Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement

Wang, Duoduo; Ortega-Salazar, Isabel B.; Blanco-Ulate, Barbara

doi:10.3390/agronomy15112641

Open AccessReview

Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement

by

Duoduo Wang

^1,*

,

Isabel B. Ortega-Salazar

²

and

Barbara Blanco-Ulate

^2,*

¹

Department of Agriculture, Nutrition and Food Systems, University of New Hampshire, Durham, NH 03824, USA

²

Plant Sciences Department, University of California, Davis, CA 95616, USA

^*

Authors to whom correspondence should be addressed.

Agronomy 2025, 15(11), 2641; https://doi.org/10.3390/agronomy15112641

Submission received: 25 October 2025 / Revised: 12 November 2025 / Accepted: 14 November 2025 / Published: 18 November 2025

(This article belongs to the Special Issue Mechanisms and Signaling Pathways of Crop Tolerance Under Stressful Conditions)

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Abstract

Homogalacturonan (HG) methylesterification is a key determinant of plant cell wall (CW) structure and function, shaping growth, morphogenesis, and responses to biotic and abiotic stresses. This review highlights recent advances in the regulation of homogalacturonan (HG) methylesterification, focusing on the coordinated roles of pectin methylesterases (PMEs), pectin methylesterase inhibitors (PMEIs), transcription factors (TFs), and hormonal signals. We examine how these regulators interact within the CW microenvironment to modulate elasticity, porosity, and remodeling dynamics. Insights from immunolocalization and biomechanical studies reveal the spatiotemporal patterning of HG de-esterification and its integration with developmental and stress-adaptive signaling. Beyond basic biology, HG methylesterification dynamics directly influence traits such as fruit firmness, pathogen resistance, and stress tolerance, positioning HG methylesterification-related genes as promising targets for molecular breeding and biotechnological interventions. By integrating mechanistic understanding with genomic and phenotypic selection approaches, breeders can precisely tailor CW properties to enhance crop resilience and quality. A comprehensive view of HG methylesterification—from enzymatic control to mechanical feedback—offers a conceptual and practical framework for guiding crop improvement and sustainable agricultural practices.

Keywords:

homogalacturonan methylesterification; cell wall mechanics; pectin methylesterase (PME); pectin methylesterase inhibitor (PMEI); plant development and stress responses; crop improvement

1. Introduction

The plant cell wall (CW) is a dynamic structure composed mainly of cellulose, hemicellulose, and pectin, together with proteins and other biomolecules. Pectin constitutes over 30% of the polysaccharide fraction in a typical dicot primary CW and comprises several structurally diverse polysaccharides, including homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II) [1]. Among these, HG is predominant and synthesized in a highly methylesterified form before secretion to the CW [2]. During plant development, pectin methylesterases (PMEs) selectively remove these methyl groups, modulating HG chemistry in a spatially and temporally controlled manner [3]. HG methylesterification is a key determinant of stiffness and flexibility. PME activity is finely regulated by pectin methylesterase inhibitor (PMEIs), transcription factors (TFs), plant hormones, and microenvironmental cues, highlighting the dynamic and integrated regulation of HG methylesterification.

The degree of methylesterification (DM) profoundly influences CW mechanical properties, wall–plasma membrane interactions, and the associations between wall polymers, collectively contributing to CW integrity (CWI) [4]. Perturbations in CWI are sensed by specialized wall-monitoring systems, triggering compensatory cellular responses through mechanochemical feedback and signaling pathways [5]. Advances in live-cell imaging, biochemical assays, and biophysical measurements have revealed that HG methylesterification is not uniform across tissues; rather, it exhibits localized patterns and oscillatory dynamics, particularly in tip-growing cells such as pollen tubes and root hairs. These dynamic patterns of demethylesterification link pectin chemistry with growth oscillations, Ca²⁺ signaling, and mechanosensitive feedback, providing a mechanistic basis for how HG methylesterificationinfluences CW remodeling.

In this review, we discuss the molecular factors controlling HG methylesterification, including the coordinated actions of PMEs and PMEIs, transcriptional regulation by TFs, and modulation by plant hormones. Beyond these classical regulators, we highlight emerging principles such as spatially localized PME/PMEI patterning, mechanochemical feedback, and oscillatory regulation in rapidly growing cells. We then explore how HG methylesterification impacts diverse wall-associated biological processes, including CWI maintenance, plant growth and morphogenesis, organ initiation, seed germination, fruit softening, and responses to biotic and abiotic stresses. Finally, we propose that future studies should integrate mechanical, biochemical, and signaling perspectives to fully understand HG demethylesterification and its interactions with other regulators in shaping plant development and adaptive responses.

2. Regulation of HG Methylesterification

HG methylesterification is tightly regulated by multiple layers, including transcriptional and post-transcriptional mechanisms, hormonal signals, and local CW pH. The regulatory relationships of PMEs and PMEIs are summarized in Table 1 and Table 2. Table 1 provides an overview of TFs and post-transcriptional modulators, while Table 2 summarizes hormonal regulation of DM and the associated physiological processes. Together, these factors coordinate PME/PMEI activity and apoplastic pH to fine-tune DM and regulate developmental processes.

2.1. PMEs and PMEIs

The degree of HG methylesterification is primarily regulated by the coordinated activities of PMEs and their protein inhibitors [6]. PMEs can remove methyl groups in either a block-wise or random pattern, depending on pH, cation availability, and CW environment. Block-wise demethylesterification generates stretches of negatively charged galacturonic acid residues (GalA) that readily crosslink with Ca²⁺, forming rigid ‘egg-box’ structures, whereas random demethylesterification produces more fragmented HG domains associated with wall loosening and increased accessibility to hydrolases [7]. Because PMEs form large multigene families with overlapping expression and context-dependent activities, determining the specific mode of action of individual isoforms remains a major challenge in CW biology [8,9].

PME activity is fine-tuned post-translationally by PMEIs, which form highly specific and reversible inhibitory complexes with PMEs. Although structurally related to invertase inhibitors, PMEIs represent a distinct protein family that regulates PME activity across a wide range of plant taxa [6,10]. Beyond simple inhibition, PMEIs function as developmental regulators by shaping spatiotemporal patterns of HG demethylesterification. Functional analyses, such as in vitro studies of AtPMEI3, demonstrate that PMEIs can control HG demethylesterification in specific tissues, thereby regulating CW stiffness, cell expansion, and developmental processes, confirming their role as developmental regulators [11]. The stability and activity of PME–PMEI complexes are sensitive to ionic strength and pH; for example, specific salt concentrations can modulate binding affinity, while extreme pH conditions may dissociate otherwise stable complexes [12,13,14,15]. Molecular simulation studies have further shown that recognition between PME and PMEI is primarily driven by weak interactions, including van der Waals forces, π-stacking, hydrogen bonds, and stable ionic bonds [16].

Deciphering the structure of PME-PMEI complex at molecular levels gives new insights into the specificity and interaction between the two proteins. Together, structural, biochemical, and functional analyses of PMEs and PMEIs illustrate how fine-tuned regulation of HG methylesterification underpins developmental control of CW mechanics and plant morphogenesis.

