Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Background and Aims

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods

Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key Results

Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions

This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.     

Potato Cell Wall Polysaccharides: Degradation with Enzymes from Phytophthora infestans

Phytophthora irtfestans culture filtrate contained 2 polygalacturonases,4 galactanases, and 2 ipectinesterases. An endo-polygalacturonase(mol. wt. 350 000) and an endo- (l, 4') ß-D-galactanase(mol. wt 55 000) were partially purified and used to degradepotato cell walls. The polygalacturon-ase released less than6% of the cell wall carbohydrate, even after galactanase treatmentor removal of the more soluble pectic fraction. Its limiteddegradative activity may be connected with the biotrophic habitof the blight fungus. The galactanase detached up to 23% ofthe cell wall, including uronic acid when calcium ions had beenremoved beforehand. Arabinose was also released, showing thatthere are arabinogalactan side-chains in the pectin. Howevergalactanase degradation of a soluble pectin fraction left 63%of the arabinose attached to large rhamnogalacturonan fragmentswith only a little galactose. Many arabinan chains are thusperhaps attached directly to rhamnogalacturonan.     

Alkaline extractability of pectic arabinan and galactan and their mobility in sugar beet and potato cell walls

The organisation of sugar beet and potato cell walls was studied using alkaline extractions following a response surface methodology, simultaneously with solid-state ¹³C NMR spectroscopy. The influence of two extraction parameters: NaOH concentration (0.05, 0.275, 0.5 M) and temperature (40, 65, 90 °C) on the composition (neutral and acidic sugars) of the residues recovered was established. Treatments of increasing harshness progressively washed off non-cellulosic polysaccharides from the cell walls. Alkaline treatments applied to sugar beet cell wall material (SB-CWM) revealed the presence of diverse pectin populations. The existence of distinct pectin populations in potato cell wall material (P-CWM) was less outstanding. Solid-state ¹³C NMR applied to SB-CWM and P-CWM and residues after treatment by 0.275 M NaOH at 65 °C revealed two fractions of pectic arabinan and galactan side chains. One fraction was highly mobile, whereas the other one displayed restricted mobility.     

Changes in the distribution of cell wall polysaccharides in early fruit pericarp and ovule, from fruit set to early fruit development, in tomato (Solanum lycopersicum)

During fruit development in tomato (Solanum lycopersicum), cell proliferation and rapid cell expansion occur after pollination. Cell wall synthesis, alteration, and degradation play important roles during early fruit formation, but cell wall composition and the extent of cell wall synthesis/degradation are poorly understood. In this study, we used immunolocalization with a range of specific monoclonal antibodies to examine the changes in cell wall composition during early fruit development in tomato. In exploring early fruit development, the ?1 day post-anthesis (DPA) ovary and fruits at 1, 3, and 5 DPA were sampled. Paraffin sections were prepared for staining and immunolabeling. The 5 DPA fruit showed rapid growth in size and an increase in both methyl-esterified pectin and de-methyl-esterified pectin content in the pericarp, suggesting rapid synthesis and de-methyl esterification of pectin during this growth period. Labeling of pectic arabinan with LM6 antibody and galactan with LM5 antibody revealed abundant amounts of both, with unique distribution patterns in the ovule and premature pericarp. These results suggest the presence of rapid pectin metabolism during the early stages of fruit development and indicate a unique distribution of pectic galactan and arabinan within the ovule, where they may be involved in embryogenesis.     

Response of the leaf cell wall to desiccation in the resurrection plant Myrothamnus flabellifolius

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.     

Isolation and partial sequence of a Kunitz-type elastase specific inhibitor from marama bean (Tylosema esculentum)

An isolation procedure utilizing ammonium sulfate fractionation and affinity chromatography was used to purify an elastase inhibitor present in large amounts in marama beans (Tylosema esculentum). The protein appeared to be heterogeneous due to carbohydrate differences, demonstrating two bands on SDS gels with molecular weights of 17.8?kDa and 20?kDa. Partial sequence, derived from mass spectrometry, indicated that the protein is a Kunitz-type inhibitor distinct from other known plant serine protease inhibitors. The marama bean inhibitor is specific for elastase, with very low K_i for both pancreatic and neutrophil elastase. The quantity of elastase inhibitor present in marama beans is many times greater than in soybean or any other bean or nut source reported to date. This raises the question of why a bean found in an arid corner of the Kalahari Desert would be so rich in a very potent elastase inhibitor.     

Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration.

Incubation of beet pulp with two arabinases (alpha-L-arabinofuranosidase and endo-arabinase), used singularly or in combination at different units of activity per gram of beet pulp, caused the hydrolysis of arabinan, which produced a hydrolyzate consisting mainly of arabinose. Pectin and a residue enriched with cellulose were subsequently separated from the incubation mixture. The best enzymatic hydrolysis results were obtained when 100 U/g of beet pulp of each enzyme worked synergistically with yields of 100% arabinose and 91.7% pectin. These yields were higher than those obtained with traditional chemical hydrolysis. The pectin fraction showed a low content of neutral sugar content and the cellulose residue contained only a small amount of pentoses. Semicontinuous hydrolysis with enzyme recycling in an ultrafiltration unit was also carried out to separate arabinose, pectin, and cellulose from beet pulp in 7 cycles of hydrolysis followed by ultrafiltration. The yields of separation were similar to those obtained in batch experiments, with an enzyme consumption reduced by 3.5 times and some significant advantages over batch processes.     

Transfer of the first arabinofuranose residue to galactan is essential for Mycobacterium smegmatis viability

The mycobacterial arabinan is an elaborate component of the cell wall with multiple glycosyl linkages and no repeating units. In Mycobacterium spp., the Emb proteins (EmbA, EmbB, and EmbC) have been identified as putative mycobacterial arabinosyltransferases implicated in the biogenesis of the cell wall arabinan. Furthermore, it is now evident that the EmbA and EmbB proteins are involved in the assembly of the nonreducing terminal motif of arabinogalactan and EmbC is involved in transferring arabinose, perhaps in the early stage of arabinan synthesis in lipoarabinomannan. It has also been shown that the Emb proteins are a target of the antimycobacterial drug ethambutol (EMB). In the search for additional mycobacterial arabinosyltransferases in addition to the Emb proteins, we disrupted MSMEG_6386 (an orthologue of Rv3792 and a gene upstream of embC) in Mycobacterium smegmatis. Allelic exchange at the chromosomal MSMEG_6386 locus of M. smegmatis could only be achieved in the presence of a rescue plasmid carrying a functional copy of MSMEG_6386 or Rv3792, strongly suggesting that MSMEG_6386 is essential. An in vitro arabinosyltransferase assay using a membrane preparation from M. smegmatis expressing Rv3792 and synthetic beta-d-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-octyl and beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-octyl showed that Rv3792 gene product can transfer an arabinose residue to the C-5 position of the internal 6-linked galactose. The reactions were insensitive to EMB, and when alpha-d-Manp-(1-->6)-alpha-D-Manp-(1-->6)-alpha-D-Manp-octylthiomethyl was used as an acceptor, no product was formed. These observations indicate that transfer of the first arabinofuranose residue to galactan is essential for M. smegmatis viability.     

The isolation of two proteins, glycoprotein I and a trypsin inhibitor, from the seeds of kidney bean (Phaseolus vulgaris)

1. The isolation of two proteins from the seeds of kidney bean is described. 2. The individual steps in the purification procedure included: extraction of the seeds at pH9.0, dialysis, first against pH9.0 and then against pH5.0 buffers, high-voltage electrophoresis of the proteins soluble at pH5.0 and chromatography on Sephadex G-200, Sephadex G-75 and DEAE-Sephadex columns. 3. Of the two proteins isolated, the first and larger component was a glycoprotein and its carbohydrate part was mainly composed of d-mannose and d-glucosamine together with smaller amounts of arabinose, xylose and fucose. 4. The second protein component isolated was a trypsin inhibitor and was almost entirely devoid of sugars but contained a firmly bound pinkish-blue pigment. 5. The amino acid composition of the two proteins was determined. 6. The glycoprotein contained very little if any cyst(e)ine but was relatively rich in aromatic amino acids, whereas the trypsin inhibitor had an unusually high cystine content (nearly 15%) but was relatively poor in valine and in aromatic amino acids.     

