A membrane preparation from tobacco
(Nicotiana tabacum L.) cells contains at least one
enzyme that is capable of transferring the methyl group from
S-adenosyl-methionine (SAM) to the C6 carboxyl of
homogalacturonan present in the membranes. This enzyme is named
homogalacturonan-methyltransferase (HGA-MT) to distinguish it from
methyltransferases that catalyze methyletherification of the pectic
polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A
trichloroacetic acid precipitation assay was used to measure HGA-MT
activity, because published procedures to recover pectic
polysaccharides via ethanol or chloroform:methanol precipitation lead
to high and variable background radioactivity in the product pellet.
Attempts to reduce the incorporation of the 14C-methyl
group from SAM into pectin by the addition of the alternative methyl
donor 5-methyltetrahydrofolate were unsuccessful, supporting the role
of SAM as the authentic methyl donor for HGA-MT. The pH optimum for
HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM
was 38 μm, and the maximum initial velocity was 0.81 pkat
mg−1 protein. At least 59% of the radiolabeled product
was judged to be methylesterified homogalacturonan, based on the
release of radioactivity from the product after a mild base treatment
and via enzymatic hydrolysis by a purified pectin methylesterase. The
released radioactivity eluted with a retention time identical to that
of methanol upon fractionation over an organic acid column. Cleavage of
the radiolabeled product by endopolygalacturonase into
fragments that migrated as small oligomers of HGA during thin-layer
Scanning electron microscopic
examination of intact tomato (Lycopersicon esculentum)
pericarp and isolated pericarp cell walls revealed pit fields and
associated radiating ridges on the inner face of cell walls. In regions
of the cell wall away from pit fields, equivalent ridges occurred in
parallel arrays. Treatment of isolated cell walls with a calcium
chelator resulted in the loss of these ridges, indicating that they
contain homogalacturonan-rich pectic polysaccharides. Immunolabeling
procedures confirmed that pit fields and associated radiating ridges
contained homogalacturonan. Epitopes of the side chains of pectic
polysaccharides were not located in the same regions as
homogalacturonan and were spatially regulated in relation to pit
fields. A (1→4)-β-galactan epitope was absent from cell walls in
regions of pit fields. A (1→5)-α-arabinan epitope occurred most
abundantly at the inner face of cell walls in regions surrounding the
The transfer of a methyl group from S-adenosyl-l-methionine onto the carboxyl group of α-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen  Plant Physiol 116: 337–347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mm dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent Km for S-adenosyl-l-methionine of 18 μm, and an apparent Vmax of 0.121 pkat mg−1 of protein. The apparent Km for HGA and for pectin is 0.1 to 0.2 mg mL−1. Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase...
The changes in cell wall polysaccharides and selected cell wall-modifying enzymes were studied during the development of green bean (Phaseolus vulgaris L.) pods. An overall increase of cell wall material on a dry-weight basis was observed during pod development. Major changes were detected in the pectic polymers. Young, exponentially growing cell walls contained large amounts of neutral, sugar-rich pectic polymers (rhamnogalacturonan), which were water insoluble and relatively tightly connected to the cell wall. During elongation, more galactose-rich pectic polymers were deposited into the cell wall. In addition, the level of branched rhamnogalacturonan remained constant, while the level of linear homogalacturonan steadily increased. During maturation of the pods, galactose-rich pectic polymers were degraded, while the accumulation of soluble homogalacturonan continued. During senescence there was an increase in the amount of ionically complexed pectins, mainly at the expense of freely soluble pectins. The most abundant of the enzymes tested for was pectin methylesterase. Peroxidase, β-galactosidase, and α-arabinosidase were also detected in appreciable amounts. Polygalacturonase was detected only in very small amounts throughout development. The relationship between endogenous enzyme levels and the properties of cell wall polymers is discussed with respect to cell wall synthesis and degradation.
Cnr (colorless non-ripening) is a pleiotropic tomato (Lycopersicon esculentum) fruit ripening mutant with altered tissue properties including weaker cell-to-cell contacts in the pericarp (A.J. Thompson, M. Tor, C.S. Barry, J. Vrebalov, C. Orfila, M.C. Jarvis, J.J. Giovannoni, D. Grierson, G.B. Seymour  Plant Physiol 120: 383–390). Whereas the genetic basis of the Cnr mutation is being identified by molecular analyses, here we report the identification of cell biological factors underlying the Cnr texture phenotype. In comparison with wild type, ripe-stage Cnr fruits have stronger, non-swollen cell walls (CW) throughout the pericarp and extensive intercellular space in the inner pericarp. Using electron energy loss spectroscopy imaging of calcium-binding capacity and anti-homogalacturonan (HG) antibody probes (PAM1 and JIM5) we demonstrate that maturation processes involving middle lamella HG are altered in Cnr fruit, resulting in the absence or a low level of HG-/calcium-based cell adhesion. We also demonstrate that the deposition of (1→5)-α-l-arabinan is disrupted in Cnr pericarp CW and that this disruption occurs prior to fruit ripening. The relationship between the disruption of (1→5)-α-l-arabinan deposition in pericarp CW and the Cnr phenotype is discussed.
