonstrates high innovation, comprehensive mechanistic insight, rigorous experimental design, and robust, reliable data. It not only delineates a complete pathway from peptide signal perception to cell wall remodeling but also introduces the novel concept of RALF4 exhibiting a stage-specific functional switch—acting both as a signaling molecule and a structural component during development. These findings represent a substantial contribution to the fields of plant developmental biology and cell signaling. However, several issues in the manuscript require attention:\n1. In Figure 1d, the three root sections shown with NBT staining do not appear to originate from the same cell layer.\n2. In Figure 2f, phosphorylation of RbohB is visibly stronger in ZH11. Although THE3ox shows a modest increase relative to the mutant, this key in vivo phosphorylation result should be repeated. If grayscale values were used to quantify phosphorylated bands, the exposure time may have been excessively long.\n3. Some inconsistency is apparent between Figure 2k and 2l. Specifically, Figure 2k suggests that NBT-stained leaves carrying the S191 mutation exhibit the weakest signal.\n4. Regarding the phosphorylation data in Figure 5d, the evidence for reduced phosphorylation is currently insufficient. These results must be replicated, and grayscale ratios (consistent with prior analyses) should be clearly reported.\n5. In Figure 5e, the addition of RALF4 did not markedly suppress O₂⁻ production. It may be useful to test a range of RALF4 concentrations to more clearly illustrate its dose-dependent effect.\n6. To determine whether increased O₂⁻ levels result from THE3-mediated phosphorylation stabilizing RbohB protein or directly enhancing its activity, further experimental validation is needed.\n7. The manuscript states that RALF4 binds THE3 and modulates its interaction with RbohB. It is therefore unclear why RALF4 treatment in RBoxthe3 plants leads to shorter roots, reduced apical meristem size, and decreased methyl-pectin content. This apparent contradiction warrants clarification.\n8. In Figure 6h, the signal intensity in the meristem of the ralf4-2 mutant appears weaker than in the wild type. What is the underlying reason?\n9. Is reactive oxygen species accumulation confined specifically to the meristem? Please justify the selection of this region for analysis.\n10. All SDS-PAGE gels should include molecular weight markers for reference.", "reviewer_3": "This is a well-written and well-presented ms describing an interesting mechanism linking a cell wall signaling module to a developmental transition in rice roots. The signaling module previously has been extensively studied in different cellular contexts, primarily in Arabidopsis thaliana, including the connection with RboH-dependent ROS production and pectin metabolism, that is reported on in this ms. The originality of this study is to connect this module to the transition of cells from the meristematic zone (MZ) to the elongation zone (EZ) in the root, and to provide an attractive framework to explain previously reported observations that the accumulation of superoxide radicals (O2-) can maintain the cells in a meristematic state.\nThe ms describes an impressive amount of data including the generation of several generations of rice transgenics and Crispr-Cas9 mutants combined with high end biochemical and imaging techniques. The use of rice remarkably allowed the observation of pronounced mutant root phenotypes for THE3, RBOHB, PME30 and RALF4 mutants, which haven’t been observed so far for mutants for putative orthologs in A. thaliana, possibly due to redundancy in the, often large, gene families.\nTo my opinion, all of this is of broad interest to the plant community and justifies publication in a journal like Nature Communications provided the remarks listed below can be taken into consideration.\nThe ms describes successively, a gradient in NBT staining as a proxy for O2- levels, going from high in the MZ to low in the EZ of the root confirming previous observations (e.g. doi:10.1038/s41586-019-1819-6). The authors identify RboHB as a major RboH in rice roots. RboHB LOF mutant and overexpressor (RBox) show reduced and enhanced NBT staining respectively. TurboID proximity labeling in rice pps using RboHB as a bait identifies THE3, a member of the CrRLKL receptor family. The interaction of RboHB with the THE3 kinase domain is confirmed by split luciferase and coIP in rice pps and pull downs. THE3-GFP accumulates in the PM in rice pps. THE3 has kinase activity in vitro and can phosphorylate the N-terminus of RboHB in vitro and in rice pp. RboHB phosphorylation is reduced in a the3 background. RboHB stability is reduced in cell free extracts of the3, relative to WT. H2DCFA staining of rice pps is higher after transformation with FER3 + RboHB than with RboHB alone. The THE3-RboHB module therefore appears to be responsible for O2- accumulation in the root.