Cell Wall Pectic Polysaccharides Research Articles

Structural characteristics of cell wall pectic polysaccharides from wampee and their decreased binding with pectinase by wampee polyphenol

To investigate the structural characteristics of cell wall pectic polysaccharides from wampee, water soluble pectin (WSP), chelator-soluble pectin (CSP) and sodium carbonate-soluble pectin (SSP) were purified. And the inhibitory effects of wampee polyphenol (WPP) on pectinase when these cell wall pectic polysaccharides were used as substrates were also explored. Purified WSP (namely PWSP) had the lowest molecular weight (8.47 × 105 Da) and the highest GalA content (33.43%). While purified CSP (called PCSP) and SSP contained more abundant rhamnogalacturonan I side chains. All of them were low-methoxy pectin (DE < 50%). Enzyme activity and kinetics analysis showed that the inhibition of pectinase by wampee polyphenol was reversible and mixed type. When SSP was used as the substrate, WPP had the strongest inhibition (IC50 = 1.96 ± 0.06 mg/mL) on pectinase. Fluorescence quenching results indicated that WPP inhibited enzyme activity by interacting with substrates and enzymes. Therefore, WPP has the application potential in controlling softening of fruits and vegetables.

Apple metabolism under oxidative stress affects plant cell wall structure and mechanical properties

Several studies have shown beneficial effects of short exposure to oxidative stress on stored fruit, such as better preservation, increased firmness, preservation of polyphenolic compounds, and reduced risk of postharvest disorders such as bitter pit and superficial scald in apples. In this study the effect of short-term oxidative stress conditions on the physiology of apple fruit was investigated. Apple fruit of three cultivars were exposed to hypoxic storage conditions of various lengths to induce anaerobiosis. The response of apple fruit to short-term oxidative stress was evaluated by means of cell wall immunolabeling and atomic force microscopy. In addition, the antioxidant capacity and antioxidative activity of apple peels was assessed. Through various techniques, it was shown that short-term oxidative stress conditions promote specific enzymatic activity that induces changes in the cell wall of apple fruit cells. Exposure to short-term stress resulted in the remodeling of cell wall pectic polysaccharides, observed as an increase in the size and complexity of extracted oxalate pectin. Structural changes in the cell wall were followed by an increase in Young’s modulus (compressive stiffness of a solid material, expressed as the relationship between stress and axial strain) of the cell wall material. The data presented in this paper show in a novel way how storage under short-term oxidative stress modifies the cell wall of apple fruit at the molecular level.

Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide defined by its repeating disaccharide backbone structure of [4)-α-D-GalA-(1,2)-α-L-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identified, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown. Here a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to reflect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into a polymeric product. RGGAT1 is a member of a new GT family categorized as GT116, which does not group into existing GT-A clades and is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan. RGGAT1 has a predicted GT-A fold structure but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type. The identification of RGGAT1 and the 8-member Arabidopsis GT116 family provides a new avenue for studying the mechanism of RG-I synthesis and the function of RG-I in plants.

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum

BackgroundTerrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed.ResultsThe total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I.ConclusionsThe significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum.

Characterization of tissue‐specific differences in cell wall pectic polysaccharides of carrot root

This study investigated the physicochemical characteristics of CWPs from epidermis, cortex, and stele of carrot. The CWPs extracted from cortex contained the highest content of galacturonic acid (GalA), as well as the highest degree of methylation. Compared to Na2CO3 extractable fraction (NEF) and water extractable fraction (WEF), chelator extractable fraction (CEF) contained higher GalA content, and it was identified with more linear homogalacturonan (HG). The highest amounts of Gal, Ara, and Rha were obtained in NEF extracted from cortex, suggesting that abundant side chains attached to pectic backbone of NEF and formed “hairy region.” In addition, gentle changes of the molar mass along with tissue zones were observed in different pectic fractions. However, the molar mass of NEF was always higher than WEF regardless of the tissues. According to FTIR, the configurations of all CWPs were similar and exhibited typical structural properties of pectic polymers. Practical applications Cell wall pectic polysaccharides (CWPs) are important macromolecules which highly correlated with food products qualities. The CWPs extracted from epidermis, cortex, and stele of carrot root were prepared and their physicochemical characteristics were evaluated. Detailed characteristics of tissue-specific differences in CWPs of carrot root could provide more fundamental information for carrot-related food processing.

