Homogalacturonan Epitopes Research Articles

Aberrant growth and expansion in Penium margaritaceum triggered by disruption of microtubules and the cell wall.

Penium margaritaceum, a unicellular zygnematophyte (Streptophyta), was employed to elucidate changes in cell expansion when cells were challenged with the fungal pectinolytic enzyme, pectate lyase, and/or the microtubule disrupting agent, amiprophos-methyl (APM). Microtubule disruption by APM results in significant swelling at expansion zones. These swollen zones provide an easy marker for the location of expansion zones, particularly in cells with altered cell wall pectin. Short term treatment with pectate lyase shows pectin degradation primarily at the isthmus expansion zone and two satellite bands, corresponding with the location of future expansion in daughter cells. When the homogalacturonan lattice of the cell wall is removed by treatment with pectate lyase during long treatments, cell division is maintained, but daughter cell products are considerably smaller. Treatment of cells with a mixture of both pectate lyase and APM results in a distinct phenotype, consisting of "dumbbell"-shaped cells, as APM-induced swelling occurs at the novel expansion centers exposed by pectate lyase treatment. These cells also possess other curious alterations including an extensive, chloroplast-free cytoplasmic zone at the center of the cell, a septum containing ß-glycan, arabinogalactan and homogalacturonan epitopes, unique stacks of ER, displaced Golgi bodies and an extensive network of vacuoles. These results provide insight into the importance of cell wall integrity in defining the location of cell growth and division in P. margaritaceum. Understanding these processes in a unicellular zygnematophyte may provide insights into steps involved in the evolution of land plants.

Disruption of a DUF247 Containing Protein Alters Cell Wall Polysaccharides and Reduces Growth in Arabidopsis.

Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth.

Bacterial decay in waterlogged archaeological compression wood varies with severity of compression wood

Abstract Bacterial decay in compression wood (CW) tracheids of waterlogged archaeological wood (WAW) was investigated using light microscopy, confocal laser scanning microscopy, transmission electron microscopy (TEM), and TEM immunogold labeling. Erosion bacteria were identified as the main degraders, and the extent of cell wall degradation differed depending on the severity of CW tracheids (mild vs. severe). Mild CW tracheids showed preferential decay in the inner S2 layer, with the locally degraded and/or fragmented S3 layer remaining. In contrast, severe CW tracheids revealed gradual degradation of the cell wall from the erosion progressing from exposed faces of the cell wall as decay progressed. The overall decay was more extensive in mild than in severe CW tracheids, and degradation of the highly lignified outer S2 layer (S2L) was only detected in mild CW tracheids. TEM immunogold labeling of 1,4-β-galactan, homogalacturonan (HG), heteroxylan, and heteromannan epitopes showed that there was no preferential degradation of pectins and hemicelluloses by action of diffusible enzymes and/or agents through the un-decayed cell wall during bacterial decay, in both mild and severe CW tracheids. Inter-CW tracheid bordered pit membranes showed higher decay resistance than CW tracheid walls. Degradation of HG and heteromannan epitopes was suppressed in pit membranes.

Tocopherols mutual balance is a key player for maintaining Arabidopsis thaliana growth under salt stress

Enhanced channeling carbon through pathways: shikimate/chorismate, benzenoid-phenylopropanoid or 2-C-methyl-D-erythritol 4-phosphate (MEP) provides a multitude of secondary metabolites and cell wall components and allows plants response to environmental stresses. Through the biosynthetic pathways, different secondary metabolites, like tocopherols (TCs), are bind to mutual dependencies and metabolic loops, that are not yet fully understood. We compared, in parallel, the influence of α- and γ-TCs on metabolites involved in osmoprotective/antioxidative response, and physico-chemical modification of plasma membrane and cell wall. We studied Arabidopsis thaliana Columbia ecotype (WT), mutant vte1 deficient in α- and γ-TCs, mutant vte4 over-accumulating γ-TC instead of α-TC, and transgenic line tmt over-accumulating α-TC; exposed to NaCl.The results indicate that salt stress activates β-carboxylation processes in WT plants and in plants with altered TCs accumulation. In α-TC-deficient plants, NaCl causes ACC decrease, but does not change SA, whose concentration remains higher than in α-TC accumulating plants. α/γ-TCs contents influence carbohydrates, poliamines, phenolic (caffeic, ferrulic, cinnamic) acids accumulation patterns. Salinity results in increased detection of the LM5 galactan and LM19 homogalacturonan epitopes in α-TC accumulating plants, and the LM6 arabinan and MAC207 AGP epitopes in α-TC deficient mutants. Parallel, plants with altered TCs composition show decreased both the cell turgor and elastic modulus determined at the individual cell level. α-TC deficient plants reveal lower values of cell turgor and elastic modulus, but higher cell hydraulic conductivity than α-TC accumulating plants. Under salt stress, α-TC shows stronger regulatory effect than γ-TC through the impact on chloroplastic biosynthetic pathways and ROS/osmotic-modulating compounds.