\nHigher and lower NBT staining is observed in roots of THE3ox and the3 KO mutant respectively. Higher NBT staining in RBox is maintained in a the3 background. This suggests that RboHB acts downstream of THE3 and is reminiscent to what has been reported for the CrRLKL receptor ANX1/2 acting upstream of RboHH/J (doi:10.1371/journal.pbio.1001719)\nrbohb and the3 mutants show reduced MZ size, The overexpression of either gene increases the MZ size. This suggests that the THE3-RboHB module regulates the MZ size.\nThe LM20 antibody against high degree of methylesterification (high-DM) pectin shows preferential labeling of roots in the MZ, whereas 2F4 (against Ca2+-crosslinked demethylesterified HG) preferentially labels cell walls in the EZ. rbohb and the3 show reduced LM20 labeling in the EZ. This is backed up by oligogalacturonide profiling of root tips by Maldi-TOF. Treatment with O2- quencher Tiron promotes demethylesterification. rbohb and the3 root tips show increased PME activity as measured with a gel diffusion assay. PME30 is identified as a major PME in the root and is highly induced in the presence of Tiron. A CrispR-Cas9 pme30 mutant shows enhanced LM20 staining in the MZ and a higher proportion of more methylesterified HG oligomers. The mutant shows a longer MZ. PME30-cMyc degradation in tobacco extracts is prevented by Tiron. Purified MBP-PME30 is turned over more quickly in the presence of O2-. This may explain how O2- produced by the PME3-RboHB module can affect the transition from MZ to EZ.\nRALF4 mRNA is highly expressed in the MZ of the root, with a positive shootward gradient. Chemically synthesized mature RALF4 binds THE3 ECD with a 6.1µM Kd as shown by MST. The interaction is confirmed by split-luciferase. RALF4 appears to inhibit the phosphorylation of RboHB by THE3-GFP in rice pps and NBT labeling of tobacco leaves infiltrated with THE3 and RboHB. ralf4 LOF mutants have longer roots and larger MZ and more NBT labeling in the MZ. The peptide appears to bind low DM pectin and colocalizes with 2F4 epitopes in the cell wall.\nBased on these data, the authors propose a model (Fig. 7) in which a positive shootward gradient of RALF4, negatively controls THE3-RboHB-dependent O2- accumulation, and in which O2- inhibits the transition of the cells from the MZ to the EZ through the inhibition of PME30 HG demethylesterification.\nMajor remarks\n1. The inhibition of PME activity by O2- produced by the THE3/RboHB module is an attractive hypothesis to explain the transition of cells from the MZ to the EZ. The ms does not address the potential effect of the the3 mutation on the surface pH and accumulation of other ROS (H202, OH., …). This should be verified using specific dyes and sensors. Indeed, changes in surface pH could equally well explain the observed effects on PME activity, given the high pH optimum of the enzymes. Other RALF peptides are known to promote the alkalinisation of the cell surface and hence PME activity, which thus leads to the transition from the MZ to the EZ. Other ROS species (e.g. H2O2), indirectly produced by RboHB, also could activate specific Ca2+ channels and inward Ca2+ currents are frequently associated with surface alkalinisation. This would provide a less biased picture of the regulation of PME activity at multiple levels (transcriptional (O2-), protein accumulation (O2-) and activity (pH).\n2. Fig. 3: it is surprising that the strong reduction in HG methylesterification in the MZ of rbohb and the3 have such a modest effect on the meristem size. Wouldn’t one expect an immediate exit of the cells from the MZ in the mutants ? The authors could discuss that in the context of potential other levels of regulation.\n3. rbohb, the3, pme30 are represented by single alleles, and no complementation data are provided. The overexpression lines are represented by a single transformant. Given past unfortunate examples in the field, I’d strongly recommend to provide additional data that validate the observations on those single alleles. It is a bit embarrassing to ask for even more data, but quantity cannot substitute for the quality in this case.