2.2. Transcriptional and Post-Transcriptional Control

Diverse TF families regulate HG methylesterification in a tissue-specific mammer, including seed coat mucilage, gynoecium, phyllotaxy, and fruit development. In Arabidopsis thaliana seed coat mucilage (A-SCM), a diverse set of TFs regulate HG methylesterification through coordinated activation or repression of PME and PMEI genes. For instance, The MADS-domain TF SEEDSTICK (STK) directly activates PMEI6, thereby promoting mucilage extrusion [17]. MYB52 enhances pectin DM in A-SCM by activating PMEI6 and PMEI14 [18]. Conversely, the transcriptional regulator LEUNIG_HOMOLOG (LUH) promotes DM by upregulating PME activity [19]. These TFs are interconnected in a complex regulatory network: LUH activates MYB52, while MYB52 in turn upregulates STK [18]. STK and MYB52 may also be indirectly repressed by BEL1-like homeodomain (BLH) proteins BLH2 and BLH4, which themselves redundantly promote HG demethylesterification by directly activating PME58 [20]. The AP2/ethylene response factor (ERF) family member ERF4 enhances HG demethylesterification by repressing PMEI genes. Notably, ERF4 physically interacts with MYB52, and the two TFs antagonize one another’s DNA-binding activity, thereby exerting opposing effects on shared downstream targets [21].

Beyond A-SCM, the auxin response factor ETTIN coordinates Arabidopsis gynoecium development by simultaneously regulating PME and PMEI expression [22]. The homeodomain TF BELLRINGER (BLR) contributes to phyllotactic patterning in Arabidopsis and directly regulates PME5 [23]. During fruit ripening, several TFs modulate HG demethylesterification by regulating PME isoforms including tomato BRI1-EMS-SUPPRESSOR1 (SlBES1) and strawberry FvMYB79, thereby contributing to softening [24,25].

In addition to transcriptional control, HG demethylesterification is also controlled post-transcriptionally. For instance, the transmembrane RING E3 ubiquitin ligase FLYING SAUCER1 (FLY1) regulates PME recycling within the endomembrane system, thereby modulating DM in A-SCM [26]. A FLY1 homolog performs redundant functions in mucilage formation [27]. Similarly, the nuclear-localized E3 ubiquitin ligase Mucilage-Defect-1 (MUD1) influences pectin DM by modulating the stability of TFs and PME-related regulators, including MYB52, LUH, SBT1.7, PMEI6, and PMEI14 [28]. Together these findings highlight that HG methylesterification is governed by multilayered regulatory networks, integrating transcriptional cascades and post-transcriptional mechanisms. Dissecting how these pathways converge to fine-tune PME/PMEI activity remains a key challenge for understanding CW remodeling during development and stress responses.

Table 1. Transcriptional and post-transcriptional regulators of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs) and their effects on homogalacturonan (HG) methylesterification. The table summarizes transcriptional and post-transcriptional regulators of PMEs and PMEIs across various plant tissues and developmental processes. Transcription factors (TFs) regulate PME/PMEI gene expression, while post-transcriptional regulators, including ubiquitin ligases FLY1 and MUD1, modulate PME recycling or the stability of TFs and PME/PMEI proteins, thus influencing the degree of methylesterification (DM) of homogalacturonan (HG).

Regulator	Type	Target Gene(s)/Mechanism	Regulatory Effect on DM	Tissue/Process	Reference
STK (MADS-domain)	TF	PMEI6	Activates PMEI6 expression, increasing DM	Seed coat mucilage	[17]
MYB52	TF	PMEI6, PMEI14	Activates PMEI expression, increasing DM	Seed coat mucilage	[18]
LUH	TF	PME	Activates PME expression, decreasing DM	Seed coat mucilage	[19]
ERF4	TF	PMEI	Represses PMEI expression, decreasing DM	Seed coat mucilage; fruit	[21]
ETTIN	TF	PME/PMEI	Activates PME activity, decreasing DM	Gynoecium morphogenesis (Arabidopsis)	[22]
SlBES1; FvMYB79	TF	PMEU1	Represses PMEU1 expression, increasing DM	Fruit softening	[24,25]
FLYING SAUCER1 (FLY1)	Transmembrane RING E3 ubiquitin ligase	Regulates PME recycling within endomembrane system	Modulates DM	Arabidopsis seed coat mucilage	[26]
FLY1 homolog	Transmembrane RING E3 ubiquitin ligase	Redundant function in PME regulation	Modulates DM	Mucilage formation	[27]
Mucilage-Defect-1 (MUD1)	Nuclear-localized E3 ubiquitin ligase	Modulates stability of TFs (MYB52, LUH) and PME/PMEI regulators (SBT1.7, PMEI6, PMEI14)	Influences DM	Seed coat mucilage	[28]

2.3. Hormone Regulation

Multiple phytohormones, including auxin, abscisic acid (ABA), brassinosteroids (BRs), gibberellins (GA), and ethylene, modulate CW remodeling by directly or indirectly regulating PME and PMEI activities.

ABA and ethylene generally promote HG demethylesterification. Heat tolerance–related PME genes such as PME34 and PME53 are ABA-responsive in Arabidopsis, linking hormone signaling to stress-induced modulation of pectin DM [29,30]. In A-SCM, ethylene signaling indirectly promotes HG demethylesterification via activation of the AP2/ERF TF ERF4, which represses multiple PMEI genes [21]. In fruit, ethylene is positively correlated with ripening-associated PME activity and pectin degradation, as observed in apricot [31]. However, in banana, ethylene also upregulates PMEI expression, suggesting a dual regulatory role depending on species and developmental context [32].

GA and BR generally negatively regulate HG demethylesterification. GA mutants with restricted hypocotyl elongation display reduced PME activity [33], while the BR pathway component BES1 represses PMEU1-mediated demethylesterification, thereby promoting tomato fruit softening [25].

Auxin exhibits context-dependent control of HG methylesterification. It promotes CW loosening to enable phyllotactic patterning [34], root emergence [35], and drive the transition from slow isotropic to rapid anisotropic elongation in dark-grown hypocotyls [36]. Conversely, auxin can indirectly inhibit demethylesterification by activating the CW sensor kinase ERULUS, which negatively regulates PME activity [37]. These contrasting outcomes underscore the tissue- and time-dependent nature of auxin signaling. For instance, the asymmetric distribution of the auxin was correlated with a differential level of HG methylesterification, which resulted in CW mechanochemical asymmetry that is essential for tissue bending [38].

Some developmental processes rely on hormone crosstalk. For instance, crosstalk between auxin and BR signaling has been shown to determine proximodistal growth in Arabidopsis leaves and sepals, with auxin-related TFs acting upstream of BR pathways to fine-tune HG methylesterification and CW mechanics [39]. Such interactions illustrate how hormone networks integrate to regulate PME/PMEI function across developmental and stress contexts.

Table 2. Hormonal regulation of homogalacturonan (HG) methylesterification. The table summarizes major plant hormones reported to regulate the expression of pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), their effects on the degree of methylesterification (DM) of HG, and the associated physiological processes. ABA: Abscisic acid; GA: Gibberellin; BR: Brassinosteroid.

Hormone	Regulatory Effect	Target(s)	Physiological Context	Reference
Auxin	Promotes or inhibits demethylesterification	PME, PMEI	Phyllotactic patterning, root emergence, hypocotyl elongation	[34,35,36,37]
ABA	Induces PME expression	PME34, PME53	Heat stress response	[29,30]
Ethylene	Represses or activates PMEI expression	PMEI genes	Fruit ripening	[31,32]
GA	Increases PME expression	PME genes	Hypocotyl elongation	[33]
BR	Represses PMEU1 expression	PME genes	Fruit softening	[25]

2.4. Apoplastic pH

Apoplastic pH is a critical determinant of PME and PMEI activity. Most PMEs function optimally at neutral to alkaline pH, whereas PMEIs are stabilized under acidic conditions, favoring PME–PMEI complex formation [7]. Structural studies highlight this dependence: in kiwi, pH above 6.0 disrupted a buried disulfide bond essential for PME inhibition [12], and the stability of the AtPME3–AtPMEI7 complex in Arabidopsis hypocotyls was shown to be pH-dependent [15,40,41].