Polygalacturonases (PGs) cleave runs of unesterified GalUA that form homogalacturonan regions along the backbone of pectin. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the wall where two adjacent cells abut and its integrity is important for cell adhesion. Transgenic apple (Malus domestica Borkh. cv Royal Gala) trees were produced that contained additional copies of a fruit-specific apple PG gene under a constitutive promoter. In contrast to previous studies in transgenic tobacco (Nicotiana tabacum) where PG overexpression had no effect on the plant (K.W. Osteryoung, K. Toenjes, B. Hall, V. Winkler, A.B. Bennett  Plant Cell 2: 1239–1248), PG overexpression in transgenic apple led to a range of novel phenotypes. These phenotypes included silvery colored leaves and premature leaf shedding due to reduced cell adhesion in leaf abscission zones. Mature leaves had malformed and malfunctioning stomata that perturbed water relations and contributed to a brittle leaf phenotype. Chemical and ultrastructural analyses were used to relate the phenotypic changes to pectin changes in the leaf cell walls. The modification of apple trees by a single PG gene has offered a new and unexpected perspective on the role of pectin and cell wall adhesion in leaf morphology and stomatal development.
The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.
Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.
Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for homogalacturonan (HG) α-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG α-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. blast analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.
The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.
Xylem hydraulic conductivity (Ks) in stems of tobacco (Nicotiana tabacum) wild-type SR1 was compared to that of PG7 and PG16, two transgenic lines with increased levels of expression of the gene encoding the Aspergillus niger endopolygalacturonase (AnPGII). Activity of AnPGII removes in planta blocks of homogalacturonan (HG) with deesterified carboxyls, thus increasing the degree of neutrality of pectins. The effect of K+ was tested in increasing stem Ks using model plants with more neutral polysaccharides in primary walls and, hence, in intervessel pit membranes. Ks measured with deionized water was compared to that with KCl solutions at increasing concentrations (ΔKs, %). Plants transformed for HG degree of neutrality showed a dwarfed phenotype, but ΔKs did not differ among the three experimental groups. The ion-mediated hydraulic effect saturated at a KCl concentration of 25 mm in SR1 plants. All the three tobacco lines showed ΔKs of around +12.5% and +17.0% when perfused with 10 and 25 mm KCl, respectively. Because modification of HG content did not influence ion-mediated hydraulic enhancement, we suggest that pectin components other than HG, like rhamnogalacturonan-I and/or rhamnogalacturonan-II, might play important roles in the hydrogel behavior of pit membranes.
Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.
Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well-defined localization of cell wall epitopes with highly esterified homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding (1) the ultrastructure of the pollen tube cell wall and (2) the immunolocalization of homogalacturonan and arabinan epitopes in 16-h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.
Pectins are complex polysaccharides that are essential components of the plant cell wall. In this study, a novel putative Arabidopsis S-adenosyl-L-methionine (SAM)-dependent methyltransferase, termed QUASIMODO 3 (QUA3, At4g00740), has been characterized and it was demonstrated that it is a Golgi-localized, type II integral membrane protein that functions in methylesterification of the pectin homogalacturonan (HG). Although transgenic Arabidopsis seedlings with overexpression, or knock-down, of QUA3 do not show altered phenotypes or changes in pectin methylation, this enzyme is highly expressed and abundant in Arabidopsis suspension-cultured cells. In contrast, in cells subjected to QUA3 RNA interference (RNAi) knock-down there is less pectin methylation as well as altered composition and assembly of cell wall polysaccharides. Taken together, these observations point to a Golgi-localized QUA3 playing an essential role in controlling pectin methylation and cell wall biosynthesis in Arabidopsis suspension cell cultures.
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.
Djarly, a natural Arabidopsis mutant defective in seed mucilage release, is shown to be defective in a pectin methylesterase inhibitor, PMEI6. Mutant and overexpressor phenotypes highlight the importance of modulating the degree of homogalacturonan methylesterification for correct primary cell wall fragmentation and pectin partitioning into adherent and soluble mucilage layers.
