Immunocytological composition of cell walls in Sapium glandulosum (Euphorbiaceae) galls reveals steps in their establishment and development

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This study investigated tissue development and cell wall dynamics in galls of Sapium glandulosum to identify key steps involved in their establishment. Samples of young, mature, and senescent galls, as well as nongalled leaves, were analyzed using structural and immunocytochemical approaches. For histology, samples were fixed, embedded in resin, sectioned, stained with toluidine blue, and mounted with Entellan®. For immunocytochemistry, resin-embedded samples were tested for epitopes of cell wall proteins, pectins, and hemicelluloses using antibodies. The leaves of S. glandulosum are glabrous, hypostomatic, and exhibit dorsiventral mesophyll. Gall development alters the typical leaf morphogenetic pattern, giving rise to structures with a parenchymatic cortex. In young galls, hypertrophy and hyperplasia were observed, followed by tissue maturation in mature galls. Senescent galls showed signs of cytoplasmic degradation in most cortical cells. Structural modifications in the side chains of rhamnogalacturonan I and increased cross-linking of pectic polymers affect cell wall properties, playing roles in both development and defense responses. Immunolabeling with JIM5 in young and mature galls suggests the suppressed activity of pectin methylesterases, which may reflect a strategy by which gall-inducing organisms inhibit host defense signaling. Xyloglucan epitopes were detected in the vascular bundles of mature galls, suggesting the reinforcement of cell walls and possibly supporting the feeding activity of the gall inducer. The combination of anatomical and immunocytochemical data provided a basis for understanding how gall induction modulates cell differentiation and cell wall composition in S. glandulosum . Feeding activity Hemicellulose Host defense Immunocytochemistry Pectin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The plant cell wall comprises a dynamic array of components, including cellulose, hemicelluloses, and pectins, as well as small amounts of proteins that perform various functions in plants (Cosgrove 2005 ; Lorenzo et al. 2019 ), such as serving as the first line of defense against invading microbes (Bacete et al. 2018 ). The physical properties of the cell walls depend on the chemical interactions among these components, which, together with turgor pressure, maintain structural equilibrium in plant cells (Anderson and Kieber 2020 ). However, biotic stress resulting from interactions between plant cells and other organisms can disrupt the cell wall and trigger multiple plant responses (Riseh et al. 2024). Galls are novel plant structures induced by the action of foreign organisms (Shorthouse et al. 2005 ; Harris and Pitzschke 2020 ) and are typically initiated by an increase in localized oxidative stress (Isaias et al. 2015). This oxidative stress is regulated by the inducing organism, which manipulates host plant tissues for its own benefit (Oliveira et al. 2016 ). Gall formation involves alterations to the plant's preexisting morphogenetic patterns, leading to a novel cellular architecture characterized by convergent processes such as cell hypertrophy, tissue hyperplasia, and cellular redifferentiation (Oliveira et al. 2010; Guedes et al. 2018 ; Ferreira et al. 2019 ). As a result, the emergence of new cell types and the associated modifications in cell wall dynamics are fundamental to gall development and reflect the specific influence of each inducing organism. Cellulose microfibrils are synthesized by the cellulose synthase complex in the plasma membrane, which uses UDP-glucose as a substrate (Albersheim et al. 2010 ). The spatial arrangement of these microfibrils in the primary cell wall determines the direction of cell elongation and hypertrophy (Kimura et al. 1999 ; Albersheim et al. 2010 ). During gall development, the reorganization of cellulose microfibrils has been associated with changes in cell shape and the formation of gall-specific structures (Magalhães et al. 2014 ). Arabinogalactan proteins (AGPs) are located within the cell wall and play key roles in cell signaling and adhesion during plant morphogenesis (Fincher et al. 1983 ; Majewska-Sawka and Nothnagel 2000 ). Extensins and other glycoproteins are incorporated into the insoluble microfibrillar matrix of primary cell walls, where they contribute to structural reinforcement, particularly in mature organs (Carpita and Gibeaut 1993 ; Brownleader et al. 1999 ; Albersheim et al. 2010 ). In galls, extensin labeling is typically associated with growth cessation, as expected (Formiga et al. 2013 ; Teixeira et al. 2017). However, some studies have reported unexpected extensin labeling in young galls, suggesting a more complex role during early gall development (Carneiro et al. 2014 ). Conversely, in Espinosa nothofagi (Hymenoptera) galls, extensin synthesis is inhibited, an effect that may be linked to the suppression of the host plant’s innate immune response, thereby facilitating gall initiation and development. This inhibition may also compromise cell wall reinforcement, despite the presence of tissues associated with mechanical support in these galls (Guedes et al. 2025 ). Another important component of cell walls, pectins, play crucial roles in regulating extensibility and growth (Showalter 1993 ; Mohnen 2008 ; Albersheim et al. 2011 ). Additionally, pectin functions as a signaling molecule, initiating immune responses such as antimicrobial compound production, cell wall reinforcement, and activation of defense-related genes (Riseh et al. 2024). Three main primary types of galacturonic acid-containing pectins are found in the cell wall: homogalacturonans (HGs), rhamnogalacturonans (RGs), and xylogalacturonans (XGAs) (Clausen et al. 2004 ; Caffall and Mohnen 2009 ). HGs are the most abundant, and their degree of methyl esterification significantly influences their functional role in the cell wall (Vincken et al. 2003 ). The degree of pectin methylesterification can also serve as an indirect indicator of the tissue developmental stage (Dolan et al. 1997 ). This parameter is modulated by the activity of pectin methylesterases (PMEs) localized in the cell walls, which alter wall stiffness and porosity, thereby introducing new functional properties during development (Jolie et al. 2010 ; Albersheim et al. 2011 ). The action of PMEs on HGs has been implicated in the formation of Manihot esculenta (Euphorbiaceae) galls, where a demethylesterification process was observed during the transition from leaf tissue to gall structures (Souza et al. 2024 ). A decrease in pectin synthesis signals the transition from primary to secondary cell wall formation, which is typically marked by the onset of lignin biosynthesis and the arrest of cell expansion. Lignification represents the final stage of differentiation in certain plant cells, contributing to increased wall rigidity and structural stability (Lawoko 2005 ). Hemicelluloses, such as heteroxylans, heteromannans, and xyloglucans, interact with cellulose microfibrils, forming crosslinks that help regulate cell wall extensibility and, consequently, cell expansion (Cosgrove 2016 ; Chen et al. 2019 ). Additionally, heteromannans can serve as carbohydrate storage compounds (Meier and Reid 1982 ; Scheller and Ulvskov 2010 ). In galls, changes in cell expansion axes and elongation patterns appear to be associated with the spatial distribution of hemicelluloses in the cell walls, which are also related to functional adaptations for nutrient acquisition by the inducing organism, as reported in various gall morphotypes of Inga ingoides (Fabaceae) (Bragança et al. 2020 ). In recent years, immunocytochemical studies of cell walls in galls have been conducted to identify patterns related to plant developmental responses (e.g., Formiga et al. 2013 ; Teixeira et al. 2017; Magalhães et al. 2014 ; Santos et al. 2024 ; Souza et al. 2024 ; Ferreira et al. 2025 ). In this study, we investigated cell wall composition via an immunocytochemical approach in a hemipteran-induced gall model: Sapium glandulosum (Euphorbiaceae) galls induced by Neolithus fasciatus (Hemiptera: Triozidae). S. glandulosum galls are globoid in shape and exhibit histological and cytological compartmentalization within the cortex, along with a high concentration of carbohydrates, which serve as an energy source for sustaining gall development (Rosa et al. 2024 ). We expect natural redifferentiation followed by maturation of cell walls, as observed in other galls, with a notable increase in structural reinforcement in cell walls (e.g., increasing xyloglucan staining) during gall development, particularly in vascular tissues since they are the inducer’s feeding site. Changes from typical cell wall development may reflect strategies to evade host defenses, such as inhibiting extensin synthesis. Materials and methods Host plant–insect system and sample preparation Nongalled leaves and galls were collected from three individuals of Sapium glandulosum in the Jataí municipality, Goiás State (17°54’03.6’’ S, 51°45’14.0’’ W), Brazil, within a Cerrado vegetation area. Nongalled leaves were sampled from the third node of the median portion, from the interveinal region. Galls were collected at three distinct developmental stages: young, mature, and senescent. The classification of gall stages was based primarily on external morphology, such as shape and size, and the developmental stage of the inducing insect, Neolithus fasciatus (Hemiptera: Triozidae). Sapium glandulosum galls develop mainly on the first nodes of the branch, between the first and tenth nodes (Fig. 1 a). Young galls (Fig. 1 b) were characterized by their small size and the presence of second- to third-instar nymphs. Mature galls (Fig. 1 c) had ceased growing and contained nymphs in the