\n4. Fig. 6c: I don’t understand how one can label the RALF4 peptide with the RED-NHS (Monolith, MOL011) method indicated in the materials and methods, given that there are no lysine residues in the sequence. Perhaps another method was used. If not, could the authors have been following the fluorescence of the unincorporate fluorophore ? Even if the peptide is labeled, and the label does not affect pectin binding of the peptide, despite its positive charge, the viscosity of the pectin solution can greatly affect the mobility of the peptide (or the fluorophore ?) in the MST capillaries. A control with another labeled protein (e.g. BSA) should be included.\n5. Indicate the RALF4 concentrations and, if relevant, duration of treatments in all the biological experiments in this ms (e.g. in Fig. 5g-j)\n6. Include phylogenetic trees of THE3, PME and RALF4 families to show the orthologous relationships with previously described Arabidopsis isoforms.\nMinor remarks\n1. Include Rice in title and abstract\n2. Line 96 ‘the RALF peptides involved in root development progression are very limited.’ What does this mean ?\n3. Line 133: rewrite “THE3 is an interacting protein of RhobB.”\n4. Line 193 and Fig. 2i : H2DCFDA is a redox sensitive probe, not specific for O2-. Why was this probe used ? This should be explained.\n5. Fig 6: Colocalisation RALF4 and pectin may not be physiological (depending on RALF4 concentration and tag used). One should observe colocalization of genetically-encoded tagged RALF4 that complements the mutant phenotype with pectin.\n6. Line 216 write “reduced” instead of “decreased”\n7. Fig. 1a", "reviewer_b": "NBT staining overlaps with the MZ of the root. How to exclude that the more intense blue staining just reflects the higher cytoplasmic content of the smaller cells in the MZ ?\n8. Figs 3 and 4: write “GalA” not “GalU”\n9. Line 249: correct “increased by 75 folds”\n10. Line 299: write “RALF4”\n11. Line 301: also mention in root hairs (Schoenaers et al 2024)\n12. Extended data Fig 2 : b write “Biochemical characterization of THE3 and subcellular localization of THE3-GFP”\n13. Fig. 2 g", "reviewer_h": "indicate on Fig. 5 g where the signal was quantified , idem for Fig 5i,j.\n21. Fig. 6b: the arrows on the scheme are confusing. Please correct. I suppose the first panel is RALF4 with 2F4, not LM20. Again, indicate the RALF4 concentrations used, here and elsewhere in the ms.\n22. Fig. 6d: which wall is shown here, the external periclinal wall of the epidermis ? The pectin is probably more organized in the anticlinal walls or the internal periclinal wall of the epidermis.\n23. Line 321ff : “dmPectin in wild-type cell walls was in a periodic banding pattern”. I don’t see anything with a periodic pattern in the image of Fig. 6d.\n24. Line 330ff: “RALF4 mutation delayed pectin demethylesterification", "reviewer_i": "include control with only THE3 or WT RboHB. Include controls to determine whether the mutations affect expression levels or RboHB protein stability\n15. Fig. 3c: include LM20/2F4 labeled image of a root grown with or without Tiron.\n16. Fig. 3d: show a larger part of the root including the EZ to see to whether the LM20 labeled zone extends in RBox\n17. Extended data Fig. 6c", "reviewer_d": "Does Tiron inhibit growth entirely ? Show root length increase after Triton or Mock treatment. If O2- controls the transition from MZ to EZ, one would still see growth and perhaps longer cells in the EZ. This could be determined as in Fig. 3b. NBT shows a patchy labeling after Tiron treatment. Any explanation ? Is this related to the cell cycle ?\n18. Extended data Fig. 8: can you rule out that the xanthine (XON)-xanthine oxidase (XOD) reaction system affects the pectin and/or agarose in the pectoplate assay, thus indirectly affecting the mobility of the enzyme ? This could be addressed by using another enzyme assay, for instance by quantifying the methanol released.\n19. Fig. 5: indicate the concentration of RALF4 used in all the bioassays throughout the ms. Also indicate the duration of the treatments in particular for Fig. 5a: indicate whether you used the ECD of THE3, for the MST\n20. Fig. 5 g", "reviewer_6": "this is not convincing. Indicate the RALF concentration. Provide a quantification. Show that the Flag-tagged RALF4 is still active and that the Flag tag does not affect pectin binding.\n26. Line 406: “super-resolution imaging demonstrated that the RALF4-dmPectin interaction occurs in expanding cells, highlighting a structural role for RALF4 in facilitating pectin condensation and patterning”: the ms does not show that endogenous RALF4 is localized in the cell wall, it shows that exogenously supplied RALF4 labelled with Alexa Fluor 647 (how was this done ? see remark above) colocalizes with 2F4 labeled pectin. The “condensation and patterning” is not really shown in this ms but is mostly deduced from the results reported for pollen tubes and root hairs (Moussu et al 2023 and Schoenaers et al 2024).