Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls

Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls Breeanna Urbanowicz, Kristen Thorne, William Barnes, Helen Hood This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8001798/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Plant cell walls are dynamic composites whose architecture determines growth, mechanics, and environmental resilience. Efforts to link pectin structure to function have been limited by the lack of molecular probes with sufficient specificity, a gap that becomes even more pronounced for the intricately branched rhamnogalacturonon-II (RG-II) subclass. Here we report the first fluorescent probes with defined specificity to RG-II, engineered from catalytic site mutants of Bacteroides thetaiotaomicron glycoside hydrolases BT1010 and BT0996. These enzyme-derived probes bind RG-II monomer with high affinity, discriminate against dimeric forms, and localize to cell corners and junctions in Arabidopsis thaliana stems, consistent with RG-II’s unique ability among wall polysaccharides to form borate-mediated, covalent crosslinkages between molecules. Application of these probes revealed spatial partitioning distinct from the homogalacturonan (HG)- and rhamnogalacturonan I (RG-I)-enriched middle lamella, highlighting functional specialization among pectic domains, with RG-II reinforcing cell junctions while HG and RG-I mediate wall flexibility. Our work establishes a generalizable framework for transforming CAZymes into high-precision imaging reagents, enabling molecular-level visualization of structurally complex polysaccharides in the cell wall. Biological sciences/Plant sciences/Plant cell biology/Cell wall Biological sciences/Biochemistry/Glycobiology Biological sciences/Biochemistry/Carbohydrates Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plants possess a remarkable ability to modulate the mechanical strength and flexibility of their cell walls, driving essential biological processes such as cellular growth and morphogenesis 1 , reproductive development 2 , 3 , and environmental and immune defense 4 . Growing cells are surrounded by primary cell walls which are composed of complex, interconnected networks of polysaccharides that assemble into highly organized macrostructures, each tailored to provide structural support in a context-dependent manner 5 , 6 . The biochemical composition and spatial arrangement of these polysaccharides are tightly regulated by both biotic and abiotic factors 7 , ensuring precise control over cell wall architecture 8 , 9 . The molecular mechanisms underlying this regulation remain poorly understood, largely due to the challenging nature of distinguishing heterogenous cell wall polymers and monitoring structural variation at the tissue and cellular level. Conferring the highest degree of structural variability among plant glycans, pectins are defined by the presence of 1,4-linked galacturonic acid (GalA) in the polymer backbone and include four main subclasses: homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (XGA) 10 . Pectins are present in the cell walls of all vascular plants, where they form extensive covalent and non-covalent interactions with each other. In particular, their enrichment in the middle lamella between adjacent primary walls underlies their key role in mediating cell–cell adhesion 11 . They have also been shown to interact with cellulose and hemicelluloses, supporting a wide range of wall architectures that enable both stability and flexibility during plant growth and development 12 , 13 . HG consists of a linear chain of α-1,4-linked GalA residues appended with O -methyl and O -acetyl substituents at the C -6 and O -2/ O -3 positions, respectively. In contrast, the RG-I backbone is based on a repeating disaccharide unit of α-1,4-GalA and α-1,2-rhamnose (Rha) residues and displays context-dependent variation in the type and degree of glycosyl substituents, which range from single sugars to branched arabinan, galactan, and arabinogalactan side-chains attached to the Rha residues 14 . Current models propose that HG and RG-I form a dynamic, covalently linked pectic network with RG-II 15 to regulate wall porosity, hydration, and adhesion between cells 16 . RG-II is the least abundant pectic domain, representing up to 5% of the polysaccharides present in primary cell walls and is often considered to be the most structurally complex polysaccharide found in Nature to date 17 , 18 . The core structure of RG-II is a backbone of at least eight 1,4-linked α- d -Gal p A residues substituted with six distinct sidechains, termed A – F, that are linked together by at least twenty-one different glycosidic linkages to form a RG-II monomer of approximately 5 kDa 18 (Fig. 1 A). These structurally conserved side chains are composed of 13 different sugars, including several unusual monosaccharides such as 3-deoxy- d -manno-oct-2-ulosonic acid ( d -Kdo) and 3-deoxy- d -lyxo-2-heptulosaric acid ( d -Dha), which are only found in RG-II and bacterial polysaccharides 19 . There are also a number of non-glycosyl substituents, including methyl-etherified GalA and xylose and methyl-esterified glucuronic acid (GlcA) in Sidechain A, methyl-etherified fucose in Sidechain B, O -acetyl groups on Sidechains A and B, and methyl-esterified GalA on the backbone 20 . More than 90% of RG-II in the cell wall presents as a cross-linked dimer, formed through a borate diester linkage between β- d -apiosyl (Api) residues of Sidechain A (Fig. 1 B) 21 , 22 . Unlike most plant polysaccharides, RG-II has a highly conserved primary structure, likely reflecting the need to maintain its unique ability to form these covalent linkages 19 , 23 . In fact, plants carrying heterozygous mutations in genes that affect RG-II synthesis and structure typically exhibit severe growth defects, while complete knockout mutations are often embryonic lethal 22 , 24 – 30 . Collectively, these studies have shown that even minor structural modifications to RG-II markedly impair its ability to dimerize, thereby compromising the integrity of the pectic network within the cell wall. Plant cell wall glycan-directed monoclonal antibodies (mAbs) are indispensable tools for visualizing the spatial organization and dynamics of wall polysaccharides. Their ability to recognize diverse glycan sub-structures, or epitopes, within their native cellular context has provided key insights into how wall composition varies across cell types, tissues, and developmental stages 31 . However, generating and characterizing mAbs against branched pectic polysaccharides remains challenging due to their structural heterogeneity, which complicates biochemical isolation and epitope assignment 32 . Although more than 80 mAbs have been generated against pectin- and protein-linked arabinogalactans (AGPs), only seven epitopes have been clearly defined 31 , and none have been conclusively characterized to target RG-II epitopes 33 – 35 . One explanation for RG-II’s apparent lack of immunogenicity is that steric hindrance from its distinctive three-dimensional (3D) structure may limit access to potential epitopes recognizable by animal immune systems 36 . Furthermore, the polysaccharides used as immunogens are frequently prepared using base extractions, which removes ester substituents. Indeed, very few glycan-directed antibodies have been successfully developed against base-labile features, including O -acetyl and methyl-ester modifications, which are critical structural elements of pectins 31 . Among these are the mannan-directed mAbs CCRC-M169 and CCRC-M170, which uniquely recognize acetylated epitopes 37 , 38 . Similarly, the HG-directed mAbs CCRC-M38, CCRC-M132, JIM5, LM18, and LM19 preferentially bind regions with low degrees of methylesterification, although each shows slight variations in tolerance for methyl substitution 32 , 39 . Together, these limitations highlight the need for alternative strategies and complementary tools to investigate how, where, and when pectins are assembled, disassembled, or relocated within plant cell walls to influence wall integrity and mechanics. Several studies have demonstrated that catalytically inactivated enzymes retain their binding specificity and can be developed for use as probes to identify specific structural motifs within heterogenous polysaccharides 40 – 43 . Unlike antibodies, such proteins can be recombinantly expressed and genetically encoded, allowing modular fusion with fluorescent tags of choice and the rapid detection of structural or chemical changes within subcellular locations of the cell wall. This makes them especially advantageous for experiments requiring live-cell imaging, co-localization with other cellular markers, or the tracking of pectin dynamics across developmental stages. Research on the gut bacterium Bacteroides thetaiotaomicron ( B. theta ) has led to the identification of a suite of Carbohydrate Active Enzymes (CAZymes) that recognize and degrade RG-II through a highly coordinated deconstruction model 44 . These enzymes, encoded in substrate-associated polysaccharide utilization loci (PUL), not only provide mechanistic insight into RG-II degradation but also represent versatile tools that can be repurposed for a multitude of applications, ranging from structural characterization, biomass conversion, tailored oligosaccharide production, and now, development of molecular probes for cell wall imaging. Here we describe an approach that leverages the linkage-level selectivity of RG-II-targeted GHs to develop recombinant, fluorescent probes directed towards Sidechains A and B of RG-II. In this work, we constructed a library of catalytically inactive GH variants and assessed their ability to recognize and bind RG-II substrates isolated from red wine. Both sets of probes preferentially bound the RG-II monomer over the dimer, providing a new means to discriminate structural conformations within this complex polysaccharide. Guided by biochemical analyses, we identified the most effective variants and applied them as recombinant probes for direct visualization of RG-II within Arabidopsis thaliana stem sections. Together, these findings introduce a new technique for mapping the spatial distribution of RG-II in native plant cell walls and outline a general strategy for repurposing PUL-encoded enzymes as molecular probes. Results Engineering catalytically inactive GH-based probes targeting RG-II sidechains We initiated our probe library by first evaluating the specificity of GHs that cleave terminal sugars from RG-II Sidechain A or Sidechain B (Fig. 1 A). These complex oligosaccharide sidechains harbor unique structural signatures and may also differ in enzymatic accessibility and reactivity between monomeric and dimeric forms. We selected the GH95 domain of BT1010 and the GH137 domain of BT0996 as candidates for developing Sidechain A– and B–targeting probes, respectively. BT1010 is an inverting α- l -galactosidase containing a GH95 catalytic module that cleaves the terminal l -Galactose ( l -Gal) from Sidechain A (Fig. 2 A). To develop probes targeted to this region, we constructed a fusion between sfGFP and the GH95 domain of BT1010 (residues 19–824), then introduced active site mutations designed to disrupt catalysis while maintaining substrate recognition (SI Fig. 1 A). Catalytic residue predictions were guided by homology between an AlphaFold predictive model of BT1010 (AF-Q8A907) and AfcA (PDB 2EAD), a structurally characterized GH95 α- l -fucosidase 45 (SI Fig. 1 B). In AfcA, Glu566 and Asp766 act as the general acid and base catalysts, while a pair of polar amino acids, Asn421 and Asn423, act as intermediates in a proton acceptor chain required for catalysis. The corresponding residues in BT1010 were selected for the generation of three variants, BT1010 N389A , BT1010 E516A , and the double mutant BT1010 N389A/E516A (Fig. 2 B, SI Fig. 1 C). The native and variant forms of BT1010 GH95 were recombinantly expressed in E. coli and purified as N-terminal His 6 –sfGFP fusion proteins (SI Fig. 2 A), with sfGFP-BT1010 N389A/E516A displaying slightly reduced expression compared to the single mutants (SI Fig. 2 B). Thermal shift analysis confirmed that the sfGFP-BT1010 N389A and sfGFP-BT1010 E516A variants retained stability comparable to the wild type sfGFP-BT1010, while sfGFP-BT1010 N389A/E516A showed a decrease in melting temperature (SI Fig. 2 B). BT0996 is a multimodular enzyme that contains two catalytic domains: a C-terminal GH2 β- d -glucuronidase and a N-terminal GH137 β- l -arabinofuranosidase, which target linkages in Sidechains A and B, respectively (Fig. 1 A). It also carries a CBM57 region that binds α-1,4-linked polygalacturonic acid 46 . To minimize off-target binding, we designed native and variant sfGFP-fusion constructs with the GH137 domain of BT0996 (residues 24–387), which cleaves the terminal l -Arabinose ( l -Ara f ) from Sidechain B (Fig. 3 A, SI Fig. 1 A). The GH137 domain of BT0996 is the founding member of its family, and predictions of catalytic residues were based on the crystal structure of the GH137 domain in complex with Sidechain B (PDB 5MUJ) (Fig. 3 B) 44 . In this structure, the terminal l -Ara f occupies a deep active site pocket, while the rest of the sidechain extends outward into solvent (SI Fig. 1 D), positioning Glu159, Glu240, and Asn227 as potential residues for catalysis. Glu159 and Glu240 were predicted to serve as the general acid and base catalysts, respectively, whereas Asn227 likely contributes indirectly to the catalytic mechanism. We recombinantly expressed the following variants in E. coli as soluble N-terminal His 6 –sfGFP fusion proteins: sfGFP-BT0996 N227A , sfGFP-BT0996 E240A , sfGFP-BT0996 E159Q , sfGFP-BT0996 N227A/E240A , and sfGFP-BT0996 E159Q/E240A (SI Fig. 2 A). The construct encoding sfGFP-BT0996 E159Q failed to yield soluble protein and was not further characterized. Thermal shift analysis revealed stable melting temperatures for the remaining variants compared to sfGFP-BT0996; however, sfGFP-BT0996 N227A/E240A and sfGFP-BT0996 E159Q/E240A expressed at reduced levels compared to sfGFP-BT0996 N227A and sfGFP-BT0996 E240A (SI Fig. 2 C). Biochemical characterization of RG-II glycosidases We evaluated the biochemical activity of all enzymes using purified RG-II monomer and dimer as saccharide substrates (SI Fig. 2 D). Previous work by Ndeh et al. (2017) established the stepwise mechanism by which B. theta PULs degrade RG-II, but most enzymes were assayed with short oligosaccharides, synthetic substrates, or crude extracts rather than the complete pectic domain. HPAEC-PAD was used to quantify the enzymatic hydrolysis of l -Gal and l -Ara f from RG-II substrates by native and variant sfGFP-BT1010 and sfGFP-BT0996, respectively (Table 1 ). In comparison to sfGFP-BT1010, the relative rate of Gal hydrolyzed from RG-II monomer decreased by 86% with sfGFP-BT1010 N389A and 99% with sfGFP-BT1010 E516A and sfGFP-BT1010 N389A/E516A (Fig. 2 C). Mutation of sfGFP-BT0996 decreased the relative rate of Ara released from RG-II monomer by 78% with sfGFP-BT0996 N227A , 97% with sfGFP-BT0996 E240A , and 99% with sfGFP-BT0996 N227A/E240A and sfGFP-BT0996 E159Q/E240A (Fig. 3 C). Both native enzymes showed limited activity on dimeric RG-II, with hydrolysis rates reduced by 94% for sfGFP-BT1010 (Fig. 2 D) and 92% for sfGFP-BT0996 (Fig. 3 D) compared to monomer. These results were reproducible across multiple independent protein preparations (SI Figs. 3 , 4 ), confirming that BT1010 GH95 and BT0996 GH137 are preferential to RG-II monomer as a substrate over dimer. Table 1 Summary of catalytic and binding data for native and variant sfGFP-BT1010 and sfGFP-BT0996 against RG-II monomer and dimer. Target ID Catalytic Activity Binding Affinity c n Relative Rate of Activity a Standard Error b n K d Confidence Std. Error of Regression d Reduced χ 2 d ng∙min − 1 ∙µM − 1 µM RG-II Monomer BT1010 GH95 3 59.110 7.375 6 5.781E + 00 2.830E + 00 1.730 0.751 BT1010 N389A 3 8.439 3.464 6 4.568E + 00 1.903E + 00 1.741 0.899 BT1010 E516A 3 0.476 0.191 6 7.940E-01 3.980E-01 1.256 0.842 BT1010 N389A/E516A 3 0.281 0.259 6 1.221E + 00 5.620E-01 1.371 1.176 BT0996 GH137 3 23.031 4.119 7 1.337E + 00 6.640E-01 1.010 0.594 BT0996 N227A 3 5.160 0.818 6 9.560E-01 4.820E-01 0.996 0.622 BT0996 E240A 3 0.733 0.896 6 8.900E-01 3.890E-01 1.010 1.029 BT0996 N227A/E240A 3 0.260 0.155 7 4.660E-01 1.460E-01 0.497 0.182 BT0996 E159Q/E240A 3 0.272 0.115 5 3.084E + 00 1.358E + 00 1.847 0.471 sfGFP - - - 6 9.491E + 02 e 1.917E + 03 0.413 0.911 RG-II Dimer BT1010 GH95 3 3.448 3.856 6 3.376E + 02 e 2.651E + 02 0.437 0.380 BT1010 N389A 3 1.665 1.352 6 1.990E + 02 e 9.248E + 01 0.346 0.405 BT1010 E516A 3 0.042 0.085 7 5.149E + 02 e 5.397E + 02 0.289 0.217 BT1010 N389A/E516A 3 0.086 0.037 6 7.944E + 02 e 7.658E + 02 0.362 0.565 BT0996 GH137 3 1.861 1.724 8 8.383E + 02 e 2.772E + 02 0.522 0.476 BT0996 N227A 3 0.524 0.266 6 4.047E + 03 e 6.578E + 03 0.627 0.816 BT0996 E240A 3 0.367 0.364 6 1.008E + 03 e 2.787E + 02 0.651 0.336 BT0996 N227A/E240A 3 0.092 0.058 6 3.178E + 03 e 4.436E + 03 0.550 1.121 BT0996 E159Q/E240A 2 0.245 0.063 - - - - - sfGFP - - - 10 1.233E + 04 e 7.836E + 03 0.537 0.642 a Relative rate of activity is calculated from the mass of hydrolyzed product detected in a 25 µL injection volume. b Standard error is calculated from the mean of 3 separate protein expressions, each calculated from the average of 4 technical replicates. c Dissociation constants (K d ) and their confidence intervals were derived from nonlinear least-squares fitting. d Standard error of regression and reduced χ 2 values were obtained from MO.Affinity Analysis Software. e Derived K d estimates represent apparent values extrapolated beyond the measurable binding range due to autofluorescence at high ligand concentrations and thus should be interpreted qualitatively as indicative of weak binding. Binding affinity of sfGFP-fused GHs to RG-II monomer and dimer Having identified variants with partial or complete catalytic inactivation, we next asked whether the extent of inactivation correlated with substrate recognition. To achieve this, we measured dissociation constants (K d ) for each sfGFP-fusion protein against RG-II monomer and dimer as substrates using MicroScale Thermophoresis (MST) (Table 1 ). Sigmoidal dose-response curves were generated from ligand concentrations ranging from 250 µM to 15.3 nM to calculate binding affinities against RG-II monomer (Fig. 4 A-B, SI Fig. 5 ). For the BT1010 GH95 l -galactosidase, native and variant proteins exhibited K d values of 5.78 ± 2.83 µM (sfGFP-BT1010), 4.57 ± 1.90 µM (sfGFP-BT1010 N389A ), 0.79 ± 0.40 µM (sfGFP-BT1010 E516A ), and 1.22 ± 0.56 µM (sfGFP-BT1010 N389A/E516A ). For the BT0996 GH137 arabinosidase, K d values were 1.34 ± 0.66 µM (sfGFP-BT0996), 0.96 ± 0.48 µM (sfGFP-BT0996 N227A ), 0.89 ± 0.39 µM (sfGFP-BT0996 E240A ), 0.47 ± 0.15 µM (sfGFP-BT0996 N227A/E240A ), and 3.08 ± 1.36 µM (sfGFP-BT0996 E159Q/E240A ). Purified sfGFP alone displayed weak or undetectable binding to RG-II monomer and dimer, confirming that substrate recognition arises from the GH domains (SI Fig. 6 ). To measure binding to dimeric RG-II, we found that ligand concentrations exceeding 500 µM were required to reach saturation, which produced autofluorescence artifacts and precluded accurate K d determination (Fig. 4 C-D, SI Fig. 7 ). Our data showed that variants with single point mutations that had the highest degree of biochemical inactivation, sfGFP-BT1010 E516A and sfGFP-BT0996 E240A , displayed higher binding affinities relative to their native or partially inactivated counterparts. We also identified two double mutants, sfGFP-BT1010 N389A/E516A and sfGFP-BT0996 E159Q/E240A , that were completely unable to hydrolyze RG-II but instead had reduced binding affinity. Based on their consistent reduction in catalytic activity while maintaining binding specificity, the BT1010 E516A and BT0996 E240A single mutant variants were selected as optimal, catalytically inactive probes to evaluate for imaging studies. Fluorescent labeling of RG-II in Arabidopsis stem cell walls We next examined whether BT1010 E516A and BT0996 E240A could function as fluorescent probes to visualize RG-II within plant cell walls. To avoid overlap with endogenous green autofluorescence 47 , each variant was fused to mRuby3, which provides improved photostability and acid tolerance relative to sfGFP 48 . For fluorescence microscopy, recombinantly expressed and purified mRuby3-BT1010 E516A and mRuby3-BT0996 E240A were applied to A. thaliana inflorescence stems. Samples were prepared using an optimized sectioning and labeling workflow that allows short incubations and requires no post-acquisition image adjustments. Stem sections were incubated for 1 hour with mRuby3-BT0996 E240A or mRuby3-BT1010 E516A , resulting in a distinct labeling of the parenchyma cell walls (Fig. 5 ). Consistent with MST analyses, in which sfGFP-BT0996 displayed more than 4-fold higher affinity for the RG-II monomer compared to sfGFP-BT1010, labeling with mRuby3-BT0996 E240A produced correspondingly stronger fluorescence signals (SI Fig. 8A). Given its superior labeling efficiency, mRuby3-BT0996 E240A was selected for subsequent studies examining RG-II localization and binding specificity in the cell wall. To confirm that the observed signal originates from the specific interaction between mRuby3-BT0996 E240A and RG-II, a series of control experiments were first conducted (Fig. 6 A, SI Fig. 8B). Incubation of stem sections with purified mRuby3 alone ruled out non-specific binding of the fluorophore to the wall (Fig. 6 B, SI Fig. 8C). Pretreatment of sections with native BT0996 GH137 greatly reduced the signal, indicating that the probe recognizes the l -Ara f epitope cleaved by the active enzyme (Fig. 6 C, SI Fig. 8D). We routinely blanched sections after enzymatic digestion to ensure that the signal loss was not due to steric blocking of the binding site by the active enzyme. Competitive inhibition by preincubating the probe with RG-II monomer (Fig. 6 D, SI Fig. 8E) or dimer (Fig. 6 E, SI Fig. 8F) prior to labeling also decreased fluorescence. Notably, preincubation with RG-II monomer (2 mM) resulted in a significantly greater reduction in signal than with even lower concentration of dimer (1 mM), further supporting the selectivity of mRuby3-BT0996 E240A to RG-II monomer. However, when sections were pretreated with higher concentrations of up to 2.5 mM of either monomer or dimer, fluorescence was completely abolished (SI Fig. 8G-H). Quantification of fluorescence intensity across multiple experiments confirmed a significant reduction in signal under all control conditions compared to direct labeling with mRuby3-BT0996 E240A , with dimer supplementation having the smallest effect (Fig. 6 F). Together, these results demonstrate that labeling depends on the integrity and accessibility of the RG-II target, supporting the site-specific binding capability of the probe. Having validated the specificity of mRuby3-BT0996 E240A for RG-II, we next compared its labeling pattern with those of established monoclonal antibodies targeting other matrix polysaccharides. To investigate the distribution of pectic domains, we used CCRC-M38, which binds de-esterified homogalacturonan, and CCRC-M14, which recognizes the RG-I backbone (Fig. 7 A–B, SI Fig. 9A-B). CCRC-M38 showed an even distribution throughout the primary cell walls of parenchyma, while CCRC-M14 labeling was distributed throughout the middle lamella and intercellular regions. Our RG-II-directed glycosidase-based probe, mRuby3-BT0996 E240A , showed intense labeling of the middle lamellar cell corners between adjoining cells, suggesting that the distribution of RG-II is distinct from that of HG and RG-I (Fig. 7 C, SI Fig. 9C). Additionally, labeling with CCRC-M1, which detects fucosylated xyloglucan, produced a pattern distinct from all three pectin-directed probes (SI Fig. 9D). These comparisons highlight the unique localization of RG-II within pectin-rich regions of the cell wall and establish mRuby3-BT0996 E240A as a valuable complement to existing probes for visualizing pectic architecture and mapping the spatial organization of different domains within distinct apoplastic regions in muro. Discussion We lack a complete toolset for investigating and visualizing carbohydrate biology in vivo , especially at the spatial and temporal scales at which it occurs. Such tools are essential to reveal the rules by which complex carbohydrates are built and how they are assembled into higher order and emergent structures, such as cell walls, and how these structures are dynamically modified to perform diverse and essential biological functions. In this study, we designed and characterized fluorescent probes using RG-II-specific GHs from B. theta . Unlike the few existing polyclonal antibodies reported to target RG-II, our enzyme-based probes developed here have defined binding sites and can distinguish between monomeric and dimeric RG-II. Furthermore, through fusion to fluorescent proteins, we demonstrated that PUL-encoded GHs can be readily repurposed for use as precise imaging tools. Although enzyme-based probes have recently been developed from mammalian glycosyltransferases (Hombu et al., 2025) or mucinases (Shon et al., 2020), CAZymes targeting plant cell wall glycans have generally been