Growth Region Impacts Cell Wall Properties and Hard-to-Cook Phenotype of Canned Navy Beans (Phaseolus vulgaris)

Cellular mechanisms underpinning the hard-to-cook phenomenon are poorly understood. Navy beans (Phaseolus vulgaris) from the same cultivar but grown in different regions of North America showed different canning characteristics. Up to 46.4% more force was required to disrupt hard beans (HB) grown in Ontario compared with soft beans (SB) grown in Manitoba. HB absorbed 12.5% and 14.7% less water during blanching and canning, respectively. Microscopic assessment revealed that canning led to swelling of middle lamella and cell walls, but preservation of cell adhesion in both types of beans. Immunofluorescence microscopy showed reduced labelling of methyl-esterified homogalacturonan (HG) and more non-methyl-esterified HG and arabinan epitopes in HB. Compositional analysis indicated that the polysaccharides from HB were less water soluble and enriched in neutral sugars, including 46.5% more arabinose and 51.8% more galactose than in SB. ELISA analysis of polymers in the canning medium supported the increased solubility of cell wall polymers in SB. In the same cultivar, growth region affects cell wall properties with consequences for processing, eating and nutritional quality.

Distribution of lignin, pectins and hemicelluloses in tension wood fibers of European ash (Fraxinus excelsior)

ABSTRACT Although there is considerable information on the chemistry of gelatinous (G) layers in tension wood (TW) fibers consisting of S1 + S2 + G cell wall structure (poplar), little is known on the chemistry of G-layers in TW fibers organized with S1+ G structure. This study investigated the distribution of lignin and non-cellulosic polysaccharides in ash TW fibers (S1+ G) using histochemistry and immunolocalization methods. TW fibers studied were fully developed (mature fibers) and obtained from two (TW-1, TW-2) mature European ash trees (Fraxinus excelsior L.). Based on differences in microfibril angle (MFA) and TW trees used, TW fibers were mainly classified into three types; 1) Type-1 fibers with MFA almost parallel to the fiber axis that were found in TW-1, 2) Type-2 fibers with 12° MFA that were abundant at the end of growth rings of TW-1 and 3) Type-3 fibers with 10° MFA that were found in TW-2. The S3 layer was absent in all TW fibers. In this study, the secondary cell wall structure of Type-1 and Type-2/Type-3 fibers were defined as G and GL (gelatinous-like) layers, respectively. Lignin with syringyl (S) units was detected in G/GL-layers, in which intensity and patterns of lignin staining likely related to the difference in MFA between G- and GL-layers. With hemicelluloses, heteroxylan and heteromannan epitopes were detected in G/GL-layers but these were much less abundant than those in S2 layers of normal wood (NW) fibers. Like lignin, distribution patterns of heteromannan epitopes in G/GL-layers likely related to differences in MFA between fiber types. Sparse xyloglucan epitopes were also detected in G/GL-layers. Homogalacturonan epitopes were absent in G/GL-layers. All fiber types showed abundant a-1, 5-arabinan epitopes in G/GL-layers. Overall results indicate that the chemistry of ash TW fibers studied differs significantly from that of other species reported previously, specifically TW fibers composed of S1 + S2 + G structure.