\n27. Extended Data Fig. 8g: indicate in material and methods how the PME-HIS construct was made, with HIS tag to N- or C-terminus ? What is the expected MW of the proprotein and mature protein. Is the protein processed in Pichia ?", "reviewer_author_response": "Response: Thank you very much for the comments on this study. Yes, although RALF-CrRLKL-ROS signaling axis and RALF-pectin interaction have been reported, here the cellular context mediated by rice RALF4 is different, which regulates the root development progression. Furthermore, the main novelty of this work lies in the elucidation of the integrated mechanistic framework for root development, especially when the previously identified modules remain disconnected.\nThanks. We sincerely appreciate your questioning. We have supplemented a series of new data to strengthen the roles of the identified signaling axis in root development. By performing phenotyping analyses on the complementary transgene of the3 and additional allelic mutants of rbohb, pme30, and ralf4, the genetic evidence has been strengthened. We added NADPH oxidase activity assay using the RbohB extracted from wild type, the3, and THE3ox roots to determine the effect of RbohB phosphorylation on oxidase activity. We also supplemented essential controls and added the statistical analysis in several key experiments. Accordingly, we reorganized the logical flow of Fig. 2, Fig. 5 and Fig. 6. Furthermore, the manuscript has undergone thoroughly revisions, highlighting its major novelty. Hope you could be satisfied with the revised manuscript.\nThanks for the suggestion. In fact, rice RALF4 is not the close homolog of Arabidopsis RALF4. It shows some similarity to Arabidopsis RALF4 and RALF22 (Moussu, S. et al., Science 382: 719–725, 2023; Schoenaers, et al., Nat Plants", "reviewer_os": "As required by Reviewe #3, in the revised version, we have included the phylogenetic trees of rice THE3, PME30 and RALF4 members to show the orthologous relationships with previously described Arabidopsis isoforms in the relevant ExtD Figs (please see the revised ExtD Fig. 3a, 9, and 11c).\nThanks for the question. We focused on RbohB is because it is the highest expressed Rboh member in rice roots. As suggested, we introduced rbhoc mutant in this study. Consequently, its defects in the root length and O – level are not as prominent as those of rbohb mutant (please see ExtD Fig. 1d–g), indicating that both gene are likely functional redundant, and RbohB is dominant in modulating root development. Although the double mutant may have a lower baseline of O – in roots, however, the generation of double mutant in rice requires at least two growth seasons (almost one year). In addition, the relevant biochemical and molecular analyses could only be examined on a single Rboh protein. We therefore focused our subsequent characterization on RbohB, the major Rboh in rice root.\nThe dataset related to Fig. 2 has been strengthened by performing following additional experiments. After determining that THE3 can phosphorylate RbohB, we performed the cell-free degradation assay by comparing the level of RbohB-His in the extracts of ZH11, the3, and THE3ox (please see Fig. 2g,h), revealing the effect of phosphorylation on RbohB stability. By optimizing the experimental details and increasing biological replicates, we re-analyzed the phosphorylation level of RbohB in ZH11, the3 and THE3ox roots using CIP treated THE3ox as a control; statistical analysis revealed that the3 has lower level of phosphorylated RbohB, whereas the THE3ox harbors increased phosphorylated RbohB (please see Fig. 2i). We further analyzed the NADPH oxidase activity using RbohB extracts from ZH11, the3 and THE3ox roots, and found that their activity exhibited similar change tendency as its phosphorylated level (please see Fig. 2i,j). These experiments established a direct link between RbohB phosphorylation and the changes in oxidase activity. Combining with NBT-staining assay, in which the tobacco leaf discs were transfected with THE3, RbohB and THE3 together with wild-type RbohB and its variants, we found that coexperssion of THE3 and RbohB variants decreased RbohB phosphorylation level, thereby decreasing O – content (please see the revised Fig. 2k–m). All those strengthened that THE3-RbohB interaction regulates the activity and stability of RbohB, influencing O – production and meristem homeostasis in RAM.