underutilised for biological imaging applications (Dornez et al., 2011). Instead, CBMs have been more commonly used as fluorescent probe scaffolds 49 ; however, their broad binding profiles often lack the structural precision needed to recognize complex polysaccharides like RG-II. In fact, the extensive branching patterns and glycosyl and non-glycosyl substitutions of plant wall glycans further limit the ability of most CBMs to discriminate among distinct pectic domains or between pectic glycostructures and other polysaccharides 46 , 50 , 51 . The exceptional complexity of RG-II makes its hydrolytic enzymes unusually well-suited for linkage-level selectivity, providing access to epitopes that are otherwise inaccessible to antibodies or CBMs. Moreover, this approach could potentially be extended to other CAZyme families, such as carbohydrate esterases, to generate novel tools for visualizing O -acetyl and methyl-ester modifications in situ . Guided by structural homology and active-site modeling, we generated multiple active site variants with the goal of disrupting hydrolysis while preserving substrate recognition. Ndeh et al. (2017) initially characterized individual B. theta enzymes using short oligosaccharides, synthetic substrates, or crude extracts a pseudo-substrates, focusing primarily on elucidating the PUL-mediated mechanism of RG-II depolymerization. Here, we used more physiologically relevant substrates to assess how specific residue substitutions influence catalytic activity and substrate selectivity, allowing more accurate predictions of each enzyme variant’s performance as a biological imaging probe (SI Fig. 2 D). Analysis of released sugars confirmed that both enzymes can efficiently hydrolyze their expected monosaccharide targets from RG-II monomer. In addition, mutations of the predicted catalytic residues markedly reduced activity on native substrates, with near-complete inactivation observed for sfGFP-BT1010 E516A and sfGFP-BT0996 E240A (Table 1 , Figs. 2 C, 3 C). These findings confirm that the chosen residues are essential for catalysis and that their modification abolishes activity without substantially perturbing overall protein structure. One notable observation from our biochemical analyses is that the native glycosidases displayed over 90% less activity when dimeric RG-II was provided as a substrate, supporting our hypothesis that covalent crosslinking of RG-II restricts enzyme accessibility (Table 1 , Figs. 2 D, 3 D). Single mutants with near-complete loss of enzymatic activity, sfGFP-BT1010 E516A and sfGFP-BT0996 E240A , did not interact with dimer but bound RG-II monomer with higher affinity than their native counterparts (Fig. 4 A-B, SI Fig. 5 ), suggesting that catalytic inactivation stabilizes enzyme–substrate interactions by eliminating turnover. Functional analysis showed that sfGFP-BT0996 N227A/E240A still bound RG-II monomer with high affinity, while sfGFP-BT1010 N389A/E516A and sfGFP-BT0996 E159Q/E240A showed weaker binding. All double mutants demonstrated reduced thermal stability and/or expression (SI Fig. 2 B-C), indicating that minor structural perturbations are occurring due to the mutations. The preference for RG-II monomer may be the result of evolutionary adaptation to the structural complexity of the polysaccharide. Because RG-II presents multiple, chemically diverse sidechains extending from a central backbone, recognition likely requires highly specific enzymes that cannot accommodate both monomeric and dimeric forms. Structural data further support this idea: the funnel-shaped active sites of Sidechain B–directed enzymes BT0986 and BT0996 encompass large portions of their respective oligosaccharide substrates, making them good probe candidates, but also suggests that binding depends on both steric accessibility and coordinated recognition within the full RG-II polymer 44 . These insights reinforce BT1010 and BT0996 as ideal candidates for probe development, as their selectivity toward unique sidechain motifs can be harnessed to achieve precise recognition of this complex pectic domain. BT1010 E516A and BT0996 E240A were shown to display high affinity for RG-II with K d values comparable to those of mAbs (K d ~10 µM) 31 and select pectin-directed CBMs (K d ~0.6–2.4 µM) 50,52,53 (Table 1 ). These results confirm our hypothesis that pectin-targeted glycosidases can be engineered into stable, high-affinity RG-II binding proteins with performance on par with established glycan-directed probes. Importantly, this strategy leverages the enzyme’s native substrate selectivity while preserving the target epitope during labeling. To confirm this, we used native BT0996 GH137 as a control to enzymatically remove the target glycosyl moiety prior to labeling. Pretreatment of plant sections with the active enzyme significantly reduced fluorescence from mRuby3-BT0996 E240A (Fig. 6 C, SI Fig. 8D), demonstrating that the fluorescent signal originates from specific recognition of intact RG-II. This result also highlights the necessity of catalytic inactivation to maintain epitope integrity during imaging. Collectively, these findings establish RG-II-directed GHs as promising scaffolds that combine enzyme-derived specificity with the binding strength of classical glyco-probes. We initially sought to identify proteins recognizing both RG-II monomer and dimer to develop complementary probes with distinct selectivity for each form. However, accurate K d values could not be determined for any enzyme when RG-II dimer was used as a substrate because the concentrations required to reach saturation generated substantial autofluorescence (Fig. 4 C-D, SI Fig. 7 ). While our results indicate weak binding, alternative methods will be necessary to quantify affinities precisely. This outcome is still consistent with our biochemical analyses, which demonstrated that all investigated proteins exhibit markedly lower affinity and glycosidic activity for RG-II dimer in vitro . The selectivity for RG-II monomer over dimer may reflect the different physiological environments encountered by dietary pectins in the human gastrointestinal tract, particularly with respect to pH, which strongly influences both microbiota composition and carbohydrate metabolism. For instance, it is well established that RG-II dimer dissociates into monomer under acidic conditions of 0.1 M HCl, pH 1 (SI Fig. 2 D) 18 . While B. theta primarily metabolizes dietary carbohydrates in the colon, where the pH ranges from 5–8, RG-II first passes through the stomach (pH 1.5–3.5) 54 , where the boron diester is likely cleaved prior to entering the gut. Thus, the observed enzymatic preference for RG-II monomer is consistent with the likelihood that these enzymes encounter RG-II that has already been hydrolyzed into its monomeric form. Interestingly, the genomes of soil bacteria such as Flavobacterium johnsoniae UW101 and Niabella soli DSM 19437 encode PULs with similar CAZyme composition, but distinct gene organization compared to B. theta 55 . Because these species do not encounter acidic environments that promote RG-II monomerization, it is possible that one or more of their PUL-encoded enzymes can recognize and hydrolyze dimeric RG-II through a different mechanism. We generated mRuby3-fusions with BT1010 E516A and BT0996 E240A to evaluate their potential as fluorescent probes to study RG-II localization in planta . Both probes successfully labeled sections from A. thaliana inflorescence stems following a 1-hour incubation, representing a considerably shorter protocol than conventional immunolabeling with mAbs (Fig. 5 ). Labeling with mRuby3-BT0996 E240A produced a slightly stronger signal than mRuby3-BT1010 E516A , which we attributed to the improved diffusion of the smaller GH137 probe into the highly interconnected regions of the primary cell wall network (SI Fig. 2 A, 8A). Intense fluorescence was observed at the middle lamellar cell corners of the primary cell wall (Fig. 5 , SI Fig. 8B, 9C), pectin-rich areas that cement together the walls of adjoining cells and contribute to the mechanical strength and elasticity of growing tissues 56 , 57 . This localization is consistent with our probe recognizing and binding a structural molecule like RG-II that can form covalent linkages between larger pectic polymers 58 . For assessing the utility of our RG-II specific probes, we performed several control experiments using plant tissue sections to validate that the observed signal arises from specific binding of mRuby3-BT0996 E240A to RG-II (Fig. 6 A, SI Fig. 8B). First, we probed stem sections with mRuby3 and showed there was no detectable fluorescence, excluding non-specific binding of the fluorophore (Fig. 6 B, SI Fig. 8C). Next, we pretreated plant tissue sections with native BT0996 GH137 to cleave the terminal l -Ara f from Sidechain B. When these sections were then labelled with mRuby3-BT0996 E240A , we observed markedly reduced fluorescence intensity, clearly demonstrating that labeling depends on the integrity of the target epitope (Fig. 6 C, SI Fig. 8D). Finally, competitive inhibition assays further supported the specificity of mRuby3-BT0996 E240A . Preincubation of the probe with purified RG-II monomer (Fig. 6 D, SI Fig. 8E) or dimer (Fig. 6 E, SI Fig. 8F) prior to labeling resulted in reduced fluorescence, with monomer preincubation causing a significantly greater decrease (Fig. 6 F). These results align with the in vitro biochemical and binding data and confirm that the probe preferentially recognizes monomeric RG-II. Collectively, our combined data demonstrate that labeling depends on the integrity and accessibility of the RG-II target and confirms that mRuby3-BT0996 E240A is a selective probe for RG-II, with a preference for monomer. To contextualize our probe labeling patterns within the broader cell wall architecture, we compared mRuby3-BT0996 E240A with established mAbs recognizing other matrix polysaccharides. Our data shows that mRuby3-BT0996 E240A and mRuby3-BT1010 E516A specifically label middle lamellar corners between adjoining cells (Fig. 5 , Fig. 7 C, SI Fig. 9C). This is quite distinct from CCRC-M38, which binds de-esterified HG, and uniformly labeled parenchyma primary cell walls (Fig. 7 A, SI Fig. 9A). We also evaluated CCRC-M14, which targets the RG-I backbone, and showed it specifically labels the middle lamella and intercellular spaces (Fig. 7 B, SI Fig. 9B). Labeling with CCRC-M1, which detects fucosylated xyloglucan, labelled regions distinct from those of all pectin-directed probes evaluated here (SI Fig. 9D). The distribution of HG and RG-I likely reflects their structural flexibility and ability to be remodeled during growth, adhesion, and abscission, processes that demand dynamic wall plasticity 59 , 60 . In contrast, RG-II localization at cell corners suggests enrichment in crosslinking hotspots that provide rigidity and cohesion 11 , 61 . These findings highlight functional partitioning among pectic domains, with RG-II reinforcing junctional integrity while HG and RG-I mediate wall flexibility. Future studies will involve using established mutants defective in aspects of RG-II synthesis, to investigate the selectivity of our probe library. However, because fully intact RG-II is essential for plant viability, relatively few mutants with modified RG-II structure have been identified and those that have are known to accumulate predominantly monomer 22 , 24 – 30 . For example, the A. thaliana murus 1 ( mur1 ) mutant lacks the ability to synthesize GDP- l -Fucose (Fuc), resulting in a ~ 50% reduction of the terminal 2- O -methyl xylose in Sidechain A and substitution of the terminal l -Fuc in Sidechain B with l -Gal 22 . Likewise, Arabidopsis lines with suppressed expression of Golgi GDP- l -galactose transporter1 ( GGLT1 ) exhibit up to a 50% reduction in Sidechain A l -Gal 26 . While such plants could serve as valuable negative controls for probes recognizing these epitopes, a key limitation is that disrupting the glycosyl sequence of RG-II impairs dimer formation, resulting in mutant lines with elevated levels of RG-II monomer. For instance, Sechet et al. (2018) found the abundance of monomer in EPG-treated AIR from gglt and mur1-1 lines increased from 23% in wild type to 52% and 94%, respectively. Because our current probes preferentially recognize monomeric RG-II, these lines may not be the best biological samples to reliably distinguish structural effects on crosslinking from those on epitope abundance, thereby complicating direct comparisons of fluorescence intensity. In conclusion, biological imaging tools are essential for elucidating the functions of individual wall components, as precise localization data can inform strategies to enhance plant properties such as biomass yield, stress tolerance, and nutrient uptake 62 – 64 . Advances in understanding pectin structure have largely centered on HG, supported by the availability of mutants affecting its biosynthesis or methylesterification, selective probes for its modification states, and the powerful spectroscopic and microscopic imaging technologies that are available. These approaches have uncovered direct links between pectin and cellulose synthesis 65 , cell wall integrity 66 – 69 , and the growth and morphogenesis of distinct cell types 70 , 71 . Yet, current tools still fall short in their ability to capture the full structural diversity and biological function of pectins within the wall. Here, we have begun to address these limitations by expanding imaging capabilities to the structurally complex domain RG-II and establish mRuby3-BT0996 E240A and mRuby3-BT1010 E516A among the first fluorescent probes with defined specificity for RG-II monomer, enabling high-precision mapping of its localization and dynamics in planta . Their application opens new opportunities for synthetic biology approaches, as these enzyme-derived probes can serve as genetically encoded biosensors for tracking RG-II remodeling during development or stress responses. When integrated with existing HG- and RG-I–directed tools, they will facilitate a systems-level view of pectin organization and function. More broadly, this strategy establishes a versatile framework for repurposing CAZymes as molecular probes, extending high-fidelity glycan imaging to the most structurally intricate components of the plant cell wall. Methods Generation of fluorescent protein constructs The genes encoding BT1010 (GenBank: AAO76117.1) and BT0996 (GenBank: AAO76103.1) were the kind gift of Dr. Harry Gilbert and had been previously cloned into pET28a(+) for recombinant expression as N-terminal His 6 -tagged fusion proteins, as described in Ndeh et al. (2017) 44 . Truncated sequences encoding the GH95 and GH137 glycoside hydrolase domains of BT1010 and BT0996, respectively, were amplified by PCR. The resulting BamHI/XhoI-restricted fragments were cloned into the pET28a T7pCONS TIR-2 sfGFP vector 73 , in-frame downstream of the gene encoding Superfolder GFP (sfGFP) 74 . The resulting expressed protein consisted of an N-terminal His 6 tag, sfGFP, and the indicated GH domain. To minimize interference from plant cell wall autofluorescence in the green channel, the T7pCONS vector was modified to encode alternative fluorescent proteins with red-shifted emission spectra. Constructs containing mRuby3 (GenBank: ATE88097.1) 48 , mScarlet-I (GenBank: APD76536.1) 75 , and TagRFP (GenBank: ABR08320.1) 76 were generated using NdeI/BamHI-restricted gene fragments synthesized by Twist Bioscience (https://www.twistbioscience.com/). Among these, mRuby fusion proteins were selected for microscopy owing to their improved brightness, photostability, and acid tolerance 77 . Site-Directed Mutagenesis Models for native and variant proteins were generated with AlphaFold 78 and analyzed using Pymol (The PyMOL Molecular Graphics System, Version 3.0.4 Schrödinger, LLC). Mutagenesis of BT1010 GH95 and BT0996 GH137 was performed using the Q5 ® Site-Directed Mutagenesis Kit (New England Biolabs) using the primers listed in SI Figure 1A. The resulting constructs were confirmed by DNA sequencing. Protein purification All chemicals and reagents were purchased from Thermo Fisher Scientific unless otherwise noted. Plasmids were transformed into Escherichia coli strains BL21(DE3) or Tuner(DE3) (Novagen) competent cells for recombinant protein expression. Cells were cultured at 37°C in Luria–Bertani (LB) medium supplemented with kanamycin (50 μg ml −1 ) to mid‐exponential phase (OD 600 nm = 0.6‐0.8). Recombinant gene expression was induced by the addition of isopropyl β-d-galactopyranoside (IPTG), with the optimal concentration, expression temperature, and induction time differing for certain proteins (SI Figure 2A). For purification of recombinant fusion proteins, cell pellets were resuspended in Buffer A (20 mM Tris-HCl, 300 mM NaCl, pH 8.0), supplemented with 10 μL of 100 mM PMSF per mL of cell suspension, and lysed using an Emulsiflex C3 (Avestin) according to manufacturer’s recommendations for E. coli . Recombinant, His-tagged fusion proteins were purified from clarified cell lysates by immobilized metal ion affinity chromatography (IMAC) using HisPur™ Cobalt Resin (Thermo Fisher Scientific). The resin was washed with Buffer A, and bound, His-tagged proteins were eluted with Buffer B (150 mM Imidazole, 20 mM Tris, 300 mM NaCl, pH 8.0). Protein purity was ascertained using SDS-PAGE. When cleaved sfGFP or mRuby3 was noted, recombinant fusion proteins were further purified by size exclusion chromatography (SEC) in 20 mM MES, 400 mM NaCl, pH 5.5, at 4°C using a HiLoad 16/600 Superdex 200 pg column (Cytiva) on an AKTA 25L system (Cytiva). Purified proteins were dialyzed into storage buffer (20 mM MES, 400 mM NaCl, pH 5.5) using D‐Tube™ Dialyzer Maxi (MWCO 6–8 kDa) or Midi (MWCO 3.5 kDa) devices (EMD Millipore). Chelex® 100 Resin (Bio-Rad) was added to all dialysis buffer to remove any remaining divalent cations. Final protein concentrations were derived with the MW and extinction coefficient of each protein, as obtained using Geneious version 2024.0 (https://www.geneious.com). Isolation, characterization, and preparation of RG-II substrates The biochemical characterization of our recombinant probe library was conducted using RG-II substrates isolated from red wine, as described in Barnes et al., 2021 18 . After extraction, crude RG-II generally consists of both monomeric and dimeric glycoforms, thus calling for in vitro monomerization and dimerization, respectively, to generate homogenous substrates 23,79 . Monomeric RG-II was prepared according to the methods of O’Neill et al. (1996). Briefly,a solution of crude wine RG-II (2.5 mg/1 mL) was prepared in 0.1 M HCl, gently mixed at room temperature for 3 hours, then dialyzed (Spectrum™ Spectra/Por™, 3500 Dalton MWCO) against deionized water and freeze dried. RG-II monomer readily dimerizes in solution, so it is important to avoid borosilicate glass, utilize Chelex® 100 Resin (Bio-Rad) during dialysis, and prepare fresh aliquots of substrate for experiments. Dimeric RG-II was prepared according to previous methods 20 . Briefly, RG-II monomer (0.2 mM; Mw 4971 Da) was dissolved in Dimerization Buffer (1 mM boric acid, 0.5 mM lead nitrate, in 50 mM NaOAc pH 3.9) and gently mixed at room temperature for 3 hours. The solution was then dialyzed (Spectrum™ Spectra/Por™, 3500 Dalton MWCO) against deionized water and freeze dried. The homogeneity of RG-II monomer and dimer preparations was confirmed by SEC (SI Figure 2D). A 50 μg sample was analyzed on a Superdex 75 Increase 10/300 GL column (Cytiva) using a 1260 Infinity II LC system (Agilent) equipped with a refractive index detector (G7162A, Agilent), as previously described 18 . High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection ( HPAEC-PAD) The optimal buffer composition for biochemical assays was determined by protein thermal shift assays using SYPROä Orange dye (Thermo Fisher) to monitor protein stabilization under varying buffer, pH, and salt conditions 80 (SI Figure 2B-C). Fluorescent signals were measured on a Bio-Rad™ CFX Real-Time PCR System and analyzed with CFX Maestro Software version 2.3 (Bio-Rad). To determine the activity of RG-II specific GHs, reactions were prepared with 1 μM protein in Reaction Buffer (20 mM MES, 100 mM NaCl, pH 5.5) and either 1 mM of RG-II monomer or 0.5 mM of RG-II dimer as saccharide substrates. Reactions were incubated at 37°C for 1 hour, followed by a 95°C heat inactivation for 5 minutes, then filtered using a 96-well filter microplate with a 0.45 µm nylon membrane (Agilent). The specific activities of native and variant BT1010 GH95 and BT0996 GH137 were determined according to the respective release of l-Gal and l-Ara f from monomeric and dimeric RG-II substrates using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Briefly, l-Gal and l-Ara f were quantified using a Dionex ICS 6000 system (Thermo Fisher Scientific) with a Dionex CarboPac PA1 column equipped with an AminoTrap guard column. After injection (25 μL), monosaccharides were separated isocratically at a rate of 1.0 mL/min with an initial flow of NaOH (0-15 min: 32 mM), followed by a multi-step gradient of sodium acetate in 100 mM NaOH (15-35 min: 0-250 mM; 35-45 min: 250-1000 mM; 45-48 min: 1000 mM; 48-51 min: 1000-0 mM; 51-60 min: 0 mM). The activity of each native and variant enzyme was quantified using 3 distinctly expressed and purified protein preparations (biological replicates, n = 3), and quadruplicate HPAEC-PAD injections. The rate of activity for each enzyme is reported as the mass of monosaccharide product released per minute per μM of protein (ng∙min -1 ∙μmol -1 ). MicroScale Thermophoresis (MST) MicroScale Thermophoresis (MST) was used to analyze the thermodynamic relationship between sfGFP-labeled native and variant enzymes and monomeric and dimeric RG-II substrates. MST parameters were optimized according to the workflow described by 81 . Fluorescent fusion proteins (sfGFP) were adjusted to a working concentration of 1 μM in MST Buffer (20 mM MES, 400 mM NaCl, pH 5.5) supplemented with 0.1% Tween 20 (Bio-Rad) to reduce aggregation and adsorption. RG-II substrates were dialyzed into 20 mM MES pH 5.5, adjusted to a starting concentration of 0.5 mM, then used to generate a serially diluted concentration gradient from 500 μM to 30.6 nM. An equal volume of prepared protein was added to each RG-II dilution, producing a final reaction concentration of 500 nM protein in 20 mM MES, 200 mM NaCl, 0.05% Tween pH 5.5 and ligand concentrations ranging from 250 μM to 15.3 nM. The samples were incubated at 37°C for 15 min and centrifuged at 15,000 x g for 5 min prior to loading reactions into standard Monolith NT.115 Capillaries (NanoTemper Technologies). MST was measured using a Monolith NT.115 instrument (NanoTemper Technologies) at an ambient temperature of 23°C. Instrument parameters were adjusted to 10-30% LED power and high MST power. Data of six independently pipetted measurements were analyzed (MO.Affinity Analysis software version 2.1.3, NanoTemper Technologies) using the signal from an MST-on time of 1.5 or 5 s (SI Figures 5-7). Statistical Analysis For HPAEC-PAD experiments, peak areas were quantified using Chromeleon™ Chromatography Data System version 7.3 (Thermo Scientific). Calculation of reaction rates (ng∙min -1 ∙μM -1 ) and subsequent statistical analyses were conducted using MATLAB R2024b (MathWorks). Technical variability was assessed from four independent reactions, reported as the standard deviation (SI Figures 3-4). Biological variability was determined from the mean of n = 3 independent protein expressions and purifications, reported as the standard error of the mean (Table 1). Significant differences between native and variant enzymes were determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test, which corrects for multiple comparisons against a single control. To compare enzyme activity toward RG-II monomer versus dimer substrates, we applied paired two-tailed t-tests for each enzyme, since the same biological replicates were assayed on both substrates. Resulting P -values were adjusted for multiple comparisons across the enzymes using the Bonferroni-Holm method. Test statistics ( F values, t values, degrees of freedom, and adjusted P values) are reported in SI Table 1. MST data was analyzed with MO.Affinity Analysis Software version 2.1.3 (NanoTemper) and fit using a K d model with fixed target concentration. Dose-response binding curves were generated from independently pipetted measurements (exact n values are provided in Table 1). Dissociation constants (K d ) and their confidence intervals were derived from nonlinear least-squares fitting, and replicate quality was assessed using software-provided parameters, including root mean squared error and reduced χ 2 (Table 1). Data are presented as the fraction bound plotted against ligand concentration, in which the change in normalized fluorescence is normalized to the curve amplitude to yield values between 0 and 1. Representative MST traces, depicting the change in fluorescence intensity of one capillary over time, are provided to demonstrate data quality and confirm the absence of aggregation and photobleaching of the target molecule (SI Figures 5-7). Plant preparation Arabidopsis thaliana ecotype Columbia-0 ( Col-0 ) was used as wild type. Sterilized seeds were stratified in the dark for 2 days at 4 °C before plating on ½ strength Murashige and Skoog medium (Sigma Aldrich), 0.1% (w/v) MES (Beantown Chemical), 1% (w/v) sucrose (Sigma Aldrich), 0.8% (w/v) agar (Dot Scientific), pH 5.6. Plates were sealed and incubated vertically at 19°C/15°C with a 16-h-light/8-h-dark photoperiod.10-day old seedlings were placed on a soil mixture consisting of a 1:1 ratio of Metromix 830 (Sungro) and Vermiculite, which was supplemented with Plant Food (Sta-Green) and Osmocote Smart-Release Plant Food Flower & Vegetable (Scotts) before being moved to a walk-in growth chamber (Conviron). Plants were grown under an 18 h light/ 6 h dark cycle at 21 °C with a light intensity of 120 μmol photons m −2 s −1 . Microscopy sample preparation Stem sections were harvested from the middle to upper regions of A. thaliana inflorescence stems using double-edged razor blades (Electron Microscopy Sciences). To inactivate endogenous cell wall–modifying enzymes, sections were blanched for 2 min at 90 °C, followed by stepwise dehydration under vacuum in 30%, 50%, and 70% ethanol (v/v) for 15 min each. Samples were then washed three times with gentle shaking for 15 min each in Wash Buffer (20 mM MES, 0.01% Tween-20, pH 5.5). To reduce nonspecific probe binding, sections were blocked overnight at 4 °C with gentle shaking in Blocking Buffer (1X Pierce™ Clear Milk Blocking Buffer (Thermo Fisher Scientific) prepared in 20 mM MES, pH 5.5). After three additional washes, samples were incubated with gentle shaking for 1 hr at 30 °C in 200 μL of 0.4 μM of mRuby3-BT0996 E240A in Blocking Buffer. Control labeling with purified mRuby3 alone was performed at the same concentration. Sections were washed three times prior to mounting on microscope slides and imaging. For enzyme digest controls, sections were pretreated with 1 μM native BT0996 GH137 in Blocking Buffer at 30 °C with gentle shaking for 1 hr, followed by blanching to inactivate the enzyme prior to blocking and labeling. Competitive inhibition controls were performed by preincubating 0.4 μM of mRuby3-BT0996 E240A with purified RG-II monomer (2 mM) or dimer (1 mM) in Blocking Buffer for 1 hr at 30 °C before adding to sections under identical labeling conditions. Immunolabeling procedure was guided by 82 and performed with minor modifications. Stem sections were fixed in 1.6% (v/v) paraformaldehyde (Electron Microscopy Sciences) in 25 mM sodium phosphate buffer, pH 7.1, for 30 min under vacuum. Fixed samples were washed three times with gentle shaking for 15 min each in 25 mM sodium phosphate buffer, pH 7.1, followed by stepwise dehydration under vacuum in 30%, 50%, and 70% ethanol (v/v) for 15 min each. Sections were rehydrated and washed three times with gentle shaking for 5 min each in KPBS (10 mM potassium phosphate buffer, pH 7.1, 500 mM NaCl), then blocked overnight at 4 °C with gentle shaking in 1X Pierce™ Clear Milk Blocking Buffer in KPBS. Primary monoclonal antibodies CCRC-M38, CCRC-M14, and CCRC-M1 (CarboSource) were used to label de-esterified HG, RG-I backbone, and fucosylated XG, respectively 32 . Sections were incubated for 1 hr at RT with gentle shaking in a 1:20 dilution of primary antibody in KPBS, washed three times, and then incubated for 1 hr in the dark with gentle shaking in a 1:100 dilution of goat anti-mouse secondary antibody conjugated to Alexa Fluor 594 (Abcam). Sections were washed three times prior to mounting and imaging. Widefield epifluorescence microscopy Fluorescence imaging was performed using a Nikon Eclipse 80i epifluorescence microscope equipped with a Nikon DS-Ri2 color camera and a Nikon Intensilight C-HGFIE light source. Excitation and emission were detected using standard DAPI, FITC, and TRITC filter blocks. Images were collected at 10X magnification using a Plan Fluor 10X DIC N1 objective (200 μm z-stack, 5.6 μm z-step, 400 ms exposure) or at 20X magnification using a Plan Fluor 20X DIC N2 objective (75 μm z-stack, 2 μm z-step, 400 ms exposure). All images were acquired at 100% transmittance with gain of 1X. Z-stacks were processed into maximum intensity projections using (Fiji Is Just) ImageJ version 2.16.0/1.54p 83 . Brightness and contrast were not adjusted prior to further analyses using MATLAB R2024b (MathWorks). A complete description of microscope hardware, optics, acquisition settings, and quality control assessment is provided in the Light Microscopy Reporting Table (SI Table 2). Image Analysis Workflow Images were processed using a custom MATLAB code available at https://github.com/kristenthorne/GHprobes.git, with usage instructions and example input and output files provided. Raw z-stacks and maximum projections (original, scaled, and masked) for all quantified images are available in Open Science Framework. For each scaled maximum intensity projection, pixel intensity values were converted to double-precision and normalized to the interval [0,1] to minimize variability in exposure and background between acquisitions. A single representative threshold was applied uniformly across all images acquired within the same experiment to ensure comparability. The average threshold was determined from images labeled with mRuby3-BT0996 E240A using Otsu’s method 84 , which minimizes within-class variance and provides a data-driven separation of signal from background. Segmentation with this average threshold generated binary masks that defined regions of positive signal. From each mask, total signal area (number of positive pixels) and a normalized area metric (square root of area) were calculated. Fluorescence intensities were quantified on the original, non-normalized images within the masked regions, extracting the following metrics: (1) mean, standard deviation, minimum, and maximum gray value of thresholded pixels; (2) raw integrated density (sum of all pixel intensities in the region); and (3) integrated density (mean gray value × area). Results were stored in structured arrays and exported for downstream statistical analysis. Raw integrated density was used as the primary metric for comparing probe signal between mRuby3-BT0996 E240A and control samples. Statistical Analysis Significant differences between the mean raw integrated densities of mRuby3-BT0996 E240A and control images were determined by ANOVA using a linear mixed-effects model, with condition as a fixed effect and experiment date as a random effect. P -values were calculated from pairwise contrasts between each fixed effect and adjusted for multiple comparisons with the Bonferroni method. Test statistics ( F values, t values, degrees of freedom, and adjusted P values) are reported in SI Table 1. References Barnes, W. J. et al. Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II. Biotechnol Biofuels 14 , 142 (2021). https://doi.org/10.1186/s13068-021-01992-0 O’Neill, M. A. et al. Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide rhamnogalacturonan II. SLAS TECHNOLOGY: Translating Life Sciences Innovation 25 , 329–344 (2020). https://doi.org/10.1177/2472630320923321 Ishii, T. et al. The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer. Journal of Biological chemistry 274 , 13098–13104 (1999). Pattathil, S. et al. A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. Plant Physiology 153 , 514–525 (2010). https://doi.org/10.1104/pp.109.151985 Ndeh, D. et al. Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature 544 , 65–70 (2017). https://doi.org/10.1038/nature21725 Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci Rep 6 , 20889 (2016). https://doi.org/10.1038/srep20889 Varki, A. et al. Symbol Nomenclature for Graphical Representations of Glycans. Glycobiology 25 , 1323–1324 (2015). https://doi.org/10.1093/glycob/cwv091 Shilling, P. J. et al. Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. Commun Biol 3 , 214 (2020). https://doi.org/10.1038/s42003-020-0939-8 Pédelacq, J.-D. et al. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24 , 79–88 (2006). https://doi.org/10.1038/nbt1172 Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nature Methods 14 , 53–56 (2017). https://doi.org/10.1038/nmeth.4074 Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nature Methods 4 , 555–557 (2007). https://doi.org/10.1038/nmeth1062 Stoddard, A. & Rolland, V. I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves. Plant Direct 3 , e00112 (2019). https://doi.org/https://doi.org/10.1002/pld3.112 Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583–589 (2021). https://doi.org/10.1038/s41586-021-03819-2 O'Neill, M. A. et al. Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. Journal of Biological Chemistry 271 , 22923–22930 (1996). https://doi.org/10.1074/jbc.271.37.22923 Huynh, K. & Partch, C. L. Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay. Current Protocols in Protein Science 79 , 28.29.21–28.29.14 (2015). https://doi.org/https://doi.org/10.1002/0471140864.ps2809s79 Wu, H., Montanier, C. Y. & Dumon, C. Quantifying CBM Carbohydrate Interactions Using Microscale Thermophoresis. Methods Mol Biol 1588 , 129–141 (2017). https://doi.org/10.1007/978-1-4939-6899-2_10 Avci, U., Pattathil, S. & Hahn, M. G. in Biomass Conversion: Methods and Protocols (ed Michael E. Himmel) 73–82 (Humana Press, 2012). Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 9 , 676–682 (2012). https://doi.org/10.1038/nmeth.2019 Otsu, N. A Threshold Selection Method from Gray-Level Histograms. IEEE Transactions on Systems, Man, and Cybernetics 9 , 62–66 (1979). https://doi.org/10.1109/TSMC.1979.4310076 Declarations Data availability Microscopy data that support the findings of this study have been deposited in Open Science Framework. Raw z-stacks, output maximum intensity projections, and annotated figure images in greyscale are available at (https://osf.io/8utvs/overview). Code availability MATLAB code used for image analysis is available at https://github.com/kristenthorne/GHprobes.git, with usage instructions and example input and output files provided. Acknowledgements Funding for research on RG-II was provided by the US Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Division, under award DESC0008472 to B.R.U. for funding structural studies of RG-II. Author contributions K. J. T: Conceptualization; Methodology (lead); Investigation (lead); Data Curation (lead); Writing – Original Draft (lead); Visualization (lead); Software (development of custom processing script). W.J.B: Conceptualization; Software (development of custom processing script); Validation; Writing – Review & Editing (critical input on methods). H.H: Investigation. B.R.U. (Corresponding Author): Conceptualization; Resources; Funding acquisition; Project administration; Supervision; Resources; Writing – Review & Editing. All authors have read and approved the final manuscript. Competing interests The authors declare no competing interests. Materials & Correspondance Supplementary Information is available for this paper. Correspondence and requests for materials should be addressed to [email protected] . References Bidhendi, A. J. & Geitmann, A. Relating the mechanics of the primary plant cell wall to morphogenesis. J Exp Bot 67 , 449–461 (2016). https://doi.org/10.1093/jxb/erv535 Lund, C. H. et al. Pectin Synthesis and Pollen Tube Growth in Arabidopsis Involves Three GAUT1 Golgi-Anchoring Proteins: GAUT5, GAUT6, and GAUT7. Frontiers in Plant Science Volume 11 - 2020 (2020). https://doi.org/10.3389/fpls.2020.585774 Cai, M. et al. ROS homeostasis and cell wall biosynthesis pathway are involved in female inflorescence development of birch (Betula platyphylla). BMC Plant Biology 25 , 708 (2025). https://doi.org/10.1186/s12870-025-06740-2 Houston, K. et al. The plant cell wall: A complex and dynamic structure as revealed by the responses of genes under stress conditions. Front Plant Sci 7 , 984 (2016). https://doi.org/10.3389/fpls.2016.00984 Chebli, Y., Bidhendi, A. J., Kapoor, K. & Geitmann, A. Cytoskeletal regulation of primary plant cell wall assembly. Current Biology 31 , R681–R695 (2021). https://doi.org/10.1016/j.cub.2021.03.092 Altartouri, B. et al. Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism1 [OPEN]. Plant Physiology 181 , 127–141 (2019). https://doi.org/10.1104/pp.19.00303 Hu, K. et al. Improvement of multiple agronomic traits by a disease resistance gene via cell wall reinforcement. Nature Plants 3 , 17009 (2017). https://doi.org/10.1038/nplants.2017.9 Zhang, Y. et al. Molecular insights into the complex mechanics of plant epidermal cell walls. Science 372 , 706–711 (2021). https://doi.org/doi:10.1126/science.abf2824 Vaahtera, L., Schulz, J. & Hamann, T. Cell wall integrity maintenance during plant development and interaction with the environment. Nature Plants 5 , 924–932 (2019). https://doi.org/10.1038/s41477-019-0502-0 Atmodjo, M. A., Hao, Z. & Mohnen, D. Evolving views of pectin biosynthesis. Annual review of plant biology 64 , 747–779 (2013). Barnes, W. J., Zelinsky, E. & Anderson, C. T. Polygalacturonase activity promotes aberrant cell separation in the quasimodo2 mutant of Arabidopsis thaliana. The Cell Surface 8 , 100069 (2022). https://doi.org/https://doi.org/10.1016/j.tcsw.2021.100069 Wang, T., Park, Y. B., Cosgrove, D. J. & Hong, M. Cellulose-pectin spatial contacts are inherent to never-dried Arabidopsis primary cell walls: Evidence from solid-state nuclear magnetic resonance Plant Physiology 168 , 871–884 (2015). https://doi.org/10.1104/pp.15.00665 Tan, L. et al. An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein. The Plant Cell 25 , 270–287 (2013). https://doi.org/10.1105/tpc.112.107334 Zhang, L. et al. The pectin puzzle: Decoding the fine structure of rhamnogalacturonan-I (RG-I) in Arabidopsis thaliana uncovers new pectin features. Carbohydrate Polymers 368 , 124161 (2025). https://doi.org/https://doi.org/10.1016/j.carbpol.2025.124161 Mohnen, D., Atmodjo, M. A. & Jayawardhane, P. in Plant Cell Walls: Research Milestones and Conceptual Insights (ed Anja Geitmann) 94–126 (CRC Press, 2023). Phyo, P., Gu, Y. & Hong, M. Impact of acidic pH on plant cell wall polysaccharide structure and dynamics: insights into the mechanism of acid growth in plants from solid-state NMR. Cellulose 26 , 291–304 (2019). https://doi.org/10.1007/s10570-018-2094-7 O'Neill, M. A., Ishii, T., Albersheim, P. & Darvill, A. G. Rhamnogalacturonan II: Structure and function of a borate cross-linked cell wall pectic polysaccharide. Annual Review of Plant Biology 55 , 109–139 (2004). https://doi.org/10.1146/annurev.arplant.55.031903.141750 Barnes, W. J. et al. Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II. Biotechnol Biofuels 14 , 142 (2021). https://doi.org/10.1186/s13068-021-01992-0 Matsunaga, T. et al. Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in Pteridophytes, Lycophytes, and Bryophytes. Implications for the evolution of vascular plants. Plant Physiology 134 , 339–351 (2004). https://doi.org/10.1104/pp.103.030072 O’Neill, M. A. et al. Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide rhamnogalacturonan II. SLAS TECHNOLOGY: Translating Life Sciences Innovation 25 , 329–344 (2020). https://doi.org/10.1177/2472630320923321 Funakawa, H. & Miwa, K. Synthesis of borate cross-linked rhamnogalacturonan II. Frontiers in Plant Science 6 , 223 (2015). https://doi.org/10.3389/fpls.2015.00223 O'Neill, M. A., Eberhard, S., Albersheim, P. & Darvill, A. G. Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science 294 , 846–849 (2001). https://doi.org/doi:10.1126/science.1062319 Ishii, T. et al. The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer. Journal of Biological chemistry 274 , 13098–13104 (1999). Ahn, J.-W. et al. Depletion of UDP-D-apiose/UDP-D-xylose Synthases Results in Rhamnogalacturonan-II Deficiency, Cell Wall Thickening, and Cell Death in Higher Plants. Journal of Biological Chemistry 281 , 13708–13716 (2006). https://doi.org/10.1074/jbc.m512403200 Delmas, F. et al. The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation. Journal of Experimental Botany 59 , 2639–2647 (2008). https://doi.org/10.1093/jxb/ern118 Sechet, J. et al. Suppression of Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan-II. The Plant Journal 96 , 1036–1050 (2018). https://doi.org/10.1111/tpj.14088 Zhao, X. et al. UDP-Api/UDP-Xyl synthases affect plant development by controlling the content of UDP-Api to regulate the RG-II-borate complex. Plant J 104 , 252–267 (2020). https://doi.org/10.1111/tpj.14921 Zhang, Y. et al. Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing which bypasses embryo lethality. Plant Physiology 195 , 2551–2565 (2024). https://doi.org/10.1093/plphys/kiae259 Voxeur, A. et al. Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato. Journal of Biological Chemistry 286 , 8014–8020 (2011). https://doi.org/10.1074/jbc.m110.198614 Dumont, M. et al. The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein. Annals of Botany 114 , 1177–1188 (2014). https://doi.org/10.1093/aob/mcu093 Thorne, K. J., Urbanowicz, B. R. & Hahn, M. G. in Plant Cell Walls: Research Milestones and Conceptual Insights (ed Anja Geitmann) (CRC Press/Taylor & Francis Group, 2023). Pattathil, S. et al. A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. Plant Physiology 153 , 514–525 (2010). https://doi.org/10.1104/pp.109.151985 Williams, M. N. et al. An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells. Plant Cell 8 , 673–685 (1996). https://doi.org/10.1105/tpc.8.4.673 Zhou, Y. et al. Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells. Bioscience, Biotechnology, and Biochemistry 81 , 899–905 (2017). https://doi.org/10.1080/09168451.2016.1270740 Zhou, Y. et al. A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots. Bioscience, Biotechnology, and Biochemistry 82 , 1780–1789 (2018). https://doi.org/10.1080/09168451.2018.1485479 Rodrı́guez-Carvajal, M. A. et al. The three-dimensional structure of the mega-oligosaccharide rhamnogalacturonan II monomer: a combined molecular modeling and NMR investigation. Carbohydrate Research 338 , 651–671 (2003). https://doi.org/https://doi.org/10.1016/S0008-6215(03)00003-X Zhang, X. et al. Understanding How the Complex Molecular Architecture of Mannan-degrading Hydrolases Contributes to Plant Cell Wall Degradation *. Journal of Biological Chemistry 289 , 2002–2012 (2014). https://doi.org/10.1074/jbc.M113.527770 Pattathil, S. et al. Immunological approaches to biomass characterization and utilization. Frontiers in Bioengineering and Biotechnology 3 , 173 (2015). https://doi.org/10.3389/fbioe.2015.00173 Verhertbruggen, Y. et al. An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydrate Research 344 , 1858–1862 (2009). https://doi.org/10.1016/j.carres.2008.11.010 Beaugrand, J. et al. Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase. Planta 222 , 246–257 (2005). https://doi.org/10.1007/s00425-005-1538-0 Dornez, E. et al. Inactive fluorescently labeled xylanase as a novel probe for microscopic analysis of arabinoxylan containing cereal cell walls. J Agric Food Chem 59 , 6369–6375 (2011). https://doi.org/10.1021/jf200746g Shon, D. J. et al. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proc Natl Acad Sci U S A 117 , 21299–21307 (2020). https://doi.org/10.1073/pnas.2012196117 Hombu, R. et al. Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform. Nature Communications 16 , 6637 (2025). https://doi.org/10.1038/s41467-025-62018-z Ndeh, D. et al. Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature 544 , 65–70 (2017). https://doi.org/10.1038/nature21725 Nagae, M. et al. Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum*. Journal of Biological Chemistry 282 , 18497–18509 (2007). https://doi.org/https://doi.org/10.1074/jbc.M702246200 Trovão, F. et al. The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome. Journal of Structural Biology: X 7 , 100084 (2023). https://doi.org/https://doi.org/10.1016/j.yjsbx.2022.100084 García-Plazaola, J. I. et al. Autofluorescence: Biological functions and technical applications. Plant Science 236 , 136–145 (2015). https://doi.org/https://doi.org/10.1016/j.plantsci.2015.03.010 Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci Rep 6 , 20889 (2016). https://doi.org/10.1038/srep20889 Paës, G. Fluorescent Probes for Exploring Plant Cell Wall Deconstruction: A Review. Molecules 19 , 9380–9402 (2014). Liu, G. et al. Discovery and Structural Analysis of a Novel Pectin-Specific Carbohydrate-Binding Module: The First Member of a New CBM Family. Journal of Agricultural and Food Chemistry 73 , 24278–24285 (2025). https://doi.org/10.1021/acs.jafc.5c07437 Dhillon, A., Sharma, K., Rajulapati, V. & Goyal, A. The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I. Archives of Biochemistry and Biophysics 654 , 194–208 (2018). https://doi.org/https://doi.org/10.1016/j.abb.2018.07.023 Abbott, D. W., Hrynuik, S. & Boraston, A. B. Identification and Characterization of a Novel Periplasmic Polygalacturonic Acid Binding Protein from Yersinia enterolitica. Journal of Molecular Biology 367 , 1023–1033 (2007). https://doi.org/https://doi.org/10.1016/j.jmb.2007.01.030 Venditto, I. et al. Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition. Proceedings of the National Academy of Sciences 113 , 7136–7141 (2016). https://doi.org/10.1073/pnas.1601558113 Brinck, J. E. et al. Intestinal pH: a major driver of human gut microbiota composition and metabolism. Nature Reviews Gastroenterology & Hepatology 22 , 639–656 (2025). https://doi.org/10.1038/s41575-025-01092-6 Terrapon, N. et al. PULDB: the expanded database of Polysaccharide Utilization Loci. Nucleic Acids Research 46 , D677–D683 (2018). https://doi.org/10.1093/nar/gkx1022 Chen, D. et al. Changes in the orientations of cellulose microfibrils during the development of collenchyma cell walls of celery (Apium graveolens L.). Planta 250 , 1819–1832 (2019). https://doi.org/10.1007/s00425-019-03262-8 Molina-Hidalgo, F. J. et al. The strawberry (Fragaria×ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae. Journal of Experimental Botany 64 , 1471–1483 (2013). https://doi.org/10.1093/jxb/ers386 Zamil, M. S. & Geitmann, A. The middle lamella—more than a glue. Physical Biology 14 , 015004 (2017). https://doi.org/10.1088/1478-3975/aa5ba5 Levesque-Tremblay, G., Pelloux, J., Braybrook, S. A. & Müller, K. Tuning of pectin methylesterification: consequences for cell wall biomechanics and development. Planta 242 , 791–811 (2015). https://doi.org/10.1007/s00425-015-2358-5 Xiao, C. & Anderson, C. T. Roles of pectin in biomass yield and processing for biofuels. Frontiers in Plant Science Volume 4 - 2013 (2013). https://doi.org/10.3389/fpls.2013.00067 Marry, M. et al. Cell-cell adhesion in fresh sugar-beet root parenchyma requires both pectin esters and calcium cross-links. Physiologia Plantarum 126 , 243–256 (2006). https://doi.org/https://doi.org/10.1111/j.1399-3054.2006.00591.x Molina, A. et al. Arabidopsis cell wall composition determines disease resistance specificity and fitness. Proceedings of the National Academy of Sciences 118 , e2010243118 (2021). https://doi.org/10.1073/pnas.2010243118 Holland, C., Ryden, P., Edwards, C. H. & Grundy, M. M. L. Plant Cell Walls: Impact on Nutrient Bioaccessibility and Digestibility. Foods 9 (2020). Uddin, N. et al. Plant cell wall biosynthesis: Immune signaling, genome editing, and physiological implications for biomass valorization. Biotechnology Advances 85 , 108714 (2025). https://doi.org/https://doi.org/10.1016/j.biotechadv.2025.108714 Du, J. et al. Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis. The Plant Cell 32 , 3576–3597 (2020). https://doi.org/10.1105/tpc.20.00252 Nicolas, W. J. et al. Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks. Current Biology 32 , 2375–2389.e2376 (2022). https://doi.org/10.1016/j.cub.2022.04.024 Wang, X., Wilson, L. & Cosgrove, D. J. Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep. Journal of Experimental Botany 71 , 2629–2640 (2020). https://doi.org/10.1093/jxb/eraa059 Feng, W. et al. The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling. Current Biology 28 , 666–675.e665 (2018). https://doi.org/10.1016/j.cub.2018.01.023 Temple, H. et al. Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation. Nature Plants 8 , 656–669 (2022). https://doi.org/10.1038/s41477-022-01156-4 Haas, K. T., Wightman, R., Meyerowitz, E. M. & Peaucelle, A. Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells. Science 367 , 1003–1007 (2020). https://doi.org/10.1126/science.aaz5103 Zhang, Q. et al. Pectin Metabolism Influences Phloem Architecture and Flowering Time in Arabidopsis Thaliana. Advanced Science n/a , e02980 (2025). https://doi.org/https://doi.org/10.1002/advs.202502980 Additional Declarations There is NO Competing Interest. Supplementary Files SIFigureandTablelegends.docx SupplementalInformationFINAL.pdf Supplemental Information: Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls Lightmicroscopyreportingtable.xlsx Light microscopy reporting table. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8001798","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":539162911,"identity":"e28aa1d9-3870-4963-8913-5c15a67d19b3","order_by":0,"name":"Breeanna Urbanowicz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACAxiDH0Ixk6BFsoFkLQYHiNViLn344IOfO+7IG99Iv/iBocI6sYGQFsu+tGTD3jPPDLfdyCmWYDiTTliLwRkeMwnetsOMQC0JEoxth4nRwv/959+2w/abZ+Qk/2D8R5QWHjZmoC2JGyTSj0kwNhChxbKHzVhatu1w8owzb9gsEo6lGxPUYs7D/PDj27bDtv3t6Y9vfKixliWoBQnwGDAkkKAcBNgfkKhhFIyCUTAKRgoAAA0pQdiqklGgAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5247-4513","institution":"University of Georgia","correspondingAuthor":true,"prefix":"","firstName":"Breeanna","middleName":"","lastName":"Urbanowicz","suffix":""},{"id":539162912,"identity":"0e1878f2-37f1-487f-8adb-325a92fa591f","order_by":1,"name":"Kristen Thorne","email":"","orcid":"https://orcid.org/0000-0002-0428-3699","institution":"TEGA Therapeutics","correspondingAuthor":false,"prefix":"","firstName":"Kristen","middleName":"","lastName":"Thorne","suffix":""},{"id":539162913,"identity":"43fda62f-58e0-4bc1-8598-f401324a597f","order_by":2,"name":"William Barnes","email":"","orcid":"","institution":"University of Georgia","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Barnes","suffix":""},{"id":539162914,"identity":"50418793-ef57-49d7-b942-b5ccddfb0510","order_by":3,"name":"Helen Hood","email":"","orcid":"","institution":"University of Georgia","correspondingAuthor":false,"prefix":"","firstName":"Helen","middleName":"","lastName":"Hood","suffix":""}],"badges":[],"createdAt":"2025-10-31 22:00:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8001798/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8001798/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96566868,"identity":"02a85c9d-05c4-453a-bae4-feec7ad8c970","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6494915,"visible":true,"origin":"","legend":"","description":"","filename":"GHprobesFINAL.docx","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/d611979e7cc407b40e58b8cf.docx"},{"id":96605527,"identity":"0ce8ba44-31c6-4c67-b443-58fa38e60f56","added_by":"auto","created_at":"2025-11-24 09:23:23","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5782,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2587891.json","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/ca8c7caf179c475e1a2048db.json"},{"id":96566867,"identity":"c5dab3b7-b9bc-433b-ac62-50d642f1b6c0","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13578,"visible":true,"origin":"","legend":"","description":"","filename":"Lightmicroscopyreportingtable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/ccd8ba42ca09be388a43f2ba.xlsx"},{"id":96566872,"identity":"86fa006d-a58b-4788-b5b4-4c278cdf887b","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4673870,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalInformationFINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/55365af88974a307a6c6e1ad.pdf"},{"id":96566876,"identity":"6517e07b-971e-435d-87a0-91e0744cc6ff","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":229619,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25878910enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/4740934dafc0d3b49dadd1b9.xml"},{"id":96566888,"identity":"10ed0ab5-1917-4f70-ab12-e9233684ba78","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":470127,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/c69d41cc459b62a472108544.jpeg"},{"id":96605820,"identity":"833841b1-b86d-4315-9e4e-357185eb299d","added_by":"auto","created_at":"2025-11-24 09:24:09","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":941758,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/025f6796c2f94d02d3118cbd.jpeg"},{"id":96605769,"identity":"494e8097-e845-4a6e-8e4a-9668d1b5da45","added_by":"auto","created_at":"2025-11-24 09:24:01","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":914633,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/b21d8269fd8b980d90930257.jpeg"},{"id":96566884,"identity":"56caa369-9e43-4924-b48c-ef4d5f2d83d1","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":536480,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/64f0b58757fed4c9b5c0da5b.jpeg"},{"id":96566877,"identity":"774c4967-22b5-41dd-a641-ab7a341c8d72","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":893758,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/658424aacc189a96c2e81bd6.jpeg"},{"id":96566889,"identity":"672c81d8-f012-44e4-9d8b-c27b88afb333","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":836335,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/fd6f3bbd6df8ee40ad43309f.jpeg"},{"id":96605963,"identity":"425bf262-c4e7-45bc-86c0-dcf0940cee22","added_by":"auto","created_at":"2025-11-24 09:24:26","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1260978,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/31c6efdca7edea45982d9825.jpeg"},{"id":96566874,"identity":"c6b33a65-f5d4-4120-a44d-94848682c3a4","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119956,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/fe2ba2fd963a593ca418fe09.png"},{"id":96566878,"identity":"d762d6e6-a54b-4136-9196-474e46fb7cde","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":236909,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/7c71dbd07f0e8560829e211f.png"},{"id":96604726,"identity":"8490615a-3916-4c7e-92e6-3e4a9d21928c","added_by":"auto","created_at":"2025-11-24 09:14:42","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198005,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/964fd3d4dad37e1258d196bf.png"},{"id":96566890,"identity":"637307af-4825-44e4-9e3c-82457a33a101","added_by":"auto","created_at":"2025-11-23 15:54:44","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121130,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/f084132780e6326dad5c03a7.png"},{"id":96566883,"identity":"077071f0-6bc0-4066-8648-c2af81e600d2","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":142798,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/ebcec7cb023c0014fec8fab3.png"},{"id":96566894,"identity":"692353b9-19c4-414e-b853-eeb1175108ad","added_by":"auto","created_at":"2025-11-23 15:54:44","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126886,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/2d5aace771f7b9f7227d5e25.png"},{"id":96605795,"identity":"ed848cff-4cc9-48cd-9ad2-8e245d49f98f","added_by":"auto","created_at":"2025-11-24 09:24:03","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":320762,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/e20999c255120148dccecb01.png"},{"id":96566892,"identity":"db0381a8-5d4d-4dc5-8de1-e90c2fe80e26","added_by":"auto","created_at":"2025-11-23 15:54:44","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":227094,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25878910structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/0198f9f51ed02162e597ed9a.xml"},{"id":96566891,"identity":"8faef67d-7992-4311-8ea0-d90963108fdc","added_by":"auto","created_at":"2025-11-23 15:54:44","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":245612,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/e64f3adb220ec5cee7b73f9d.html"},{"id":96566865,"identity":"49a5981e-c5d4-4808-b7a9-0b3702a0e954","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":283376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic structures of RG-II monomer and dimer. a, \u003c/strong\u003eThe RG-II monomer is represented using the symbol nomenclature for glycans\u003csup\u003e72\u003c/sup\u003e. The four conserved oligosaccharide sidechains (A, B, C, and D) and two distinct α-l-Ara\u003cem\u003ef\u003c/em\u003e substituents (E and F) attached to the (1→4)-linked α-d-GalpA backbone are shown. Arrows indicate the linkages in Sidechain A and B that are targeted by the \u003cem\u003eBacteroides thetaiotaomicron \u003c/em\u003eglycoside hydrolases (GHs) BT1010 and BT0996. \u003cstrong\u003eb,\u003c/strong\u003e Schematic structure of RG-II dimer highlighting the single, site-specific borate di-ester linkage between the apiosyl residues of Sidechain A.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/3226313770745e177141dd3d.png"},{"id":96605219,"identity":"0ce0442d-febb-4f00-b04e-91ba1b4adc75","added_by":"auto","created_at":"2025-11-24 09:21:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":585619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical characterization of sfGFP-fusion proteins containing native or variant BT1010 GH95. a, \u003c/strong\u003eSchematic representation of the construct design for native or variant BT1010 GH95 fusion proteins targeting the terminal l-Galactose (l-Gal) of Sidechain A. \u003cstrong\u003eb, \u003c/strong\u003eResidues selected for mutagenesis are labeled in modeled structure of BT1010 GH95 (AF-Q8A907) with a cavity map illustrating the predicted substrate-binding pocket. \u003cstrong\u003ec,\u003c/strong\u003e Quantification of l-Gal released from 1 mM RG-II monomer by native or variant sfGFP-BT1010 GH95. Data are presented as mean values of \u003cem\u003en\u003c/em\u003e= 3 independent experiments using three separate protein expressions and purifications. Overlaid points indicate the mean of 4 independent HPAEC-PAD injections and error bars correspond to s.e.m across three biological replicates. Asterisks indicate significant differences between variants and native enzyme, as defined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003ed,\u003c/strong\u003e Quantification of l-Gal released from 0.5 mM RG-II dimer by native or variant sfGFP-BT1010 GH95. Data are presented as means of independent experiments (\u003cem\u003en\u003c/em\u003e = 3) using three separate protein expressions and purifications. Overlaid points indicate the mean of 4 independent HPAEC-PAD injections and error bars correspond to s.e.m across three biological replicates. Asterisks indicate a significant difference between monomer and dimer substrates for native BT1010 GH95, defined by a paired t-test followed by Bonferroni-Holm correction for multiple comparisons *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/2b4b6ae42c4cedd6b2fcc180.png"},{"id":96604909,"identity":"adb74f37-b5ab-44be-810b-a379257b0e2b","added_by":"auto","created_at":"2025-11-24 09:15:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":582308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical characterization of sfGFP-fusion proteins containing native or variant BT0996 GH137. a, \u003c/strong\u003eSchematic representation of the construct design for native or variant BT0996 GH137 fusion proteins targeting the terminal l-Arabinose (l-Ara\u003cem\u003ef\u003c/em\u003e) of Sidechain B. \u003cstrong\u003eb, \u003c/strong\u003eResidues selected for mutagenesis are indicated in the crystal structure of the BT0996 GH137 domain in complex with Sidechain B (PDB 5MUJ). \u003cstrong\u003ec,\u003c/strong\u003e Quantification of l-Ara\u003cem\u003ef\u003c/em\u003e released from 1 mM RG-II monomer by native or variant sfGFP-BT0996 GH137. Data are presented as mean values of \u003cem\u003en\u003c/em\u003e = 3 independent experiments using three separate protein expressions and purifications. Overlaid points indicate the mean of 4 independent HPAEC-PAD injections and error bars correspond to s.e.m across three biological replicates. Asterisks indicate significant differences between variants and native BT0996 GH137, as defined by one-way analysis of variance (ANOVA) followed byDunnett’s multiple-comparison test. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001. \u003cstrong\u003ed,\u003c/strong\u003e Quantification of l-Ara\u003cem\u003ef\u003c/em\u003e released from 0.5 mM RG-II dimer by native or variant sfGFP-BT0996 GH137. Data are presented as mean values of \u003cem\u003en\u003c/em\u003e = 3 independent experiments using three separate protein expressions and purifications (\u003cem\u003en\u003c/em\u003e = 2 for E159Q/E240A). Overlaid points indicate the mean of 4 independent HPAEC-PAD injections and error bars correspond to s.e.m across biological replicates. Asterisks indicate a significant difference between monomer and dimer substrates for native or variant BT0996 GH137, defined by a paired t-test followed by Bonferroni-Holm correction for multiple comparisons *\u003cem\u003eP\u003c/em\u003e£0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/33b412d7f88815175493d4e0.png"},{"id":96605154,"identity":"569209b6-e7e0-417d-9478-12f3a18e94a9","added_by":"auto","created_at":"2025-11-24 09:20:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":370827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMST analysis of sfGFP-fusion proteins against RG-II monomer and dimer. a, \u003c/strong\u003eDose-response curves between native or variant sfGFP-BT1010 GH95 and RG-II monomer. Respective K\u003csub\u003ed\u003c/sub\u003e values for sfGFP-BT1010 (red) and variants N389A (purple), E516A (blue), and double mutant N389A/E516A (green) = 5.78 ± 2.83 μM, 4.57 ± 1.90 μM, 0.79 ± 0.40 μM, 1.22 ± 0.56 μM. \u003cstrong\u003eb, \u003c/strong\u003eDose-response curves between native or variant sfGFP-BT0996 GH137 and RG-II monomer. Respective K\u003csub\u003ed\u003c/sub\u003e values for sfGFP-BT0996 (orange) and variants N227A (purple), E240A (blue), and double mutants N227A/E240A (magenta) and E159Q/E240A (navy) = 1.34 ± 0.66 μM, 0.96 ± 0.48 μM, 0.89 ± 0.39 μM, 0.47 ± 0.15 μM, 3.08 ± 1.36 μM. \u003cstrong\u003ec-d, \u003c/strong\u003eDose-response curves for native or variant \u003cstrong\u003ec, \u003c/strong\u003esfGFP-BT1010 GH95 and \u003cstrong\u003ed,\u003c/strong\u003e sfGFP-BT0996 GH137 with RG-II dimer as the ligand did not reach saturation, and the derived K\u003csub\u003ed \u003c/sub\u003eestimates represent apparent values extrapolated beyond the measurable binding range due to autofluorescence at high ligand concentrations. Respective apparent K\u003csub\u003ed \u003c/sub\u003evalues are reported in Table 1 but should be interpreted qualitatively as indicative of weak binding. All dose response curves were generated from the means of independently pipetted measurements (\u003cem\u003en\u003c/em\u003e indicated) with error bars representing standard deviation.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/65441f326c34b793809cd8e4.png"},{"id":96566881,"identity":"a10814b6-69b0-4470-be4c-cfb6601c05c2","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":461660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescent labeling of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e stem sections with mRuby3-BT0996\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eE240A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and mRuby3-BT1010\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eE516A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e. a, \u003c/strong\u003e(left) Maximum projection image of parenchyma cells labeled with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e. (right) Contrast image with pixel intensities rescaled to the range [0,1] according to the minimum and maximum values in the image. \u003cstrong\u003eb, \u003c/strong\u003e(left) Maximum projection image of parenchyma cells labeled with mRuby3-BT1010\u003csup\u003eE516A\u003c/sup\u003e. (right) Contrast image with pixel intensities rescaled to the range [0,1] according to the minimum and maximum values in the image. Color scales indicate the normalized maximum [+] and minimum [-] pixel intensities (left). Scale bar = 25 μm.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/6155531ced4fb416c41f6eb2.png"},{"id":96566871,"identity":"83c62c0b-566d-4ed0-ba00-b1fffbc6d0d0","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":421942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescence microscopy controls to confirm binding of mRuby3-BT0996\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eE240A\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e to RG-II. a, \u003c/strong\u003eMaximum projection images of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e stem sections labeled with 0.4 μM mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e, \u003cstrong\u003eb, \u003c/strong\u003e0.4 μM mRuby3, \u003cstrong\u003ec,\u003c/strong\u003e 0.4 μM mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e after pretreatment with 1 μM native BT0996 GH137, \u003cstrong\u003ed,\u003c/strong\u003e 0.4 μM mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e after preincubation with 2 mM RG-II monomer, and \u003cstrong\u003ee,\u003c/strong\u003e 0.4 μM mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e after preincubation with 1 mM RG-II dimer. Scale bar = 25 μm. \u003cstrong\u003ef\u003c/strong\u003e, Comparison of fluorescence intensities between mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and controls. Boxplots represent raw integrated densities, where the inner line is the sample median while the top and bottom edges are the upper and lower quartiles, respectively. Whiskers extend to maximum and minimum values. Overlaid points indicate individual images and are colored by experiment. Asterisks indicate a significant difference between the mean fluorescence of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and controls. Statistical significance was assessed using a linear mixed-effects model with the condition as a fixed effect and experiment date as a random effect. Pairwise contrasts were tested and \u003cem\u003eP\u003c/em\u003e-values adjusted by Bonferroni-Holm correction. *\u003cem\u003eP\u003c/em\u003e£0.05, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/6f3a30180ff3e53eff8d0501.png"},{"id":96566886,"identity":"b10e016e-d5c8-4d9b-a3c0-cd83f1015929","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":689174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistinct labeling patterns of pectic domains in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e stem cell walls. a\u003c/strong\u003e, Fluorescence image showing labeling with CCRC-M38 (de-esterified HG), \u003cstrong\u003eb\u003c/strong\u003e, CCRC-M14 (RG-I backbone), and \u003cstrong\u003ec\u003c/strong\u003e, mRuby3–BT0996\u003csup\u003eE240A\u003c/sup\u003e (RG-II). \u003cstrong\u003ed\u003c/strong\u003e, Model summarizing the inferred spatial organization of HG, RG-I, and RG-II within the primary wall and middle lamella, highlighting distinct but overlapping pectic microdomains.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/07c9323e95d2341a0260f5f3.png"},{"id":96708126,"identity":"c25d8304-0846-4ea7-bbdf-b6f086e8ab05","added_by":"auto","created_at":"2025-11-25 09:57:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5131167,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/548c31c3-3fc0-4025-a3cb-2048e477d365.pdf"},{"id":96566863,"identity":"3caa0ad1-e890-4c04-b9f9-b8a011e03db1","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17128,"visible":true,"origin":"","legend":"","description":"","filename":"SIFigureandTablelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/c3adfda326a45388e2e474de.docx"},{"id":96566869,"identity":"c5025701-c660-462d-bd37-1c6e10c7b945","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4673870,"visible":true,"origin":"","legend":"Supplemental Information: Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls","description":"","filename":"SupplementalInformationFINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/54d9cd1e06cc2de7815c09ce.pdf"},{"id":96566870,"identity":"847814b8-1dbb-4b4a-a98b-070535ef5bb7","added_by":"auto","created_at":"2025-11-23 15:54:43","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13578,"visible":true,"origin":"","legend":"Light microscopy reporting table.","description":"","filename":"Lightmicroscopyreportingtable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8001798/v1/459cdeec8a8a5a49020cf80c.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants possess a remarkable ability to modulate the mechanical strength and flexibility of their cell walls, driving essential biological processes such as cellular growth and morphogenesis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, reproductive development\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and environmental and immune defense\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Growing cells are surrounded by primary cell walls which are composed of complex, interconnected networks of polysaccharides that assemble into highly organized macrostructures, each tailored to provide structural support in a context-dependent manner\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The biochemical composition and spatial arrangement of these polysaccharides are tightly regulated by both biotic and abiotic factors\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, ensuring precise control over cell wall architecture\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The molecular mechanisms underlying this regulation remain poorly understood, largely due to the challenging nature of distinguishing heterogenous cell wall polymers and monitoring structural variation at the tissue and cellular level.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eConferring the highest degree of structural variability among plant glycans, pectins are defined by the presence of 1,4-linked galacturonic acid (GalA) in the polymer backbone and include four main subclasses: homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (XGA)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Pectins are present in the cell walls of all vascular plants, where they form extensive covalent and non-covalent interactions with each other. In particular, their enrichment in the middle lamella between adjacent primary walls underlies their key role in mediating cell\u0026ndash;cell adhesion\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. They have also been shown to interact with cellulose and hemicelluloses, supporting a wide range of wall architectures that enable both stability and flexibility during plant growth and development\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. HG consists of a linear chain of α-1,4-linked GalA residues appended with \u003cem\u003eO\u003c/em\u003e-methyl and \u003cem\u003eO\u003c/em\u003e-acetyl substituents at the \u003cem\u003eC\u003c/em\u003e-6 and \u003cem\u003eO\u003c/em\u003e-2/\u003cem\u003eO\u003c/em\u003e-3 positions, respectively. In contrast, the RG-I backbone is based on a repeating disaccharide unit of α-1,4-GalA and α-1,2-rhamnose (Rha) residues and displays context-dependent variation in the type and degree of glycosyl substituents, which range from single sugars to branched arabinan, galactan, and arabinogalactan side-chains attached to the Rha residues\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Current models propose that HG and RG-I form a dynamic, covalently linked pectic network with RG-II \u003csup\u003e15\u003c/sup\u003e to regulate wall porosity, hydration, and adhesion between cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRG-II is the least abundant pectic domain, representing up to 5% of the polysaccharides present in primary cell walls and is often considered to be the most structurally complex polysaccharide found in Nature to date\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The core structure of RG-II is a backbone of at least eight 1,4-linked α-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-Gal\u003cem\u003ep\u003c/em\u003eA residues substituted with six distinct sidechains, termed A \u0026ndash; F, that are linked together by at least twenty-one different glycosidic linkages to form a RG-II monomer of approximately 5 kDa \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These structurally conserved side chains are composed of 13 different sugars, including several unusual monosaccharides such as 3-deoxy-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-manno-oct-2-ulosonic acid (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-Kdo) and 3-deoxy-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-lyxo-2-heptulosaric acid (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-Dha), which are only found in RG-II and bacterial polysaccharides\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. There are also a number of non-glycosyl substituents, including methyl-etherified GalA and xylose and methyl-esterified glucuronic acid (GlcA) in Sidechain A, methyl-etherified fucose in Sidechain B, \u003cem\u003eO\u003c/em\u003e-acetyl groups on Sidechains A and B, and methyl-esterified GalA on the backbone\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. More than 90% of RG-II in the cell wall presents as a cross-linked dimer, formed through a borate diester linkage between β-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-apiosyl (Api) residues of Sidechain A (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Unlike most plant polysaccharides, RG-II has a highly conserved primary structure, likely reflecting the need to maintain its unique ability to form these covalent linkages\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In fact, plants carrying heterozygous mutations in genes that affect RG-II synthesis and structure typically exhibit severe growth defects, while complete knockout mutations are often embryonic lethal\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Collectively, these studies have shown that even minor structural modifications to RG-II markedly impair its ability to dimerize, thereby compromising the integrity of the pectic network within the cell wall.