According to the acid growth theory, auxin lowers apoplastic pH by stimulating plasma membrane H⁺-ATPases, promoting CW loosening [42,43]. This pH shift directly modulates PME/PMEI activity, while protons released during HG demethylesterification may further acidify specific wall domains. Acidic conditions also enhance polygalacturonase (PG) activity, facilitating the release of oligogalacturonides (OGs), which feedback to dampen auxin signaling and prevent excessive acidification [44].

Apoplastic pH is further buffered by Ca²⁺ fluxes. Acidification triggers Ca²⁺ influx and wall deposition, which raises pH and promotes Ca²⁺–HG crosslinking [36]. Depending on Ca²⁺ availability, demethylesterified HG can either be stabilized through crosslinking or degraded by PGs [10]. Together, H⁺ release, Ca²⁺ dynamics, and enzymatic activities create feedback loops that stabilize extracellular pH and maintain wall integrity. Thus, pH-dependent PME/PMEI dynamics form a rapid and reversible layer of regulation that connects metabolism, ion fluxes, and growth responses.

HG methylesterification is regulated through multiple interconnected layers, including the activities of PMEs and PMEIs, transcriptional and post-transcriptional control, hormone signaling, and apoplastic pH dynamics. These factors together shape the spatial and temporal patterns of HG demethylesterification. The regulation of pectin demethylesterification in Arabidopsis is summarized in Figure 1, which illustrates how HG methylesterification is modulated by these factors.

3. HG Methylesterification and CW Mechanics

Having outlined the transcriptional, hormonal, and pH-dependent regulation of PME/PMEI activity, we next examine how these molecular and biochemical factors translate into CW mechanics, shaping HG methylesterification patterns and influencing tissue-level wall properties.

3.1. Biomechanical Models and Pectin-Cellulose Interactions

The traditional “tethered network” model of the primary CW emphasized xyloglucan as a major determinant of wall mechanics [45]. This view was later refined by the “biomechanical hotspot” model, in which cellulose-xyloglucan interactions primarily dictate wall mechanics, while pectin functions as a “glue” stabilizing the network [46,47]. Subsequent studies revealed that pectin is extensively linked to other polysaccharide domains, including cellulose and xylan [48,49,50,51]. Together, these studies reposition pectin from a passive filler to an active biomechanical regulator.

Pectin’s DM strongly influences the strength and viscoelasticity of pectin-Ca²⁺ gels, thereby affecting cellulose network micro- and nanostructure [52]. Mutations in QUA2, encoding a pectin methyltransferase, impair cellulose biosynthesis and disrupt cellulose organization in Arabidopsis [53]. Solid-state nuclear magnetic resonance (ssNMR) of qua2 mutants revealed tighter packing between cellulose and pectic backbones, highlighting functional pectin-cellulose interactions during wall assembly [54]. Conversely, overexpression of PG increases pectin esterification, weakens HG–cellulose interactions, and promotes cell expansion [55].

Beyond polysaccharide interactions, HG methylesterification shapes CW-protein interactions. Partially demethylesterified pectin platforms created by PMEI6 recruit PEROXIDASE36, enabling local wall loosening during seed imbibition in Arabidopsis [56]. Similarly, unesterified pectin domains scaffold apoplastic isoperoxidase (APRX) in zucchini, which binds Ca²⁺-pectate, a specific pectin conformation in defined CW regions [57,58]. Specific detection of pectin DM using CW probes allows the mapping of these dynamic microdomains [59]. Recent evidence also suggests a feedback mechanism in which wall mechanics influence PME activity, highlighting a self-organizing system of biochemical and biomechanical regulation [38,60].

3.2. PME/PMEI-Mediated Wall Regulation

Spatially and temporally controlled HG methylesterification also governs seed germination and organ morphogenesis. In cotton and Arabidopsis, GhPMEI53 and AtPMEI19 inhibit PME activity, increasing HG methylesterification, softening radicle walls, and promoting protrusion at the appropriate developmental stage [61]. In the parasitic plant Phtheirospermum japonicum, demethylesterified pectins accumulate in outer haustorial cells to aid invasion, while highly methylesterified pectins are enriched in inner vascular tissues. Disrupting PME activity or overexpressing PMEI delays haustorium development, highlighting the importance of localized pectin modification in organ morphogenesis [62].

3.3. Integration with Hormone Signaling and Feedback Loops

PME/PMEI-mediated wall modifications intersect with phytohormone pathways to create complex regulatory loops. For instance, in seed germination, wall softening via increased methylesterification modulates ABA and GA signaling, which in turn feeds back to regulate PME/PMEI activity [61]. Similarly, overexpression of PMEI5 reduces PME activity, altering HG methylesterification and activating BR signaling via receptor-like protein RLP44, leading to defects in CW placement during cytokinesis independent of organ-level growth [63,64,65]. CWI also feeds back into hormone networks during organ morphogenesis. In Arabidopsis, the qua2 mutant and cellulose-inhibited seedlings fail to form apical hooks, showing disrupted auxin asymmetry and reduced expression of HLS1, encoding a key regulator of differential growth, and PIF4, encoding a TF integrating light and hormone signals. These defects can be alleviated by GA signaling, HLS1 overexpression, or turgor manipulation, indicating that turgor-dependent wall integrity cues converge on the PIF4–HLS1 module to regulate auxin/GA-driven hook curvature [66].

Collectively, these studies illustrate a self-organizing mechanochemical system in which PME/PMEI-mediated pectin modifications integrate with ionic signals and hormonal pathways. This dynamic interplay orchestrates localized wall stiffness, tissue-specific growth, and complex morphogenesis across developmental contexts.

3.4. Integration with Other CW Components and Signaling

PMEs frequently cooperate with other CW–modifying enzymes, such as PGs and pectate lyases (PLs), to dynamically regulate plant CWs. This cooperation is often isoform-specific, with PME–PMEI interactions fine-tuned to modulate enzyme activity without compromising plant growth. In plant–pathogen interactions, pathogen-secreted PMEs can synergize with other CW–degrading enzymes to promote infection. For example, in soybean (Glycine max), Phytophthora sojae secretes PsPME1, which works together with PsPG1 to weaken CW [67]. The plant inhibitor GmPMI1, and its modified form GmPMI1R designed using AlphaFold, selectively blocks pathogen PMEs while sparing plant PMEs, enhancing resistance without affecting growth [67].

In Nicotiana tabacum L., PME activity is sensed by wall-associated receptors: NtPMEI21 regulates HG methylesterification, activating immunity-related genes via Wall-Associated Kinase 2 (WAK2) [68], and Arabidopsis WAK-like protein RFO1 directly binds de-methylated pectin, controlling mitogen-activated protein kinase (MAPK) activation to coordinate BR-mediated growth and early defense against Fusarium oxysporum [69]. Similarly, in resistant Camellia japonica, upregulation of CjPME28 and CjPG1 links pectin modification to phenylpropanoid and antioxidant pathways; and their overexpression in tobacco promoted secondary wall reinforcement through lignin biosynthesis [70]. These examples highlight how PME activity integrates with other wall enzymes and receptor-mediated signaling to balance growth and immunity.