\nWe retrieved the original images and found that saturated exposure is due to incorrect focus plane. In the revised manuscript, we provided new images and quantified the NBT length accordingly (please see the revised Fig. 5l,m).\nAs suggested, we reorganized Fig. 5 and Fig. 6. The phenotypes of ralf4 mutants were moved to Fig. 5h–k. In addition, the performances of ralf4 mutants were included in Fig. 5l–n (previous Fig. 5j). All those demonstrate the role of RALF4 in signaling.\nWe revised the statement of “banding’’ to “pectin condensation”.\nThanks. We substantively revised the discussion in the revised manuscript.\nThank you very much. In the revised manuscript, we supplemented a series of experiments. Accordingly, we updated the manuscript with the new data and provided a thorough discussion. We appreciate all the comments and suggestions you provided, which help us to improve the manuscript. We sincerely hope that you will find the revised version being greatly improved.\nThank you very much for your constructive comments.\nWe provided the new images to ensure that they were taken by focusing on the similar cell layer (please see Fig. 1d).\nBy optimizing the experimental details and increasing the biological replicates, we re-analyzed the phosphorylation level of RbohB in the ZH11, the3 and THE3ox roots using CIP treated THE3ox as a control. Based on essential replicates, we calculated the relative level of phosphorylated RbohB. THE3ox exhibited enhanced RbohB phosphorylation level compared to ZH11 (please see Fig. 2i).\nWe repeated this experiment. According to the in vitro phosphorylation assay in ExtD Fig. 5c, S140 and S191 mutations reduced the phosphorylation of RbohB to comparable levels, while S278 mutation slightly reduced RbohB phosphorylation. In the current data, the changes in NBT level are consistent with that of RbohB phosphorylation in vivo and in vitro analyses (Fig. 2k–m and ExtD Fig. 5c).\nWe increased the replicates of this experiment, and quantified the grayscale ratios accordingly to indicate the phosphorylation level of RbohB (please see the lower panel of Fig. 5d and the source data of Fig. 5). The addition of RALF4 significantly reduced the phosphorylation level of RbohB.\nThank you for the constructive suggestion. As suggested, we treated the tobacco leaves with different concentrations of RALF4 and examined the O ⁻ level of the treated leaf discs. The inhibitory effect of RALF4 on O – is dose-dependent (please see Fig. 5e and ExtD Fig. 12a). We repeated NBT-staining assay of N. benthamiana leaves transfected with THE3 and/or RbohB. The current data clearly showed that the addition of 2 μM RALF4 markedly suppresses O ⁻ production (please see Fig. 5f,g).\nAs suggested by you and Reviewer #1, we strengthened this part of dataset. After determining that THE3 can phosphorylate RbohB, we performed the cell-free degradation assay by comparing the level of RbohB-His in the protein extracts of ZH11, the3, and THE3ox (please see Fig. 2g,h), revealing the effect of phosphorylation on RbohB stability. By optimizing the experimental details and increasing biological replicates, we re-analyzed the phosphorylation level of RbohB in ZH11, the3 and THE3ox roots; statistical analysis revealed that the3 has lower level of phosphorylated RbohB, whereas the THE3ox harbors increased phosphorylated RbohB (please see Fig. 2i). We further analyzed the NADPH oxidase activity using RbohB extracts from ZH11, the3 and THE3ox roots, and found that their activity exhibited similar change tendency as its phosphorylation level (please see Fig. 2i,j). These experiments established a direct link between RbohB phosphorylation and the changes in oxidase activity. We further performed NBT-staining assay, in which the tobacco leaf discs were transfected with THE3, RbohB and THE3 together with wild-type RbohB and its variants. We found that coexperssion of THE3 and RbohB variants decreased RbohB phosphorylation level, thereby decreasing O – content (please see Fig. 2k–m). All those strengthened that THE3-RbohB interaction regulates the activity and stability of RbohB. The increase in O – levels likely results from both the enhanced RbohB activity and stability mediated by phosphorylation.