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePlant cell wall glycan-directed monoclonal antibodies (mAbs) are indispensable tools for visualizing the spatial organization and dynamics of wall polysaccharides. Their ability to recognize diverse glycan sub-structures, or epitopes, within their native cellular context has provided key insights into how wall composition varies across cell types, tissues, and developmental stages\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, generating and characterizing mAbs against branched pectic polysaccharides remains challenging due to their structural heterogeneity, which complicates biochemical isolation and epitope assignment\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Although more than 80 mAbs have been generated against pectin- and protein-linked arabinogalactans (AGPs), only seven epitopes have been clearly defined\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and none have been conclusively characterized to target RG-II epitopes\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOne explanation for RG-II\u0026rsquo;s apparent lack of immunogenicity is that steric hindrance from its distinctive three-dimensional (3D) structure may limit access to potential epitopes recognizable by animal immune systems\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, the polysaccharides used as immunogens are frequently prepared using base extractions, which removes ester substituents. Indeed, very few glycan-directed antibodies have been successfully developed against base-labile features, including \u003cem\u003eO\u003c/em\u003e-acetyl and methyl-ester modifications, which are critical structural elements of pectins\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Among these are the mannan-directed mAbs CCRC-M169 and CCRC-M170, which uniquely recognize acetylated epitopes\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Similarly, the HG-directed mAbs CCRC-M38, CCRC-M132, JIM5, LM18, and LM19 preferentially bind regions with low degrees of methylesterification, although each shows slight variations in tolerance for methyl substitution\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Together, these limitations highlight the need for alternative strategies and complementary tools to investigate how, where, and when pectins are assembled, disassembled, or relocated within plant cell walls to influence wall integrity and mechanics.\u003c/p\u003e\u003cp\u003eSeveral studies have demonstrated that catalytically inactivated enzymes retain their binding specificity and can be developed for use as probes to identify specific structural motifs within heterogenous polysaccharides\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Unlike antibodies, such proteins can be recombinantly expressed and genetically encoded, allowing modular fusion with fluorescent tags of choice and the rapid detection of structural or chemical changes within subcellular locations of the cell wall. This makes them especially advantageous for experiments requiring live-cell imaging, co-localization with other cellular markers, or the tracking of pectin dynamics across developmental stages. Research on the gut bacterium \u003cem\u003eBacteroides thetaiotaomicron\u003c/em\u003e (\u003cem\u003eB. theta\u003c/em\u003e) has led to the identification of a suite of Carbohydrate Active Enzymes (CAZymes) that recognize and degrade RG-II through a highly coordinated deconstruction model\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These enzymes, encoded in substrate-associated polysaccharide utilization loci (PUL), not only provide mechanistic insight into RG-II degradation but also represent versatile tools that can be repurposed for a multitude of applications, ranging from structural characterization, biomass conversion, tailored oligosaccharide production, and now, development of molecular probes for cell wall imaging. Here we describe an approach that leverages the linkage-level selectivity of RG-II-targeted GHs to develop recombinant, fluorescent probes directed towards Sidechains A and B of RG-II. In this work, we constructed a library of catalytically inactive GH variants and assessed their ability to recognize and bind RG-II substrates isolated from red wine. Both sets of probes preferentially bound the RG-II monomer over the dimer, providing a new means to discriminate structural conformations within this complex polysaccharide. Guided by biochemical analyses, we identified the most effective variants and applied them as recombinant probes for direct visualization of RG-II within \u003cem\u003eArabidopsis thaliana\u003c/em\u003e stem sections. Together, these findings introduce a new technique for mapping the spatial distribution of RG-II in native plant cell walls and outline a general strategy for repurposing PUL-encoded enzymes as molecular probes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEngineering catalytically inactive GH-based probes targeting RG-II sidechains\u003c/h2\u003e\u003cp\u003eWe initiated our probe library by first evaluating the specificity of GHs that cleave terminal sugars from RG-II Sidechain A or Sidechain B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These complex oligosaccharide sidechains harbor unique structural signatures and may also differ in enzymatic accessibility and reactivity between monomeric and dimeric forms. We selected the GH95 domain of BT1010 and the GH137 domain of BT0996 as candidates for developing Sidechain A\u0026ndash; and B\u0026ndash;targeting probes, respectively. BT1010 is an inverting α-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-galactosidase containing a GH95 catalytic module that cleaves the terminal \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Galactose (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gal) from Sidechain A (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To develop probes targeted to this region, we constructed a fusion between sfGFP and the GH95 domain of BT1010 (residues 19\u0026ndash;824), then introduced active site mutations designed to disrupt catalysis while maintaining substrate recognition (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Catalytic residue predictions were guided by homology between an AlphaFold predictive model of BT1010 (AF-Q8A907) and AfcA (PDB 2EAD), a structurally characterized GH95 α-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-fucosidase \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In AfcA, Glu566 and Asp766 act as the general acid and base catalysts, while a pair of polar amino acids, Asn421 and Asn423, act as intermediates in a proton acceptor chain required for catalysis. The corresponding residues in BT1010 were selected for the generation of three variants, BT1010\u003csup\u003eN389A\u003c/sup\u003e, BT1010\u003csup\u003eE516A\u003c/sup\u003e, and the double mutant BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The native and variant forms of BT1010 GH95 were recombinantly expressed in \u003cem\u003eE. coli\u003c/em\u003e and purified as N-terminal His\u003csub\u003e6\u003c/sub\u003e\u0026ndash;sfGFP fusion proteins (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), with sfGFP-BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e displaying slightly reduced expression compared to the single mutants (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Thermal shift analysis confirmed that the sfGFP-BT1010\u003csup\u003eN389A\u003c/sup\u003e and sfGFP-BT1010\u003csup\u003eE516A\u003c/sup\u003e variants retained stability comparable to the wild type sfGFP-BT1010, while sfGFP-BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e showed a decrease in melting temperature (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBT0996 is a multimodular enzyme that contains two catalytic domains: a C-terminal GH2 β-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-glucuronidase and a N-terminal GH137 β-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-arabinofuranosidase, which target linkages in Sidechains A and B, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It also carries a CBM57 region that binds α-1,4-linked polygalacturonic acid\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To minimize off-target binding, we designed native and variant sfGFP-fusion constructs with the GH137 domain of BT0996 (residues 24\u0026ndash;387), which cleaves the terminal \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Arabinose (\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ara\u003cem\u003ef\u003c/em\u003e) from Sidechain B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The GH137 domain of BT0996 is the founding member of its family, and predictions of catalytic residues were based on the crystal structure of the GH137 domain in complex with Sidechain B (PDB 5MUJ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB)\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In this structure, the terminal \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ara\u003cem\u003ef\u003c/em\u003e occupies a deep active site pocket, while the rest of the sidechain extends outward into solvent (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), positioning Glu159, Glu240, and Asn227 as potential residues for catalysis. Glu159 and Glu240 were predicted to serve as the general acid and base catalysts, respectively, whereas Asn227 likely contributes indirectly to the catalytic mechanism. We recombinantly expressed the following variants in \u003cem\u003eE. coli\u003c/em\u003e as soluble N-terminal His\u003csub\u003e6\u003c/sub\u003e\u0026ndash;sfGFP fusion proteins: sfGFP-BT0996\u003csup\u003eN227A\u003c/sup\u003e, sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e, sfGFP-BT0996\u003csup\u003eE159Q\u003c/sup\u003e, sfGFP-BT0996\u003csup\u003eN227A/E240A\u003c/sup\u003e, and sfGFP-BT0996\u003csup\u003eE159Q/E240A\u003c/sup\u003e (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The construct encoding sfGFP-BT0996\u003csup\u003eE159Q\u003c/sup\u003e failed to yield soluble protein and was not further characterized. Thermal shift analysis revealed stable melting temperatures for the remaining variants compared to sfGFP-BT0996; however, sfGFP-BT0996\u003csup\u003eN227A/E240A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE159Q/E240A\u003c/sup\u003e expressed at reduced levels compared to sfGFP-BT0996\u003csup\u003eN227A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBiochemical characterization of RG-II glycosidases\u003c/h3\u003e\n\u003cp\u003eWe evaluated the biochemical activity of all enzymes using purified RG-II monomer and dimer as saccharide substrates (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Previous work by Ndeh et al. (2017) established the stepwise mechanism by which \u003cem\u003eB. theta\u003c/em\u003e PULs degrade RG-II, but most enzymes were assayed with short oligosaccharides, synthetic substrates, or crude extracts rather than the complete pectic domain. HPAEC-PAD was used to quantify the enzymatic hydrolysis of \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gal and \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ara\u003cem\u003ef\u003c/em\u003e from RG-II substrates by native and variant sfGFP-BT1010 and sfGFP-BT0996, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In comparison to sfGFP-BT1010, the relative rate of Gal hydrolyzed from RG-II monomer decreased by 86% with sfGFP-BT1010\u003csup\u003eN389A\u003c/sup\u003e and 99% with sfGFP-BT1010\u003csup\u003eE516A\u003c/sup\u003e and sfGFP-BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Mutation of sfGFP-BT0996 decreased the relative rate of Ara released from RG-II monomer by 78% with sfGFP-BT0996\u003csup\u003eN227A\u003c/sup\u003e, 97% with sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e, and 99% with sfGFP-BT0996\u003csup\u003eN227A/E240A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE159Q/E240A\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Both native enzymes showed limited activity on dimeric RG-II, with hydrolysis rates reduced by 94% for sfGFP-BT1010 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and 92% for sfGFP-BT0996 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) compared to monomer. These results were reproducible across multiple independent protein preparations (SI Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), confirming that BT1010 GH95 and BT0996 GH137 are preferential to RG-II monomer as a substrate over dimer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary of catalytic and binding data for native and variant sfGFP-BT1010 and sfGFP-BT0996 against RG-II monomer and dimer.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eTarget ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eCatalytic Activity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eBinding Affinity \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eRelative Rate of Activity\u003c/strong\u003e \u003csup\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard Error\u003c/strong\u003e \u003csup\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eK\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eConfidence\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eStd. Error of Regression\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eReduced \u0026chi;\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e \u003cstrong\u003ed\u003c/strong\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eng∙min\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e∙\u0026micro;M\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026micro;M\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"9\"\u003e\n \u003cp\u003eRG-II Monomer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 GH95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e59.110\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e7.375\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.781E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.830E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.751\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 \u003csup\u003eN389A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8.439\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.464\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.568E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.903E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.741\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.899\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 \u003csup\u003eE516A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.476\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.191\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.940E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.980E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.256\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.842\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 \u003csup\u003eN389A/E516A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.281\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.221E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.620E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.371\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.176\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 GH137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e23.031\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.337E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.640E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.594\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eN227A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e5.160\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.560E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.820E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.622\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eE240A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.733\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.896\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.900E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.890E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.029\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eN227A/E240A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.260\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.660E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.460E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.182\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eE159Q/E240A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.272\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.084E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.358E\u0026thinsp;+\u0026thinsp;00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.847\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.471\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esfGFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.491E\u0026thinsp;+\u0026thinsp;02 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.917E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.413\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.911\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"9\"\u003e\n \u003cp\u003eRG-II Dimer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 GH95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e3.448\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.856\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.376E\u0026thinsp;+\u0026thinsp;02 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.651E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.437\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.380\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 \u003csup\u003eN389A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e1.665\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.352\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.990E\u0026thinsp;+\u0026thinsp;02 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.248E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.405\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 \u003csup\u003eE516A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.042\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.149E\u0026thinsp;+\u0026thinsp;02 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.397E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.289\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.217\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT1010 \u003csup\u003eN389A/E516A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.086\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.944E\u0026thinsp;+\u0026thinsp;02 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.658E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.362\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.565\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 GH137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e1.861\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.383E\u0026thinsp;+\u0026thinsp;02 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.772E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.522\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.476\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eN227A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.524\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.047E\u0026thinsp;+\u0026thinsp;03 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.578E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.816\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eE240A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.367\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.364\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.008E\u0026thinsp;+\u0026thinsp;03 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.787E\u0026thinsp;+\u0026thinsp;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.651\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.336\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eN227A/E240A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.092\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.058\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.178E\u0026thinsp;+\u0026thinsp;03 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.436E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.550\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.121\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBT0996 \u003csup\u003eE159Q/E240A\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e0.245\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esfGFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.233E\u0026thinsp;+\u0026thinsp;04 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.836E\u0026thinsp;+\u0026thinsp;03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.537\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.642\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"10\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Relative rate of activity is calculated from the mass of hydrolyzed product detected in a 25 \u0026micro;L injection volume. \u003csup\u003eb\u003c/sup\u003e Standard error is calculated from the mean of 3 separate protein expressions, each calculated from the average of 4 technical replicates. \u003csup\u003ec\u003c/sup\u003e Dissociation constants (K\u003csub\u003ed\u003c/sub\u003e) and their confidence intervals were derived from nonlinear least-squares fitting. \u003csup\u003ed\u003c/sup\u003e Standard error of regression and reduced \u0026chi;\u003csup\u003e2\u003c/sup\u003e values were obtained from MO.Affinity Analysis Software. \u003csup\u003ee\u003c/sup\u003e Derived K\u003csub\u003ed\u003c/sub\u003e estimates represent apparent values extrapolated beyond the measurable binding range due to autofluorescence at high ligand concentrations and thus should be interpreted qualitatively as indicative of weak binding.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eBinding affinity of sfGFP-fused GHs to RG-II monomer and dimer\u003c/p\u003e\n\u003cp\u003eHaving identified variants with partial or complete catalytic inactivation, we next asked whether the extent of inactivation correlated with substrate recognition. To achieve this, we measured dissociation constants (K\u003csub\u003ed\u003c/sub\u003e) for each sfGFP-fusion protein against RG-II monomer and dimer as substrates using MicroScale Thermophoresis (MST) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Sigmoidal dose-response curves were generated from ligand concentrations ranging from 250 \u0026micro;M to 15.3 nM to calculate binding affinities against RG-II monomer (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, SI Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). For the BT1010 GH95 \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-galactosidase, native and variant proteins exhibited K\u003csub\u003ed\u003c/sub\u003e values of 5.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83 \u0026micro;M (sfGFP-BT1010), 4.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 \u0026micro;M (sfGFP-BT1010\u003csup\u003eN389A\u003c/sup\u003e), 0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 \u0026micro;M (sfGFP-BT1010\u003csup\u003eE516A\u003c/sup\u003e), and 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 \u0026micro;M (sfGFP-BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e). For the BT0996 GH137 arabinosidase, K\u003csub\u003ed\u003c/sub\u003e values were 1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 \u0026micro;M (sfGFP-BT0996), 0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48 \u0026micro;M (sfGFP-BT0996\u003csup\u003eN227A\u003c/sup\u003e), 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 \u0026micro;M (sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e), 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u0026micro;M (sfGFP-BT0996\u003csup\u003eN227A/E240A\u003c/sup\u003e), and 3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 \u0026micro;M (sfGFP-BT0996\u003csup\u003eE159Q/E240A\u003c/sup\u003e). Purified sfGFP alone displayed weak or undetectable binding to RG-II monomer and dimer, confirming that substrate recognition arises from the GH domains (SI Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). To measure binding to dimeric RG-II, we found that ligand concentrations exceeding 500 \u0026micro;M were required to reach saturation, which produced autofluorescence artifacts and precluded accurate K\u003csub\u003ed\u003c/sub\u003e determination (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-D, SI Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eOur data showed that variants with single point mutations that had the highest degree of biochemical inactivation, sfGFP-BT1010\u003csup\u003eE516A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e, displayed higher binding affinities relative to their native or partially inactivated counterparts. We also identified two double mutants, sfGFP-BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE159Q/E240A\u003c/sup\u003e, that were completely unable to hydrolyze RG-II but instead had reduced binding affinity. Based on their consistent reduction in catalytic activity while maintaining binding specificity, the BT1010\u003csup\u003eE516A\u003c/sup\u003e and BT0996\u003csup\u003eE240A\u003c/sup\u003e single mutant variants were selected as optimal, catalytically inactive probes to evaluate for imaging studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescent labeling of RG-II in\u003c/strong\u003e \u003cstrong\u003eArabidopsis\u003c/strong\u003e \u003cstrong\u003estem cell walls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next examined whether BT1010\u003csup\u003eE516A\u003c/sup\u003e and BT0996\u003csup\u003eE240A\u003c/sup\u003e could function as fluorescent probes to visualize RG-II within plant cell walls. To avoid overlap with endogenous green autofluorescence\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, each variant was fused to mRuby3, which provides improved photostability and acid tolerance relative to sfGFP\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For fluorescence microscopy, recombinantly expressed and purified mRuby3-BT1010\u003csup\u003eE516A\u003c/sup\u003e and mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e were applied to \u003cem\u003eA. thaliana\u003c/em\u003e inflorescence stems. Samples were prepared using an optimized sectioning and labeling workflow that allows short incubations and requires no post-acquisition image adjustments. Stem sections were incubated for 1 hour with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e or mRuby3-BT1010\u003csup\u003eE516A\u003c/sup\u003e, resulting in a distinct labeling of the parenchyma cell walls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Consistent with MST analyses, in which sfGFP-BT0996 displayed more than 4-fold higher affinity for the RG-II monomer compared to sfGFP-BT1010, labeling with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e produced correspondingly stronger fluorescence signals (SI Fig.