PMEs also function cooperatively during symbiotic interactions. In ectomycorrhizal fungi–Laccaria bicolor, LbPME1 and LbGH28A loosen host root CWs to facilitate Hartig net formation [71]. During legume–rhizobial interactions, host PMEs (e.g., SyPME1) produce unesterified pectins that serve as substrates for PLs, and PME inhibition phenocopies Nep1-like protein (NPL) mutants, demonstrating coordinated PME–PL activity for infection thread progression [72]. In actinorhizal symbioses with Frankia, host PLs and PMEIs remodel CWs to compensate for limited symbiont-derived enzymes [73].

Collectively, these studies illustrate that PME activity is tightly integrated with other CW–modifying enzymes and sensed by receptor kinases, coordinating CW remodeling with growth, immunity, and symbiotic signaling.

4. HG Methylesterification in Development and Environmental Interactions

The cellular mechanisms of HG methylesterification and PME/PMEI-mediated wall mechanics described above form the foundation for more complex developmental processes. In the following section, we explore how HG methylesterification regulates diverse physiological processes, including plant growth, morphogenesis, organ initiation, and responses to environmental stresses

4.1. Cell Growth

Pectin demethylesterification regulates plant cell elongation and growth transitions. In Arabidopsis GA-deficient mutant, low DM correlates with reduced cell elongation in hypocotyls, whereas increased DM promotes elongation, as observed in root cells in transgenic lines overexpressing AtPMEI1 and AtPMEI2 as well as AtPME17 mutant [33,74,75]. Pectin demethylesterification also controls the timing of the growth transition in dark-grown Arabidopsis hypocotyls [76]. More recent work showed that neighbor-proximity (shade avoidance) triggers upregulation of HG methylesterification in hypocotyls, which is required for shade-induced elongation; mutants with reduced methylesterification fail to elongate normally under low red/far red light [77]. In Arabidopsis, constitutive reduction in methylesterification by expressing Aspergillus nidulans PME genes causes morphological defects, dwarfism and shortened roots, confirming that reduced DM limits cell elongation [78]. Overexpression of a homogalacturonan methyltransferase (HGMT) gene CGR2, which adds methyl groups to HG during pectin biosynthesis in the Golgi, increases DM, leading to expanded leaf size and enhanced organ growth, whereas cgr2/cgr3 double mutants, with lower DM, show reduced growth and altered leaf morphology [79]. Interestingly, under heavy-metal stress, reduced PME activity maintains higher DM, yet growth is impaired; exogenous proline restores PME activity, locally reduces DM where needed, and rescues root elongation [80]. Together, these findings indicate that the spatial-temporal regulation of DM—not simply high or low DM—is essential for proper cell elongation, with effects depending on tissue type, growth mode, and environmental conditions.

4.2. HG Methylesterification in Tip Growth and Morphogenesis

4.2.1. Localized PME/PMEI Activity in Tip-Growing Cells

Plants generate highly specific spatial patterns of methylesterification across tissues or even within single CW, creating micro-domains with distinct mechanical properties. Such patterning enables differential growth—one region of the wall expands while an adjacent region remains rigid—which is crucial for complex morphogenesis.

Spatiotemporal regulation of HG methylesterificationPM by PME and PMEI is a common mechanism for controlling CW dynamics in plant development. In pollen tubes, AtPPME1 regulates tip growth, while AtPMEI2 locally inhibits it at the apex to maintain flexibility and directional elongation [81,82]. A similar mechanism operates in Arabidopsis root hairs, where PME17 and its inhibitor PMEI4 generate a flexible tip and rigid shank to sustain polarized growth [83]. These activities interact with Ca²⁺ signaling [84]. PMEs create binding sites for Ca²⁺ cross-linking, stiffening subapical walls while the apex remains flexible; Oscillatory cytosolic Ca²⁺ gradients further coordinate vesicle trafficking, exocytosis, and PME/PMEI activity, creating a dynamic feedback loop in which PME-generated Ca²⁺ binding sites modulate wall stiffness, and Ca²⁺ levels in turn regulate the spatial and temporal activity of PMEs [85,86]. Together, PME/PMEI dynamics and Ca²⁺ signaling establish mechanical gradients that drive polarized and oscillatory tip growth.

4.2.2. Apical-Basal Gradients and Wall Mechanics

Tip-growing systems, such as pollen tubes and root hairs, rely on tightly coordinated oscillatory cycles of methylesterification and Ca²⁺-pectate crosslinking to sustain rapid pulsatile growth. Localized PME/PMEI activity and vesicle trafficking establish the apical gradient of highly methyl-esterified “soft” pectin at the tip and demethylated “hard” pectin in the distal region, such that modulation of HG chemistry—not cellulose orientation alone—governs wall mechanics and growth dynamics.

4.2.3. Integration with Plant Morphogenesis

HG demethylesterification is a major driver of plant morphogenesis, challenging the traditional creep-dependent growth model which views that cellulose microfibril orientation dominates morphogenesis [87,88]. Atomic force microscopy (AFM) studies demonstrated that asymmetric CW loosening was triggered by selective pectin demethylesterification rather than changes in turgor pressure caused by cellulose micro-fibrils [89]. The ‘expanding beam’ model further suggests the expansion of pectin nano-filaments alone can drive cell shape changes without turgor-driven growth [90]. Multiple regulatory layers have recently been uncovered: peptide signals such as RALF4–LRX–FER rapidly halt pollen tube elongation by altering reactive oxygen species (ROS) and Ca²⁺ fluxes at the apex [91]; The tobacco LORELEI-like glycosylphosphatidylinositol-anchored protein 4 (NtLLG4)-mediated unconventional polar exocytosis delivers the NtPPME1 specifically to the growing tip, ensuring localized wall remodeling [92]; and exogenous PG treatments trigger pollen tube rupture followed by a redistribution of PME activity to restore wall integrity [93]. Together, these findings highlight that spatial–temporal regulation of HG demethylesterification is a central biophysical mechanism driving tip morphogenesis.

4.3. Organ Initiation, and Seed Germination

Demethylesterification reduces CW stiffness and increases tissue elasticity, facilitating organ initiation.

Increased wall elasticity correlates with demethylesterified HG during organogenesis in Arabidopsis [94]. In seeds, tissue-specific HG methylesterification determines germination dynamics: overexpression of seed-specific PMEI5 accelerates germination in Arabidopsis by maintaining higher DM [95], whereas HG demethylesterification in SCM is essential for mucilage release and seed gemination, as mutations in the HIGHLY METHYL ESTERIFIED SEEDS (HMS) gene of Arabidopsis impaired a PME isoform abundant during mucilage secretion, thereby disrupting CW loosening and embryo cell expansion [96]. Recent studies in cotton and Arabidopsis further highlight the role of PMEIs in seed germination. Overexpression of GhPMEI53 in cotton decreased PME activity, increased HG methylesterification, and softened seed CWs, promoting radicle protrusion and germination. The Arabidopsis homologue, AtPMEI19, performed a similar function, suggesting a conserved mechanism. These PMEIs also affected ABA and GA pathways, indicating that PME/PMEI-mediated regulation of HG methylesterification integrates CW mechanics with phytohormone signaling during germination [61].

DM also regulates rhythmic developmental events: balanced HG esterification around lateral root primordia is required for root clock function in Arabidopsis [97], and increased DM softens rice lodicule walls, promoting early diurnal flower opening and regulating flower-opening time [98]. Recent advances now extend these findings to the single-cell level: live-confocal tracking of Arabidopsis sepals enables monitoring of cell growth dynamics across tissue layers [99], providing tools to directly test how local wall modifications drive morphogenesis, while single-nucleus transcriptomics of meristems reveals spatially restricted expression of PMEs/PMEIs, confirming that local control of HG methylesterification is imposed at the single-cell level during organ initiation [100]. Together, these studies highlight that finely tuned HG methylesterification provides a flexible regulatory mechanism linking CW mechanics to developmental transitions, ensuring precise control of organ initiation, seed germination, and rhythmic developmental processes across species.