Galling insects as phenotype manipulators of cell wall composition during the development of galls induced on leaves of Aspidosperma tomentosum (Apocynaceae)

The gall structure is built from redifferentiation of host plant tissues, changing the cell wall proprieties and associated functions. Because of the oxidative stress imposed by the galling insect on the plant tissues, some phenotypic traits associated with cell wall metabolism may be suppressed during gall development. Using an immunocytochemical approach we investigated the pectin and glycoprotein composition of the cell walls during non-galled leaf development and galls induced on Aspidosperma tomentosum (Apocynaceae). Embedding in historesin was used for anatomical characterization, as well as for immunocytochemical analyses. The samples were probed with the primary monoclonal antibodies JIM5, JIM7, LM1, LM2, LM5 and LM6. The gall develops especially from cell hypertrophy and hyperplasia of the spongy and palisade parenchyma, which determine its shape. Epitopes of β-D-galactan and α-L-arabinan in the galls were totally suppressed or less expressed. The increased labeling of homogalacturonan epitopes, especially in senescent galls, is a clear indicator that pectin methylesterase activity is maintained despite oxidative stresses imposed by the galling insect. Arabinogalactan proteins were not detected in any non-galled leaf or gall stages. The galling insect deeply changed the structure and deposition pattern of the cell wall, promoting new functionalities in the gall tissue compartments.

Boron and calcium deficiency disturbing the growth of trifoliate rootstock seedlings (Poncirus trifoliate L.) by changing root architecture and cell wall

Boron (B) and calcium (Ca) are essential elements for plant growth. Both deficiencies inhibit root growth. However, the mechanism of inhibition is not well clear. Morphological characteristics of roots and changes in root cell wall grown at different B and Ca deficiencies were examined by using a hydroponic culture system. Both B and Ca deficiencies caused reduced plant biomass and root growth. Ca deficiency significantly decreased the fresh weight of root, stem, and leaves by 47%, 50%, and 62%, respectively, while B deficiency only reduced root fresh weight. The PCA combined with Pearson correlation analysis showed that there was significant different correlation among root parameters under B and Ca deficiency treatments when compared to control. The results of observation of transmission electron microscope showed that Ca deficiency reduced but B deprivation increased the thickness of the cell wall. Combining these technologies like X-ray diffraction, fourier transform infrared spectroscopy, homogalacturonan epitopes (JIM5 and JIM7), we confirmed that those changes above may be due to different changes in the degree of methyl esterification of pectin and glycoprotein of the cell wall. Taken together, we concluded that B deficiency can promote the formation of more low methyl esterified pectin to increase cell wall thickness, and then affect the morphological development of root system, while the formation of more highly methyl esterified pectin to increase cell wall degradation under Ca deficiency, which inhibited root elongation and formation of root branches.

Callose and homogalacturonan epitope distribution in stomatal complexes of Zea mays and Vigna sinensis.

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function.

Boron increases root elongation by reducing aluminum induced disorganized distribution of HG epitopes and alterations in subcellular cell wall structure of trifoliate orange roots

Aluminum toxicity limits the plant growth by inducing inhibition of root elongation. Although several mechanisms have been proposed regarding the phytotoxic effects of aluminum on inhibition of root elongation; the primary causes of aluminum induced root inhibition and its mitigation by boron (B) are still elusive. The present study was carried out to explore the mechanisms of B induced mitigation of aluminum toxicity and to investigate the changes in well wall structure under aluminum toxicity coupled with the techniques of confocal laser microscope, lumogallion and transmission electron microscope. The results revealed that aluminum toxicity severely hampered the root elongation and plant biomass. Moreover, alteration in subcellular structure were observed under aluminum toxicity, however, such negative effects were further exacerbated with B deficiency. Aluminum toxicity indicated disorganized distribution of HG (homogalacturonan) epitopes with higher accumulation of apoplastic aluminum. Nevertheless, B supply improved root elongation, and reduced the aluminum uptake. Taken together, it is concluded that B application can reduce aluminum toxicity and improve root elongation by decreasing Al3+ accumulation to cell wall, alteration in the cell wall structure and reducing the distribution of HG epitopes in the roots of trifoliate (Poncirus trifoliate) orange.