\u0026nbsp;8A).\u003c/p\u003e\n\u003cp\u003eGiven its superior labeling efficiency, mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e was selected for subsequent studies examining RG-II localization and binding specificity in the cell wall. To confirm that the observed signal originates from the specific interaction between mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and RG-II, a series of control experiments were first conducted (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, SI Fig.\u0026nbsp;8B). Incubation of stem sections with purified mRuby3 alone ruled out non-specific binding of the fluorophore to the wall (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, SI Fig.\u0026nbsp;8C). Pretreatment of sections with native BT0996 GH137 greatly reduced the signal, indicating that the probe recognizes the \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ara\u003cem\u003ef\u003c/em\u003e epitope cleaved by the active enzyme (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, SI Fig.\u0026nbsp;8D). We routinely blanched sections after enzymatic digestion to ensure that the signal loss was not due to steric blocking of the binding site by the active enzyme. Competitive inhibition by preincubating the probe with RG-II monomer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD, SI Fig.\u0026nbsp;8E) or dimer (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, SI Fig.\u0026nbsp;8F) prior to labeling also decreased fluorescence. Notably, preincubation with RG-II monomer (2 mM) resulted in a significantly greater reduction in signal than with even lower concentration of dimer (1 mM), further supporting the selectivity of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e to RG-II monomer. However, when sections were pretreated with higher concentrations of up to 2.5 mM of either monomer or dimer, fluorescence was completely abolished (SI Fig.\u0026nbsp;8G-H). Quantification of fluorescence intensity across multiple experiments confirmed a significant reduction in signal under all control conditions compared to direct labeling with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e, with dimer supplementation having the smallest effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). Together, these results demonstrate that labeling depends on the integrity and accessibility of the RG-II target, supporting the site-specific binding capability of the probe.\u003c/p\u003e\n\u003cp\u003eHaving validated the specificity of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e for RG-II, we next compared its labeling pattern with those of established monoclonal antibodies targeting other matrix polysaccharides. To investigate the distribution of pectic domains, we used CCRC-M38, which binds de-esterified homogalacturonan, and CCRC-M14, which recognizes the RG-I backbone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;B, SI Fig.\u0026nbsp;9A-B). CCRC-M38 showed an even distribution throughout the primary cell walls of parenchyma, while CCRC-M14 labeling was distributed throughout the middle lamella and intercellular regions. Our RG-II-directed glycosidase-based probe, mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e, showed intense labeling of the middle lamellar cell corners between adjoining cells, suggesting that the distribution of RG-II is distinct from that of HG and RG-I (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC, SI Fig.\u0026nbsp;9C). Additionally, labeling with CCRC-M1, which detects fucosylated xyloglucan, produced a pattern distinct from all three pectin-directed probes (SI Fig.\u0026nbsp;9D). These comparisons highlight the unique localization of RG-II within pectin-rich regions of the cell wall and establish mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e as a valuable complement to existing probes for visualizing pectic architecture and mapping the spatial organization of different domains within distinct apoplastic regions \u003cem\u003ein muro.\u003c/em\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe lack a complete toolset for investigating and visualizing carbohydrate biology \u003cem\u003ein vivo\u003c/em\u003e, especially at the spatial and temporal scales at which it occurs. Such tools are essential to reveal the rules by which complex carbohydrates are built and how they are assembled into higher order and emergent structures, such as cell walls, and how these structures are dynamically modified to perform diverse and essential biological functions. In this study, we designed and characterized fluorescent probes using RG-II-specific GHs from \u003cem\u003eB. theta\u003c/em\u003e. Unlike the few existing polyclonal antibodies reported to target RG-II, our enzyme-based probes developed here have defined binding sites and can distinguish between monomeric and dimeric RG-II. Furthermore, through fusion to fluorescent proteins, we demonstrated that PUL-encoded GHs can be readily repurposed for use as precise imaging tools. Although enzyme-based probes have recently been developed from mammalian glycosyltransferases (Hombu et al., 2025) or mucinases (Shon et al., 2020), CAZymes targeting plant cell wall glycans have generally been underutilised for biological imaging applications (Dornez et al., 2011). Instead, CBMs have been more commonly used as fluorescent probe scaffolds\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e; however, their broad binding profiles often lack the structural precision needed to recognize complex polysaccharides like RG-II. In fact, the extensive branching patterns and glycosyl and non-glycosyl substitutions of plant wall glycans further limit the ability of most CBMs to discriminate among distinct pectic domains or between pectic glycostructures and other polysaccharides\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The exceptional complexity of RG-II makes its hydrolytic enzymes unusually well-suited for linkage-level selectivity, providing access to epitopes that are otherwise inaccessible to antibodies or CBMs. Moreover, this approach could potentially be extended to other CAZyme families, such as carbohydrate esterases, to generate novel tools for visualizing \u003cem\u003eO\u003c/em\u003e-acetyl and methyl-ester modifications \u003cem\u003ein situ\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eGuided by structural homology and active-site modeling, we generated multiple active site variants with the goal of disrupting hydrolysis while preserving substrate recognition. Ndeh et al. (2017) initially characterized individual \u003cem\u003eB. theta\u003c/em\u003e enzymes using short oligosaccharides, synthetic substrates, or crude extracts a pseudo-substrates, focusing primarily on elucidating the PUL-mediated mechanism of RG-II depolymerization. Here, we used more physiologically relevant substrates to assess how specific residue substitutions influence catalytic activity and substrate selectivity, allowing more accurate predictions of each enzyme variant\u0026rsquo;s performance as a biological imaging probe (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Analysis of released sugars confirmed that both enzymes can efficiently hydrolyze their expected monosaccharide targets from RG-II monomer. In addition, mutations of the predicted catalytic residues markedly reduced activity on native substrates, with near-complete inactivation observed for sfGFP-BT1010\u003csup\u003eE516A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings confirm that the chosen residues are essential for catalysis and that their modification abolishes activity without substantially perturbing overall protein structure.\u003c/p\u003e\u003cp\u003eOne notable observation from our biochemical analyses is that the native glycosidases displayed over 90% less activity when dimeric RG-II was provided as a substrate, supporting our hypothesis that covalent crosslinking of RG-II restricts enzyme accessibility (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Single mutants with near-complete loss of enzymatic activity, sfGFP-BT1010\u003csup\u003eE516A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE240A\u003c/sup\u003e, did not interact with dimer but bound RG-II monomer with higher affinity than their native counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B, SI Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that catalytic inactivation stabilizes enzyme\u0026ndash;substrate interactions by eliminating turnover. Functional analysis showed that sfGFP-BT0996\u003csup\u003eN227A/E240A\u003c/sup\u003e still bound RG-II monomer with high affinity, while sfGFP-BT1010\u003csup\u003eN389A/E516A\u003c/sup\u003e and sfGFP-BT0996\u003csup\u003eE159Q/E240A\u003c/sup\u003e showed weaker binding. All double mutants demonstrated reduced thermal stability and/or expression (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C), indicating that minor structural perturbations are occurring due to the mutations. The preference for RG-II monomer may be the result of evolutionary adaptation to the structural complexity of the polysaccharide. Because RG-II presents multiple, chemically diverse sidechains extending from a central backbone, recognition likely requires highly specific enzymes that cannot accommodate both monomeric and dimeric forms. Structural data further support this idea: the funnel-shaped active sites of Sidechain B\u0026ndash;directed enzymes BT0986 and BT0996 encompass large portions of their respective oligosaccharide substrates, making them good probe candidates, but also suggests that binding depends on both steric accessibility and coordinated recognition within the full RG-II polymer\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These insights reinforce BT1010 and BT0996 as ideal candidates for probe development, as their selectivity toward unique sidechain motifs can be harnessed to achieve precise recognition of this complex pectic domain.\u003c/p\u003e\u003cp\u003eBT1010\u003csup\u003eE516A\u003c/sup\u003e and BT0996\u003csup\u003eE240A\u003c/sup\u003e were shown to display high affinity for RG-II with K\u003csub\u003ed\u003c/sub\u003e values comparable to those of mAbs (K\u003csub\u003ed\u003c/sub\u003e~10 \u0026micro;M) \u003csup\u003e31\u003c/sup\u003eand select pectin-directed CBMs (K\u003csub\u003ed\u003c/sub\u003e ~0.6\u0026ndash;2.4 \u0026micro;M) \u003csup\u003e50,52,53\u003c/sup\u003e(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results confirm our hypothesis that pectin-targeted glycosidases can be engineered into stable, high-affinity RG-II binding proteins with performance on par with established glycan-directed probes. Importantly, this strategy leverages the enzyme\u0026rsquo;s native substrate selectivity while preserving the target epitope during labeling. To confirm this, we used native BT0996 GH137 as a control to enzymatically remove the target glycosyl moiety prior to labeling. Pretreatment of plant sections with the active enzyme significantly reduced fluorescence from mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, SI Fig.\u0026nbsp;8D), demonstrating that the fluorescent signal originates from specific recognition of intact RG-II. This result also highlights the necessity of catalytic inactivation to maintain epitope integrity during imaging. Collectively, these findings establish RG-II-directed GHs as promising scaffolds that combine enzyme-derived specificity with the binding strength of classical glyco-probes.\u003c/p\u003e\u003cp\u003eWe initially sought to identify proteins recognizing both RG-II monomer and dimer to develop complementary probes with distinct selectivity for each form. However, accurate K\u003csub\u003ed\u003c/sub\u003e values could not be determined for any enzyme when RG-II dimer was used as a substrate because the concentrations required to reach saturation generated substantial autofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D, SI Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). While our results indicate weak binding, alternative methods will be necessary to quantify affinities precisely. This outcome is still consistent with our biochemical analyses, which demonstrated that all investigated proteins exhibit markedly lower affinity and glycosidic activity for RG-II dimer \u003cem\u003ein vitro\u003c/em\u003e. The selectivity for RG-II monomer over dimer may reflect the different physiological environments encountered by dietary pectins in the human gastrointestinal tract, particularly with respect to pH, which strongly influences both microbiota composition and carbohydrate metabolism. For instance, it is well established that RG-II dimer dissociates into monomer under acidic conditions of 0.1 M HCl, pH 1 (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. While \u003cem\u003eB. theta\u003c/em\u003e primarily metabolizes dietary carbohydrates in the colon, where the pH ranges from 5\u0026ndash;8, RG-II first passes through the stomach (pH 1.5\u0026ndash;3.5)\u003csup\u003e54\u003c/sup\u003e, where the boron diester is likely cleaved prior to entering the gut. Thus, the observed enzymatic preference for RG-II monomer is consistent with the likelihood that these enzymes encounter RG-II that has already been hydrolyzed into its monomeric form. Interestingly, the genomes of soil bacteria such as \u003cem\u003eFlavobacterium johnsoniae\u003c/em\u003e UW101 and \u003cem\u003eNiabella soli\u003c/em\u003e DSM 19437 encode PULs with similar CAZyme composition, but distinct gene organization compared to \u003cem\u003eB. theta\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Because these species do not encounter acidic environments that promote RG-II monomerization, it is possible that one or more of their PUL-encoded enzymes can recognize and hydrolyze dimeric RG-II through a different mechanism.\u003c/p\u003e\u003cp\u003eWe generated mRuby3-fusions with BT1010\u003csup\u003eE516A\u003c/sup\u003e and BT0996\u003csup\u003eE240A\u003c/sup\u003e to evaluate their potential as fluorescent probes to study RG-II localization \u003cem\u003ein planta\u003c/em\u003e. Both probes successfully labeled sections from \u003cem\u003eA. thaliana\u003c/em\u003e inflorescence stems following a 1-hour incubation, representing a considerably shorter protocol than conventional immunolabeling with mAbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Labeling with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e produced a slightly stronger signal than mRuby3-BT1010\u003csup\u003eE516A\u003c/sup\u003e, which we attributed to the improved diffusion of the smaller GH137 probe into the highly interconnected regions of the primary cell wall network (SI Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, 8A). Intense fluorescence was observed at the middle lamellar cell corners of the primary cell wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, SI Fig.\u0026nbsp;8B, 9C), pectin-rich areas that cement together the walls of adjoining cells and contribute to the mechanical strength and elasticity of growing tissues\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. This localization is consistent with our probe recognizing and binding a structural molecule like RG-II that can form covalent linkages between larger pectic polymers\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor assessing the utility of our RG-II specific probes, we performed several control experiments using plant tissue sections to validate that the observed signal arises from specific binding of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e to RG-II (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, SI Fig.\u0026nbsp;8B). First, we probed stem sections with mRuby3 and showed there was no detectable fluorescence, excluding non-specific binding of the fluorophore (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, SI Fig.\u0026nbsp;8C). Next, we pretreated plant tissue sections with native BT0996 GH137 to cleave the terminal \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Ara\u003cem\u003ef\u003c/em\u003e from Sidechain B. When these sections were then labelled with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e, we observed markedly reduced fluorescence intensity, clearly demonstrating that labeling depends on the integrity of the target epitope (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, SI Fig.\u0026nbsp;8D). Finally, competitive inhibition assays further supported the specificity of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e. Preincubation of the probe with purified RG-II monomer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, SI Fig.\u0026nbsp;8E) or dimer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, SI Fig.\u0026nbsp;8F) prior to labeling resulted in reduced fluorescence, with monomer preincubation causing a significantly greater decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results align with the \u003cem\u003ein vitro\u003c/em\u003e biochemical and binding data and confirm that the probe preferentially recognizes monomeric RG-II. Collectively, our combined data demonstrate that labeling depends on the integrity and accessibility of the RG-II target and confirms that mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e is a selective probe for RG-II, with a preference for monomer.\u003c/p\u003e\u003cp\u003eTo contextualize our probe labeling patterns within the broader cell wall architecture, we compared mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e with established mAbs recognizing other matrix polysaccharides. Our data shows that mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and mRuby3-BT1010\u003csup\u003eE516A\u003c/sup\u003e specifically label middle lamellar corners between adjoining cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, SI Fig.\u0026nbsp;9C). This is quite distinct from CCRC-M38, which binds de-esterified HG, and uniformly labeled parenchyma primary cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, SI Fig.\u0026nbsp;9A). We also evaluated CCRC-M14, which targets the RG-I backbone, and showed it specifically labels the middle lamella and intercellular spaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, SI Fig.\u0026nbsp;9B). Labeling with CCRC-M1, which detects fucosylated xyloglucan, labelled regions distinct from those of all pectin-directed probes evaluated here (SI Fig.\u0026nbsp;9D). The distribution of HG and RG-I likely reflects their structural flexibility and ability to be remodeled during growth, adhesion, and abscission, processes that demand dynamic wall plasticity\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In contrast, RG-II localization at cell corners suggests enrichment in crosslinking hotspots that provide rigidity and cohesion\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. These findings highlight functional partitioning among pectic domains, with RG-II reinforcing junctional integrity while HG and RG-I mediate wall flexibility.\u003c/p\u003e\u003cp\u003eFuture studies will involve using established mutants defective in aspects of RG-II synthesis, to investigate the selectivity of our probe library. However, because fully intact RG-II is essential for plant viability, relatively few mutants with modified RG-II structure have been identified and those that have are known to accumulate predominantly monomer\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. For example, the \u003cem\u003eA. thaliana murus 1\u003c/em\u003e (\u003cem\u003emur1\u003c/em\u003e) mutant lacks the ability to synthesize GDP-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Fucose (Fuc), resulting in a\u0026thinsp;~\u0026thinsp;50% reduction of the terminal 2-\u003cem\u003eO\u003c/em\u003e-methyl xylose in Sidechain A and substitution of the terminal \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Fuc in Sidechain B with \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gal\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Likewise, \u003cem\u003eArabidopsis\u003c/em\u003e lines with suppressed expression of \u003cem\u003eGolgi GDP-\u003c/em\u003e\u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e\u003cem\u003e-galactose transporter1\u003c/em\u003e (\u003cem\u003eGGLT1\u003c/em\u003e) exhibit up to a 50% reduction in Sidechain A \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-Gal\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. While such plants could serve as valuable negative controls for probes recognizing these epitopes, a key limitation is that disrupting the glycosyl sequence of RG-II impairs dimer formation, resulting in mutant lines with elevated levels of RG-II monomer. For instance, Sechet et al. (2018) found the abundance of monomer in EPG-treated AIR from \u003cem\u003egglt\u003c/em\u003e and \u003cem\u003emur1-1\u003c/em\u003e lines increased from 23% in wild type to 52% and 94%, respectively. Because our current probes preferentially recognize monomeric RG-II, these lines may not be the best biological samples to reliably distinguish structural effects on crosslinking from those on epitope abundance, thereby complicating direct comparisons of fluorescence intensity.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn conclusion, biological imaging tools are essential for elucidating the functions of individual wall components, as precise localization data can inform strategies to enhance plant properties such as biomass yield, stress tolerance, and nutrient uptake\u003csup\u003e\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Advances in understanding pectin structure have largely centered on HG, supported by the availability of mutants affecting its biosynthesis or methylesterification, selective probes for its modification states, and the powerful spectroscopic and microscopic imaging technologies that are available. These approaches have uncovered direct links between pectin and cellulose synthesis\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, cell wall integrity\u003csup\u003e\u003cspan additionalcitationids=\"CR67 CR68\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, and the growth and morphogenesis of distinct cell types\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Yet, current tools still fall short in their ability to capture the full structural diversity and biological function of pectins within the wall.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eHere, we have begun to address these limitations by expanding imaging capabilities to the structurally complex domain RG-II and establish mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and mRuby3-BT1010\u003csup\u003eE516A\u003c/sup\u003e among the first fluorescent probes with defined specificity for RG-II monomer, enabling high-precision mapping of its localization and dynamics \u003cem\u003ein planta\u003c/em\u003e. Their application opens new opportunities for synthetic biology approaches, as these enzyme-derived probes can serve as genetically encoded biosensors for tracking RG-II remodeling during development or stress responses. When integrated with existing HG- and RG-I\u0026ndash;directed tools, they will facilitate a systems-level view of pectin organization and function. More broadly, this strategy establishes a versatile framework for repurposing CAZymes as molecular probes, extending high-fidelity glycan imaging to the most structurally intricate components of the plant cell wall.