In vitro fermentation of sugar beet arabinan and arabino-oligosaccharides by the human gut microflora

AIMS: To determine the fermentation profiles by human gut bacteria of arabino-oligosaccharides of varying degree of polymerization. MATERIALS AND METHODS: Sugar beet arabinan was hydrolyzed with a commercial pectinase and eight fractions, of varying molecular weight, were isolated by gel-filtration chromatography. Hydrolysis fractions, arabinose, arabinan and fructo-oligosaccharides were fermented anaerobically by gut bacteria. Total bacteria, bifidobacteria, bacteroides, lactobacilli and the Clostridium perfringens/histolyticum sub. grp. were enumerated using fluorescent in situ hybridization. RESULTS: Bifidobacteria were stimulated to different extents depending on molecular weight, i.e. maximum increase in bifidobacteria after 48 h was seen on the lower molecular weight fractions. Lactobacilli fluctuated depending on the initial inoculum levels. Bacteroides numbers varied according to fraction; arabinan, arabinose and higher oligosaccharides (degree of polymerization, dp > 8) resulted in significant increases at 24 h. Only carbohydrate mixtures with dp of 1-2 resulted in significant increases at 48 h (log 8.77 +/- 0.23). Clostridia decreased on all substrates. CONCLUSIONS: Arabino-oligosaccharides can be considered as potential prebiotics. Significance and Impact of the Study: Arabinan is widely available as it is a component of sugar beet pulp, a co-product from the sugar beet industry. Generation of prebiotic functionality from arabinan would represent significant added value to a renewable resource.     

Isolation and analysis of sacculi from Streptococcus sanguis.

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.     

Cell wall polysaccharide distribution in Sandersonia aurantiaca flowers using immuno-detection

The localization of cell wall polysaccharides of the fused petals of monocotyledonous Sandersonia aurantiaca flowers has been identified using antibodies directed to pectin and xyloglucan epitopes and detection by fluorescence microscopy. Cross sections of the petal tissue were taken from cut flowers in bud and at various stages of maturity and senescence. Patterns of esterification in pectin backbones were identified by JIM5 and 2F4 labelling. Pectic galactan and arabinan side branches were detected by LM5 and LM6, respectively, while fucosylated xyloglucan was identified by CCRC-M1. The labelling patterns highlighted compositional differences between walls of the outer/inner epidermis compared to the spongy parenchyma cells of the interior mesophyll for fucosylated xyloglucan and arabinan. Partially esterified homogalacturonan was present in the junction zones of the outer epidermis and points of contact between cells of the mesophyll, and persisted throughout senescence. Pectic galactans were ubiquitous in the outer and inner epidermal cell walls and walls of the interior mesophyll at flower opening, whereas pectic arabinan was found predominantly in the epidermal cells. Galactan was lost from walls of all cells as flowers began to senesce, while fucosylated xyloglucan appeared to increase over this time. Such differences in the location of polysaccharides and the timing of changes suggest distinct combinations of certain polysaccharides offer mechanical and rheological advantages that may assist with flower opening and senescence.     

Isolation of polysaccharides from callus culture of Lemna minor L

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minor L.) with water and ammonium oxalate. Residues of galactose and arabinose in the 2.0-2.5:1 ratio were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glucuronic acids, galactose, and arabinose. The percentage of arabinogalactan and pectin was similar. The yield of polysaccharide fractions did not depend on the method for their isolation. Extraction with water, treatment of the biomass with an aqueous solution of formalin and diluted hydrochloric acid, and extraction with an aqueous solution of ammonium oxalate allowed us to obtain the highest-purity pectin polysaccharide.     

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.     

Arabinan synthase and xylan synthase activities of Phaseolus vulgaris. Subcellular localization and possible mechanism of action.

Membrane fractions from bean hypocotyl or suspension cultures incorporated arabinose from UDP-beta-L-arabinose into arabinan and xylose from UDP-alpha-D-xylose in vitro; the level of each activity was dependent on the state of differentiation of the cells. These activities may be due to single transglycosylases, since no lipid or proteinaceous intermediate acceptors were found in either case. Subcellular fractionation studies showed that enzyme activity in vitro was localized in both Golgi-derived membranes and endoplasmic reticulum in similar amounts. However, incorporation into the polymers in vivo in suspension culture cells incubated with [1-3H]arabinose was considerably greater in the Golgi-derived membranes. Thus, although these enzymes may be translated and inserted at the level of the endoplasmic reticulum, their activities are under other levels of control, so that most of the activity in vivo is confined to the Golgi apparatus. Initiation of glycosylation in the endoplasmic activity may, however, occur.     