fifth-instar stage. Senescent galls (Fig. 1 d) were identified by the confirmed absence of the inducer within the nymphal chamber, indicating the end of gall activity. After collection, fragments of nongalled leaves and galls at different developmental stages were fixed in FAA solution (formalin, acetic acid, and 50% ethanol at a 1:1:18 v/v/v ratio) following Johansen (1940). After 48 hours of fixation, the samples were transferred to and stored in 70% ethanol. Anatomical analyses Fragments of nongalled leaves (n = 3) and galls (n = 3 per developmental stage) were dehydrated through an ethanol series and embedded in 2-hydroxyethyl methacrylate (Historesin®, Leica Instruments, Heidelberg). Five-micron-thick transverse sections were obtained via a rotary microtome (Leica® RM2235) and stained with 0.05% toluidine blue at pH 4.7 (O’Brien et al. 1964 ). The slides were mounted with Entellan® (Kraus and Arduin 1997 ) and photographed under a light microscope (Leica® DM750) equipped with a digital camera (Leica® ICC50 HD). Immunocytochemical analyses The histological sections obtained from the Historesin®-embedded samples were used without staining or permanent mounting. For hemicellulose labeling, the sections were pretreated with a solution containing 10 µg mL⁻¹ pectate lyase (Sigma‒Aldrich) in 2 mM CaCl₂ and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer (Sigma‒Aldrich, USA), pH 10, for 2 hours. Following pretreatment, all the samples underwent the same procedure: the sections were immersed in a blocking solution of Molico® powdered milk in phosphate-buffered saline (PBS) for 30 minutes. The samples were then incubated for 2 hours with primary monoclonal antibodies against LM1, LM2, JIM5, JIM7, LM5, LM6, LM11, LM15, and LM21 (Centre for Plant Sciences, University of Leeds, UK) (Table 1 ). After being washed in PBS, the sections were incubated in the dark with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (diluted 1:100 in 3% milk/PBS) for 2 hours. A section from the leaf and each gall developmental stage was processed without primary antibody incubation as a control for nonspecific binding and autofluorescence. After a final PBS wash, the sections were mounted in 50% glycerin. Fluorescence analysis was performed via a Leica® DM4000 LED fluorescence microscope with an HD monochromatic camera (DFC3000 G) and Leica® analysis software. Quantitative fluorescence measurements were obtained via ImageJ® software (version 1.51k; http://rsb.info.nih.gov/ij ) , with the fluorescence intensity evaluated via a grayscale analysis method. The fluorescence intensity in the cell walls was categorized as (i) weak ( 30 Gy value) based on the range of observed values. Table 1 Monoclonal antibodies, epitopes and respective references. Monoclonal antibodies Epitopes References Proteins LM1 Extensins Smallwood et al. (1996) LM2 Arabinogalactans Smallwood et al. (1996); Yates et al. (1996) Pectins LM5 (1 → 4) β-D-galactans Jones et al. (1997); Andersen et al. 2016 ) LM6 (1 → 5) α-L-arabinans Williats et al. (1998); Willats et al. 2001 ); Verhertbruggen et al. (2009) JIM5 Homogalacturanans (HGs) partially methylesterified up to 40% (termed as low methylesterified HGs) Clausen et al. ( 2004 ) JIM7 Methylesterified HGs – 15% to 80% (termed as methylesterified HGs) Clausen et al. ( 2004 ) Hemicelluloses LM11 Heteroxylans McCartney et al. ( 2005 ) LM15 XXXG motif of xyloglucans Marcus et al. ( 2008 ) LM21 Heteromannans Marcus et al. ( 2010 ) Results Anatomical profile The mature nongalled leaf has a uniseriate epidermis characterized by a thin cuticle and rectangular cells (Fig. 2 a). The leaf is glabrous, hypostomatic, and displays dorsiventral mesophyll organization (Fig. 2 a). The mesophyll consists of a single, slightly elongated layer of palisade parenchyma and 4 to 5 layers of spongy parenchyma with few intercellular spaces (Fig. 2 a, b). The vascular bundles are collateral and well differentiated, accompanied by intrusive growth laticifers (Fig. 2 b, c). The young gall exhibits a uniseriate epidermis with anticlinally elongated cells undergoing hyperplasia (Fig. 2 d) and stomata in formation. The cortex is parenchymatic and organized into two distinct regions: (i) an outer cortex composed of elongated cells containing chloroplasts concentrated in the peripheral layers and (ii) an inner cortex made up of smaller, densely packed cells with a cytoplasm-rich profile (Fig. 2 d, e). Large and conspicuous nuclei are present in both cortical and epidermal cells (Fig. 2 e, f). Vascular bundles begin to differentiate in the median region of the cortex (Fig. 2 d-f). The nymphal chamber was visible as a developing central cavity (Fig. 2 d-f). The mature gall features a uniseriate epidermis with flattened, tabular cells that are more periclinally elongated than those in young galls (Fig. 2 g). The cortex remains parenchymatous and is divided into two regions (Fig. 2 g). Nuclei were less evident in all the tissues (Fig. 2 h, i). The collateral vascular bundles are centrally located (Fig. 2 h), with associated laticifers. The nymphal chamber was still one nymphal chamber (Fig. 2 i). In the senescent stage of the gall, the epidermis shows no major structural differences compared with the mature stage (Fig. 2 j). The cortex and vascular tissues retain their previous organization, but the cytoplasmic content in most cortical cells becomes inconspicuous (Fig. 2 k, l). At this stage, both cell division and hypertrophy ceased completely (Fig. 2 j-l). Immunocytochemistry In nongalled leaves, low-methylesterified homogalacturonan (HG) epitopes, recognized by JIM5, were detected in both epidermal and mesophyll cells (Fig. 3 ; Fig. 4 A). Xyloglucan epitopes, recognized by LM15, showed moderate to intense labeling in the epidermis and mesophyll cell walls (Fig. 3 ; 4 b). The (1→4)-β-D-galactan epitopes recognized by LM5 were moderately to intensely labeled across all the leaf tissues (Fig. 3 ; 4 c). In contrast, the (1→5)-α-L-arabinan epitopes, which are recognized by LM6, exhibited moderate labeling restricted to epidermal cell walls (Fig. 3 ; 4 d). In young galls, epitopes recognized by JIM5 were weakly detected in the cortical parenchyma cell walls (Fig. 3 ; 4 e), while JIM7 moderately detected the epitopes in the epidermis and weakly in the cortex (Fig. 3 ; 4 f). Epitopes of (1→4)-β-D-galactan and (1→5)-α-L-arabinan, recognized by LM5 and LM6, respectively, were present only in the cortical parenchyma cell walls, with moderate intensity in both cases (Fig. 3 ; 4 g, 4 h). In mature galls, epitopes recognized by JIM5 were detected weakly in the epidermis and cortical parenchyma cell walls (Fig. 3 ; 5 a). In contrast, JIM7 strongly marked the epitopes in the parenchyma cell walls and weakly in the vascular bundles (Fig. 3 ; 5 b). The epitopes recognized by LM5 showed moderate labeling in the parenchyma cell walls (Fig. 3 ; 5 c), whereas the epitopes recognized by LM6 were not detected at this developmental stage (Fig. 3 ). Epitopes recognized by LM15 were detected throughout all the gall tissues, with moderate labeling in the epidermis, weak labeling in the parenchyma, and intense labeling in the vascular bundles (Fig. 3 ; 5 d). The senescent galls exhibited widespread labeling for both pectic and hemicellulosic epitopes, generally with moderate intensity across tissues (Fig. 3 ). The epitopes recognized by JIM5 were intensely labeled in the epidermis cell walls and moderately labeled in the cortical parenchyma and vascular bundles (Fig. 3 ; 5 e), whereas the epitopes recognized by JIM7 were moderately labeled throughout all the tissues (Fig. 3 ; 5 e). The epitopes detected by LM6 were moderately labeled in both the parenchyma and vascular bundle cell walls (Fig. 3 ; 5 g). Epitopes of xyloglucan epitopes, recognized by LM15, also display moderate labeling in the cortex and vascular bundle cell walls at this developmental stage (Fig. 3 ; 5 h). Extensin and arabinogalactan protein (AGP) epitopes, which are recognized by LM1 and LM2, respectively, were not labeled in either nongalled leaf tissues or galls. Similarly, heteromannan epitopes recognized by LM21 were also not detected in any of the analyzed tissues (Fig. 3 ). Discussion The development of Sapium glandulosum galls induced by Neolithus fasciatus (Hemiptera: Triozidae) involves the redifferentiation of host leaf tissues, resulting in the formation of a parenchymatic gall, a pattern commonly observed in galls induced by Hemiptera (Ferreira et al. 2019 ; Rosa et al. 2024 ). Although these galls are classified as nonnutritive, they exhibit cytological features resembling the nutritive tissues typically found in galls induced by other insect taxa, such as galls induced by Cecidomyiidae in Manihot esculenta (Euphorbiaceae) (Silva et al. 2024). In this context, the response of host tissue to biotic stress and subsequent gall development is driven by modifications in cell wall composition, ultimately resulting in the formation of globoid galls composed of cells with novel functionalities. These changes were primarily associated with variations in the degree of pectin methylesterification, which is influenced by the activity of pectin methylesterases (PMEs). PMEs play a crucial role in plant immune responses during biotic interactions by modulating cell wall degradation, thereby contributing to induced resistance in plant tissues (Riseh et al. 2024). Additionally, structural modifications in the side chains of rhamnogalacturonan I (RG-I) and the cross-linking of pectic polymers further impact cell wall properties and defense mechanisms. The preferred sites for gall induction on S. glandulosum are young leaves located at the first nodes. These young leaves exhibit conspicuous nuclei across all tissues, suggesting the persistence of meristematic characteristics (Meier et al. 2017 ). This observation aligns with the notion that gall-inducing organisms typically target developmentally responsive regions in