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eGeneration of fluorescent protein constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genes encoding BT1010 (GenBank: AAO76117.1) and BT0996 (GenBank: AAO76103.1) were the kind gift of Dr. Harry Gilbert and had been previously cloned into pET28a(+) for recombinant expression as N-terminal His\u003csub\u003e6\u003c/sub\u003e-tagged\u003csub\u003e\u0026nbsp;\u003c/sub\u003efusion proteins, as described in Ndeh et al. (2017)\u003csup\u003e44\u003c/sup\u003e. Truncated sequences encoding the GH95 and GH137 glycoside hydrolase domains of BT1010 and BT0996, respectively, were amplified by PCR. The resulting BamHI/XhoI-restricted fragments were cloned into the pET28a T7pCONS TIR-2 sfGFP vector\u003csup\u003e73\u003c/sup\u003e, in-frame downstream of the gene encoding Superfolder GFP (sfGFP)\u003csup\u003e74\u003c/sup\u003e. The resulting expressed protein consisted of an N-terminal His\u003csub\u003e6\u003c/sub\u003e tag, sfGFP, and the indicated GH domain. To minimize interference from plant cell wall autofluorescence in the green channel, the T7pCONS vector was modified to encode alternative fluorescent proteins with red-shifted emission spectra. Constructs containing mRuby3 (GenBank: ATE88097.1)\u003csup\u003e48\u003c/sup\u003e, mScarlet-I (GenBank: APD76536.1)\u003csup\u003e75\u003c/sup\u003e, and TagRFP (GenBank: ABR08320.1) \u003csup\u003e76\u003c/sup\u003ewere generated using NdeI/BamHI-restricted gene fragments synthesized by Twist Bioscience (https://www.twistbioscience.com/). Among these, mRuby fusion proteins were selected for microscopy owing to their improved brightness, photostability, and acid tolerance\u003csup\u003e77\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSite-Directed Mutagenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eModels for native and variant proteins were generated with AlphaFold \u003csup\u003e78\u003c/sup\u003e and analyzed using Pymol (The PyMOL Molecular Graphics System, Version 3.0.4 Schrödinger, LLC). Mutagenesis of BT1010 GH95 and BT0996 GH137 was performed using the Q5\u003csup\u003e®\u003c/sup\u003e Site-Directed Mutagenesis Kit (New England Biolabs) using the primers listed in SI Figure 1A. The resulting constructs were confirmed by DNA sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemicals and reagents were purchased from Thermo Fisher Scientific unless otherwise noted. Plasmids were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e strains BL21(DE3) or Tuner(DE3) (Novagen) competent cells for recombinant protein expression. Cells were cultured at 37°C in Luria–Bertani (LB) medium supplemented with kanamycin (50 μg ml\u003csup\u003e−1\u003c/sup\u003e) to mid‐exponential phase (OD\u003csub\u003e600 nm\u003c/sub\u003e = 0.6‐0.8). Recombinant gene expression was induced by the addition of isopropyl β-d-galactopyranoside (IPTG), with the optimal concentration, expression temperature, and induction time differing for certain proteins (SI Figure 2A). For purification of recombinant fusion proteins, cell pellets were resuspended in Buffer A (20 mM Tris-HCl, 300 mM NaCl, pH 8.0), supplemented with 10 μL of 100 mM PMSF per mL of cell suspension, and lysed using an Emulsiflex C3 (Avestin) according to manufacturer’s recommendations for \u003cem\u003eE. coli\u003c/em\u003e. Recombinant, His-tagged fusion proteins were purified from clarified cell lysates by immobilized metal ion affinity chromatography (IMAC) using HisPur™ Cobalt Resin (Thermo Fisher Scientific). The resin was washed with Buffer A, and bound, His-tagged proteins were eluted with Buffer B (150 mM Imidazole, 20 mM Tris, 300 mM NaCl, pH 8.0). Protein purity was ascertained using SDS-PAGE. When cleaved sfGFP or mRuby3 was noted, recombinant fusion proteins were further purified by size exclusion chromatography (SEC) in 20 mM MES, 400 mM NaCl, pH 5.5, at 4°C using a HiLoad 16/600 Superdex 200 pg column (Cytiva) on an AKTA 25L system (Cytiva). Purified proteins were dialyzed into storage buffer (20 mM MES, 400 mM NaCl, pH 5.5) using D‐Tube™ Dialyzer Maxi (MWCO 6–8 kDa) or Midi (MWCO 3.5 kDa) devices (EMD Millipore). Chelex® 100 Resin (Bio-Rad) was added to all dialysis buffer to remove any remaining divalent cations. Final protein concentrations were derived with the MW and extinction coefficient of each protein, as obtained using Geneious version 2024.0 (https://www.geneious.com).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, characterization, and preparation of RG-II substrates\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biochemical characterization of our recombinant probe library was conducted using RG-II substrates isolated from red wine, as described in Barnes et al., 2021\u003csup\u003e18\u003c/sup\u003e. After extraction, crude RG-II generally consists of both monomeric and dimeric glycoforms, thus calling for \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003emonomerization and dimerization, respectively, to generate homogenous substrates\u003csup\u003e23,79\u003c/sup\u003e. Monomeric RG-II was prepared according to the methods of O’Neill et al. (1996). Briefly,a solution of crude wine RG-II (2.5 mg/1 mL) was prepared in 0.1 M HCl, gently mixed at room temperature for 3 hours, then dialyzed (Spectrum™ Spectra/Por™, 3500 Dalton MWCO) against deionized water and freeze dried. RG-II monomer readily dimerizes in solution, so it is important to avoid borosilicate glass, utilize Chelex® 100 Resin (Bio-Rad) during dialysis, and prepare fresh aliquots of substrate for experiments. Dimeric RG-II was prepared according to previous methods\u003csup\u003e20\u003c/sup\u003e. Briefly, RG-II monomer (0.2 mM; Mw 4971 Da) was dissolved in Dimerization Buffer (1 mM boric acid, 0.5 mM lead nitrate, in 50 mM NaOAc pH 3.9) and gently mixed at room temperature for 3 hours. The solution was then dialyzed (Spectrum™ Spectra/Por™, 3500 Dalton MWCO) against deionized water and freeze dried. The homogeneity of RG-II monomer and dimer preparations was confirmed by SEC (SI Figure 2D). A 50 μg sample was analyzed on a Superdex 75 Increase 10/300 GL column (Cytiva) using a 1260 Infinity II LC system (Agilent) equipped with a refractive index detector (G7162A, Agilent), as previously described\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection\u003c/strong\u003e (\u003cstrong\u003eHPAEC-PAD)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe optimal buffer composition for biochemical assays was determined by protein thermal shift assays using SYPROä Orange dye (Thermo Fisher) to monitor protein stabilization under varying buffer, pH, and salt conditions \u003csup\u003e80\u003c/sup\u003e (SI Figure 2B-C). Fluorescent signals were measured on a Bio-Rad™ CFX Real-Time PCR System and analyzed with CFX Maestro Software version 2.3 (Bio-Rad). To determine the activity of RG-II specific GHs, reactions were prepared with 1 μM protein in Reaction Buffer (20 mM MES, 100 mM NaCl, pH 5.5) and either 1 mM of RG-II monomer or 0.5 mM of RG-II dimer as saccharide substrates. Reactions were incubated at 37°C for 1 hour, followed by a 95°C heat inactivation for 5 minutes, then filtered using a 96-well filter microplate with a 0.45 µm nylon membrane (Agilent). The specific activities of native and variant BT1010 GH95 and BT0996 GH137 were determined according to the respective release of l-Gal and l-Ara\u003cem\u003ef\u0026nbsp;\u003c/em\u003efrom monomeric and dimeric RG-II substrates using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Briefly, l-Gal and l-Ara\u003cem\u003ef\u003c/em\u003e were quantified using a Dionex ICS 6000 system (Thermo Fisher Scientific) with a Dionex CarboPac PA1 column equipped with an AminoTrap guard column. After injection (25 μL), monosaccharides were separated isocratically at a rate of 1.0 mL/min with an initial flow of NaOH (0-15 min: 32 mM), followed by a multi-step gradient of sodium acetate in 100 mM NaOH (15-35 min: 0-250 mM; 35-45 min: 250-1000 mM; 45-48 min: 1000 mM; 48-51 min: 1000-0 mM; 51-60 min: 0 mM). The activity of each native and variant enzyme was quantified using 3 distinctly expressed and purified protein preparations (biological replicates, \u003cem\u003en\u0026nbsp;\u003c/em\u003e= 3), and quadruplicate HPAEC-PAD injections. The rate of activity for each enzyme is reported as the mass of monosaccharide product released per minute per μM of protein (ng∙min\u003csup\u003e-1\u003c/sup\u003e∙μmol\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroScale Thermophoresis (MST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroScale Thermophoresis (MST) was used to analyze the thermodynamic relationship between sfGFP-labeled native and variant enzymes and monomeric and dimeric RG-II substrates. MST parameters were optimized according to the workflow described by \u003csup\u003e81\u003c/sup\u003e. Fluorescent fusion proteins (sfGFP) were adjusted to a working concentration of 1 μM in MST Buffer (20 mM MES, 400 mM NaCl, pH 5.5) supplemented with 0.1% Tween 20 (Bio-Rad) to reduce aggregation and adsorption. RG-II substrates were dialyzed into 20 mM MES pH 5.5, adjusted to a starting concentration of 0.5 mM, then used to generate a serially diluted concentration gradient from 500 μM to 30.6 nM. An equal volume of prepared protein was added to each RG-II dilution, producing a final reaction concentration of 500 nM protein in 20 mM MES, 200 mM NaCl, 0.05% Tween pH 5.5 and ligand concentrations ranging from 250 μM to 15.3 nM. The samples were incubated at 37°C for 15 min and centrifuged at 15,000 \u003cem\u003ex g\u003c/em\u003e for 5 min prior to loading reactions into standard Monolith NT.115 Capillaries (NanoTemper Technologies). MST was measured using a Monolith NT.115 instrument (NanoTemper Technologies) at an ambient temperature of 23°C. Instrument parameters were adjusted to 10-30% LED power and high MST power. Data of six independently pipetted measurements were analyzed (MO.Affinity Analysis software version 2.1.3, NanoTemper Technologies) using the signal from an MST-on time of 1.5 or 5 s (SI Figures 5-7).\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor HPAEC-PAD experiments, peak areas were quantified using Chromeleon™ Chromatography Data System version 7.3 (Thermo Scientific). Calculation of reaction rates (ng∙min\u003csup\u003e-1\u003c/sup\u003e∙μM\u003csup\u003e-1\u003c/sup\u003e) and subsequent statistical analyses were conducted using MATLAB R2024b (MathWorks). Technical variability was assessed from four independent reactions, reported as the standard deviation (SI Figures 3-4). Biological variability was determined from the mean of \u003cem\u003en\u003c/em\u003e = 3 independent protein expressions and purifications, reported as the standard error of the mean (Table 1). Significant differences between native and variant enzymes were determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test, which corrects for multiple comparisons against a single control. To compare enzyme activity toward RG-II monomer versus dimer substrates, we applied paired two-tailed t-tests for each enzyme, since the same biological replicates were assayed on both substrates. Resulting \u003cem\u003eP\u003c/em\u003e-values were adjusted for multiple comparisons across the enzymes using the Bonferroni-Holm method. Test statistics (\u003cem\u003eF\u003c/em\u003e values, t values, degrees of freedom, and adjusted \u003cem\u003eP\u003c/em\u003e values) are reported in SI Table 1.\u003c/p\u003e\n\u003cp\u003eMST data was analyzed with MO.Affinity Analysis Software version 2.1.3 (NanoTemper) and fit using a K\u003csub\u003ed\u003c/sub\u003e model with fixed target concentration. Dose-response binding curves were generated from independently pipetted measurements (exact \u003cem\u003en\u003c/em\u003e values are provided in Table 1). Dissociation constants (K\u003csub\u003ed\u003c/sub\u003e) and their confidence intervals were derived from nonlinear least-squares fitting, and replicate quality was assessed using software-provided parameters, including root mean squared error and reduced χ\u003csup\u003e2\u003c/sup\u003e (Table 1). Data are presented as the fraction bound plotted against ligand concentration, in which the change in normalized fluorescence is normalized to the curve amplitude to yield values between 0 and 1. Representative MST traces, depicting the change in fluorescence intensity of one capillary over time, are provided to demonstrate data quality and confirm the absence of aggregation and photobleaching of the target molecule (SI Figures 5-7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype \u003cem\u003eColumbia-0\u003c/em\u003e (\u003cem\u003eCol-0\u003c/em\u003e) was used as wild type. Sterilized seeds were stratified in the dark for 2 days at 4 °C before plating on ½ strength Murashige and Skoog medium (Sigma Aldrich), 0.1% (w/v) MES (Beantown Chemical), 1% (w/v) sucrose (Sigma Aldrich), 0.8% (w/v) agar (Dot Scientific), pH 5.6. Plates were sealed and incubated vertically at 19°C/15°C with a 16-h-light/8-h-dark photoperiod.10-day old seedlings were placed on a soil mixture consisting of a 1:1 ratio of Metromix 830 (Sungro) and Vermiculite, which was supplemented with Plant Food (Sta-Green) and Osmocote Smart-Release Plant Food Flower \u0026amp; Vegetable (Scotts) before being moved to a walk-in growth chamber (Conviron). Plants were grown under an 18 h light/ 6 h dark cycle at 21 °C with a light intensity of 120 μmol photons m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy sample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStem sections were harvested from the middle to upper regions of \u003cem\u003eA. thaliana\u003c/em\u003e inflorescence stems using double-edged razor blades (Electron Microscopy Sciences). To inactivate endogenous cell wall–modifying enzymes, sections were blanched for 2 min at 90 °C, followed by stepwise dehydration under vacuum in 30%, 50%, and 70% ethanol (v/v) for 15 min each. Samples were then washed three times with gentle shaking for 15 min each in Wash Buffer (20 mM MES, 0.01% Tween-20, pH 5.5). To reduce nonspecific probe binding, sections were blocked overnight at 4 °C with gentle shaking in Blocking Buffer (1X Pierce™ Clear Milk Blocking Buffer (Thermo Fisher Scientific) prepared in 20 mM MES, pH 5.5). After three additional washes, samples were incubated with gentle shaking for 1 hr at 30 °C in 200 μL of 0.4 μM of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e in Blocking Buffer. Control labeling with purified mRuby3 alone was performed at the same concentration. Sections were washed three times prior to mounting on microscope slides and imaging.\u003c/p\u003e\n\u003cp\u003eFor enzyme digest controls, sections were pretreated with 1 μM native BT0996 GH137 in Blocking Buffer at 30 °C with gentle shaking for 1 hr, followed by blanching to inactivate the enzyme prior to blocking and labeling. Competitive inhibition controls were performed by preincubating 0.4 μM of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e with purified RG-II monomer (2 mM) or dimer (1 mM) in Blocking Buffer for 1 hr at 30 °C before adding to sections under identical labeling conditions.\u003c/p\u003e\n\u003cp\u003eImmunolabeling procedure was guided by \u003csup\u003e82\u003c/sup\u003e and performed with minor modifications. Stem sections were fixed in 1.6% (v/v) paraformaldehyde (Electron Microscopy Sciences) in 25 mM sodium phosphate buffer, pH 7.1, for 30 min under vacuum. Fixed samples were washed three times with gentle shaking for 15 min each in 25 mM sodium phosphate buffer, pH 7.1, followed by stepwise dehydration under vacuum in 30%, 50%, and 70% ethanol (v/v) for 15 min each. Sections were rehydrated and washed three times with gentle shaking for 5 min each in KPBS (10 mM potassium phosphate buffer, pH 7.1, 500 mM NaCl), then blocked overnight at 4 °C with gentle shaking in 1X Pierce™ Clear Milk Blocking Buffer in KPBS.\u003c/p\u003e\n\u003cp\u003ePrimary monoclonal antibodies CCRC-M38, CCRC-M14, and CCRC-M1 (CarboSource) were used to label de-esterified HG, RG-I backbone, and fucosylated XG, respectively\u003csup\u003e32\u003c/sup\u003e. Sections were incubated for 1 hr at RT with gentle shaking in a 1:20 dilution of primary antibody in KPBS, washed three times, and then incubated for 1 hr in the dark with gentle shaking in a 1:100 dilution of goat anti-mouse secondary antibody conjugated to Alexa Fluor 594 (Abcam). Sections were washed three times prior to mounting and imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWidefield epifluorescence microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence imaging was performed using a Nikon Eclipse 80i epifluorescence microscope equipped with a Nikon DS-Ri2 color camera and a Nikon Intensilight C-HGFIE light source. Excitation and emission were detected using standard DAPI, FITC, and TRITC filter blocks. Images were collected at 10X magnification using a Plan Fluor 10X DIC N1 objective (200 μm z-stack, 5.6 μm z-step, 400 ms exposure) or at 20X magnification using a Plan Fluor 20X DIC N2 objective (75 μm z-stack, 2 μm z-step, 400 ms exposure). All images were acquired at 100% transmittance with gain of 1X. Z-stacks were processed into maximum intensity projections using (Fiji Is Just) ImageJ version 2.16.0/1.54p\u003csup\u003e83\u003c/sup\u003e. Brightness and contrast were not adjusted prior to further analyses using MATLAB R2024b (MathWorks). A complete description of microscope hardware, optics, acquisition settings, and quality control assessment is provided in the Light Microscopy Reporting Table (SI Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage Analysis Workflow\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages were processed using a custom MATLAB code available at https://github.com/kristenthorne/GHprobes.git, with usage instructions and example input and output files provided. Raw z-stacks and maximum projections (original, scaled, and masked) for all quantified images are available in Open Science Framework. For each scaled maximum intensity projection, pixel intensity values were converted to double-precision and normalized to the interval [0,1] to minimize variability in exposure and background between acquisitions. A single representative threshold was applied uniformly across all images acquired within the same experiment to ensure comparability. The average threshold was determined from images labeled with mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e using Otsu’s method\u003csup\u003e84\u003c/sup\u003e, which minimizes within-class variance and provides a data-driven separation of signal from background. Segmentation with this average threshold generated binary masks that defined regions of positive signal. From each mask, total signal area (number of positive pixels) and a normalized area metric (square root of area) were calculated. Fluorescence intensities were quantified on the original, non-normalized images within the masked regions, extracting the following metrics: (1) mean, standard deviation, minimum, and maximum gray value of thresholded pixels; (2) raw integrated density (sum of all pixel intensities in the region); and (3) integrated density (mean gray value × area). Results were stored in structured arrays and exported for downstream statistical analysis. Raw integrated density was used as the primary metric for comparing probe signal between mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and control samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificant differences between the mean raw integrated densities of mRuby3-BT0996\u003csup\u003eE240A\u003c/sup\u003e and control images were determined by ANOVA using a linear mixed-effects model, with condition as a fixed effect and experiment date as a random effect. \u003cem\u003eP\u003c/em\u003e-values were calculated from pairwise contrasts between each fixed effect and adjusted for multiple comparisons with the Bonferroni method. Test statistics (\u003cem\u003eF\u003c/em\u003e values, t values, degrees of freedom, and adjusted \u003cem\u003eP\u003c/em\u003e values) are reported in SI Table 1.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003col start = \"18\"\u003e\n\u003cli\u003eBarnes, W. J.\u003cem\u003e et al.\u003c/em\u003e Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II. \u003cem\u003eBiotechnol Biofuels\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 142 (2021). https://doi.org/10.1186/s13068-021-01992-0\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Neill, M. A.\u003cem\u003e et al.\u003c/em\u003e Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide rhamnogalacturonan II. \u003cem\u003eSLAS TECHNOLOGY: Translating Life Sciences Innovation\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 329\u0026ndash;344 (2020). https://doi.org/10.1177/2472630320923321\u003c/li\u003e\n\u003cli\u003eIshii, T.\u003cem\u003e et al.\u003c/em\u003e The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer. \u003cem\u003eJournal of Biological chemistry\u003c/em\u003e \u003cstrong\u003e274\u003c/strong\u003e, 13098\u0026ndash;13104 (1999). \u003c/li\u003e\n\u003cli\u003ePattathil, S.\u003cem\u003e et al.\u003c/em\u003e A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e153\u003c/strong\u003e, 514\u0026ndash;525 (2010). https://doi.org/10.1104/pp.109.151985\u003c/li\u003e\n\u003cli\u003eNdeh, D.\u003cem\u003e et al.\u003c/em\u003e Complex pectin metabolism by gut bacteria reveals novel catalytic functions. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e544\u003c/strong\u003e, 65\u0026ndash;70 (2017). https://doi.org/10.1038/nature21725\u003c/li\u003e\n\u003cli\u003eBajar, B. T.\u003cem\u003e et al.\u003c/em\u003e Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 20889 (2016). https://doi.org/10.1038/srep20889\u003c/li\u003e\n\u003cli\u003eVarki, A.\u003cem\u003e et al.\u003c/em\u003e Symbol Nomenclature for Graphical Representations of Glycans. \u003cem\u003eGlycobiology\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1323\u0026ndash;1324 (2015). https://doi.org/10.1093/glycob/cwv091\u003c/li\u003e\n\u003cli\u003eShilling, P. J.\u003cem\u003e et al.\u003c/em\u003e Improved designs for pET expression plasmids increase protein production yield in Escherichia coli. \u003cem\u003eCommun Biol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 214 (2020). https://doi.org/10.1038/s42003-020-0939-8\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;delacq, J.-D.\u003cem\u003e et al.\u003c/em\u003e Engineering and characterization of a superfolder green fluorescent protein. \u003cem\u003eNature Biotechnology\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 79\u0026ndash;88 (2006). https://doi.org/10.1038/nbt1172\u003c/li\u003e\n\u003cli\u003eBindels, D. S.\u003cem\u003e et al.\u003c/em\u003e mScarlet: a bright monomeric red fluorescent protein for cellular imaging. \u003cem\u003eNature Methods\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 53\u0026ndash;56 (2017). https://doi.org/10.1038/nmeth.4074\u003c/li\u003e\n\u003cli\u003eMerzlyak, E. M.\u003cem\u003e et al.\u003c/em\u003e Bright monomeric red fluorescent protein with an extended fluorescence lifetime. \u003cem\u003eNature Methods\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 555\u0026ndash;557 (2007). https://doi.org/10.1038/nmeth1062\u003c/li\u003e\n\u003cli\u003eStoddard, A. \u0026amp; Rolland, V. I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves. \u003cem\u003ePlant Direct\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, e00112 (2019). https://doi.org/https://doi.org/10.1002/pld3.112\u003c/li\u003e\n\u003cli\u003eJumper, J.\u003cem\u003e et al.\u003c/em\u003e Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e596\u003c/strong\u003e, 583\u0026ndash;589 (2021). https://doi.org/10.1038/s41586-021-03819-2\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Neill, M. A.\u003cem\u003e et al.\u003c/em\u003e Rhamnogalacturonan-II, a pectic polysaccharide in the walls of growing plant cell, forms a dimer that is covalently cross-linked by a borate ester. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e271\u003c/strong\u003e, 22923\u0026ndash;22930 (1996). https://doi.org/10.1074/jbc.271.37.22923\u003c/li\u003e\n\u003cli\u003eHuynh, K. \u0026amp; Partch, C. L. Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay. \u003cem\u003eCurrent Protocols in Protein Science\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 28.29.21\u0026ndash;28.29.14 (2015). https://doi.org/https://doi.org/10.1002/0471140864.ps2809s79\u003c/li\u003e\n\u003cli\u003eWu, H., Montanier, C. Y. \u0026amp; Dumon, C. Quantifying CBM Carbohydrate Interactions Using Microscale Thermophoresis. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e1588\u003c/strong\u003e, 129\u0026ndash;141 (2017). https://doi.org/10.1007/978-1-4939-6899-2_10\u003c/li\u003e\n\u003cli\u003eAvci, U., Pattathil, S. \u0026amp; Hahn, M. G. in \u003cem\u003eBiomass Conversion: Methods and Protocols\u003c/em\u003e (ed Michael E. Himmel) 73\u0026ndash;82 (Humana Press, 2012).\u003c/li\u003e\n\u003cli\u003eSchindelin, J.\u003cem\u003e et al.\u003c/em\u003e Fiji: an open-source platform for biological-image analysis. \u003cem\u003eNature Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 676\u0026ndash;682 (2012). https://doi.org/10.1038/nmeth.2019\u003c/li\u003e\n\u003cli\u003eOtsu, N. A Threshold Selection Method from Gray-Level Histograms. \u003cem\u003eIEEE Transactions on Systems, Man, and Cybernetics\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 62\u0026ndash;66 (1979). https://doi.org/10.1109/TSMC.1979.4310076\u003c/li\u003e\n\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroscopy data that support the findings of this study have been deposited in Open Science Framework. Raw z-stacks, output maximum intensity projections, and annotated figure images in greyscale are available at (https://osf.io/8utvs/overview). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMATLAB code used for image analysis is available at https://github.com/kristenthorne/GHprobes.git, with usage instructions and example input and output files provided.