Boron and calcium, essential inorganic constituents of pectic polysaccharides in higher plant cell walls

Among 16 essential elements of higher plants, Ca²⁺ and B have been termed as apoplastic elements. This is mainly because of their localization in cell walls, however, it has turned to be highly likely that these two elements significantly contribute to maintain the integrity of cell walls through binding to pectic polysaccharides. Boron in cell walls exclusively forms a complex with rhamnogalacturonan II (RG-II), and the B-RG-II complex is ubiquitous in higher plants. Analysis of the structure of the B-RG-II complex revealed that the complex contains two molecules boric acid, two molecules Ca²⁺ and two chains of monomeric RG-II. This result indicates that pectic chains are cross-linked covalently with boric acid at their RG-II regions. The complex was reconstitutedin vitro only by mixing monomeric RG-II and boric acid, however, the complex decomposed spontaneously unless Ca²⁺ was supplemented. Furthermore, the native complex decomposed when it was incubated withtrans-1,2-diaminocyclohexane-N, N, N′, N′-tetraacetic acid (CDTA) which chelates Ca²⁺. When radish root cell walls were washed with a buffered 1.5% (w/v) sodium dodesyl sulfate (SDS) solution (pH 6.5), 96%, 13% and 6% of Ca²⁺, B and pectic polysaccharides of the cell walls, respectively, were released and the cell wall swelled twice. Subsequent extraction with 50 mM CDTA (pH 6.5) of the SDS-washed cell walls further released 4%, 80% and 61% of Ca²⁺, B and pectic polysaccharides, respectively. Pectinase hydrolysis of the SDS-treated cell walls yielded a B-RG-II complex and almost all the remaining Ca²⁺ was recovered in the complex. This result suggests that cell-wall bound Ca²⁺ is divided into at least two fractions, one anchors the CDTA-soluble pectic polysaccharides into cell walls together with B, and the other may control the properties of the pectic gel. These studies demonstrate that B functions to retain CDTA-soluble pectic polysaccharides in cell walls through its binding to the RG-II regions in collaboration with Ca²⁺.     

Germanium does not substitute for boron in cross-linking of rhamnogalacturonan II in pumpkin cell walls

Boron (B)-deficient pumpkin (Cucurbita moschata Duchesne) plants exhibit reduced growth, and their tissues are brittle. The leaf cell walls of these plants contain less than one-half the amount of borate cross-linked rhamnogalacturonan II (RG-II) dimer than normal plants. Supplying germanium (Ge), which has been reported to substitute for B, to B-deficient plants does not restore growth or reduce tissue brittleness. Nevertheless, the leaf cell walls of the Ge-treated plants accumulated considerable amounts of Ge. Dimeric RG-II (dRG-II) accounted for between 20% and 35% of the total RG-II in the cell walls of the second to fourth leaves from Ge-treated plants, but only 2% to 7% of the RG-II was cross-linked by germanate (dRG-II-Ge). The ability of RG-II to form a dimer is not reduced by Ge treatment because approximately 95% of the monomeric RG-II generated from the walls of Ge-treated plants is converted to dRG-II-Ge in vitro in the presence of germanium oxide and lead acetate. However, dRG-II-Ge is unstable and is converted to monomeric RG-II when the Ge is removed. Therefore, the content of dRG-II-Ge and dRG-II-B described above may not reflect the actual ratio of these in muro. (10)B-Enriched boric acid and Ge are incorporated into the cell wall within 10 min after their foliar application to B-deficient plants. Foliar application of (10)B but not Ge results in an increase in the proportion of dRG-II in the leaf cell wall. Taken together, our results suggest that Ge does not restore the growth of B-deficient plants.     

Formation of rhamnogalacturonan II-borate dimer in pectin determines cell wall thickness of pumpkin tissue

Boron (B) deficiency results in inhibition of pumpkin (Cucurbia moschata Duchesne) growth that is accompanied by swelling of the cell walls. Monomeric rhamnogalacturonan II (mRG-II) accounted for 80% to 90% of the total RG-II in B-deficient walls, whereas the borate ester cross-linked RG-II dimer (dRG-II-B) accounted for more than 80% of the RG-II in control plants. The results of glycosyl residue and glycosyl linkage composition analyses of the RG-II from control and B-deficient plants were similar. Thus, B deficiency does not alter the primary structure of RG-II. The addition of (10)B-enriched boric acid to B-deficient plants resulted within 5 h in the conversion of mRG-II to dRG-II-(10)B. The wall thickness of the (10)B-treated plants and control plants was similar. The formation and possible functions of a borate ester cross-linked RG-II in the cell walls are discussed.     

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.     