Boron deprivation induced inhibition of root elongation is provoked by oxidative damage, root injuries and changes in cell wall structure

Boron (B) is one of the essential micronutrients for the growth and development of higher plants and its deprivation inhibits root elongation. However, little is known about the mechanisms underlying inhibition of root elongation under B deficiency. The present study was carried out with three B levels (0, 5 and 10 μM B) to investigate the effect of B deprivation on the growth of trifoliate orange seedlings and to explore the possible mechanisms involved in the B deprivation induced root growth inhibition. The results indicated that B deprivation severely hampered primary and lateral root elongation with visible symptoms of leaf chlorosis. The B deprivation led to the accumulation of reactive oxygen species, membrane peroxidation and root cell injuries. Boron deprivation not only exhibited reduced concentrations of glutathione but also inhibited the activities of major antioxidant enzymes. Moreover, an elevated accumulation of Ca2+ ion and altered subcellular cell wall structure and disorganized homogalacturonan epitopes were observed in B deficient plants. Taken together, it is concluded that B deprivation leads to root cell injuries, changes in the cell wall structure and accumulates oxidative damage that could be possible causes of root growth inhibition of trifoliate orange under B deprivation.

Structural network of arabinogalactan proteins (AGPs) and pectins in apple fruit during ripening and senescence processes

The cell wall is an essential framework determining the overall form of the plant cell. Our study was focused on the distribution of arabinogalactan proteins (AGPs), arabinan, and homogalacturonan in fruit cells during ripening and storage with emphasis on quantitative analysis of their presence in particular regions of the cell wall - plasma membrane. The localization of the examined compounds was determined with immunohistochemistry techniques and immunogold labelling. Spatio-temporal colocalization between AGPs epitopes - [βGlcA(1→3)-αGalA(1→2)Rha] recognized by JIM13 and MAC207 antibodies, and arabinan labelled by the LM16 antibody was detected in the inner cell wall layer, in association with the plasma membrane. The specific arrangement of AGP and arabinan epitopes differentiated them from homogalacturonan epitopes, consisting of GalA residues recognized by LM19 and LM20 antibodies in all the examined fruit maturity stages. The disruption of cell wall - plasma membrane continuum, observed during ripening-associated softening process, was associated with both the substantial decrease of AGPs, pectins content and with remodeling of their arrangement. The results indicate that the textural properties of fruit during growth and postharvest storage, an attribute of fruit quality becoming selection criteria for consumers, depend on the existence of dynamic network organizing polysaccharides and glycoproteins in the extracellular matrix.

Heterogeneous distribution of pectin and hemicellulose epitopes in the phloem of four hardwood species

Key messageDistributional patterns of pectin and hemicellulose epitopes in the phloem of four hardwoods vary between cell types including sieve tube elements, companion cells, parenchyma and sclerenchyma and between tree species.Using immunolocalization methods combined with monoclonal antibodies, the distribution of pectin and hemicellulose epitopes was examined in the secondary phloem of two diffuse porous (birch, aspen)- and two ring porous (oak, ash) hardwoods with a focus on sieve tube elements (SEs), companion cells (CCs), axial/ray parenchyma cells, and sclerenchyma cells (sclereids and phloem fibers). In all tree species, rhamnogalacturonan-I (RG-I), homogalacturonan (HG), and xyloglucan epitopes were common in cell walls of SEs, CCs, and axial/ray parenchyma cells. However, the amount of these epitopes varied greatly between cell types and between hardwood species. Apart from aspen, heteroxylan or/and heteromannan epitopes were detected in SEs, but were not detected in CCs and parenchyma cells. With sclerenchyma cells, RG-I, HG, and xyloglucan epitopes were common in compound middle lamellae (CML) of sclereids and phloem fibers. Except for oak, heteromannan epitopes were also detected in CML of sclereids. Distributional patterns of epitopes in CML of birch and ash sclereids varied greatly depending on anatomical structure of CML. Secondary cell walls of sclereids and phloem fibers revealed abundant heteroxylan epitopes, but showed no heteromannan epitopes. Some phloem fibers also showed sparse xyloglucan epitopes in secondary cell walls. Together, results suggest that there are great variations in distributional patterns of pectin and hemicellulose epitopes in hardwood phloem between cell types and between tree species.