host plants (Oliveira et al. 2016 ). In terms of cell wall properties, there appears to be a balance between flexibility and rigidity in most leaf cells. Flexibility is indicated by moderate to intense labeling of (1→4)-β-D-galactans across all tissues, whereas rigidity is demonstrated by strong labeling of low methyl-esterified homogalacturonans (HGs) in the epidermis and chlorophyll parenchyma (McCartney et al. 2005 ; Willats et al. 2001 ). The low labeling of methyl-esterified HGs indicates a natural cell maturation process driven by pectin methylesterases (PMEs), which increase cell wall stiffness (Jolie et al. 2010 ). The epitope labeling of S. glandulosum leaves suggests that the cell walls are still undergoing maturation, even in more developed nodes. Following gall induction in S. glandulosum , there is a notable reduction in the labeling of low methyl-esterified HGs by the JIM5 antibody, which becomes weak in the cell walls of both the epidermis and parenchyma. Concurrently, methyl-esterified HGs begin to appear in the parenchyma, accompanied by the continued moderate labeling of (1→4)-β-D-galactans. During cell development, the activity of PMEs promotes the demethylesterification of HGs in the cell wall, leading to new functional properties such as increased stiffness and porosity (Jolie et al. 2010 ). This enzymatic process has been observed in the leaf galls of Psidium cattleianum (Carneiro et al. 2014 ). In contrast, PME activity appears to be inhibited in the leaf galls of Baccharis dracunculifolia (Oliveira et al. 2014 ) and Croton floribundus (Teixeira et al. 2017), resulting in persistent labeling of methylesterified HGs throughout gall development. Interestingly, S. glandulosum does not conform to either of these patterns. In this gall, intense JIM7 labeling, indicative of highly methylesterified HGs, was observed exclusively in the parenchyma cell walls at the mature stage, suggesting an atypical synthesis of methylesterified HGs in differentiated cells. This pattern of HG labeling indicates the retention of cell wall plasticity and totipotency, which may enable the parenchyma to have the capacity for future structural modifications. Such parenchyma plasticity has also been documented in eight gall morphotypes of Croton floribundus (Teixeira et al. 2022 ), where parenchyma underwent the most significant structural changes compared with nongalled leaf tissues. M. esculenta galls, which are methylesterified HGs recognized by LM20, are intensely labeled in nutritive cells, which is also related to their capacity for cell expansion (Souza et al. 2024 ). HGs also play a crucial role in the initial defense response of plants to biotic stress by promoting the activity of cell wall-degrading enzymes (CWDEs), which facilitate the breakdown of the cell wall. Among these, PMEs are key enzymes that remove methyl ester groups from pectins, thereby increasing the accessibility of other CWDEs (Pagorelko et al. 2013; Wojtasik et al. 2011 ; Wolf 2022 ). The degradation of pectins leads to the formation of oligogalacturonides (OGs), which serve as elicitors of various immune responses in the host plant (Riseh et al., 2024). In this context, the reduced presence of low methyl-esterified HGs in both young and mature galls suggest the suppression of PME activity. This phenomenon is likely to reflect a strategy by which the gall-inducing organism inhibits host defense signaling, thereby facilitating gall development. In Matayba guianensis (Sapindaceae) leaf galls, the demethyl-esterified HGs were also related to increased vulnerability of galls to the action of pathogens (Silva et al. 2021 ), as reported in previous studies (Tans-Kersten et al. 1998 ; Wydra and Beri 2006 ). This discussion offers a novel perspective for investigating cell wall dynamics and defense modulation during gall formation. The maintenance of epitopes associated with cell wall flexibility, together with the emergence of methyl-esterified HGs, indicates increased responsiveness of the cell walls in S. glandulosum young galls, especially with increasing cell wall elongation capacity (see Albersheim et al. 2010 ). Our immunocytochemical approach contrasts the cytological features of S. glandulosum young galls, which exhibit an advanced stage of cell differentiation, with large vacuoles, peripheral cytoplasm, and fully developed chloroplasts (Rosa et al. 2024 ). Therefore, although the protoplast characteristics of young galls in S. glandulosum indicate the onset of cellular differentiation, the cell wall remains responsive to growth. The maturation of S. glandulosum galls was accompanied by structural reinforcement of the cell walls and a reduced capacity for cell elongation in the epidermis and vascular bundles, as evidenced by moderate to intense labeling of xyloglucans with the LM15 antibody. Xyloglucans are the most abundant hemicelluloses in the primary cell walls of eudicots (Scheller and Ulvskov 2010 ), forming strong associations with cellulose that contribute to the structural integrity of the cell walls (Somerville et al. 2004 ; Albersheim et al. 2010 ). This polysaccharide is associated with reduced sliding of cellulose microfibrils, thereby limiting cell wall expansion (Voiniciuc et al. 2018 ). In galls, xyloglucans are associated with the nutrition of galling herbivores, especially galling inducers that feed on nutritive tissues, as shown for galls of Inga ingoides (Fabaceae) (Bragança et al. 2020 ), Manihot esculenta (Euphorbiaceae) (Silva et al. 2024) and Macairea radula (Melastomataceae) (Santos et al. 2024 ). Rosa et al. ( 2024 ) demonstrated that S. glandulosum galls contain abundant neoformed vascular bundles, which serve to increase the number of feeding sites available to the sap-sucking gall inducer (Burckhardt 2005 ; Carneiro and Isaias 2015 ), as well as to increase the supply of water and nutrients to the gall tissues. Furthermore, the intense labeling of xyloglucans in the vascular bundles of S. glandulosum mature galls appears to reinforce their cell walls, providing structural support to withstand the feeding activity of the galling inducer. Additionally, sap-sucking insects are known to secrete pectinases and hemicellulases to facilitate penetration into plant tissues (Cherqui and Tjallingii 2000 ; Calderón-Cortés et al. 2012 ), thereby releasing pentoses, hexoses, and other sugars (Aro et al. 2005 ). In the context of Hemiptera-induced galls, the continuous feeding activity of the insect may lead to the progressive degradation of cell walls, resulting in the release of sugars into the phloem. These sugars may then become available to insects during feeding, as is viable in the case of gall-inducing N. fasciatus . During the development of S. glandulosum galls, rhamnogalacturonan I (RG-I), which contains (1→4) β-D-galactan side chains, was particularly prominent. LM5 labeled this epitope from moderate to intense in the epidermal and parenchyma cell walls of young and mature galls. RG-I constitutes approximately 20–35% of the pectic matrix in the cell wall and can perform diverse functions, especially depending on the composition of its side chains (Pérez et al. 2003 ). The presence of (1→4) β-D-galactan epitopes in cell walls, detected by LM5, has previously been associated with tissue flexibility and structural reconfiguration during gall formation, such as in the “kidney-shaped” galls of Baccharis dracunculifolia (Oliveira et al. 2014 ). In S. glandulosum galls, sustained LM5 labeling suggests that the cell walls retain expansion capacity, similar to what is observed in nongalled leaves. This finding reinforces the idea of high tissue plasticity within the gall structure (Ferreira et al. 2019 ). In senescent galls, however, the (1→4) β-D-galactan epitopes are gradually replaced by (1→5) α-L-arabinan epitopes, as indicated by LM6 labeling. This shift likely confers a reinforcement in the cell wall and an increase in cell-to-cell adhesion (Brummell et al. 2004 ). Similar LM6 labeling has been reported in the globoid galls of Croton floribundus (Teixeira et al. 2017) and Psidium myrtoides (Carneiro et al. 2014 ). Finally, the low labeling of most epitopes used here in the senescent galls may indicate the end of the gall cycle, following the protoplast degradations shown by Rosa et al. ( 2024 ). Main conclusions The formation and development of galls on Sapium glandulosum leaves followed a series of key steps. Initially, the presence of (1→4)-β-D-galactan epitopes in young gall tissues likely contributes to cell elongation, thereby promoting early gall growth. As the galls mature, the labeling of xyloglucans in the vascular bundles suggests a role in reinforcing cell walls, providing the structural support necessary for the feeding activity of the gall-inducing organism. Additionally, the consistently reduced levels of low methyl-esterified homogalacturonans revealed in both young and mature galls indicate suppressed pectin methylesterase activity—a potential strategy to inhibit host defense signaling and thereby facilitate gall development. Declarations Author contribution Vinícius Coelho Kuster and Maraíza Sousa Silva conceived the study. Maraíza Sousa Silva conducted the material collection and preparation. Anatomical analyses were performed by Maraíza Sousa Silva, Lorena Moreira Pires Rosa, and Maísa Barbosa Santos. Immunocytochemical analyses were carried out by Vinícius Coelho Kuster, Lana Laene Lima Dias, and Denis Coelho de Oliveira. The first draft of the manuscript was written by Maraíza Sousa Silva and Vinícius Coelho Kuster, with subsequent revisions refined by Vinícius Coelho Kuster, Denis Coelho de Oliveira, and Maísa Barbosa Santos. All authors read and approved the final manuscript. Funding The current study was financed in part by “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq) for financial support (403159/2023-7) and fellowship for Denis Coelho de Oliveira (303691/2022-0). We also thank the “Fundação de Amparo à Pesquisa do Estado de Minas Gerais” (FAPEMIG) and “Fundação de Amparo à Pesquisa do Estado de Goiás” (FAPEG) for financial support. Data availability The datasets generated during the current study are available from the corresponding author upon request. Conflict of interest The authors declare no competing interests. References Albersheim P, Darvill A, Roberts K, et al (2010) Plant Cell Walls. Garland Science, Londres, England. Albersheim P, Darvill A, O’Neill M, Schols H, and Voragen A (2011). An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants. In: Visser J and Voragen A (eds) Pectins and pectinases. Elsevier Sciences, Amsterdam, Netherlands, pp. 47–53. 