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca²⁺. The complex formed in the presence of Ca²⁺ was more stable than that without Ca²⁺. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca²⁺ and B. Removal of the Ca²⁺ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca²⁺ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca²⁺ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca²⁺ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca²⁺_, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca²⁺ but also borate and Ca²⁺ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.Boron (B) is an essential element for higher plant growth, although its primary function is not known (Loomis and Durst, 1992). Determining the sites of B in cells is required to identify its function. In cultured tobacco cells more than 80% of cellular B is in the cell wall (Matoh et al., 1993), whereas the membrane fraction (Kobayashi et al., 1997) and protoplasts (Matoh et al., 1992) do not contain a significant amount of B. In radish (Raphanus sativus L. cv Aokubi-daikon) root cell walls, B cross-links two RG-II regions of pectic polysaccharides through a borate-diol ester (Kobayashi et al., 1995, 1996). The association of B with RG-II has been confirmed in sugar beet (Ishii and Matsunaga, 1996), bamboo (Kaneko et al., 1997), sycamore and pea (O''Neill et al., 1996), and red wine (Pellerin et al., 1996). In cultured tobacco cells the B associated with RG-II accounts for about 80% of the cell wall B (Kobayashi et al., 1997) and RG-II may be the exclusive carrier of B in higher plant cell walls (Matoh et al., 1996). Germanic acid, which partly substitutes for B in the growth of the B-deprived plants (Skok, 1957), also cross-links two RG-II chains (Kobayashi et al., 1997). These results suggest that the physiological role of B is to cross-link cell wall pectic polysaccharides in the RG-II region and thereby form a pectic network.It is believed that in the cell wall pectic polysaccharides are cross-linked with Ca²⁺, which binds to carboxyl groups of the polygalacturonic acid regions (Jarvis, 1984). Thus, the ability of B and Ca²⁺ to cross-link cell wall pectic polysaccharides needs to be evaluated. In this report we describe the B-RG-II complex of radish root and the role of B-RG-II and Ca²⁺ in the formation of a pectic network.     

Boron Nutrition of Cultured Tobacco BY-2 Cells. II. Characterization of the Boron-Polysaccharide Complex

In cultured tobacco BY-2 cells, more than 90% of the cellularboron (B) occurs in the cell wall and a negligible amount ofB is detected in the membrane fraction. Nearly 80% of the cellwall B binds to rhamnogalacturonan II (RG-II) to form a borate-dimericRG-II complex. Mono-meric RG-II is not detected in the cellwall, but it is detected in the extracellular polysaccharides.The complex is reconstituted spontaneously in vitro simply bymixing mon-omeric RG-II and boric acid at pH 4. Germanic acid,which partially substitutes for B in the growth of the B-deprivedplants, also induces dimerization of RG-II. These results suggeststhat B may fulfill its essential function as forming the B-RG-IIcomplex in cell walls. ¹ Present address: Kasai Experimental Farm, Sumitomo ChemicalCo., Ltd., Kasai, Hyogo, 675%ndash;23 Japan     

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.     

Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.     

An NMR solution study of the mega-oligosaccharide, rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic mega-oligosaccharide that is released enzymatically from the primary cell wall of higher plants. It contains roughly 30 monosaccharide units (MW 5 kDa) including very unusual residues such as Kdo, Dha, aceric acid and apiose. Previous studies have demonstrated that these monomers are arranged into four structurally well-defined oligosaccharide side chains (A–D), linked to a homogalacturonan mainchain, but the specific attachment sites of these branches on the pectic backbone have not yet been elucidated. In the present work, fairly complete assignments of the 750 MHz ¹ H NMR spectra and partial assignments of the ¹³ C NMR spectra of the sodium-borohydride-reduced RG-II monomer were obtained for a 5 mM sample isolated from red wine. On the whole, these data corroborate the primary structures of the sidechains previously established by methylation analysis, partial hydrolysis and FAB-MS spectrometry but some heterogeneity has been demonstrated (partial substitution at B5, B6, and A5). The preferred orientations of the majority of the sidechain glycosidic linkages in the RG-II monomer have been determined from the sequential nOe data and the solution structure is generally in good agreement with the stable conformers previously obtained by molecular modeling (MM3) of the disaccharide and sidechain oligosaccharide building blocks. All of a two-residue, a three-residue, and a four-residue segment of the backbone have been tentatively identified from long range interactions between sidechain protons as well as in the mainchain. Taking into account the length of the 9-mer galacturonan mainchain described in prior work, these building blocks constitute almost the complete structure of RG-II (Scheme 2).     

Silencing of the GDP-D-mannose 3,5-epimerase affects the structure and cross-linking of the pectic polysaccharide rhamnogalacturonan II and plant growth in tomato

L-galactose (L-Gal), a monosaccharide involved in L-ascorbate and rhamnogalacturonan II (RG-II) biosynthesis in plants, is produced in the cytosol by a GDP-D-mannose 3,5-epimerase (GME). It has been recently reported that the partial inactivation of GME induced growth defects affecting both cell division and cell expansion (Gilbert, L., Alhagdow, M., Nunes-Nesi, A., Quemener, B., Guillon, F., Bouchet, B., Faurobert, M., Gouble, B., Page, D., Garcia, V., Petit, J., Stevens, R., Causse, M., Fernie, A. R., Lahaye, M., Rothan, C., and Baldet, P. (2009) Plant J. 60, 499-508). In the present study, we show that the silencing of the two GME genes in tomato leaves resulted in approximately a 60% decrease in terminal L-Gal content in the side chain A of RG-II as well as in a lower capacity of RG-II to perform in muro cross-linking. In addition, we show that unlike supplementation with L-Gal or ascorbate, supplementation of GME-silenced lines with boric acid was able to restore both the wild-type growth phenotype of tomato seedlings and an efficient in muro boron-mediated cross-linking of RG-II. Our findings suggest that developmental phenotypes in GME-deficient lines are due to the structural alteration of RG-II and further underline the crucial role of the cross-linking of RG-II in the formation of the pectic network required for normal plant growth and development.     