A multienzyme NSP product solubilises and degrades NSP structures in canola and mediates protein solubilisation and degradation in vitro

To conclusively assess the value enhancing effect of enzymes targeting antinutritional non–starch polysaccharides in a specific feed source, in depth understanding of the substrate is required, to use the right enzyme for the relevant polysaccharides. In the current study a commercial non-starch polysaccharide multienzyme product obtained by submerged fermentation of Aspergillus aculeatus was supplemented to canola non-starch polysaccharides. The enzyme effects studied included substrate viscosity reduction and solubilisation of non-starch polysaccharides and crude protein as well as peptide cleavage. Results were compared with the effects obtained by a monocomponent protease and a combination of protease and the multienzyme product. Viscosity reduction of solutions of xyloglucan and galactomannan solution clearly showed the presence of xyloglucanase and galactomannan activity in the product. On enzyme supplementation, there was an increase (P<0.05) in the solubilisation of rhamnose, fucose, arabinose, xylose, galactose and glucose non-starch polysaccharide residues. In a separate experiment using a different measuring system (HPLC) the product was also shown to solubilise galacturonic acid from canola meal. Highest concentrations of solubilised crude protein and peptides measured by combining the protease and the multienzyme product together.Antibodies (LM25, LM19 and LM 6) targeting different cell wall components were used in microscopy studies. Monoclonal antibody LM25, directed towards xyloglucan, bound abundantly to the intercellular spaces on the cytoplasmic side of the cell wall lining. On enzyme treatment with the multienzyme product the LM25 binding disappeared, indicating a reduction of xyloglucans as also was observed in a polysaccharide analysis of enzyme incubated canola meal.Monoclonal antibody LM19 recognizes de-esterified homogalacturonan epitopes in pectin also bound to the uronic acid residues in canola meal. The LM 19 signal was weak in samples treated only with buffer, but became stronger when the xyloglucan layer was removed by a purified experimental xyloglucanase.Monoclonal antibody LM6, directed towards arabinan, also became more visible in canola meal after the removal of the xyloglucan layer by the experimental xyloglucanase.Treatment with the commercial non starch polysaccharide multienzyme product resulted in degradation of arabinan and thereby also a disappearance of the LM6 signal. Both the pectin and the arabinan signal were visible mainly/only after removal of the xyloglucan layer in both the seed and the meal.

Cotton fiber tips have diverse morphologies and show evidence of apical cell wall synthesis.

Cotton fibers arise through highly anisotropic expansion of a single seed epidermal cell. We obtained evidence that apical cell wall synthesis occurs through examining the tips of young elongating Gossypium hirsutum (Gh) and G. barbadense (Gb) fibers. We characterized two tip types in Gh fiber (hemisphere and tapered), each with distinct apical diameter, central vacuole location, and distribution of cell wall components. The apex of Gh hemisphere tips was enriched in homogalacturonan epitopes, including a relatively high methyl-esterified form associated with cell wall pliability. Other wall components increased behind the apex including cellulose and the α-Fuc-(1,2)-β-Gal epitope predominantly found in xyloglucan. Gb fibers had only one narrow tip type featuring characters found in each Gh tip type. Pulse-labeling of cell wall glucans indicated wall synthesis at the apex of both Gh tip types and in distal zones. Living Gh hemisphere and Gb tips ruptured preferentially at the apex upon treatment with wall degrading enzymes, consistent with newly synthesized wall at the apex. Gh tapered tips ruptured either at the apex or distantly. Overall, the results reveal diverse cotton fiber tip morphologies and support primary wall synthesis occurring at the apex and discrete distal regions of the tip.