\nThanks for the question. RALF4 treatment did not reduced root growth and apical meristem size in the3 mutant, indicating that THE3 is a major receptor of RALF4. Overexpression of RbohB in the3 background has rescued O – level and methyl-pectin content, as well as root growth (please see Fig. 1k, 2c and 3d). The reason for why RALF4 treatment suppressed the NBT staining and MZ size, leading to shorter roots in RBoxthe3 roots, is probable due to the constitutive expression of RbohB. RbohB may respond to exogenous RALF4 through receptors other than THE3 in RBoxthe3 plants. Under exogenous RALF4 treatment, the subtle phenotypic differences between rbohb and the3 can support this speculation. To avoid any confusion, we replaced RBoxthe3 with Comthe3, and performed the relevant analyses in Comthe3 (please see the revised Fig. 5l–n).\nEdU staining marks the region undergoing vigorous cell division, indicating meristem homeostasis. Our focus is on measuring the length of the EdU-positive region. The weak intensity in original Fig. 6h might be due to differences between individual seedlings. We repeated EdU staining assay and provided a new image for ralf4-2 (please see Fig 5j)\nROS, as an important second messengers, are involved in myriad basic processes of plant growth and the stress adaptations of various tissues. We selected the meristem region for O – analysis because several important literatures have reported it as the intrinsic signal for establishing MZ size, whereas H O is abundant in the EZ to promote stem cell differentiation (Tsukagoshi, H. et al., Cell 143: 606–616 2010; Yamada, M. et al., Nature 577: 85–88, 2020; Zeng, J. et al., EMBO J. 36: 2844–2855, 2017). Since the focus of this manuscript lies in exploring the connections between O – and the signaling pathway mediated by cell surface receptor THE3 in root meristem, we overlooked the changes of H O in this study.\nThanks. We have included the indication of molecular weight markers in all SDS-PAGE gels.\nThank you very much for your detailed commentary, which help us to ravel out the novelty of this study and think deeply about the rationale and reasonable interpretation behind each set of experiment.\nSpecific dyes for pH examination, such as HPTS, are not suitable for analyzing rice roots because the roots are too thick to image. We thus examined the surface pH in the MZ and EZ of the ZH11, the3 and THE3ox roots using the mainstream method Non-invasive Micro-test Technology (NMT) (Sa et al., New Phytol. 222: 1951–1964, 2019). We consequently found that the difference of pH values in the MZ and EZ is very small and negligible (MZ: pH5.9; EZ: pH6.0) in rice roots. Mutation in THE3 does not cause significant pH changes, and overexpression of THE3 leads to a consistent slight drop in the MZ and EZ (please see below). Given such small alterations, we decided not to discuss the influence of pH value in this manuscript. However, we do not deny its importance on PME activity.\nSince the focus of this manuscript lies in exploring the connection between O – and the signaling pathway mediated by cell surface receptor THE3 in the root meristem, we overlooked the changes of H O in this study\nIn rice roots, the total pectin content and its methylesterification level are relatively low. The HG methylesterification in the RAM of rbohb allelic mutants and the3 is further reduced. The meristem size of these mutants showed modest alterations, suggesting a nonlinear correlation between the degree of pectic methylesterification and the MZ size. Considering the significance of MZ homeostasis, it is plausible that additional layers of regulation exist. As suggested, we have conducted a moderate discussion about this.\nThanks for your suggestion. As requested, we added complementary data for the3, and supplied additional allelic mutants for rbohb, pme30 and ralf4. Moreover, additional overexpression lines for RbohB were included. We carried out the relevant phenotypic analyses on these additional genetic materials. The consistent results confirm the reliability of our study.\nRALF4 peptide analyzed in our experiment indeed does not contain lysine residues, but has free terminal amino group (-NH₂) that can serve as the site for labeling. The labeled RALF4 peptides were rinsed and collected by ultrafiltration to remove unincorporated free fluorophore. The operational details were included in the method.\nTo improve the credibility of MST assay, we labeled pectins using the same method and supplied BSA as an additional negative control in the assay (please see ExtD Fig. 13i).\nWe included the information regarding to RALF4 concentration and treatment duration in the figures or the relevant figure legends.\nAs requested, we included the phylogenetic trees of rice THE3, PME30 and RALF4 families to show the orthologous relationships with previously described Arabidopsis isoforms (please see ExtD Fig. 3a, 9, and 11c).