4.4. HG in the Middle Lamella (ML) Influences Plant Development

Beyond individual CW, HG methylesterification in the ML controls adhesion versus separation, influencing organ cohesion and fruit ripening. The ML is a pectin-rich layer deposited during cytokinesis that cements adjacent cells together. It is especially enriched in HG, making it a key determinant of cell–cell adhesion and separation [101] Within the ML, tricellular junctions (TCJs)—the points where three CW converge—represent structural hotspots for wall remodeling. Esterified HG is distributed broadly throughout the ML, whereas de-esterified HG preferentially accumulates at TCJ tips, where it facilitates the controlled formation of intercellular spaces [101].

The mechanical properties of the ML are closely tied to developmental events. Modulating the DM of HG in the ML, particularly at TCJs, is crucial for balancing adhesion versus separation. In Arabidopsis, overexpression of PMEIs or knockout of PMEs leads to stronger cell–cell adhesion, as reduced demethylesterified HG prevents wall loosening [102]. In crop systems, PME- and PMEI-mediated adjustments of HG DM in the ML contribute to fruit softening during ripening, as summarized in Table 3. In woodland strawberry (Fragaria vesca ‘Hawaii 4’), a total of 54 PMEs were identified, among which FvPME38 and FvPME39 were highly associated with ripening. RNA interference (RNAi)-silencing of FvPME38 and FvPME39 delayed fruit softening, while their overexpression accelerated it, indicating that PME activity promotes pectin modification and CW loosening during ripening [103]. In contrast, antibody-based analyses in loquat revealed that PME-mediated demethylesterification and Ca²⁺-associated egg-box structure formation were linked to increased fruit firmness during postharvest storage [104]. These studies demonstrate that PME-mediated HG demethylesterification plays distinct roles in modulating fruit ripening in a species-dependent manner.

Cell adhesion regulation in the ML is increasingly recognized as more complex than previously assumed, involving crosstalk between HG demethylesterification and other pectin-remodeling enzymes. For example, in Arabidopsis, loss of the HG methyltransferase QUA2 increases PG activity, causing excessive degradation of demethylesterified HG in the ML. This leads to insufficient Ca²⁺-mediated crosslinking and results in adhesion defects [105]. Beyond mechanical regulation, cell adhesion is also modulated by feedback from endogenous OGs. In wild type and HG methylation mutants (qua2-1, esmd1-1, qua2-1/esmd1-1), seven distinct OGs were identified, differing in polymerization and substitution. Changes in HG esterification patterns correlated with expression of pectin-modifying enzymes (PME, PMEI, pectin acetylesterase) and adhesion phenotypes, suggesting that OGs signal to fine-tune CW structure and pectin-modifying gene expression, forming a feedback loop that maintains cell adhesion [106]. Together, these findings indicate that dynamic regulation of HG methylesterification in the ML—particularly at TCJs—functions as a molecular switch that balances adhesion and separation, underpinning developmental processes from intercellular space formation to fruit ripening

4.5. Plant Resistance to Abiotic Stresses

The HG methylesterification status of the CW influences plant resistance to diverse abiotic stresses, including cold, heat, drought, heavy metals, and salinity. During cold acclimation, a decrease in HG DM increases tissue tensile strength, enhancing freezing resistance in leaves [107]. Recent work on Chorispora bungeana illustrates that PMEI-mediated demethylesterification leads to a trade-off between stress tolerance and growth. Overexpression of a cold-induced PMEI, CbPMEI1, and its homolog AtPMEI13 in Arabidopsis decreased freezing tolerance, while enhancing salt tolerance and root growth under cold conditions. This case, as highlighted in Table 3 highlights the complexity and dynamic control of HG methylesterification in modulating plant responses to abiotic stress in a context-dependent manner [108]. Low-methylated HG likely promotes Ca²⁺-mediated crosslinking, decreasing CW porosity and impeding ice spread.

Under heat stress, PMEs contribute to thermotolerance by maintaining CW flexibility. In Arabidopsis, PME53 facilitates Ca²⁺-HG crosslink reconstitution, supporting stomatal movement and heat resistance [30]. The heat-sensitive pme34 mutant displays impaired stomatal function and altered PME and PG activities, indicating that PME34 participates in thermotolerance through coordinated CW remodeling [29].

HG methylesterification also modulates drought tolerance by affecting water retention and osmotic stress. Overexpression of PtoPME35 from Populus tomentosa enhanced drought tolerance in Arabidopsis by reducing stomatal opening, limiting water loss, and inducing drought-responsive genes [109]. More recently, PME-mediated demethylesterification was found to play a crucial role in enhancing tolerance to heavy metal stress in rice roots. As summarized in Table 3, the below studies represent key cases of PME-mediated regulation in heavy metal tolerance. A high cadmium (Cd)-accumulating (HA) rice line was associated with greater PME activity and a lower DM of pectin in a comparative study. The same group later reported that nitric oxide (NO) further promoted HG demethylesterification, leading to increased Cd binding in the rice CW. These findings suggest that the CW serves as a critical barrier and storage compartment for Cd detoxification through HG–Cd complexation [110,111].

The role of HG methylesterification in salinity stress is more complex. Overexpression of AtPMEI13 and AtPMEI17 increased HG DM and enhanced salt tolerance during germination and root growth [108,112], whereas AtPME31 expression positively affected salt stress tolerance, likely via induction of salinity-responsive genes [113]. These contrasting findings suggest that the impact of CW mechanics and stress-triggered intracellular signaling must be further investigated.

Overall, modulation of HG methylesterification enables plants to adjust CW mechanics, Ca²⁺ crosslinking, and biochemical interactions, contributing to resistance against multiple abiotic stresses. The specific outcomes depend on stress type, tissue context, and the dynamic coordination of PME, PMEI, and other CW–modifying enzymes.

4.6. Plant Resistance to Pathogens and Insects

The plant CW serves as the first barrier against pathogen invasion. The ML and TCJs, enriched in HG, are likely initial targets of pathogen attack. The DM in these regions affects CW porosity, elasticity, and expansion capacity, influencing pathogen penetration. Dynamic changes in HG methylesterification during plant–pathogen interactions are tightly regulated by the localized expression of PMEs and PMEIs [114]. Arabidopsis AtPME17, which is induced in response to Botrytis cinerea (B. cinerea), has been reported to contribute to resistance against the pathogen by triggering the jasmonic acid (JA)–ethylene signaling pathway. Its biochemical mechanism was characterized using the Pichia pastoris expression system, and it was found to perform a blockwise pattern of pectin de-methylesterification, forming ‘egg-box’ structures between HG molecules. This modification likely strengthens the CW, limits pathogen invasion, and demonstrates how precise regulation of PME activity can dynamically modulate plant defense responses [115].

Cooperation between plant and pathogen PMEs can facilitate HG backbone degradation by other pectin-degrading enzymes [114]. For example, increased expression and activity of AtPME3 are linked to susceptibility to B. cinerea and Pectobacterium carotovorum [116], whereas AtPMEI1, AtPMEI2, AtPMEI10–12 enhance resistance to B. cinerea by regulating HG methylesterification levels and patterns [74,117]. Similar HG methylesterification–resistance relationships have been observed in other crops [118,119,120].