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12:17:21","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1063677,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/09b41262f2d2ad0b65370e4d.jpg"},{"id":92715263,"identity":"6d9abced-cc4a-46eb-b04d-d1329a0dba94","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":888317,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/a69367381332785ad06ca126.jpg"},{"id":92715275,"identity":"c5f31cab-032f-4e33-8487-6854ec7247e4","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1665925,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/d9b2fb9043b5f564cf640f57.png"},{"id":92715277,"identity":"7a9ae8a3-a38f-4bf3-97dc-2f9a1d3117fc","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2623762,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/107c8fa032a37ec0fa12b65e.png"},{"id":92715272,"identity":"478af8ab-475e-4fbe-a928-365d7bea6b69","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":187587,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/245fd75b07c89fcec4181d78.png"},{"id":92715270,"identity":"8a352a59-5d38-44ea-9137-f504fd8c841b","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1042834,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/4697af06b91548805b75fb46.png"},{"id":92715985,"identity":"9c3ee849-0ad7-4868-8bad-11ffd8461c38","added_by":"auto","created_at":"2025-10-03 12:25:22","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":826460,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/c4f6558e7c061b9f66a1e05a.png"},{"id":92715973,"identity":"b31b0c78-5ddd-473a-8231-53f85a70509a","added_by":"auto","created_at":"2025-10-03 12:25:21","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147649,"visible":true,"origin":"","legend":"","description":"","filename":"9902e9f7480948ee830b719911b7f83d1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/3897c18e27d02d4eaf10bdc8.xml"},{"id":92716215,"identity":"d2943f13-659a-40e4-81f3-f96ef021b2f6","added_by":"auto","created_at":"2025-10-03 12:33:21","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157757,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/4162e9131a896b4022e4cb7c.html"},{"id":92716214,"identity":"eca833c6-c20c-4a21-9e3a-cedd6fba8bfa","added_by":"auto","created_at":"2025-10-03 12:33:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1128600,"visible":true,"origin":"","legend":"\u003cp\u003eStructural features of \u003cem\u003eSapium glandulosum \u003c/em\u003egalls. A- Branch with galls; B- Young gall; C- Mature gall; D- Open senescent gall with nymphal chamber (NC) pointed out.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/0f52451b133cafd64b7fe979.jpg"},{"id":92715970,"identity":"4481dcd6-652c-4c72-9278-c8c9af6ea024","added_by":"auto","created_at":"2025-10-03 12:25:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2497120,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomical features of the nongalled leaves and galls of \u003cem\u003eSapium glandulosum\u003c/em\u003e. A-C- Nongalled leaf, with dorsiventral mesophyll, collateral bundles, and stomata (arrow) on the abaxial surface; D-F- Young gall, with parenchymal cortex in formation and a single nymphal chamber; G-I- Mature gall, with parenchymal cortex formed, as well as collateral vascular bundles; J-L- Senescent gall, with similarity of tissue organization to the mature gall. \u003cem\u003eAbbreviations:\u003c/em\u003eEAB- Epidermis on the abaxial surface; EAD- Epidermis on the adaxial surface; Co-Cortex; La-Laticifer; NC- Nymphal chamber; Ph- Phloem; PP- Palisade parenchyma; SP- Spongy parenchyma; VB- Vascular bundle; Xy- Xylem.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/147792d4f130d3ab474313aa.jpg"},{"id":92715253,"identity":"a362dd20-7e67-4577-af1f-e49a0ea79093","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":391500,"visible":true,"origin":"","legend":"\u003cp\u003eIntensity of immunofluorescence reactions in tissues of nongalled leaves and galls in young, mature and senescent stages of \u003cem\u003eSapium glandulosum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/0492d4974546028e3b27a552.jpg"},{"id":92715254,"identity":"9c0f3301-872f-4531-b648-d54c08a8fb4f","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1063677,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of pectic and hemicellulosic epitopes in nongalled leaves (A-D) and young galls (E-H) of \u003cem\u003eSapium glandulosum\u003c/em\u003e. A, E- Low methylesterification HGs, identified by JIM5; B- Xyloglucan epitopes, recognized by LM15; C, G- (1→ 4) β-D-galactan epitopes, labeled by LM5; D, H- (1 → 5) α-L-arabinan epitopes, labeled by LM6; F- Methylesterified HGs, marked by JIM7. \u003cem\u003eAbbreviations\u003c/em\u003e: Co-Cortex; Ep- Epidermis; EAB- Epidermis on the abaxial surface; EAD- Epidermis on the adaxial surface; PP- Palisade parenchyma; SP- Spongy parenchyma; Xy- Xylem.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/e0592df18e7bcdb574820f43.jpg"},{"id":92715257,"identity":"88cb3275-0375-4629-8fb5-ec4deabd7ead","added_by":"auto","created_at":"2025-10-03 12:17:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":888317,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of pectic and hemicellulosic epitopes in mature (A-D) and senescent (E-H) galls of \u003cem\u003eSapium glandulosum\u003c/em\u003e. A, E- Low methylesterification HGs identified by JIM5; B, F- Methylesterified HGs labeled by JIM7; C- (1→ 4) β-D-galactan epitopes recognized by LM5; D, H- Xyloglucan epitopes recognized by LM15; G- (1 → 5) α-L-arabinan epitopes labeled by LM6. \u003cem\u003eAbbreviations\u003c/em\u003e: Co- Cortex; Ep- Epidermis; VB- Vascular bundle.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/9b83488458afa56bc3965f51.jpg"},{"id":102785335,"identity":"7620b885-1b9b-4634-9e8d-6494b5867a30","added_by":"auto","created_at":"2026-02-16 16:05:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6638410,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7601682/v1/67a5911b-1c87-4235-a878-0dca7709c52e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunocytological composition of cell walls in Sapium glandulosum (Euphorbiaceae) galls reveals steps in their establishment and development","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe plant cell wall comprises a dynamic array of components, including cellulose, hemicelluloses, and pectins, as well as small amounts of proteins that perform various functions in plants (Cosgrove \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lorenzo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), such as serving as the first line of defense against invading microbes (Bacete et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The physical properties of the cell walls depend on the chemical interactions among these components, which, together with turgor pressure, maintain structural equilibrium in plant cells (Anderson and Kieber \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, biotic stress resulting from interactions between plant cells and other organisms can disrupt the cell wall and trigger multiple plant responses (Riseh et al. 2024). Galls are novel plant structures induced by the action of foreign organisms (Shorthouse et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Harris and Pitzschke \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and are typically initiated by an increase in localized oxidative stress (Isaias et al. 2015). This oxidative stress is regulated by the inducing organism, which manipulates host plant tissues for its own benefit (Oliveira et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Gall formation involves alterations to the plant's preexisting morphogenetic patterns, leading to a novel cellular architecture characterized by convergent processes such as cell hypertrophy, tissue hyperplasia, and cellular redifferentiation (Oliveira et al. 2010; Guedes et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As a result, the emergence of new cell types and the associated modifications in cell wall dynamics are fundamental to gall development and reflect the specific influence of each inducing organism.