\nWe included “rice” in the title and abstract.\nWe have deleted this sentence in the revised manuscript.\nThis sentence has been rephrased.\nWe removed the results regarding to H2DCFDA.\nWe agree that the solid colocalization of RALF4 should derive from the analysis of the genetic complementary transgene harboring the tagged RALF4 in the ralf4 mutant. However, such transgene is unavailable and the generation of this transgenic rice plants will take more than half a year. We therefore transiently transfected tobacco leaves using RALF4-GFP and found that RALF4 can target to the cell walls (please see ExtD Fig. 13g). Moreover, the dSTORM analysis of dmPectin in ZH11 and ralf4 mutant ascertained the structure role of RALF4 in pectin organization.\nWe have amended this sentence.\nNBT staining can nicely indicate the content of O₂⁻ in root cells. In our experience, the blue staining is not significantly correlated with the high cytoplasmic content, as we did not observe the pronounced NBT staining signal in the wild-type root tips after Tiron treatment (please see ExtD Fig. 6h).\nWe have amended this mistake throughout the manuscript.\nWe have amended this statement.\nWe have amended this mistake.\nWe supplied this information and cited this reference.\nWe amended the title of current ExtD Fig 3.\nWe repeated the cell-free degradation assays and added the sample of THE3ox. As suggested, we included statistical analysis based on more quantitative data (please see Fig. 2g.h).\nWe included the controls with only THE3 and WT RbohB as suggested. We also performed gene expression and protein blotting to determine the levels of phosphorated and total RbohB in the tobacco leaf discs transfected with THE3 and the mutated and WT RbohB (please see Fig. 2k–m). In this experiment, we found that the abundance of RbohB and its variants expressed in tobacco leaf discs were almost comparable, whereas the mutations decreased RbohB phosphorylation level, thereby decreasing O – content. However, the heterologous, transient expression system in tobacco poses challenges for comparing protein stability, due to the difficulty in maintaining consistent initial protein concentrations. Therefore, this experiment demonstrates that THE3 mediated RbohB phosphorylation affects its activity and O – level.\nWe included the LM20 image and the root growth with and without Tiron treatment as requested (please see ExD Fig. 6c–e).\nWe provided the larger view of the root sections that include the EZ of the examined genotypes (please see Fig. 3d).\nYes, Tiron treatment almost completely inhibited root growth (please see ExtD Fig. 6e). Since the root growth was almost suppressed by Tiron treatment, we did not examine the cell length in the EZ.\nWe repeated the NBT-staining assay, and did not observe the patchy signal (please see ExD Fig. 6h). The previous patchy might be caused by the damage or wounding during the staining process. We therefore provided a new image to avoid confusion.\nSince the released O₂⁻ is highly unstable in aqueous solution and rapidly converts to H O , we developed the XON-XOD reaction system in the pectoplate assay to enable immediate interaction between the released O₂⁻ and the added PME proteins. In the revised manuscript, we supplemented additional controls by including XOD, XON, or the reaction by-product urea in the pectoplate (please see ExtD Fig. 8c). PME activity was not affected in these tests. Therefore, we can rule out the possibility that the xanthine (XON)-xanthine oxidase (XOD) reaction system affects the pectin and/or agarose in the pectoplate assay.\nWe indicated the concentrations of RALF4 and the duration of the treatments in the relevant figures, figure legends, or method section as requested.\nIn the MST assays, we used the ECD of THE3 to examine the binding affinity to RALF4 polypeptide. It has been labeled in the relevant figure panels.\nIn the revised manuscript, we marked the place for signal quantification in the root (please see Fig. 5l). We also indicated MZ site in ExD Fig. 12e for the quantification in Fig. 5n.\nThanks. We corrected the label mistake. The concentrations of RALF4 were labeled in the relevant figures.\nThanks for the information. Fig. 6d showed the wall of the elongating cortex cells in the EZ region. The relevant information has been included in the figure legend.\nWe rephrased this sentence to “dmPectin formed random bundles in wild-type cell walls”.