Cell wall composition during expansion, ripening and postharvest water loss of red bell peppers (Capsicum annuum L.)

Abstract We have investigated the cell wall polysaccharides of a commercially grown blocky-type red bell pepper (Capsicum annuum L. ‘Funky’) during on-plant growth and ripening, and after postharvest water loss. There was no evidence of substantial depolymerisation of cell wall pectic polysaccharides or hemicelluloses at any ripening stage. The onset of fruit ripening was accompanied by a slightly increased solubilisation of very high molecular weight polyuronides. Measurable polygalacturonase activity was absent, despite an endo-polygalacturonase mRNA being present as fruit turned red. The largest cell wall change was a substantial loss of galactose (primarily from Na2CO3-soluble rhamnogalacturonan-I) as green fruit approached full size, which could not be directly correlated with conventionally assayed β-galactosidase/galactanase activities. The majority of β-galactosidase/galactanase gene expression and enzyme activity was found in fully ripe fruit, although by this stage galactose loss was comparatively minimal. This suggests that the upregulation in activity has significance beyond wall rheology, or is vestigial. Severe water-stress applied to harvested ripe fruit did not result in any cell wall changes suggestive of cell wall breakdown, compared with cell walls of fruit stored under high humidity conditions. We conclude that this commercial line of bell pepper, which was bred for intensive glasshouse production, has wall characteristics that result in retention of typical crisp texture and extended postharvest storage life.

Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD4)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD4 reduction to identify 3-, 4-, and 3,4-O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD4-reduced counterparts, suggest that methyl etherification of the β-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

Characteristics of cell wall pectic polysaccharides affect textural properties of instant controlled pressure drop dried carrot chips derived from different tissue zone

To better understanding the role of cell wall pectic polysaccharides (CWPs) on the formation of textural properties of carrot chips dried by instant controlled pressure drop technology (French for Détente Instantanée Contrôlée, DIC), the characteristics of CWPs from ground tissue (GT), junction of ground and vascular tissue (JT), and vascular tissue (VT) of carrot were investigated. Larger expansion volume was obtained in the carrot chips derived from GT, which accompanied with superior textural qualities compared with the chips derived from JT and VT. Remarkable differences were obtained in the amount of pectic fractions, galacturonic acid content, degree of methoxylation, sugar composition and linearity of CWPs that fractionated from different tissue zones of raw carrots. The characteristics of CWPs was confirmed to be a substantial factor that significantly affected the expansion ratio and textural properties of the DIC-dried carrot chips, which providing a mechanistic insight of the relationship between variation in CWPs and the expanding behaviors of DIC-dried fruits and vegetables.

Active proton efflux, nutrient retention and boron-bridging of pectin are related to greater tolerance of proton toxicity in the roots of two Erica species

Background and aimsTolerance to soil acidity was studied in two species of Ericaceae that grow in mine-contaminated soils (S Portugal, SW Spain) to find out if there are interspecific variations in H+ tolerance which might be related to their particular location. MethodsTolerance to H+ toxicity was tested in nutrient solutions using seeds collected in SW Spain. Plant growth and nutrient contents in leaves, stems and roots were determined. Viability tests and proton exchange were studied in roots exposed, short-term, to acidic conditions. Membrane ATPase activity and the cell-wall pectic polysaccharide domain rhamnogalacturonan-II (RG-II) were analysed to find out interspecific differences. ResultsVariation in survival, growth and mineral composition was found between species. The H+-tolerant species (Erica andevalensis) showed greater concentration of nutrients than E. australis. Very low pH (pH 2) produced a significant loss of root nutrients (K, P, Mg) in the sensitive species. Root ATPase activity was slightly higher in the tolerant species with a correspondingly greater H+ efflux capacity. In both species, the great majority of the RG-II domains were in their boron-bridged dimeric form. However, shifting to a medium of pH 2 caused some of the boron bridges to break in the sensitive species. ConclusionsVariation in elements linked to the cell wall-membrane complex and the stability of their components (RG-II, H+-ATPases) are crucial for acid stress tolerance. Thus, by maintaining root cell structure, active proton efflux avoided toxic H+ build-up in the cytoplasm and supported greater nutrient acquisition in H+-tolerant species.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a β-1,6-galactosyl substitution of β-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear β-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