\u003c/p\u003e\u003cp\u003eCellulose microfibrils are synthesized by the cellulose synthase complex in the plasma membrane, which uses UDP-glucose as a substrate (Albersheim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The spatial arrangement of these microfibrils in the primary cell wall determines the direction of cell elongation and hypertrophy (Kimura et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Albersheim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). During gall development, the reorganization of cellulose microfibrils has been associated with changes in cell shape and the formation of gall-specific structures (Magalh\u0026atilde;es et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Arabinogalactan proteins (AGPs) are located within the cell wall and play key roles in cell signaling and adhesion during plant morphogenesis (Fincher et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Majewska-Sawka and Nothnagel \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Extensins and other glycoproteins are incorporated into the insoluble microfibrillar matrix of primary cell walls, where they contribute to structural reinforcement, particularly in mature organs (Carpita and Gibeaut \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Brownleader et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Albersheim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In galls, extensin labeling is typically associated with growth cessation, as expected (Formiga et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Teixeira et al. 2017). However, some studies have reported unexpected extensin labeling in young galls, suggesting a more complex role during early gall development (Carneiro et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Conversely, in \u003cem\u003eEspinosa nothofagi\u003c/em\u003e (Hymenoptera) galls, extensin synthesis is inhibited, an effect that may be linked to the suppression of the host plant\u0026rsquo;s innate immune response, thereby facilitating gall initiation and development. This inhibition may also compromise cell wall reinforcement, despite the presence of tissues associated with mechanical support in these galls (Guedes et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAnother important component of cell walls, pectins, play crucial roles in regulating extensibility and growth (Showalter \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Mohnen \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Albersheim et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, pectin functions as a signaling molecule, initiating immune responses such as antimicrobial compound production, cell wall reinforcement, and activation of defense-related genes (Riseh et al. 2024). Three main primary types of galacturonic acid-containing pectins are found in the cell wall: homogalacturonans (HGs), rhamnogalacturonans (RGs), and xylogalacturonans (XGAs) (Clausen et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Caffall and Mohnen \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). HGs are the most abundant, and their degree of methyl esterification significantly influences their functional role in the cell wall (Vincken et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The degree of pectin methylesterification can also serve as an indirect indicator of the tissue developmental stage (Dolan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). This parameter is modulated by the activity of pectin methylesterases (PMEs) localized in the cell walls, which alter wall stiffness and porosity, thereby introducing new functional properties during development (Jolie et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Albersheim et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The action of PMEs on HGs has been implicated in the formation of \u003cem\u003eManihot esculenta\u003c/em\u003e (Euphorbiaceae) galls, where a demethylesterification process was observed during the transition from leaf tissue to gall structures (Souza et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A decrease in pectin synthesis signals the transition from primary to secondary cell wall formation, which is typically marked by the onset of lignin biosynthesis and the arrest of cell expansion. Lignification represents the final stage of differentiation in certain plant cells, contributing to increased wall rigidity and structural stability (Lawoko \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Hemicelluloses, such as heteroxylans, heteromannans, and xyloglucans, interact with cellulose microfibrils, forming crosslinks that help regulate cell wall extensibility and, consequently, cell expansion (Cosgrove \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, heteromannans can serve as carbohydrate storage compounds (Meier and Reid \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Scheller and Ulvskov \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In galls, changes in cell expansion axes and elongation patterns appear to be associated with the spatial distribution of hemicelluloses in the cell walls, which are also related to functional adaptations for nutrient acquisition by the inducing organism, as reported in various gall morphotypes of \u003cem\u003eInga ingoides\u003c/em\u003e (Fabaceae) (Bragan\u0026ccedil;a et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, immunocytochemical studies of cell walls in galls have been conducted to identify patterns related to plant developmental responses (e.g., Formiga et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Teixeira et al. 2017; Magalh\u0026atilde;es et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Santos et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this study, we investigated cell wall composition via an immunocytochemical approach in a hemipteran-induced gall model: \u003cem\u003eSapium glandulosum\u003c/em\u003e (Euphorbiaceae) galls induced by \u003cem\u003eNeolithus fasciatus\u003c/em\u003e (Hemiptera: Triozidae). \u003cem\u003eS. glandulosum\u003c/em\u003e galls are globoid in shape and exhibit histological and cytological compartmentalization within the cortex, along with a high concentration of carbohydrates, which serve as an energy source for sustaining gall development (Rosa et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We expect natural redifferentiation followed by maturation of cell walls, as observed in other galls, with a notable increase in structural reinforcement in cell walls (e.g., increasing xyloglucan staining) during gall development, particularly in vascular tissues since they are the inducer\u0026rsquo;s feeding site. Changes from typical cell wall development may reflect strategies to evade host defenses, such as inhibiting extensin synthesis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHost plant\u0026ndash;insect system and sample preparation\u003c/h2\u003e\u003cp\u003eNongalled leaves and galls were collected from three individuals of \u003cem\u003eSapium glandulosum\u003c/em\u003e in the Jata\u0026iacute; municipality, Goi\u0026aacute;s State (17\u0026deg;54\u0026rsquo;03.6\u0026rsquo;\u0026rsquo; S, 51\u0026deg;45\u0026rsquo;14.0\u0026rsquo;\u0026rsquo; W), Brazil, within a Cerrado vegetation area. Nongalled leaves were sampled from the third node of the median portion, from the interveinal region. Galls were collected at three distinct developmental stages: young, mature, and senescent. The classification of gall stages was based primarily on external morphology, such as shape and size, and the developmental stage of the inducing insect, \u003cem\u003eNeolithus fasciatus\u003c/em\u003e (Hemiptera: Triozidae). \u003cem\u003eSapium glandulosum\u003c/em\u003e galls develop mainly on the first nodes of the branch, between the first and tenth nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Young galls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) were characterized by their small size and the presence of second- to third-instar nymphs. Mature galls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) had ceased growing and contained nymphs in the fifth-instar stage. Senescent galls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) were identified by the confirmed absence of the inducer within the nymphal chamber, indicating the end of gall activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter collection, fragments of nongalled leaves and galls at different developmental stages were fixed in FAA solution (formalin, acetic acid, and 50% ethanol at a 1:1:18 v/v/v ratio) following Johansen (1940). After 48 hours of fixation, the samples were transferred to and stored in 70% ethanol.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnatomical analyses\u003c/h3\u003e\n\u003cp\u003eFragments of nongalled leaves (n\u0026thinsp;=\u0026thinsp;3) and galls (n\u0026thinsp;=\u0026thinsp;3 per developmental stage) were dehydrated through an ethanol series and embedded in 2-hydroxyethyl methacrylate (Historesin\u0026reg;, Leica Instruments, Heidelberg). Five-micron-thick transverse sections were obtained via a rotary microtome (Leica\u0026reg; RM2235) and stained with 0.05% toluidine blue at pH 4.7 (O\u0026rsquo;Brien et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1964\u003c/span\u003e). The slides were mounted with Entellan\u0026reg; (Kraus and Arduin \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and photographed under a light microscope (Leica\u0026reg; DM750) equipped with a digital camera (Leica\u0026reg; ICC50 HD).\u003c/p\u003e\n\u003ch3\u003eImmunocytochemical analyses\u003c/h3\u003e\n\u003cp\u003eThe histological sections obtained from the Historesin\u0026reg;-embedded samples were used without staining or permanent mounting. For hemicellulose labeling, the sections were pretreated with a solution containing 10 \u0026micro;g mL⁻\u0026sup1; pectate lyase (Sigma‒Aldrich) in 2 mM CaCl₂ and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer (Sigma‒Aldrich, USA), pH 10, for 2 hours. Following pretreatment, all the samples underwent the same procedure: the sections were immersed in a blocking solution of Molico\u0026reg; powdered milk in phosphate-buffered saline (PBS) for 30 minutes. The samples were then incubated for 2 hours with primary monoclonal antibodies against LM1, LM2, JIM5, JIM7, LM5, LM6, LM11, LM15, and LM21 (Centre for Plant Sciences, University of Leeds, UK) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After being washed in PBS, the sections were incubated in the dark with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (diluted 1:100 in 3% milk/PBS) for 2 hours. A section from the leaf and each gall developmental stage was processed without primary antibody incubation as a control for nonspecific binding and autofluorescence. After a final PBS wash, the sections were mounted in 50% glycerin. Fluorescence analysis was performed via a Leica\u0026reg; DM4000 LED fluorescence microscope with an HD monochromatic camera (DFC3000 G) and Leica\u0026reg; analysis software. Quantitative fluorescence measurements were obtained via ImageJ\u0026reg; software (version 1.51k; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsb.info.nih.gov/ij\u003c/span\u003e\u003cspan address=\"http://rsb.info.nih.gov/ij\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e, with the fluorescence intensity evaluated via a grayscale analysis method. The fluorescence intensity in the cell walls was categorized as (i) weak (\u0026lt;\u0026thinsp;15 Gy value), (ii) moderate (15\u0026ndash;30 Gy value), or (iii) intense (\u0026gt;\u0026thinsp;30 Gy value) based on the range of observed values.