HG demethylesterification can strengthen the CW and improve resistance. Host PME activity induced during the Myzus persicae–Arabidopsis interaction increased Ca²⁺-crosslinked HG, modulating aphid behavior [121]. Methanol and OGs generated from HG demethylesterification and degradation act as chemical signals known as damage-associated molecular patterns (DAMPs). Exogenous methanol can induce cytosolic Ca²⁺ changes, ROS production, anion channel regulation, and ethylene synthesis, thereby activating early defense responses [122]. In monocots, methanol is a potent elicitor of MAPK signaling, while in dicots, methanol alone has weak effects but strongly modulates MAPK responses in the presence of other DAMPs or pathogen-associated molecular patterns (PAMPs) [123]. OGs also function as DAMPs, with their DM influencing defense responses. Overexpression of the fruit-specific FaPE1 in strawberry reduced OG esterification and enhanced resistance to B. cinerea, likely via constitutive salicylic acid (SA) signaling activation [118]. A recent study developed a highly sensitive method to profile OGs during Arabidopsis–B. cinerea interactions and found that most OGs produced during infection were acetyl- and methylesterified and generated by fungal pectin lyases (PLs). These chemically modified OGs act as defense elicitors, revealing the dynamic enzymatic “arms race” between plants and pathogens [124]. While OGs enhance resistance, excessive OG levels can negatively affect plant growth, highlighting the need for precise regulation to balance immunity and development [125].

HG methylesterification status also influences the localization and activity of pattern-recognition receptors (PRRs) involved in CW damage sensing. In Arabidopsis, the dominant effect of WAK2 is suppressed in a PME3 null mutant, indicating that PME3-mediated HG de-esterification is required for WAK2 activation [126]. Moreover, PRRs—including WAK1, WAK2, and FERONIA (FER)—preferentially bind demethylesterified pectin, linking HG status to receptor function [126,127]. Pattern-triggered immunity (PTI) is subsequently activated upon perception of these signals through PRRs [128,129]. In summary, the degree and pattern of HG methylesterification govern both the physical properties of the CW and its role as a signaling hub. Through mechanical reinforcement, chemical signaling (methanol, OGs), and modulation of PRR-mediated immunity, HG methylesterification is central to plant resistance against pathogens and insect herbivores.

HG methylesterification modulates CW rigidity, porosity, and extensibility, thereby influencing various physiological processes such as cell growth, fruit softening, and stress responses. A simplified model illustrating how HG-modifying enzymes regulate CW properties and related biological processes is presented in Figure 2.

Table 3. Roles of Homogalacturonan (HG) methylesterification in plant development and interactions with the environment. The table summarizes selected representative roles of HG methylesterification in plant development and environmental interactions. Arrows indicate changes in HG degree of methylesterification (DM) (↑ increase, ↓ decrease). OG: Oligogalacturonide; DAMP: Damage-associated molecular pattern; MAPK: Mitogen-activated protein kinase; PRR: Pattern recognition receptor; WAK: Wall-associated kinase; JA: Jasmonic acid; SA: Salicylic acid.

Biological Process/Context	Plant/Tissue	PME/PMEI/Enzyme Involved	HG Methylesterification Change	Outcome/Phenotype	Reference
Cell elongation/growth	Arabidopsishypocotyls	AtPMEI1, AtPMEI2, AtPME17	↑ DM	Promotes cell elongation	[33,74,75]
Shade avoidance	Arabidopsishypocotyls	-	↑ DM	Required for shade-induced elongation	[77]
Organogenesis/leaf growth	Arabidopsis	CGR2 (HGMT)	↑ DM	Expanded leaves, enhanced organ growth	[79]
Seed germination	Arabidopsis seeds	PMEI5	↑ DM	Accelerates germination	[95]
Seed coat mucilage release	Arabidopsis	PME isoform	↓ DM	Enables mucilage release	[96]
Tip growth (pollen tube/root hairs)	Arabidopsis/tobacco	NtPPME1, PMEs/PMEIs	Localized DM gradients	Sustained pulsatile growth	[92,93]
Fruit softening	Strawberry/loquat	PMEs/PMEIs	↓ DM	Softening of fruit/tissue	[103,104]
Cold stress tolerance	Arabidopsis/Chorispora bungeana	CbPMEI1, AtPMEI13	↑ DM—freezing resistance decreased	Reduced freezing tolerance	[108]
Heat stress tolerance	Arabidopsis	PME53/PME34	Maintains Ca²⁺-HG crosslinks	Supports stomatal movement and thermotolerance	[29,30]
Drought tolerance	Arabidopsis/Populus tomentosa	PtoPME35	↓ DM	Reduced stomatal opening, enhanced drought tolerance	[109]
Heavy metal stress	Rice roots	PMEs	↓ DM	Cd sequestration in cell walls	[110,111]
Salt stress	Arabidopsis	AtPMEI13/AtPMEI17	↑ or ↓ DM depending on context	Modulates salt tolerance	[108,112]
Pathogen resistance	Arabidopsis	AtPMEI13/AtPME17	Localized DM changes	Modulates Botrytis susceptibility/resistance, aphid behavior	[115,121]
DAMP/OG signaling	Arabidopsis/strawberry	PMEs/FaPE1	↓ DM (OGs)	Activates defense signaling (MAPK, JA, SA pathways)	[118,124]
PRR activation	Arabidopsis	AtPME3	Required for WAK activity	Pattern-triggered immunity activation	[126]

5. HG Methylesterification as a Target for Crop Improvement and Breeding

The regulatory complexity of HG methylesterification underpins both developmental plasticity and stress responses, offering new opportunities for crop improvement. Translating mechanistic insights on HG methylesterification into applied breeding is increasingly feasible, with multiple routes available to exploit DM-related variation.

Conventional and marker-assisted selection (MAS) can leverage natural allelic variation in PME/PMEI loci. HG methylesterification influences both crop quality and stress resistance, making these genes attractive targets for breeding. Forward-genetic approaches, including genome-wide association study (GWAS) and genomic prediction, have identified HG methylesterification-related loci that influence adaptive traits and can prioritize candidate PME/PMEI genes associated with agronomic traits. GWAS revealed allelic variation in Trichome Birefringence (TBR) that modulates root CW HG methylesterification, reducing Zn binding and enhancing tolerance—a mechanism conserved across dicots and monocots and relevant for breeding heavy metal–tolerant crops [130]. Fine-mapping and pan-genome analyses in maize revealed that unilateral cross-incompatibility (UCI) systems such as Ga1 and Ga2 are governed by PME genes mediating pollen–silk recognition, providing tools for precise hybrid seed production [131,132,133]. Similarly, fine mapping of drought-resistant tomato mutants identified Slpmei27, a CW–expressed PMEI gene; silencing Slpmei27 enhanced drought tolerance by modifying CW structure, stomatal conductance, water loss, and ROS scavenging, highlighting HG methylesterification’s role in stress resilience [134].

Biotechnological manipulation, including overexpression, RNAi, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, offers rapid functional testing of candidate PME/PMEI genes and the creation of alleles tailored for agronomic traits. By demonstrating that FaPE1-mediated modulation of OGs methylation enhances both fruit size and resistance to B. cinerea in strawberry, this study provides a practical framework for breeding programs aimed at simultaneously improving productivity and disease resistance [119]. Collectively, these findings illustrate HG methylesterification modulation as a promising avenue for improving crop resilience, compatibility, and quality.