Identification of a Novel L-rhamnose Uptake Transporter in the Filamentous Fungus Aspergillus niger.

The study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. Most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. Pectin is another important constituent of plant cell walls, but has received less attention. In relation to the uptake of pectic building blocks, fungal transporters for the uptake of galacturonic acid recently have been reported in Aspergillus niger and Neurospora crassa. However, not a single L-rhamnose (6-deoxy-L-mannose) transporter has been identified yet in fungi or in other eukaryotic organisms. L-rhamnose is a deoxy-sugar present in plant cell wall pectic polysaccharides (mainly rhamnogalacturonan I and rhamnogalacturonan II), but is also found in diverse plant secondary metabolites (e.g. anthocyanins, flavonoids and triterpenoids), in the green seaweed sulfated polysaccharide ulvan, and in glycan structures from viruses and bacteria. Here, a comparative plasmalemma proteomic analysis was used to identify candidate L-rhamnose transporters in A. niger. Further analysis was focused on protein ID 1119135 (RhtA) (JGI A. niger ATCC 1015 genome database). RhtA was classified as a Family 7 Fucose: H+ Symporter (FHS) within the Major Facilitator Superfamily. Family 7 currently includes exclusively bacterial transporters able to use different sugars. Strong indications for its role in L-rhamnose transport were obtained by functional complementation of the Saccharomyces cerevisiae EBY.VW.4000 strain in growth studies with a range of potential substrates. Biochemical analysis using L-[3H(G)]-rhamnose confirmed that RhtA is a L-rhamnose transporter. The RhtA gene is located in tandem with a hypothetical alpha-L-rhamnosidase gene (rhaB). Transcriptional analysis of rhtA and rhaB confirmed that both genes have a coordinated expression, being strongly and specifically induced by L-rhamnose, and controlled by RhaR, a transcriptional regulator involved in the release and catabolism of the methyl-pentose. RhtA is the first eukaryotic L-rhamnose transporter identified and functionally validated to date.

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.

High hydrostatic pressure induced physiological changes and physical damages in asparagus spears

High hydrostatic pressure (HHP) has been applied as a postharvest treatment in fresh fruits and vegetables for certain purpose, e.g. as a phytosanitary approach to control quarantine insect pests. However, detailed information on HHP-induced physiological changes of plant tissues in a broad range of pressure is limited. In this context, asparagus spears were treated by HHP at mild (10–200MPa) and high pressure (400–600MPa) for 0.5 to 30min. At pressure lower than 200MPa, the CO2 production rate of asparagus spears increased with raising pressure and extending treatment times, while no sign of respiration was found at treatment pressures higher than 400MPa. A shift from aerobic respiration to fermentation was noticed in the sample treated at mild pressure, which might be attributed to the HPP-induced hypoxia stress, enzyme activation, and cell damage. Generally, HHP treatment resulted in a decreasing firmness; however, slight but substantial recovery in firmness was observed in the treatments at pressure higher than 100MPa for 20min, which might be caused by fortification of intercellular adhesion through formation of new ionic linkages in cell wall pectic polysaccharides. Overall, it was suggested that 200MPa could be a threshold for causing instant lethal metabolic disorder and severe physical damage in asparagus spears.

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture.