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMonoclonal antibodies, epitopes and respective references.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMonoclonal antibodies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEpitopes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReferences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProteins\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExtensins\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSmallwood et\u0026nbsp;al. (1996)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArabinogalactans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSmallwood et\u0026nbsp;al. (1996); Yates et\u0026nbsp;al. (1996)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePectins\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(1 \u0026rarr; 4) β-D-galactans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJones et\u0026nbsp;al. (1997); Andersen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(1 \u0026rarr; 5) α-L-arabinans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWilliats et\u0026nbsp;al. (1998); Willats et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2001\u003c/span\u003e); Verhertbruggen et\u0026nbsp;al. (2009)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eJIM5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHomogalacturanans (HGs) partially methylesterified up to 40% (termed as low methylesterified HGs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClausen et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eJIM7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMethylesterified HGs \u0026ndash; 15% to 80% (termed as methylesterified HGs)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eClausen et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHemicelluloses\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHeteroxylans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMcCartney et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eXXXG motif of xyloglucans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMarcus et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLM21\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHeteromannans\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMarcus et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eAnatomical profile\u003c/h2\u003e\u003cp\u003eThe mature nongalled leaf has a uniseriate epidermis characterized by a thin cuticle and rectangular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The leaf is glabrous, hypostomatic, and displays dorsiventral mesophyll organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The mesophyll consists of a single, slightly elongated layer of palisade parenchyma and 4 to 5 layers of spongy parenchyma with few intercellular spaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). The vascular bundles are collateral and well differentiated, accompanied by intrusive growth laticifers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe young gall exhibits a uniseriate epidermis with anticlinally elongated cells undergoing hyperplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) and stomata in formation. The cortex is parenchymatic and organized into two distinct regions: (i) an outer cortex composed of elongated cells containing chloroplasts concentrated in the peripheral layers and (ii) an inner cortex made up of smaller, densely packed cells with a cytoplasm-rich profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Large and conspicuous nuclei are present in both cortical and epidermal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). Vascular bundles begin to differentiate in the median region of the cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f). The nymphal chamber was visible as a developing central cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f).\u003c/p\u003e\u003cp\u003eThe mature gall features a uniseriate epidermis with flattened, tabular cells that are more periclinally elongated than those in young galls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). The cortex remains parenchymatous and is divided into two regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Nuclei were less evident in all the tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, i). The collateral vascular bundles are centrally located (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh), with associated laticifers. The nymphal chamber was still one nymphal chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003eIn the senescent stage of the gall, the epidermis shows no major structural differences compared with the mature stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). The cortex and vascular tissues retain their previous organization, but the cytoplasmic content in most cortical cells becomes inconspicuous (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek, l). At this stage, both cell division and hypertrophy ceased completely (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej-l).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunocytochemistry\u003c/h2\u003e\u003cp\u003eIn nongalled leaves, low-methylesterified homogalacturonan (HG) epitopes, recognized by JIM5, were detected in both epidermal and mesophyll cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Xyloglucan epitopes, recognized by LM15, showed moderate to intense labeling in the epidermis and mesophyll cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The (1\u0026rarr;4)-β-D-galactan epitopes recognized by LM5 were moderately to intensely labeled across all the leaf tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In contrast, the (1\u0026rarr;5)-α-L-arabinan epitopes, which are recognized by LM6, exhibited moderate labeling restricted to epidermal cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn young galls, epitopes recognized by JIM5 were weakly detected in the cortical parenchyma cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), while JIM7 moderately detected the epitopes in the epidermis and weakly in the cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Epitopes of (1\u0026rarr;4)-β-D-galactan and (1\u0026rarr;5)-α-L-arabinan, recognized by LM5 and LM6, respectively, were present only in the cortical parenchyma cell walls, with moderate intensity in both cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh).\u003c/p\u003e\u003cp\u003eIn mature galls, epitopes recognized by JIM5 were detected weakly in the epidermis and cortical parenchyma cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In contrast, JIM7 strongly marked the epitopes in the parenchyma cell walls and weakly in the vascular bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). The epitopes recognized by LM5 showed moderate labeling in the parenchyma cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), whereas the epitopes recognized by LM6 were not detected at this developmental stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Epitopes recognized by LM15 were detected throughout all the gall tissues, with moderate labeling in the epidermis, weak labeling in the parenchyma, and intense labeling in the vascular bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe senescent galls exhibited widespread labeling for both pectic and hemicellulosic epitopes, generally with moderate intensity across tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The epitopes recognized by JIM5 were intensely labeled in the epidermis cell walls and moderately labeled in the cortical parenchyma and vascular bundles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee), whereas the epitopes recognized by JIM7 were moderately labeled throughout all the tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). The epitopes detected by LM6 were moderately labeled in both the parenchyma and vascular bundle cell walls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). Epitopes of xyloglucan epitopes, recognized by LM15, also display moderate labeling in the cortex and vascular bundle cell walls at this developmental stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e\u003cp\u003eExtensin and arabinogalactan protein (AGP) epitopes, which are recognized by LM1 and LM2, respectively, were not labeled in either nongalled leaf tissues or galls. Similarly, heteromannan epitopes recognized by LM21 were also not detected in any of the analyzed tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of \u003cem\u003eSapium glandulosum\u003c/em\u003e galls induced by \u003cem\u003eNeolithus fasciatus\u003c/em\u003e (Hemiptera: Triozidae) involves the redifferentiation of host leaf tissues, resulting in the formation of a parenchymatic gall, a pattern commonly observed in galls induced by Hemiptera (Ferreira et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rosa et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although these galls are classified as nonnutritive, they exhibit cytological features resembling the nutritive tissues typically found in galls induced by other insect taxa, such as galls induced by Cecidomyiidae in \u003cem\u003eManihot esculenta\u003c/em\u003e (Euphorbiaceae) (Silva et al. 2024). In this context, the response of host tissue to biotic stress and subsequent gall development is driven by modifications in cell wall composition, ultimately resulting in the formation of globoid galls composed of cells with novel functionalities. These changes were primarily associated with variations in the degree of pectin methylesterification, which is influenced by the activity of pectin methylesterases (PMEs). PMEs play a crucial role in plant immune responses during biotic interactions by modulating cell wall degradation, thereby contributing to induced resistance in plant tissues (Riseh et al. 2024). Additionally, structural modifications in the side chains of rhamnogalacturonan I (RG-I) and the cross-linking of pectic polymers further impact cell wall properties and defense mechanisms.