Immunocytochemical studies of axial resin canals. II. Localization of non-cellulosic polysaccharides in epithelium and subsidiary cells of Scots pine

Axial resin canals in wood are distinguished into two types based on the morphology of epithelial cells; resin canals with narrow canals and thick-walled epithelial cells (Type I), and resin canals with wide canals and thin-walled epithelial cells (Type II). Following studies on Norway spruce (Type I), the distribution of non-cellulosic polysaccharides in axial resin canals of Scots pine (Type II) is reported here using cytochemical and immunocytochemical methods. The distribution of (1→4)-β-galactan (LM5), (1→5)-α-arabinan (LM6), homogalacturonan (LM19, LM20), xyloglucan (LM15), xylan (LM10, LM11) and mannan (LM21, LM22) epitopes were examined. Axial resin canal complexes in the xylem were composed of canal, epithelium and subsidiary cells (parenchyma and strand tracheids). Strand tracheids were absent in axial resin canals in the phloem. Strand tracheids showed a completely different ultrastructure and chemistry from normal mature tracheids and other types of axial resin canal cells. Immunolocalization of non-cellulosic polysaccharides in axial resin canals showed an overall similar cell wall composition in epithelial cells and subsidiary parenchyma between the xylem and phloem. All types of axial resin canal cells in both xylem and phloem contained homogalacturonan (HG), rhamnogalacturonan-I (RG-I) and xyloglucan with a high variation in amount and chemical structure depending on cell wall region and between cell types. In particular, epithelial cell walls facing the canal showed significant differences in HG distribution from other epithelial cell wall regions. No xylan and mannan epitopes were detected in any of axial resin canal cells. Together, our results suggest that the chemistry of axial resin canal cells in Scots pine may be highly compartmentalized depending on functional differences between both cell types and cell wall regions.

Heterogeneity and Glycan Masking of Cell Wall Microstructures in the Stems of Miscanthus x giganteus, and Its Parents M. sinensis and M. sacchariflorus

Plant cell walls, being repositories of fixed carbon, are important sources of biomass and renewable energy. Miscanthus species are fast growing grasses with a high biomass yield and they have been identified as potential bioenergy crops. Miscanthus x giganteus is the sterile hybrid between M. sinensis and M. sacchariflorus, with a faster and taller growth than its parents. In this study, the occurrence of cell wall polysaccharides in stems of Miscanthus species has been determined using fluorescence imaging with sets of cell wall directed monoclonal antibodies. Heteroxylan and mixed linkage-glucan (MLG) epitopes are abundant in stem cell walls of Miscanthus species, but their distributions are different in relation to the interfascicular parenchyma and these epitopes also display different developmental dynamics. Detection of pectic homogalacturonan (HG) epitopes was often restricted to intercellular spaces of parenchyma regions and, notably, the high methyl ester LM20 HG epitope was specifically abundant in the pith parenchyma cell walls of M. x giganteus. Some cell wall probes cannot access their target glycan epitopes because of masking by other polysaccharides. In the case of Miscanthus stems, masking of xyloglucan by heteroxylan and masking of pectic galactan by heteroxylan and MLG was detected in certain cell wall regions. Knowledge of tissue level heterogeneity of polysaccharide distributions and molecular architectures in Miscanthus cell wall structures will be important for both understanding growth mechanisms and also for the development of potential strategies for the efficient deconstruction of Miscanthus biomass.

Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv.

The formation of an extracellular matrix surface network (ECMSN), and associated changes in the distribution of arabinogalactan-protein and pectin epitopes, have been studied during somatic embryogenesis (SE) and callogenesis of Trifolium nigrescens Viv. Scanning electron microscopy observations revealed the occurrence of an ECMSN on the surface of cotyledonary-staged somatic embryos as well as on the peripheral, non-regenerating callus cells. The occurrence of six AGP (JIM4, JIM8, JIM13, JIM16, LM2, MAC207) and four pectin (JIM5, JIM7, LM5, LM6) epitopes was analysed during early stages of SE, in cotyledonary-staged somatic embryos and in non-embryogenic callus using monoclonal antibodies. The JIM5 low methyl-esterified homogalacturonan (HG) epitope localized to ECMSN on the callus surface but none of the epitopes studied were found to localize to ECMSN over mature somatic embryos. The LM2 AGP epitope was detected during the development of somatic embryos and was also observed in the cell walls of meristematic cells from which SE was initiated. The pectic epitopes JIM5, JIM7, LM5 and LM6 were temporally regulated during SE. The LM6 arabinan epitope, carried by side chains of rhamnogalacturonan-I (RG-I), was detected predominantly in cells of embryogenic swellings, whilst the LM5 galactan epitope of RG-I was uniformly distributed throughout the ground tissue of cotyledonary-staged embryoids but not detected at the early stages of SE. Differences in the distribution patterns of low and high methyl-esterified HG were detected: low ester HG (JIM5 epitope) was most abundant during the early steps of embryo formation and highly methyl-esterified form of HG (JIM7 epitope) became prevalent during embryoid maturation.