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

Escherichia coli Common Pilus (ECP) Targets Arabinosyl Residues in Plant Cell Walls to Mediate Adhesion to Fresh Produce Plants

Outbreaks of verotoxigenic Escherichia coli are often associated with fresh produce. However, the molecular basis to adherence is unknown beyond ionic lipid-flagellum interactions in plant cell membranes. We demonstrate that arabinans present in different constituents of plant cell walls are targeted for adherence by E. coli common pilus (ECP; or meningitis-associated and temperature-regulated (Mat) fimbriae) for E. coli serotypes O157:H7 and O18:K1:H7. l-Arabinose is a common constituent of plant cell wall that is rarely found in other organisms, whereas ECP is widespread in E. coli and other environmental enteric species. ECP bound to oligosaccharides of at least arabinotriose or longer in a glycan array, plant cell wall pectic polysaccharides, and plant glycoproteins. Recognition overlapped with the antibody LM13, which binds arabinanase-sensitive pectic epitopes, and showed a preferential affinity for (1→5)-α-linked l-arabinosyl residues and longer chains of arabinan as demonstrated with the use of arabinan-degrading enzymes. Functional adherence in planta was mediated by the adhesin EcpD in combination with the structural subunit, EcpA, and expression was demonstrated with an ecpR-GFP fusion and ECP antibodies. Spinach was found to be enriched for ECP/LM13 targets compared with lettuce. Specific recognition of arabinosyl residues may help explain the persistence of E. coli in the wider environment and association of verotoxigenic E. coli with some fresh produce plants by exploitation of a glycan found only in plant, not animal, cells.

Structural characterisation of the polysaccharides from endemic Mongolian desert plants and their effect on the intestinal absorption of ovalbumin

Using successive extractions with water and 0.7% aqueous ammonium oxalate, pectic polysaccharides were isolated from the following plants growing in the arid climate of Mongolia (Gobi): saxaul Haloxylon ammodendron Maxim., rhubarb Rheum nanum Sievers, Nitraria sibirica Pall., Peganum harmala L. and almond Amygdalus mongolica Maxim. The data obtained exhibited the primary synthesis of the cell wall pectic polysaccharides but not the middle lamellae water-soluble pectins in plants growing in the dry climatic zone.Both α-(1→4)-d-galacturonan and α-(1→4)-d-galacturonan, which was substituted with methyl groups, were found to be backbone of pectins. The l-arabinofuranose residues were identified as the main components of ramified regions. The pectins from almond differed from other pectins due to a high arabinose content. The data from NMR spectroscopy and methylation analyses demonstrated that pectic polysaccharides from almond included terminal, (1→5)-, (1→3)-linked and 3,5-substituted l-arabinofuranose residues and a small terminal d-galactopyranose and 2,5- and 2,3,5-substituted l-arabinofuranose residue content.The pectic polysaccharides were found to decrease the absorption of ovalbumin (OVA) in the blood from the gut lumen. The serum OVA level was lower in mice fed with OVA mixed with the pectins compared with the control group, which was administered OVA alone.

Galacturonosyltransferase (GAUT)1 and GAUT7 are the core of a plant cell wall pectin biosynthetic homogalacturonan:galacturonosyltransferase complex