\u003c/p\u003e\u003cp\u003eThe preferred sites for gall induction on \u003cem\u003eS. glandulosum\u003c/em\u003e are young leaves located at the first nodes. These young leaves exhibit conspicuous nuclei across all tissues, suggesting the persistence of meristematic characteristics (Meier et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This observation aligns with the notion that gall-inducing organisms typically target developmentally responsive regions in host plants (Oliveira et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In terms of cell wall properties, there appears to be a balance between flexibility and rigidity in most leaf cells. Flexibility is indicated by moderate to intense labeling of (1\u0026rarr;4)-β-D-galactans across all tissues, whereas rigidity is demonstrated by strong labeling of low methyl-esterified homogalacturonans (HGs) in the epidermis and chlorophyll parenchyma (McCartney et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Willats et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The low labeling of methyl-esterified HGs indicates a natural cell maturation process driven by pectin methylesterases (PMEs), which increase cell wall stiffness (Jolie et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The epitope labeling of \u003cem\u003eS. glandulosum\u003c/em\u003e leaves suggests that the cell walls are still undergoing maturation, even in more developed nodes.\u003c/p\u003e\u003cp\u003eFollowing gall induction in \u003cem\u003eS. glandulosum\u003c/em\u003e, there is a notable reduction in the labeling of low methyl-esterified HGs by the JIM5 antibody, which becomes weak in the cell walls of both the epidermis and parenchyma. Concurrently, methyl-esterified HGs begin to appear in the parenchyma, accompanied by the continued moderate labeling of (1\u0026rarr;4)-β-D-galactans. During cell development, the activity of PMEs promotes the demethylesterification of HGs in the cell wall, leading to new functional properties such as increased stiffness and porosity (Jolie et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This enzymatic process has been observed in the leaf galls of \u003cem\u003ePsidium cattleianum\u003c/em\u003e (Carneiro et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In contrast, PME activity appears to be inhibited in the leaf galls of \u003cem\u003eBaccharis dracunculifolia\u003c/em\u003e (Oliveira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and \u003cem\u003eCroton floribundus\u003c/em\u003e (Teixeira et al. 2017), resulting in persistent labeling of methylesterified HGs throughout gall development. Interestingly, \u003cem\u003eS. glandulosum\u003c/em\u003e does not conform to either of these patterns. In this gall, intense JIM7 labeling, indicative of highly methylesterified HGs, was observed exclusively in the parenchyma cell walls at the mature stage, suggesting an atypical synthesis of methylesterified HGs in differentiated cells. This pattern of HG labeling indicates the retention of cell wall plasticity and totipotency, which may enable the parenchyma to have the capacity for future structural modifications. Such parenchyma plasticity has also been documented in eight gall morphotypes of \u003cem\u003eCroton floribundus\u003c/em\u003e (Teixeira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), where parenchyma underwent the most significant structural changes compared with nongalled leaf tissues. \u003cem\u003eM. esculenta\u003c/em\u003e galls, which are methylesterified HGs recognized by LM20, are intensely labeled in nutritive cells, which is also related to their capacity for cell expansion (Souza et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHGs also play a crucial role in the initial defense response of plants to biotic stress by promoting the activity of cell wall-degrading enzymes (CWDEs), which facilitate the breakdown of the cell wall. Among these, PMEs are key enzymes that remove methyl ester groups from pectins, thereby increasing the accessibility of other CWDEs (Pagorelko et al. 2013; Wojtasik et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wolf \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The degradation of pectins leads to the formation of oligogalacturonides (OGs), which serve as elicitors of various immune responses in the host plant (Riseh et al., 2024). In this context, the reduced presence of low methyl-esterified HGs in both young and mature galls suggest the suppression of PME activity. This phenomenon is likely to reflect a strategy by which the gall-inducing organism inhibits host defense signaling, thereby facilitating gall development. In \u003cem\u003eMatayba guianensis\u003c/em\u003e (Sapindaceae) leaf galls, the demethyl-esterified HGs were also related to increased vulnerability of galls to the action of pathogens (Silva et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), as reported in previous studies (Tans-Kersten et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Wydra and Beri \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This discussion offers a novel perspective for investigating cell wall dynamics and defense modulation during gall formation.\u003c/p\u003e\u003cp\u003eThe maintenance of epitopes associated with cell wall flexibility, together with the emergence of methyl-esterified HGs, indicates increased responsiveness of the cell walls in \u003cem\u003eS. glandulosum\u003c/em\u003e young galls, especially with increasing cell wall elongation capacity (see Albersheim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Our immunocytochemical approach contrasts the cytological features of \u003cem\u003eS. glandulosum\u003c/em\u003e young galls, which exhibit an advanced stage of cell differentiation, with large vacuoles, peripheral cytoplasm, and fully developed chloroplasts (Rosa et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, although the protoplast characteristics of young galls in \u003cem\u003eS. glandulosum\u003c/em\u003e indicate the onset of cellular differentiation, the cell wall remains responsive to growth.\u003c/p\u003e\u003cp\u003eThe maturation of \u003cem\u003eS. glandulosum\u003c/em\u003e galls was accompanied by structural reinforcement of the cell walls and a reduced capacity for cell elongation in the epidermis and vascular bundles, as evidenced by moderate to intense labeling of xyloglucans with the LM15 antibody. Xyloglucans are the most abundant hemicelluloses in the primary cell walls of eudicots (Scheller and Ulvskov \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), forming strong associations with cellulose that contribute to the structural integrity of the cell walls (Somerville et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Albersheim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This polysaccharide is associated with reduced sliding of cellulose microfibrils, thereby limiting cell wall expansion (Voiniciuc et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In galls, xyloglucans are associated with the nutrition of galling herbivores, especially galling inducers that feed on nutritive tissues, as shown for galls of \u003cem\u003eInga ingoides\u003c/em\u003e (Fabaceae) (Bragan\u0026ccedil;a et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eManihot esculenta\u003c/em\u003e (Euphorbiaceae) (Silva et al. 2024) and \u003cem\u003eMacairea radula\u003c/em\u003e (Melastomataceae) (Santos et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Rosa et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated that \u003cem\u003eS. glandulosum\u003c/em\u003e galls contain abundant neoformed vascular bundles, which serve to increase the number of feeding sites available to the sap-sucking gall inducer (Burckhardt \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Carneiro and Isaias \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), as well as to increase the supply of water and nutrients to the gall tissues. Furthermore, the intense labeling of xyloglucans in the vascular bundles of \u003cem\u003eS. glandulosum\u003c/em\u003e mature galls appears to reinforce their cell walls, providing structural support to withstand the feeding activity of the galling inducer. Additionally, sap-sucking insects are known to secrete pectinases and hemicellulases to facilitate penetration into plant tissues (Cherqui and Tjallingii \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Calder\u0026oacute;n-Cort\u0026eacute;s et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), thereby releasing pentoses, hexoses, and other sugars (Aro et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In the context of Hemiptera-induced galls, the continuous feeding activity of the insect may lead to the progressive degradation of cell walls, resulting in the release of sugars into the phloem. These sugars may then become available to insects during feeding, as is viable in the case of gall-inducing \u003cem\u003eN. fasciatus\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eDuring the development of \u003cem\u003eS. glandulosum\u003c/em\u003e galls, rhamnogalacturonan I (RG-I), which contains (1\u0026rarr;4) β-D-galactan side chains, was particularly prominent. LM5 labeled this epitope from moderate to intense in the epidermal and parenchyma cell