However, practical deployment of HG methylesterification-based breeding must consider context-dependent trade-offs. PME/PMEI activity is highly tissue- and stage-specific; constitutive or whole-plant manipulation may cause unintended phenotypes, such as altered growth or organ development. Moreover, changes in HG methylesterification status can have opposite effects depending on stress type, tissue, or species. Thus, breeding strategies should prioritize tissue-specific edits, coupled with molecular assays, high-throughput phenotyping, and multi-omics validation to avoid compromising plant performance. The future of HG methylesterification-centric crop improvement is promising. By integrating mechanistic knowledge of PME/PMEI regulation with high-throughput phenomics, breeders can move beyond simple phenotypes toward precise control over more complex agronomic traits. Advanced CRISPR/Cas technologies offer the potential to target traits accurately and reduce time-to-cultivar for traits previously limited to traditional breeding. Ultimately, understanding and manipulating HG methylesterification at molecular, biochemical and biomechanical levels opens new horizons for designing cultivars that balance growth, quality, and resilience.

6. Future Perspectives

Recent advances have deepened our understanding of how HG methylesterification influences CW mechanics and associated biological processes (Table 3). Yet, many fundamental questions remain unanswered, presenting opportunities for future research across scales—from molecular regulation to crop-level applications.

6.1. Molecular Regulation, Enzymatic Mechanisms, and Structural Complexity

HG methylesterification is regulated by a diverse set of factors, including PME/PMEI isoforms, TFs, phytohormones, and apoplastic pH. Yet the full regulatory network remains far from understood. Additional modulators likely operate in specific cellular contexts, and their interactions and feedback mechanisms are still poorly defined. A key challenge is to clarify the precise biochemical functions of individual PME and PMEI isoforms, including their substrate specificities, modes of action, and potential roles in forming isoform-specific complexes.

Recombinant protein studies have laid the groundwork for dissecting isoform-level activities, but higher-resolution structural insights are needed to capture how PME–PMEI complexes form and function dynamically, particularly during developmental transitions or plant–pathogen interactions. Omics-based approaches—such as transcriptomics, proteomics, and emerging single-cell or spatial omics—offer powerful means to map the expression, localization, and interaction networks of PME/PMEI isoforms across tissues and developmental stages.

Importantly, HG methylesterification regulation often operates with strong spatial precision—for example, at the growing tips of pollen tubes and root hairs, where localized demethylesterification controls wall extensibility and directional growth. Integrating biochemical, structural, and omics data with spatiotemporal analyses will be essential for linking molecular mechanisms to emergent growth behaviors.

6.2. Crosstalk in CW Remodeling

HG demethylesterification exerts contrasting effects across developmental and stress contexts, suggesting the presence of yet unidentified molecular modulators. Future research should investigate how demethylesterification interacts with other CW-modifying enzymes, such as peroxidases and oxidoreductases, and how this crosstalk contributes to the spatial organization of CW microdomains. Importantly, HG methylesterification status is also closely linked to broader signaling networks, including ROS production, Ca²⁺ oscillations, and phytohormone pathways. These interactions likely serve as checkpoints that balance growth with defense, allowing plants to fine-tune wall remodeling in response to environmental cues. Addressing this complexity will require integrative approaches. Omics and systems biology can help identify hidden regulators and network-level connections, while live-cell imaging and biosensors can capture the dynamics of these interactions in real time. Ultimately, clarifying how crosstalk coordinates localized biochemical changes with tissue- and organ-scale growth could provide insights for agriculture—for example, by identifying ways to manipulate CW remodeling to improve stress resilience without compromising growth.

6.3. Analytical Tools and Technological Advances

Advances in analytical techniques will be essential for dissecting HG methylesterification dynamics at multiple scales. While immunolabeling has enabled visualization of HG demethylesterification, current methods are limited in capturing interactions among wall components at microdomain resolution. Complementary technologies such as AFM, Raman microscopy, Fourier transform infrared spectroscopy (FTIR), in vivo sensors, and micro-mechanical assays provide finer-scale insights into CW mechanics but are often applied in isolation. Future research should prioritize integrative approaches that combine molecular imaging, biomechanical measurements, and computational modeling to bridge molecular regulation with emergent tissue- and organ-level growth patterns.

Emerging technologies also hold considerable promise. Single-cell and spatial omics can link gene expression and protein localization with local mechanical states. High-resolution live-cell imaging, paired with fluorescent biosensors, can reveal dynamic changes in pectin status in real time. Meanwhile, machine learning (ML) and artificial intelligence (AI)-assisted image analysis offer new opportunities for extracting quantitative information from complex datasets. Applying these methods across developmental contexts and species will not only improve mechanistic understanding but also enable predictive models of CW remodeling with direct relevance to crop improvement.

6.4. Comparative Insights Beyond Arabidopsis

Most current knowledge of HG methylesterification and CW regulation derives from Arabidopsis, but this narrow focus limits broader biological and agricultural insights. Expanding investigations to crops and non-model species will be critical for uncovering both conserved mechanisms and lineage-specific innovations. Comparative studies across phylogenetically diverse plants—including mosses, ferns, monocots, and dicots—could reveal how HG methylesterification regulation evolved and how it contributes to species-specific developmental processes. As shown in Table 4, PME and PMEI gene families are generally larger and vary widely across plant species, reflecting evolutionary diversification and potential functional specialization.

In crops, dynamic changes in HG methylesterification are closely linked with key agronomic traits such as plant growth, fruit ripening, organ initiation, stress resilience, and biomass quality. Looking forward, combining comparative genomics, multi-scale modeling, and experimental data could reveal conserved regulatory roles of HG methylesterification across species, enabling predictive frameworks for CW remodeling and associated developmental processes. Comparative omics allow us to dissect species-specific differences and predict evolutionary events in PME/PMEI families. Forward genetics, combined with reverse genetic tools, allows candidate identification and functional analysis of specific isoforms. Emerging studies have revealed the importance of the spatiotemporal coordination of PME/PMEI isoforms in modulating fundamental plant morphogenesis, growth, and stress adaptation. Exploring spatial and temporal dynamics using advanced tools such as single-cell omics, high-resolution imaging, and AI-assisted modeling offers a particularly promising approach to reveal CW dynamics during specific developmental stages. Altogether, these insights can accelerate discovery and guide targeted breeding to optimize CW properties and other important agronomic traits using modern technologies, including genome editing and synthetic biology strategies.

Table 4. Comparative overview of pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in representative plant species.

Species	No. of PME Genes	No. of PMEI Genes	Key References
Arabidopsis thaliana	66	71	[135]
Oryza sativa (rice)	43	49	[136,137]
Solanum lycopersicum (tomato)	57	48	[138,139]
Glycine max (soybean)	127	170	[140,141]
Zea mays (maize)	43	49	[142]

Author Contributions

Conceptualization, D.W. and B.B.-U.; Methodology, D.W.; Investigation, D.W. and B.B.-U.; Data curation, D.W. and I.B.O.-S.; Writing—original draft, D.W.; Writing, review and editing, D.W., I.B.O.-S. and B.B.-U.; Supervision, D.W.; Visualization, D.W.; Resources, D.W., I.B.O.-S. and B.B.-U.; Project administration, D.W.; All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Acknowledgments

The authors used ChatGPT (version: ChatGPT-5 Mini) solely for language polishing and readability improvements.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:

CW	Cell wall
HG	Homogalacturonan
RG-I	Rhamnogalacturonan I
RG-II	Rhamnogalacturonan II
PMEs	Pectin methylesterases
PMEIs	PME inhibitors
TFs	Transcription factors
DM	Degree of methylesterification
CWI	Cell wall integrity
GalA	Galacturonic acid residues
A-SCM	Arabidopsis seed coat mucilage
STK	SEEDSTICK
LUH	LEUNIG_HOMOLOG
BLH	BEL1-Like homeodomain
ERF	Ethylene response factor
BLR	BELLRINGER
FLY1	FLYING SAUCER1
MUD1	Mucilage-Defect-1
ABA	Abscisic acid
BRs	Brassinosteroids
GA	Gibberellic acid
BES1	BRI1-EMS-SUPPRESSOR1
OGs	Oligogalacturonides
ssNMR	Solid-state nuclear magnetic resonance (ssNMR)
PG	Polygalacturonase
APRX	Apoplastic isoperoxidase
PL	Pectate lyase
HGMT	Homogalacturonan methyltransferase
NPL	Nep1-like protein
AFM	Atomic force microscopy
NtLLG4	LORELEI-like glycosylphosphatidylinositol-anchored protein 4
ROS	Reactive oxygen species
HMS	HIGHLY METHYL ESTERIFIED SEEDS
TCJ	Tricellular junction
ML	Middle lamella
Cd	Cadmium
DAMPs	Damage-associated molecular patterns
MAPK	Mitogen-activated protein kinase
PAMPs	Pathogen-associated molecular patterns
PRRs	Pattern-recognition receptors
WAK2	Wall associated kinase 2
WAK1	Wall associated kinase 1
SA	Salicylic acid
FER	FERONIA
PTI	Pattern-triggered immunity
TBR	Trichome birefringence
MAS	Marker-assisted selection
GWAS	Genome-wide association study
UCI	Unilateral cross-incompatibility
RNAi	RNA interference
CRISPR	Clustered regularly interspaced short palindromic repeats
FTIR	Fourier transform infrared spectroscopy
ML	Machine learning
AI	Artificial intelligence
JA	Jasmonic acid

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Figure 1. Regulation of homogalacturonan (HG) methylesterification in the plant cell wall (CW) in Arabidopsis. This schematic illustrates the multilayered regulation of HG methylesterification in the CW of the model plant Arabidopsis. The degree of methylesterification (DM) is modulated by numerous transcription factors (TFs) (LUH, BLH2, BLH4, ERF4, ETTIN, BLR, STK, and MYB52) that directly regulate pectin methylesterase (PME)/pectin methylesterase inhibitor (PMEI) expression, as well as by ubiquitin ligases (FLY1, FLY2, MUD1) that control PME recycling and stability. Hormones also modulate HG methylesterification. Ethylene and abscisic acid (ABA) promote HG demethylesterification, while gibberellic acid (GA) and brassinosteroids (BR) inhibit it. Auxin can either promote or inhibit HG demethylesterification, which might be determined by DM. In addition, apoplastic pH influences the enzymatic function of PME/PMEI through acidification driven by H⁺-ATPase, proton pump embedded in the plasma membrane (PM). Red arrows indicate positive regulation, blue arrow indicate negative regulation, and dashed indicated regulatory relationships that are not fully confirmed. The multiple layers described above-including TFs, post-transcriptional regulators, hormones, and apoplastic pH—are interwoven to form a complex network controlling HG methylesterification. Arrows indicate positive (red) or negative (blue) regulation. The red arrows with double heads refer to indirect positive regulation. Black dotted lines suggest interactions that are not fully characterized or confirmed.

Figure 2. Regulation and functional consequences of homogalacturonan (HG) demethylesterification in the primary cell wall (CW) and middle lamella (ML) of Arabidopsis. This schematic illustrates how HG methylesterification is dynamically regulated by pectin methylesterases (PMEs) and pectin methylesterase inhibitors (PMEIs), leading to distinct CW mechanical properties and biological outcomes. Highly methylesterified HG backbones can be demethylesterified by PMEs using two different modes of action. Block-wise demethylesterification generates large stretches of demethylesterified galacturonic acid residues (GalA), which can form ‘egg-boxes’ in the presence of calcium ions. The ‘egg-boxes’ structure increases CW stiffness. In the random mode of action, PMEs cleave O-methyl groups at random sites, producing low methylesterified HGs, which are subject to degradation by pectin-degrading enzymes such as polygalacturonase (PG) and pectin lyase (PL). This mode of action usually produces oligogalacturonides (OGs), which induce CW loosening. The activity of PMEs is modulated by PMEIs and is pH-dependent, with apoplastic acidification modulating their function. The degree of methylesterification (DM) can be detected by specific monoclonal antibodies (e.g., CCRC-M130/M34, JIM7, LM20 for high DM; JIM5, LM19, LM18 for low DM). Changes in HG methylesterification alter biomechanical properties of the wall—including hydration, porosity, adhesion, permeability, and elasticity—by affecting pectin–polymer interactions, cellulose mobility, and pectin–calcium gel network formation. Wall mechanical changes further contribute to multiple developmental events, including cell expansion-related processes such as hypocotyl and root hair growth, organ initiation, pollen tube elongation, seed mucilage release, seed germination, and embryo development, as well as cell separation–related processes including fruit softening, organ abscission, and cell division. Moreover, HG methylesterification status affects plant responses to abiotic stresses (drought, cold, heat, metal ions, salt) and biotic stresses, particularly pathogen infection. Symbols: blue line, demethylesterified HG; red dot, O-methyl group; orange circle, PME; green hexagon, PMEI; yellow triangle, Ca²⁺; green star, PG; purple triangle, PL.

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Wang, D.; Ortega-Salazar, I.B.; Blanco-Ulate, B. Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement. Agronomy 2025, 15, 2641. https://doi.org/10.3390/agronomy15112641

AMA Style

Wang D, Ortega-Salazar IB, Blanco-Ulate B. Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement. Agronomy. 2025; 15(11):2641. https://doi.org/10.3390/agronomy15112641

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Wang, Duoduo, Isabel B. Ortega-Salazar, and Barbara Blanco-Ulate. 2025. "Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement" Agronomy 15, no. 11: 2641. https://doi.org/10.3390/agronomy15112641

APA Style

Wang, D., Ortega-Salazar, I. B., & Blanco-Ulate, B. (2025). Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement. Agronomy, 15(11), 2641. https://doi.org/10.3390/agronomy15112641

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Homogalacturonan Methylesterification and Cell Wall Regulation: Integrating Biochemistry, Mechanics, and Developmental Signaling for Crop Improvement

Abstract

1. Introduction

2. Regulation of HG Methylesterification

2.1. PMEs and PMEIs

2.2. Transcriptional and Post-Transcriptional Control

2.3. Hormone Regulation

2.4. Apoplastic pH

3. HG Methylesterification and CW Mechanics

3.1. Biomechanical Models and Pectin-Cellulose Interactions

3.2. PME/PMEI-Mediated Wall Regulation

3.3. Integration with Hormone Signaling and Feedback Loops

3.4. Integration with Other CW Components and Signaling

4. HG Methylesterification in Development and Environmental Interactions

4.1. Cell Growth

4.2. HG Methylesterification in Tip Growth and Morphogenesis

4.2.1. Localized PME/PMEI Activity in Tip-Growing Cells

4.2.2. Apical-Basal Gradients and Wall Mechanics

4.2.3. Integration with Plant Morphogenesis

4.3. Organ Initiation, and Seed Germination

4.4. HG in the Middle Lamella (ML) Influences Plant Development

4.5. Plant Resistance to Abiotic Stresses

4.6. Plant Resistance to Pathogens and Insects

5. HG Methylesterification as a Target for Crop Improvement and Breeding

6. Future Perspectives

6.1. Molecular Regulation, Enzymatic Mechanisms, and Structural Complexity

6.2. Crosstalk in CW Remodeling

6.3. Analytical Tools and Technological Advances

6.4. Comparative Insights Beyond Arabidopsis

Author Contributions

Funding

Data Availability Statement

Acknowledgments

Conflicts of Interest

Abbreviations

References

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