Plant cell wall pectic polysaccharides are arguably the most complex carbohydrates in nature. Progress in understanding pectin synthesis has been slow due to its complex structure and difficulties in purifying and expressing the low-abundance, Golgi membrane-bound pectin biosynthetic enzymes. Arabidopsis galacturonosyltransferase (GAUT) 1 is an α-1,4-galacturonosyltransferase (GalAT) that synthesizes homogalacturonan (HG), the most abundant pectic polysaccharide. We now show that GAUT1 functions in a protein complex with the homologous GAUT7. Surprisingly, although both GAUT1 and GAUT7 are type II membrane proteins with single N-terminal transmembrane-spanning domains, the N-terminal region of GAUT1, including the transmembrane domain, is cleaved in vivo. This raises the question of how the processed GAUT1 is retained in the Golgi, the site of HG biosynthesis. We show that the anchoring of GAUT1 in the Golgi requires association with GAUT7 to form the GAUT1:GAUT7 complex. Proteomics analyses also identified 12 additional proteins that immunoprecipitate with the GAUT1:GAUT7 complex. This study provides conclusive evidence that the GAUT1:GAUT7 complex is the catalytic core of an HG:GalAT complex and that cell wall matrix polysaccharide biosynthesis occurs via protein complexes. The processing of GAUT1 to remove its N-terminal transmembrane domain and its anchoring in the Golgi by association with GAUT7 provides an example of how specific catalytic domains of plant cell wall biosynthetic glycosyltransferases could be assembled into protein complexes to enable the synthesis of the complex and developmentally and environmentally plastic plant cell wall.

ABA signalling modulates the detection of the LM6 arabinan cell wall epitope at the surface of Arabidopsis thaliana seedling root apices

• The hormonal and physiological regulations underpinning the cell contexts of structural features of the heterogeneous cell wall pectic polysaccharide rhamnogalacturonan-I are far from being understood. • The effect of the modulation of abscisic acid (ABA) concentrations and sensitivity on the detection of the LM6 1,5-arabinan epitope at the surface of Arabidopsis thaliana seedling root apices was assessed by means of fluorescence imaging. • Treatment with 50 nM ABA resulted in an increase in the detection of the LM6 epitope at the root surface in the region of the meristem. An inhibitor of ABA biosynthesis and introduction of the ABA synthesis mutation aba3-2 resulted in reduced epitope detection. The same ABA application resulted in an increase in the number of epidermal root meristem cells and both this and LM6 epitope detection were specifically disrupted in the abi4 ABA-insensitive mutant. These two effects were uncoupled with the application of higher ABA concentrations, which resulted in a reduction in the number of epidermal root meristem cells but increased LM6 epitope detection. • This work demonstrates a role for ABI4-mediated ABA signalling in the modulation of pectic arabinan occurrence at the A. thaliana root meristem.

Fractional changes of pectic polysaccharides in different tissue zones of developing guava ( Psidium guajava L.) fruits

Cell-wall pectic polysaccharides were fractionated by sequential extraction with water, 1% potassium oxalate at room temperature, 0.05 M HCl at 100 °C and 0.05 M NaOH at 4 °C from the three specific tissue zones, i.e., outer pericarp (OP), middle pericarp (MP) and inner pericarp (IP) of the guava fruits during their development and ripening. The water and oxalate-soluble pectic fractions were found to increase, while acid and alkali-soluble pectic fractions had a decreasing trend at ripening phase of fruit, irrespective of the different tissue zones. However, the extent of pectic solubilization was more pronounced in IP region as compared to OP and MP zone of the fruit tissue. The differential degradation of pectic polymers thus points out that ripening as well as tissue softening of guava fruits are centrifugally expressed as evidenced by higher accumulation of sugar and soluble pectic polysaccharides in IP zone of the fruits.

Structural characterisation of the pectic polysaccharide rhamnogalacturonan II using an acidic fingerprinting methodology

Rhamnogalacturonan II (RG-II) is a structurally complex cell wall pectic polysaccharide. Despite its complexity, both the structure of RG-II and its ability to dimerise via a borate diester are conserved in vascular plants suggesting that RG-II has a fundamental role in primary cell wall organisation and function. The selection and analysis of new mutants affected in RG-II formation represents a promising strategy to unravel these functions and to identify genes encoding enzymes involved in RG-II biosynthesis. In this paper, a novel fingerprinting strategy is described for the screening of RG-II mutants based on the mild acid hydrolysis of RG-II coupled to the analysis of the resulting fragments by mass spectrometry. This methodology was developed using RG-II fractions isolated from citrus pectins and then validated for RG-II isolated from the Arabidopsis mur1 mutant and irx10 irx10-like double mutant.