walls of young and mature galls. RG-I constitutes approximately 20\u0026ndash;35% of the pectic matrix in the cell wall and can perform diverse functions, especially depending on the composition of its side chains (P\u0026eacute;rez et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The presence of (1\u0026rarr;4) β-D-galactan epitopes in cell walls, detected by LM5, has previously been associated with tissue flexibility and structural reconfiguration during gall formation, such as in the \u0026ldquo;kidney-shaped\u0026rdquo; galls of \u003cem\u003eBaccharis dracunculifolia\u003c/em\u003e (Oliveira et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In \u003cem\u003eS. glandulosum\u003c/em\u003e galls, sustained LM5 labeling suggests that the cell walls retain expansion capacity, similar to what is observed in nongalled leaves. This finding reinforces the idea of high tissue plasticity within the gall structure (Ferreira et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In senescent galls, however, the (1\u0026rarr;4) β-D-galactan epitopes are gradually replaced by (1\u0026rarr;5) α-L-arabinan epitopes, as indicated by LM6 labeling. This shift likely confers a reinforcement in the cell wall and an increase in cell-to-cell adhesion (Brummell et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Similar LM6 labeling has been reported in the globoid galls of \u003cem\u003eCroton floribundus\u003c/em\u003e (Teixeira et al. 2017) and \u003cem\u003ePsidium myrtoides\u003c/em\u003e (Carneiro et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Finally, the low labeling of most epitopes used here in the senescent galls may indicate the end of the gall cycle, following the protoplast degradations shown by Rosa et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Main conclusions","content":"\u003cp\u003eThe formation and development of galls on \u003cem\u003eSapium glandulosum\u003c/em\u003e leaves followed a series of key steps. Initially, the presence of (1\u0026rarr;4)-β-D-galactan epitopes in young gall tissues likely contributes to cell elongation, thereby promoting early gall growth. As the galls mature, the labeling of xyloglucans in the vascular bundles suggests a role in reinforcing cell walls, providing the structural support necessary for the feeding activity of the gall-inducing organism. Additionally, the consistently reduced levels of low methyl-esterified homogalacturonans revealed in both young and mature galls indicate suppressed pectin methylesterase activity\u0026mdash;a potential strategy to inhibit host defense signaling and thereby facilitate gall development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVin\u0026iacute;cius Coelho Kuster and Mara\u0026iacute;za Sousa Silva conceived the study. Mara\u0026iacute;za Sousa Silva conducted the material collection and preparation. Anatomical analyses were performed by Mara\u0026iacute;za Sousa Silva, Lorena Moreira Pires Rosa, and Ma\u0026iacute;sa Barbosa Santos. Immunocytochemical analyses were carried out by Vin\u0026iacute;cius Coelho Kuster, Lana Laene Lima Dias, and Denis Coelho de Oliveira. The first draft of the manuscript was written by Mara\u0026iacute;za Sousa Silva and Vin\u0026iacute;cius Coelho Kuster, with subsequent revisions refined by Vin\u0026iacute;cius Coelho Kuster, Denis Coelho de Oliveira, and Ma\u0026iacute;sa Barbosa Santos. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study was financed in part by \u0026ldquo;Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico\u0026rdquo; (CNPq) for financial support (403159/2023-7) and fellowship for Denis Coelho de Oliveira (303691/2022-0). We also thank the \u0026ldquo;Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Minas Gerais\u0026rdquo; (FAPEMIG) and \u0026ldquo;Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de Goi\u0026aacute;s\u0026rdquo; (FAPEG) for financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlbersheim P, Darvill A, Roberts K, et al (2010) Plant Cell Walls. Garland Science, Londres, England.\u003c/li\u003e\n\u003cli\u003eAlbersheim P, Darvill A, O\u0026rsquo;Neill M, Schols H, and Voragen A (2011). An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants. In: Visser J and Voragen A (eds) Pectins and pectinases. Elsevier Sciences, Amsterdam, Netherlands, pp. 47\u0026ndash;53.\u003c/li\u003e\n\u003cli\u003eAndersen MCF, Boos I, Marcus SE, Kračun SK, Rydahl MG, Willats WGT, Knox JP, Clausen MH (2016) Characterization of the LM5 pectic galactan epitope with synthetic analogues of \u0026beta;-1,4- d-galactotetraose. Carbohydr Res 436:36\u0026ndash;40. https://doi.org/10. 1016/j.carres.2016.10.012\u003c/li\u003e\n\u003cli\u003eAnderson CT, Kieber JJ (2020) Dynamic construction, perception, and remodeling of plant cell walls. Annu Rev Plant Biol 71:39\u0026ndash;69. https://doi.org/10.1146/annurev-arplant-081519-035846\u003c/li\u003e\n\u003cli\u003eAro N, Pakula T, Penttil\u0026auml; M (2005) Transcriptional regulation of plant cell wall degradation by filamentous fungi. FEMS Microbiol Rev 29:719\u0026ndash;739. https://doi.org/10.1016/j.femsre.2004.11.006\u003c/li\u003e\n\u003cli\u003eBacete L, M\u0026eacute;lida H, Miedes E, Molina A (2018) Plant cell wall-mediated immunity: cell wall changes trigger disease resistance responses. Plant J 93:614\u0026ndash;636. https://doi.org/10.1111/tpj.13807\u003c/li\u003e\n\u003cli\u003eBragan\u0026ccedil;a GPP, Alencar CF, Freitas MSC, Isaias RMS (2020) Hemicelluloses and associated compounds determine gall functional traits. Plant Biol (Stuttg) 22:981\u0026ndash;991. https://doi.org/10.1111/plb.13151\u003c/li\u003e\n\u003cli\u003eBrownleader MD, Jackson P, Mobasheri A, et al (1999) Molecular aspects of cell wall modifications during fruit ripening. 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J Exp Bot 48:713\u0026ndash;720. https://doi.org/10.1093/jxb/48.3.713\u003c/li\u003e\n\u003cli\u003eFerreira BG, \u0026Aacute;lvarez R, Bragan\u0026ccedil;a GP, et al (2019) Feeding and other gall facets: Patterns and determinants in gall structure. Bot Rev 85:78\u0026ndash;106. https://doi.org/10.1007/s12229-019-09207-w\u003c/li\u003e\n\u003cli\u003eFerreira BG, Oliveira DC, Isaias RMS (2025) New functional tissues induced by a galling lepidopteran on stems of \u003cem\u003eMarcetia taxifolia\u003c/em\u003e (Melastomataceae): An analysis based on cell wall composition and dynamics. Acta Bot Brasilica 39:. https://doi.org/10.1590/1677-941x-abb-2024-0146\u003c/li\u003e\n\u003cli\u003eFincher GB, Stone BA, Clarke AE (1983) Arabinogalactan-proteins: Structure, biosynthesis, and function. 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Glycobiology 6:131\u0026ndash;139. https://doi.org/10.1093/glycob/6.2.131\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Feeding activity, Hemicellulose, Host defense, Immunocytochemistry, Pectin","lastPublishedDoi":"10.21203/rs.3.rs-7601682/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7601682/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGalls alter the tissue organization of host plants, including modifications in cell wall composition. This study investigated tissue development and cell wall dynamics in galls of \u003cem\u003eSapium glandulosum\u003c/em\u003e to identify key steps involved in their establishment. Samples of young, mature, and senescent galls, as well as nongalled leaves, were analyzed using structural and immunocytochemical approaches. For histology, samples were fixed, embedded in resin, sectioned, stained with toluidine blue, and mounted with Entellan\u0026reg;. For immunocytochemistry, resin-embedded samples were tested for epitopes of cell wall proteins, pectins, and hemicelluloses using antibodies. The leaves of \u003cem\u003eS. glandulosum\u003c/em\u003e are glabrous, hypostomatic, and exhibit dorsiventral mesophyll. Gall development alters the typical leaf morphogenetic pattern, giving rise to structures with a parenchymatic cortex. In young galls, hypertrophy and hyperplasia were observed, followed by tissue maturation in mature galls. Senescent galls showed signs of cytoplasmic degradation in most cortical cells. Structural modifications in the side chains of rhamnogalacturonan I and increased cross-linking of pectic polymers affect cell wall properties, playing roles in both development and defense responses. Immunolabeling with JIM5 in young and mature galls suggests the suppressed activity of pectin methylesterases, which may reflect a strategy by which gall-inducing organisms inhibit host defense signaling. Xyloglucan epitopes were detected in the vascular bundles of mature galls, suggesting the reinforcement of cell walls and possibly supporting the feeding activity of the gall inducer. The combination of anatomical and immunocytochemical data provided a basis for understanding how gall induction modulates cell differentiation and cell wall composition in \u003cem\u003eS. glandulosum\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Immunocytological composition of cell walls in Sapium glandulosum (Euphorbiaceae) galls reveals steps in their establishment and development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 12:17:16","doi":"10.21203/rs.3.rs-7601682/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b65f915c-aba2-46cc-92af-a48cd32ec892","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:02:12+00:00","versionOfRecord":{"articleIdentity":"rs-7601682","link":"https://doi.org/10.1007/s00709-026-02162-5","journal":{"identity":"protoplasma","isVorOnly":false,"title":"Protoplasma"},"publishedOn":"2026-02-10 15:59:15","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-10-03 12:17:16","video":"","vorDoi":"10.1007/s00709-026-02162-5","vorDoiUrl":"https://doi.org/10.1007/s00709-026-02162-5","workflowStages":[]},"version":"v1","identity":"rs-7601682","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7601682","identity":"rs-7601682","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}