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding for research on RG-II was provided by the US Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Division, under award DESC0008472 to B.R.U. for funding structural studies of RG-II.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK. J. T: Conceptualization; Methodology (lead); Investigation (lead); Data Curation (lead); Writing \u0026ndash; Original Draft (lead); Visualization (lead); Software (development of custom processing script). W.J.B: Conceptualization; Software (development of custom processing script); Validation; Writing \u0026ndash; Review \u0026amp; Editing (critical input on methods). H.H: Investigation. B.R.U. (Corresponding Author): Conceptualization; Resources; Funding acquisition; Project administration; Supervision; Resources; Writing \u0026ndash; Review \u0026amp; Editing. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials \u0026amp; Correspondance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Information is available for this paper.\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to [email protected].\u0026nbsp;\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBidhendi, A. J. \u0026amp; Geitmann, A. Relating the mechanics of the primary plant cell wall to morphogenesis. \u003cem\u003eJ Exp Bot\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 449\u0026ndash;461 (2016). https://doi.org/10.1093/jxb/erv535\u003c/li\u003e\n\u003cli\u003eLund, C. H.\u003cem\u003e et al.\u003c/em\u003e Pectin Synthesis and Pollen Tube Growth in Arabidopsis Involves Three GAUT1 Golgi-Anchoring Proteins: GAUT5, GAUT6, and GAUT7. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e \u003cstrong\u003eVolume 11 - 2020\u003c/strong\u003e (2020). https://doi.org/10.3389/fpls.2020.585774\u003c/li\u003e\n\u003cli\u003eCai, M.\u003cem\u003e et al.\u003c/em\u003e ROS homeostasis and cell wall biosynthesis pathway are involved in female inflorescence development of birch (Betula platyphylla). \u003cem\u003eBMC Plant Biology\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 708 (2025). https://doi.org/10.1186/s12870-025-06740-2\u003c/li\u003e\n\u003cli\u003eHouston, K.\u003cem\u003e et al.\u003c/em\u003e The plant cell wall: A complex and dynamic structure as revealed by the responses of genes under stress conditions. \u003cem\u003eFront Plant Sci\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 984 (2016). https://doi.org/10.3389/fpls.2016.00984\u003c/li\u003e\n\u003cli\u003eChebli, Y., Bidhendi, A. J., Kapoor, K. \u0026amp; Geitmann, A. Cytoskeletal regulation of primary plant cell wall assembly. \u003cem\u003eCurrent Biology\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, R681\u0026ndash;R695 (2021). https://doi.org/10.1016/j.cub.2021.03.092\u003c/li\u003e\n\u003cli\u003eAltartouri, B.\u003cem\u003e et al.\u003c/em\u003e Pectin Chemistry and Cellulose Crystallinity Govern Pavement Cell Morphogenesis in a Multi-Step Mechanism1 [OPEN]. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e181\u003c/strong\u003e, 127\u0026ndash;141 (2019). https://doi.org/10.1104/pp.19.00303\u003c/li\u003e\n\u003cli\u003eHu, K.\u003cem\u003e et al.\u003c/em\u003e Improvement of multiple agronomic traits by a disease resistance gene via cell wall reinforcement. \u003cem\u003eNature Plants\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 17009 (2017). https://doi.org/10.1038/nplants.2017.9\u003c/li\u003e\n\u003cli\u003eZhang, Y.\u003cem\u003e et al.\u003c/em\u003e Molecular insights into the complex mechanics of plant epidermal cell walls. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e372\u003c/strong\u003e, 706\u0026ndash;711 (2021). https://doi.org/doi:10.1126/science.abf2824\u003c/li\u003e\n\u003cli\u003eVaahtera, L., Schulz, J. \u0026amp; Hamann, T. Cell wall integrity maintenance during plant development and interaction with the environment. \u003cem\u003eNature Plants\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 924\u0026ndash;932 (2019). https://doi.org/10.1038/s41477-019-0502-0\u003c/li\u003e\n\u003cli\u003eAtmodjo, M. A., Hao, Z. \u0026amp; Mohnen, D. Evolving views of pectin biosynthesis. \u003cem\u003eAnnual review of plant biology\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 747\u0026ndash;779 (2013). \u003c/li\u003e\n\u003cli\u003eBarnes, W. J., Zelinsky, E. \u0026amp; Anderson, C. T. Polygalacturonase activity promotes aberrant cell separation in the quasimodo2 mutant of Arabidopsis thaliana. \u003cem\u003eThe Cell Surface\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 100069 (2022). https://doi.org/https://doi.org/10.1016/j.tcsw.2021.100069\u003c/li\u003e\n\u003cli\u003eWang, T., Park, Y. B., Cosgrove, D. J. \u0026amp; Hong, M. Cellulose-pectin spatial contacts are inherent to never-dried Arabidopsis primary cell walls: Evidence from solid-state nuclear magnetic resonance \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e168\u003c/strong\u003e, 871\u0026ndash;884 (2015). https://doi.org/10.1104/pp.15.00665\u003c/li\u003e\n\u003cli\u003eTan, L.\u003cem\u003e et al.\u003c/em\u003e An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein. \u003cem\u003eThe Plant Cell\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 270\u0026ndash;287 (2013). https://doi.org/10.1105/tpc.112.107334\u003c/li\u003e\n\u003cli\u003eZhang, L.\u003cem\u003e et al.\u003c/em\u003e The pectin puzzle: Decoding the fine structure of rhamnogalacturonan-I (RG-I) in Arabidopsis thaliana uncovers new pectin features. \u003cem\u003eCarbohydrate Polymers\u003c/em\u003e \u003cstrong\u003e368\u003c/strong\u003e, 124161 (2025). https://doi.org/https://doi.org/10.1016/j.carbpol.2025.124161\u003c/li\u003e\n\u003cli\u003eMohnen, D., Atmodjo, M. A. \u0026amp; Jayawardhane, P. in \u003cem\u003ePlant Cell Walls: Research Milestones and Conceptual Insights\u003c/em\u003e (ed Anja Geitmann) 94\u0026ndash;126 (CRC Press, 2023).\u003c/li\u003e\n\u003cli\u003ePhyo, P., Gu, Y. \u0026amp; Hong, M. Impact of acidic pH on plant cell wall polysaccharide structure and dynamics: insights into the mechanism of acid growth in plants from solid-state NMR. \u003cem\u003eCellulose\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 291\u0026ndash;304 (2019). https://doi.org/10.1007/s10570-018-2094-7\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Neill, M. A., Ishii, T., Albersheim, P. \u0026amp; Darvill, A. G. Rhamnogalacturonan II: Structure and function of a borate cross-linked cell wall pectic polysaccharide. \u003cem\u003eAnnual Review of Plant Biology\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 109\u0026ndash;139 (2004). https://doi.org/10.1146/annurev.arplant.55.031903.141750\u003c/li\u003e\n\u003cli\u003eBarnes, W. J.\u003cem\u003e et al.\u003c/em\u003e Protocols for isolating and characterizing polysaccharides from plant cell walls: a case study using rhamnogalacturonan-II. \u003cem\u003eBiotechnol Biofuels\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 142 (2021). https://doi.org/10.1186/s13068-021-01992-0\u003c/li\u003e\n\u003cli\u003eMatsunaga, T.\u003cem\u003e et al.\u003c/em\u003e Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in Pteridophytes, Lycophytes, and Bryophytes. Implications for the evolution of vascular plants. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, 339\u0026ndash;351 (2004). https://doi.org/10.1104/pp.103.030072\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Neill, M. A.\u003cem\u003e et al.\u003c/em\u003e Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide rhamnogalacturonan II. \u003cem\u003eSLAS TECHNOLOGY: Translating Life Sciences Innovation\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 329\u0026ndash;344 (2020). https://doi.org/10.1177/2472630320923321\u003c/li\u003e\n\u003cli\u003eFunakawa, H. \u0026amp; Miwa, K. Synthesis of borate cross-linked rhamnogalacturonan II. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 223 (2015). https://doi.org/10.3389/fpls.2015.00223\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Neill, M. A., Eberhard, S., Albersheim, P. \u0026amp; Darvill, A. G. Requirement of borate cross-linking of cell wall rhamnogalacturonan II for Arabidopsis growth. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, 846\u0026ndash;849 (2001). https://doi.org/doi:10.1126/science.1062319\u003c/li\u003e\n\u003cli\u003eIshii, T.\u003cem\u003e et al.\u003c/em\u003e The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer. \u003cem\u003eJournal of Biological chemistry\u003c/em\u003e \u003cstrong\u003e274\u003c/strong\u003e, 13098\u0026ndash;13104 (1999). \u003c/li\u003e\n\u003cli\u003eAhn, J.-W.\u003cem\u003e et al.\u003c/em\u003e Depletion of UDP-D-apiose/UDP-D-xylose Synthases Results in Rhamnogalacturonan-II Deficiency, Cell Wall Thickening, and Cell Death in Higher Plants. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e281\u003c/strong\u003e, 13708\u0026ndash;13716 (2006). https://doi.org/10.1074/jbc.m512403200\u003c/li\u003e\n\u003cli\u003eDelmas, F.\u003cem\u003e et al.\u003c/em\u003e The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 2639\u0026ndash;2647 (2008). https://doi.org/10.1093/jxb/ern118\u003c/li\u003e\n\u003cli\u003eSechet, J.\u003cem\u003e et al.\u003c/em\u003e Suppression of Arabidopsis GGLT1 affects growth by reducing the L-galactose content and borate cross-linking of rhamnogalacturonan-II. \u003cem\u003eThe Plant Journal\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 1036\u0026ndash;1050 (2018). https://doi.org/10.1111/tpj.14088\u003c/li\u003e\n\u003cli\u003eZhao, X.\u003cem\u003e et al.\u003c/em\u003e UDP-Api/UDP-Xyl synthases affect plant development by controlling the content of UDP-Api to regulate the RG-II-borate complex. \u003cem\u003ePlant J\u003c/em\u003e \u003cstrong\u003e104\u003c/strong\u003e, 252\u0026ndash;267 (2020). https://doi.org/10.1111/tpj.14921\u003c/li\u003e\n\u003cli\u003eZhang, Y.\u003cem\u003e et al.\u003c/em\u003e Putative rhamnogalacturonan-II glycosyltransferase identified through callus gene editing which bypasses embryo lethality. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e195\u003c/strong\u003e, 2551\u0026ndash;2565 (2024). https://doi.org/10.1093/plphys/kiae259\u003c/li\u003e\n\u003cli\u003eVoxeur, A.\u003cem\u003e et al.\u003c/em\u003e Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e286\u003c/strong\u003e, 8014\u0026ndash;8020 (2011). https://doi.org/10.1074/jbc.m110.198614\u003c/li\u003e\n\u003cli\u003eDumont, M.\u003cem\u003e et al.\u003c/em\u003e The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein. \u003cem\u003eAnnals of Botany\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 1177\u0026ndash;1188 (2014). https://doi.org/10.1093/aob/mcu093\u003c/li\u003e\n\u003cli\u003eThorne, K. J., Urbanowicz, B. R. \u0026amp; Hahn, M. G. in \u003cem\u003ePlant Cell Walls: Research Milestones and Conceptual Insights\u003c/em\u003e (ed Anja Geitmann) (CRC Press/Taylor \u0026amp; Francis Group, 2023).\u003c/li\u003e\n\u003cli\u003ePattathil, S.\u003cem\u003e et al.\u003c/em\u003e A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. \u003cem\u003ePlant Physiology\u003c/em\u003e \u003cstrong\u003e153\u003c/strong\u003e, 514\u0026ndash;525 (2010). https://doi.org/10.1104/pp.109.151985\u003c/li\u003e\n\u003cli\u003eWilliams, M. N.\u003cem\u003e et al.\u003c/em\u003e An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells. \u003cem\u003ePlant Cell\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 673\u0026ndash;685 (1996). https://doi.org/10.1105/tpc.8.4.673\u003c/li\u003e\n\u003cli\u003eZhou, Y.\u003cem\u003e et al.\u003c/em\u003e Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells. \u003cem\u003eBioscience, Biotechnology, and Biochemistry\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 899\u0026ndash;905 (2017). https://doi.org/10.1080/09168451.2016.1270740\u003c/li\u003e\n\u003cli\u003eZhou, Y.\u003cem\u003e et al.\u003c/em\u003e A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots. \u003cem\u003eBioscience, Biotechnology, and Biochemistry\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 1780\u0026ndash;1789 (2018). https://doi.org/10.1080/09168451.2018.1485479\u003c/li\u003e\n\u003cli\u003eRodrı́guez-Carvajal, M. A.\u003cem\u003e et al.\u003c/em\u003e The three-dimensional structure of the mega-oligosaccharide rhamnogalacturonan II monomer: a combined molecular modeling and NMR investigation. \u003cem\u003eCarbohydrate Research\u003c/em\u003e \u003cstrong\u003e338\u003c/strong\u003e, 651\u0026ndash;671 (2003). https://doi.org/https://doi.org/10.1016/S0008-6215(03)00003-X\u003c/li\u003e\n\u003cli\u003eZhang, X.\u003cem\u003e et al.\u003c/em\u003e Understanding How the Complex Molecular Architecture of Mannan-degrading Hydrolases Contributes to Plant Cell Wall Degradation *. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 2002\u0026ndash;2012 (2014). https://doi.org/10.1074/jbc.M113.527770\u003c/li\u003e\n\u003cli\u003ePattathil, S.\u003cem\u003e et al.\u003c/em\u003e Immunological approaches to biomass characterization and utilization. \u003cem\u003eFrontiers in Bioengineering and Biotechnology\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 173 (2015). https://doi.org/10.3389/fbioe.2015.00173\u003c/li\u003e\n\u003cli\u003eVerhertbruggen, Y.\u003cem\u003e et al.\u003c/em\u003e An extended set of monoclonal antibodies to pectic homogalacturonan. \u003cem\u003eCarbohydrate Research\u003c/em\u003e \u003cstrong\u003e344\u003c/strong\u003e, 1858\u0026ndash;1862 (2009). https://doi.org/10.1016/j.carres.2008.11.010\u003c/li\u003e\n\u003cli\u003eBeaugrand, J.\u003cem\u003e et al.\u003c/em\u003e Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase. \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e222\u003c/strong\u003e, 246\u0026ndash;257 (2005). https://doi.org/10.1007/s00425-005-1538-0\u003c/li\u003e\n\u003cli\u003eDornez, E.\u003cem\u003e et al.\u003c/em\u003e Inactive fluorescently labeled xylanase as a novel probe for microscopic analysis of arabinoxylan containing cereal cell walls. \u003cem\u003eJ Agric Food Chem\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 6369\u0026ndash;6375 (2011). https://doi.org/10.1021/jf200746g\u003c/li\u003e\n\u003cli\u003eShon, D. J.\u003cem\u003e et al.\u003c/em\u003e An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e117\u003c/strong\u003e, 21299\u0026ndash;21307 (2020). https://doi.org/10.1073/pnas.2012196117\u003c/li\u003e\n\u003cli\u003eHombu, R.\u003cem\u003e et al.\u003c/em\u003e Engineering glycosyltransferases into glycan binding proteins using a mammalian surface display platform. \u003cem\u003eNature Communications\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 6637 (2025). https://doi.org/10.1038/s41467-025-62018-z\u003c/li\u003e\n\u003cli\u003eNdeh, D.\u003cem\u003e et al.\u003c/em\u003e Complex pectin metabolism by gut bacteria reveals novel catalytic functions. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e544\u003c/strong\u003e, 65\u0026ndash;70 (2017). https://doi.org/10.1038/nature21725\u003c/li\u003e\n\u003cli\u003eNagae, M.\u003cem\u003e et al.\u003c/em\u003e Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-\u0026alpha;-L-Fucosidase from Bifidobacterium bifidum*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e282\u003c/strong\u003e, 18497\u0026ndash;18509 (2007). https://doi.org/https://doi.org/10.1074/jbc.M702246200\u003c/li\u003e\n\u003cli\u003eTrov\u0026atilde;o, F.\u003cem\u003e et al.\u003c/em\u003e The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome. \u003cem\u003eJournal of Structural Biology: X\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 100084 (2023). https://doi.org/https://doi.org/10.1016/j.yjsbx.2022.100084\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Plazaola, J. I.\u003cem\u003e et al.\u003c/em\u003e Autofluorescence: Biological functions and technical applications. \u003cem\u003ePlant Science\u003c/em\u003e \u003cstrong\u003e236\u003c/strong\u003e, 136\u0026ndash;145 (2015). https://doi.org/https://doi.org/10.1016/j.plantsci.2015.03.010\u003c/li\u003e\n\u003cli\u003eBajar, B. T.\u003cem\u003e et al.\u003c/em\u003e Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 20889 (2016). https://doi.org/10.1038/srep20889\u003c/li\u003e\n\u003cli\u003ePa\u0026euml;s, G. Fluorescent Probes for Exploring Plant Cell Wall Deconstruction: A Review. \u003cem\u003eMolecules\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 9380\u0026ndash;9402 (2014). \u003c/li\u003e\n\u003cli\u003eLiu, G.\u003cem\u003e et al.\u003c/em\u003e Discovery and Structural Analysis of a Novel Pectin-Specific Carbohydrate-Binding Module: The First Member of a New CBM Family. \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 24278\u0026ndash;24285 (2025). https://doi.org/10.1021/acs.jafc.5c07437\u003c/li\u003e\n\u003cli\u003eDhillon, A., Sharma, K., Rajulapati, V. \u0026amp; Goyal, A. The multi-ligand binding first family 35 Carbohydrate Binding Module (CBM35) of Clostridium thermocellum targets rhamnogalacturonan I. \u003cem\u003eArchives of Biochemistry and Biophysics\u003c/em\u003e \u003cstrong\u003e654\u003c/strong\u003e, 194\u0026ndash;208 (2018). https://doi.org/https://doi.org/10.1016/j.abb.2018.07.023\u003c/li\u003e\n\u003cli\u003eAbbott, D. W., Hrynuik, S. \u0026amp; Boraston, A. B. Identification and Characterization of a Novel Periplasmic Polygalacturonic Acid Binding Protein from Yersinia enterolitica. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e367\u003c/strong\u003e, 1023\u0026ndash;1033 (2007). https://doi.org/https://doi.org/10.1016/j.jmb.2007.01.030\u003c/li\u003e\n\u003cli\u003eVenditto, I.\u003cem\u003e et al.\u003c/em\u003e Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 7136\u0026ndash;7141 (2016). https://doi.org/10.1073/pnas.1601558113\u003c/li\u003e\n\u003cli\u003eBrinck, J. E.\u003cem\u003e et al.\u003c/em\u003e Intestinal pH: a major driver of human gut microbiota composition and metabolism. \u003cem\u003eNature Reviews Gastroenterology \u0026amp; Hepatology\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 639\u0026ndash;656 (2025). https://doi.org/10.1038/s41575-025-01092-6\u003c/li\u003e\n\u003cli\u003eTerrapon, N.\u003cem\u003e et al.\u003c/em\u003e PULDB: the expanded database of Polysaccharide Utilization Loci. \u003cem\u003eNucleic Acids Research\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, D677\u0026ndash;D683 (2018). https://doi.org/10.1093/nar/gkx1022\u003c/li\u003e\n\u003cli\u003eChen, D.\u003cem\u003e et al.\u003c/em\u003e Changes in the orientations of cellulose microfibrils during the development of collenchyma cell walls of celery (Apium graveolens L.). \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e250\u003c/strong\u003e, 1819\u0026ndash;1832 (2019). https://doi.org/10.1007/s00425-019-03262-8\u003c/li\u003e\n\u003cli\u003eMolina-Hidalgo, F. J.\u003cem\u003e et al.\u003c/em\u003e The strawberry (Fragaria\u0026times;ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 1471\u0026ndash;1483 (2013). https://doi.org/10.1093/jxb/ers386\u003c/li\u003e\n\u003cli\u003eZamil, M. S. \u0026amp; Geitmann, A. The middle lamella\u0026mdash;more than a glue. \u003cem\u003ePhysical Biology\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 015004 (2017). https://doi.org/10.1088/1478-3975/aa5ba5\u003c/li\u003e\n\u003cli\u003eLevesque-Tremblay, G., Pelloux, J., Braybrook, S. A. \u0026amp; M\u0026uuml;ller, K. Tuning of pectin methylesterification: consequences for cell wall biomechanics and development. \u003cem\u003ePlanta\u003c/em\u003e \u003cstrong\u003e242\u003c/strong\u003e, 791\u0026ndash;811 (2015). https://doi.org/10.1007/s00425-015-2358-5\u003c/li\u003e\n\u003cli\u003eXiao, C. \u0026amp; Anderson, C. T. Roles of pectin in biomass yield and processing for biofuels. \u003cem\u003eFrontiers in Plant Science\u003c/em\u003e \u003cstrong\u003eVolume 4 - 2013\u003c/strong\u003e (2013). https://doi.org/10.3389/fpls.2013.00067\u003c/li\u003e\n\u003cli\u003eMarry, M.\u003cem\u003e et al.\u003c/em\u003e Cell-cell adhesion in fresh sugar-beet root parenchyma requires both pectin esters and calcium cross-links. \u003cem\u003ePhysiologia Plantarum\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 243\u0026ndash;256 (2006). https://doi.org/https://doi.org/10.1111/j.1399-3054.2006.00591.x\u003c/li\u003e\n\u003cli\u003eMolina, A.\u003cem\u003e et al.\u003c/em\u003e Arabidopsis cell wall composition determines disease resistance specificity and fitness. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, e2010243118 (2021). https://doi.org/10.1073/pnas.2010243118\u003c/li\u003e\n\u003cli\u003eHolland, C., Ryden, P., Edwards, C. H. \u0026amp; Grundy, M. M. L. Plant Cell Walls: Impact on Nutrient Bioaccessibility and Digestibility. \u003cem\u003eFoods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e (2020).\u003c/li\u003e\n\u003cli\u003eUddin, N.\u003cem\u003e et al.\u003c/em\u003e Plant cell wall biosynthesis: Immune signaling, genome editing, and physiological implications for biomass valorization. \u003cem\u003eBiotechnology Advances\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 108714 (2025). https://doi.org/https://doi.org/10.1016/j.biotechadv.2025.108714\u003c/li\u003e\n\u003cli\u003eDu, J.\u003cem\u003e et al.\u003c/em\u003e Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis. \u003cem\u003eThe Plant Cell\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 3576\u0026ndash;3597 (2020). https://doi.org/10.1105/tpc.20.00252\u003c/li\u003e\n\u003cli\u003eNicolas, W. J.\u003cem\u003e et al.\u003c/em\u003e Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks. \u003cem\u003eCurrent Biology\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 2375\u0026ndash;2389.e2376 (2022). https://doi.org/10.1016/j.cub.2022.04.024\u003c/li\u003e\n\u003cli\u003eWang, X., Wilson, L. \u0026amp; Cosgrove, D. J. Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 2629\u0026ndash;2640 (2020). https://doi.org/10.1093/jxb/eraa059\u003c/li\u003e\n\u003cli\u003eFeng, W.\u003cem\u003e et al.\u003c/em\u003e The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling. \u003cem\u003eCurrent Biology\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 666\u0026ndash;675.e665 (2018). https://doi.org/10.1016/j.cub.2018.01.023\u003c/li\u003e\n\u003cli\u003eTemple, H.\u003cem\u003e et al.\u003c/em\u003e Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation. \u003cem\u003eNature Plants\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 656\u0026ndash;669 (2022). https://doi.org/10.1038/s41477-022-01156-4\u003c/li\u003e\n\u003cli\u003eHaas, K. T., Wightman, R., Meyerowitz, E. M. \u0026amp; Peaucelle, A. Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e367\u003c/strong\u003e, 1003\u0026ndash;1007 (2020). https://doi.org/10.1126/science.aaz5103\u003c/li\u003e\n\u003cli\u003eZhang, Q.\u003cem\u003e et al.\u003c/em\u003e Pectin Metabolism Influences Phloem Architecture and Flowering Time in Arabidopsis Thaliana. \u003cem\u003eAdvanced Science\u003c/em\u003e \u003cstrong\u003en/a\u003c/strong\u003e, e02980 (2025). https://doi.org/https://doi.org/10.1002/advs.202502980\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8001798/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8001798/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Plant cell walls are dynamic composites whose architecture determines growth, mechanics, and environmental resilience. Efforts to link pectin structure to function have been limited by the lack of molecular probes with sufficient specificity, a gap that becomes even more pronounced for the intricately branched rhamnogalacturonon-II (RG-II) subclass. Here we report the first fluorescent probes with defined specificity to RG-II, engineered from catalytic site mutants of Bacteroides thetaiotaomicron glycoside hydrolases BT1010 and BT0996. These enzyme-derived probes bind RG-II monomer with high affinity, discriminate against dimeric forms, and localize to cell corners and junctions in Arabidopsis thaliana stems, consistent with RG-II’s unique ability among wall polysaccharides to form borate-mediated, covalent crosslinkages between molecules. Application of these probes revealed spatial partitioning distinct from the homogalacturonan (HG)- and rhamnogalacturonan I (RG-I)-enriched middle lamella, highlighting functional specialization among pectic domains, with RG-II reinforcing cell junctions while HG and RG-I mediate wall flexibility. Our work establishes a generalizable framework for transforming CAZymes into high-precision imaging reagents, enabling molecular-level visualization of structurally complex polysaccharides in the cell wall.","manuscriptTitle":"Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-23 15:54:38","doi":"10.21203/rs.3.rs-8001798/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7b17d417-742a-489f-bd0c-502741bfca6e","owner":[],"postedDate":"November 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57338531,"name":"Biological sciences/Plant sciences/Plant cell biology/Cell wall"},{"id":57338532,"name":"Biological sciences/Biochemistry/Glycobiology"},{"id":57338533,"name":"Biological sciences/Biochemistry/Carbohydrates"}],"tags":[],"updatedAt":"2025-12-24T10:40:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-23 15:54:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8001798","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8001798","identity":"rs-8001798","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}