Wound-induced pectin methylesterases enhance banana (Musa spp. AAA) susceptibility to Fusarium oxysporum f. sp. cubense

Recent studies suggest that plant pectin methylesterases (PMEs) are directly involved in plant defence besides their roles in plant development. However, the molecular mechanisms of PME action on pectins are not well understood. In order to understand how PMEs modify pectins during banana (Musa spp.)–Fusarium interaction, the expression and enzyme activities of PMEs in two banana cultivars, highly resistant or susceptible to Fusarium, were compared with each other. Furthermore, the spatial distribution of PMEs and their effect on pectin methylesterification of 10 individual homogalacturonan (HG) epitopes with different degrees of methylesterification (DMs) were also examined. The results showed that, before pathogen treatment, the resistant cultivar displayed higher PME activity than the susceptible cultivar, corresponding well to the lower level of pectin DM. A significant increase in PME expression and activity and a decrease in pectin DM were observed in the susceptible cultivar but not in the resistant cultivar when plants were wounded, which was necessary for successful infection. With the increase of PME in the wounded susceptible cultivar, the JIM5 antigen (low methyestrified HGs) increased. Forty-eight hours after pathogen infection, the PME activity and expression in the susceptible cultivar were higher than those in the resistant cultivar, while the DM was lower. In conclusion, the resistant and the susceptible cultivars differ significantly in their response to wounding. Increased PMEs and thereafter decreased DMs acompanied by increased low methylesterified HGs in the root vascular cylinder appear to play a key role in determination of banana susceptibility to Fusarium.

DEVELOPMENTAL LOCALIZATION OF HOMOGALACTURONAN AND XYLOGLUCAN EPITOPES IN PIT MEMBRANES VARIES BETWEEN PIT TYPES IN TWO POPLAR SPECIES

Localization of homogalacturonan (HG) and xyloglucan epitopes in developing and mature pit membranes from different pit types in xylem of Populus tremula L. × P. tremuloides Michx. (hybrid aspen) and Populus tremula L. (European aspen) was investigated using immunogold labeling. Pit types not mediated by ray parenchyma (intervessel- and fiber pits) showed significant developmental changes in HG epitope localization. Both low- and high methyl-esterified HG epitopes (recognized by LM19 and LM20, respectively) were detected in developing pit membranes of intervessel- and fiber pits until late stages of xylem formation, whereas no HG- and high methyl-esterified HG epitopes were detected in mature intervessel (except for annulus regions of pit membranes)- and mature fiber pit membranes, respectively. In contrast, no notable developmental changes in HG epitope localization were detected in pit types mediated by ray parenchyma (vessel-ray-, ray- and fiber-ray pits) during pit maturation. Vesselray- and fiber-ray pits showed abundant low- and high methyl-esterified HG epitopes in pit membranes, while ray pits showed presence of primarily low methyl-esterified HG epitope during all stages of pit development including at maturity. With xyloglucan (recognized by LM15), specific developmental changes in epitope localization were detected in vessel-ray pits. Xyloglucan epitope was detected in developing vessel-ray pit membranes, but was almost absent in mature pit membranes. Instead, xyloglucan was detected in the protective layers of vessel-ray pits showing completely different localization pattern than homogalacturonan, which was only detected in pit membranes. Together, our results suggest that the chemistry of pit membranes varies depending on both the developmental stage and pit type.