\nWe rephrased this sentence to “immuno-labeling of the root tips using the LM20 antibodies revealed the extended mPectin pattern in ralf4 mutants”, and provided the quantitative data of the region lengths of fluorescent signal along the root tip of WT and ralf4 (please see Fig. 6h).\nWe performed MST assay and physiological treatment using the AF647 labeled RALF4 and Flag-tagged RALF4. Both labeled RALF4 indeed inhibited the root growth of ZH11, and the AF647 labeled RALF4 can bind dmPectin as revealed by MST assay (please see ExD Fig. 13a−e). These analyses demonstrate that addition of these tags to RALF4 peptides does not affect its function. Anti-Flag antibodies were used in immuno-labeling to assess RALF4 expression levels in the rice protoplasts.\nSince the transgenic plants harboring a tag-labeled RALF4 is unavailable, we are unable to analyze its in-planta localization. In the revised manuscript, we performed transient transformation of RALF4-GFP in N. benthamiana leaves and demonstrated that RALF4 can target to cell walls. Furthermore, by analyzing dmPectin in the EZ cells of wild-type and ralf4 roots using dSTROM, we observed pectin condensation in the wild-type, whereas fine dmPectin particles were observed in the ralf4 mutant. Therefore, the conclusion regarding the RALF4’s role in pectin structure was drawn based on analysis of dmPectins in wild type and ralf4-1. To avoid overstated, we revised the relevant interpretations.\nYes, the PME30-His proteins were prepared in Pichia. The His tag was fused at the C-terminus of PME30; the mature protein of PME30 was approximately 50 kD. All those have been described in the figure legend. As suggested, we included the essential information, as well as how the PME-His construct was prepared, in the figure legend and the method section."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_2": "The revised manuscript is much improved and adequately addresses my previous concerns. The authors have put significant effort into addressing both my comments and those of the other referees. However, there are still some remaining issues with the revised version:\n1.The interaction between THE3 and RbohB regulates O2- homeostasis in the meristem. RALF4 acts as a ligand for THE3, inhibiting THE3-RbohB signaling, while in the elongation zone, RALF4 binds to dmPectin to promote pectin condensation and cell expansion. Given the distinct expression patterns of RALF4 in the meristem versus the elongation zone, is there a clear functional distinction between its roles in these two regions?\n2. Although the phenotype images in Figure 1 have been modified according to the suggestions, several details remain imperfect. Fig.1g is not labeled within the figure; In Figure 1j, the meaning of the white triangles (arrowheads) is not defined in the figure legend, and one triangle appears to be missing; The term \"Comthe3\" used in the revised version is not defined in the figure legend; the main text must also explicitly clarify what specific material or line this name represents.\n3. There is an apparent error in Figure 5d. Are the statistical results for the three replicates in the fourth lane exactly identical? Furthermore, the RbohBN bands appear fused together, how was the gray value quantification performed accurately on these merged bands?", "reviewer_3": "The authors have now provided satisfactory answers to the reviewer's comments"}, "author_response": "Response: Thank you very much for your constructive comments.\nThank your very much for the comments. OsRALF4 serves both as a ligand for THE3 and as a structural component binding to dmPectin. Its functional duality is likely dictated by the availability of OsRALF4 and its interacting partners. According to the expression profile of OsRALF4 in developing root, OsRALF4 mainly plays signaling role in the meristem zone. With the increase of OsRALF4 in the transition region, both functions are enhanced. Its role in binding to dmPectin is further enhanced in the elongation zone due to the activity of PMEs, which promotes pectin condensation and cell expansion. Therefore, we accordingly revised the interpretations in the results and discussion of this version (please see L428-432).\nThanks for pointing out these mistakes. We added the labels in these figures and defined them in the figure legend and main text as requested.\nWe repeated this experiment and strictly controlled the experimental details, including the exposure time. The quantification of gray value has been improved based on the new replicates (please see Fig. 5d).\nResponse: Thank you very much for your constructive comments, which have helped us improve the manuscript."}]

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