Differential patterns of arabinosylation by membranes of suspension-cultured cells of Phaseolus vulgaris (French bean) after subculture or elicitation.

Suspension-cultured cells of Phaseolus vulgaris (French bean) incorporated [1-3H] arabinose in vivo into high-Mr polymers that could be separated into glycoprotein and polysaccharide. Microsomal membranes from suspension-cultured cells of beans incorporated arabinose from UDP-beta-L-arabinose in vitro into both polysaccharide and glycoprotein. The enzyme involved in arabinan synthesis, arabinan synthase, appeared to be immunologically distinct from the protein:arabinosyltransferase system. Both these activities are inducible, but behave differently with either plant-growth-regulator or fungal-elicitor treatments. After subculture of cells entering the stationary growth phase the arabinan synthase activity reaches much higher values than does that of the protein transferase system during the initial period of cell division and growth, whereas after elicitation at the same growth stage, all the increased incorporation of arabinose occurs into glycoprotein of Mr higher than 200 000 and to a greater extent into a specific glycoprotein of Mr 42 500. Preliminary characterization of these glycoproteins prepared under non-reducing conditions and after acid and alkaline hydrolysis suggests that the high-Mr glycoprotein material is similar to arabinogalactan protein, whereas the lower-Mr material may be a hydroxyproline-rich protein existing as a dimer and that specifically increases during the hypersensitive response of the cells to the fungal elicitor from Colletotrichum lindemuthianum.     

Extraction and characterization of pectic substances from pulp of grape berries

Pectic substances were successively extracted from the alcohol-insoluble residue (AIR) of the pulp of grape berries, by water (WSP), oxalate (OXP), hot dilute HCl (HP) and cold dilute NaOH (OHP). Pectins (WSP, OXP, HP) were purified by ion-exchange chromatography (DEAE-Sephacel) or precipitation with cupric ions (OHP). Total pectic substances represent 20·8% (w/w) of the AIR, WSP and HP being the main components (6% and 12% of the AIR, respectively). An alternative to oxalate extraction of the water-insoluble pectin was extraction with NaCl solutions of increasing concentrations, which had released small amounts of pectins. Each of the fractions contained mainly galacturonic acid, arabinose and galactose, lower amounts of rhamnose and xylose, and minor amounts of glucose and mannose. Ion-exchange chromatography was performed on DEAE-Sephacel, M_w distribution was checked by gel permeation on Sepharose CL-2B, and M_v was determined as well as degrees of methylesterification (DM), acetylation (DA), and protein content.     

Isolation of Polysaccharides from the Callus Culture of Lemna minor L.

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minorL.) with water and ammonium oxalate. Residues of galactose and arabinose (ratio, (2.0–2.5) : 1) were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glycuronic acids, galactose, and arabinose. The percentages of arabinogalactan and pectin were similar. The yield of polysaccharide fractions did not depend on the method used for their isolation. Extraction with water, treatment of the biomass with aqueous formalin and dilute hydrochloric acid, and extraction with aqueous ammonium oxalate allowed us to obtain the pectin polysaccharide with the highest purity.     

Cell-wall antigens in mesophyll cells and mesophyll-derived protoplasts of sugar beet: possible implication in protoplast recalcitrance?

We have investigated the possible relation between plant cell-wall constituents and the recalcitrance of the cell to regenerate organs and whole plants in vitro. A temporal and spatial expression of several carbohydrate epitopes was observed both within leaf tissue used for protoplast isolation and within new walls reformed by recalcitrant mesophyll protoplasts of sugar beet ( Beta vulgaris L.); these include four pectic epitopes, one xyloglucan (rhamnogalacturonan I) epitope, two carbohydrate motifs of arabinogalactan proteins (AGPs) and callose. The walls of mesophyll cells and newly formed walls of protoplasts were similar with respect to the presence of large amounts of pectins recognized by JIM7 antibodies, the scarcity of JIM5-pectins and the complete absence of LM5-responding pectin molecules. Their main differences were the significantly higher accumulation of LM6-recognizing pectins and the very conspicuous greater accumulation of AGPs and callose in walls deposited by protoplasts than in those synthesized by donor cells.