Studies on angiosperm plants have shown that homogalacturonan present in the extracellular matrix of pistils plays an important role in the interaction with the male gametophyte. However, in gymnosperms, knowledge on the participation of HG in the pollen–ovule interaction is limited, and only a few studies on male gametophytes have been reported. Thus, the aim of this study was to determine the distribution of HG in male gametophytes and ovules during their interaction in Larix decidua Mill. The distribution of HG in pollen grains and unpollinated and pollinated ovules was investigated by immunofluorescence techniques using monoclonal antibodies that recognise high methyl-esterified HG (JIM7), low methyl-esterified HG (JIM5) and calcium cross-linked HG (2F4). All studied categories of HG were detected in the ovule. Highly methyl-esterified HG was present in the cell walls of all cells throughout the interaction; however, the distribution of low methyl-esterified and calcium cross-linked HG changed during the course of interaction. Both of these categories of HG appeared only in the apoplast and the extracellular matrix of the ovule tissues, which interact with the male gametophyte. This finding suggests that in L. decidua, low methyl-esterified and calcium cross-linked HG play an important role in pollen–ovule interaction. The last category of HG is most likely involved in adhesion between the pollen and the ovule and might provide an optimal calcium environment for pollen grain germination and pollen tube growth.
Two natural homogalacturonan (HG) pectins (MW
ca. 20 kDa) were isolated from green tea based on their immunomodulatory activity. The crude tea polysaccharides (TPS1 and TPS2) were obtained from green tea leaves by hot water extraction and followed by 40% and 70% ethanol precipitation, respectively. Two homogenous water soluble polysaccharides (TPS1-2a and TPS1-2b) were obtained from TPS1 after purification with gel permeation, which gave a higher phagocytic effect than TPS2. A combination of composition, methylation and configuration analyses, as well as NMR (nuclear magnetic resonance) spectroscopy revealed that TPS1-2a and TPS1-2b were homogalacturonan (HG) pectins consisting of a backbone of 1,4-linked α-d-galacturonic acid (GalA) residues with 28.4% and 26.1% of carboxyl groups as methyl ester, respectively. The immunological assay results demonstrated that TPS1-2, which consisted mainly of HG pectins, showed phagocytosis-enhancing activity in HL-60 cells.
A Vassoura-de-bruxa é uma das principais doenças que acometem o cacaueiro (Theobroma cacao L.). Ela é causada pelo fungo hemibiotrófico Moniliophthora perniciosa. Durante a fase biotrófica, o patógeno coloniza o espaço intercelular e estimula a formação de ramos hipertróficos/ hiperplásicos denominados vassoura verde. No estágio avançado da doença, o fungo passa a colonizar também o interior das células e observa-se a morte do tecido originando a vassoura seca. Homogalacturonano (HG) é um domínio estrutural péctico que constitui a matriz da parede celular primária e define suas propriedades funcionais e estruturais. Durante sua síntese, HG é altamente metilesterificado pela ação de pectinas metiltransferases (PMT) que utilizam S-adenosil-L-metionina (SAM) como fonte de grupos metil. Após ser depositado na parede celular, o padrão de metilesterificações de HG é modelado por pectina metilesterases (PME). Alterações no padrão de metilesterificação de HG são relacionadas à má formação de tecidos, complicações durante o desenvolvimento vegetal e à resistência contra a degradação da parede celular por enzimas microbianas. Neste trabalho, a expressão tecido-específica de genes envolvidos nas reações de síntese de SAM e o grau de metilesterificação do HG foram analisadas em plântulas de T. cacao sadias e infectadas. ESTs (expressed sequence tags) de T. cacao foram utilizadas como molde para a síntese de sondas de RNA específicas para os genes: glicosiltransferase...
This is the final version of the article. It first appeared from Springer via http://dx.doi.org/10.1007/s00425-015-2358-5; Pectins are highly complex polysaccharides abundant in plant primary cell walls. New analytical and microscopy techniques are revealing the composition and mechanical properties of the cell wall and increasing our knowledge on the topic. Progress in plant physiological research supports a link between cell wall pectin modifications and plant development and interactions with the environment. Homogalacturonan pectins, which are major components of the primary cell wall, have a potential for modifications such as methylesterification, as well as an ability to form cross-linked structures with divalent cations. This contributes to changing the mechanical properties of the cell wall. This review aims to give a comprehensive overview of the pectin component homogalacturonan, including its synthesis, modification, regulation and role in the plant cell wall.; The financial support from the NSERC Postgraduate Scholarships (to G.L.-T.), the Institut Universitaire de France (IUF) to J.P. and a Marie Curie International Outgoing Fellowship to K.M. is gratefully acknowledged.