Boron deficiency: How does the defect in cell wall damage the cells?

Boron (B) is an essential micronutrient for vascular plants. Boron plays a structural role in cell walls through binding to pectic polysaccharides. It still remains unclear how B deficiency, and hence probably alterations in cell wall structure, leads to various metabolic disorders and cell death. To understand the process, we analyzed the physiological changes in suspension- cultured tobacco (Nicotiana tabacum) BY-2 cells under B deficiency. The results indicated that the cells deprived of B did not undergo a typical programmed cell death process. Oxidative damage was proven to be the direct and major cause of cell death. We discuss possible mechanisms for the generation and accumulation of reactive oxygen species under B deprivation.Key words: boron deficiency, cell death, cell wall, oxidative damage, pectic polysaccharides, rhamnogalacturonan II, tobaccoBoron (B) deficiency is the most widespread micronutrient deficiency around the world and causes large losses in crop production both quantitatively and qualitatively.¹ Boron deficiency affects vegetative and reproductive growth of plants resulting in inhibition of cell expansion, death of meristem and reduced fertility.²Plants contain B both in a water-soluble and insoluble form. In intact plants, the amount of water-soluble B fluctuates with the quantity of B supplied, while insoluble B does not.³ The appearance of B deficiency symptoms coincides with the decrease of water-insoluble B, from which it is concluded that the insoluble B is the functional form while the soluble B represents the surplus. We found at least 98% of the insoluble B in tobacco cells bound to the cell wall,⁴ and identified their molecular entity as the borate diester with rhamnogalacturonan II (RG-II) regions of pectic polysaccharides.⁵ The diester crosslinks pectic polysaccharides to form a network and thereby contributes to construction of a supramolecular cell wall structure.⁶ Mutant plants with altered RG-II structures are dwarf and sterile, indicating that the B-RG-II complex is essential for normal plant growth and development.⁷ Increasing evidence indicates that B is also essential for animals.⁸ The requirement for B in organisms lacking cell walls implies that B may also have additional roles in plants. To date, however, no molecule other than apiosyl residues in pectic polysaccharides has been demonstrated to form a borate ester which could be stable enough under physiological conditions. Thus it is reasonable to consider that B functions primarily, if not exclusively, as a structural component of the cell wall, and B deficiency symptoms arise from disturbance of the cell wall structure. How, then, does the disturbed cell wall structure lead to the damage and cell death that are observed under B deficiency? To understand the linkage, we have analyzed physiological changes of suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells under B deficiency.When cells at the log phase of growth were transferred to B-free media, cell death was detectable as early as 12 h after the treatment. As cell walls play pivotal roles in plant development and growth, we assumed that the B deprivation, which probably causes aberrant cell wall structure, might induce programmed cell death (PCD) as an active response to eliminate damaged cells. Then we examined if the known biochemical hallmark of PCD could be observed in cells deprived of B (hereafter referred to as -B cells). However, internucleosomal DNA fragmentation, decrease in antioxidant content and antioxidant enzyme expression,⁹ or protection from death by cycloheximide, were not detected in these cells, suggesting that the cell death is necrosis. We found oxidative damage to be the direct and major cause of cell death, because -B cells contained more reactive oxygen species (ROS) than control cells, and because cell death was effectively suppressed by supplementing the media with lipophilic antioxidants. The deprivation treatment did not induce an oxidative burst, as the extracellular H₂O₂ concentration was not significantly different between -B and control cells at all time points examined. Resupply of B immediately suppressed cell death. Collectively, these results suggest that low but persistent ROS production occurred under the -B condition.In the study described above, we demonstrated that B deprivation, and hence probably a defective cell wall structure, leads to oxidative damage. How and why B deprivation induces ROS overproduction remains to be clarified. We hypothesize that ROS are originally produced as a signal for disturbance of the cell wall structure, and build up to a toxic level unless B is resupplied and the cell wall structure is restored. It has been reported that the mechanical strength of the squash root cell wall decreases within minutes after B deprivation.^10,11 The mechanical change could be brought about by insufficient crosslinking of pectic polysaccharides at RG-II regions, as the B-RG-II complex significantly contributes to the wall tensile strength.¹² If the cell wall becomes weaker and less resistant to turgor, then the plasma membrane would stretch. The change may lead to opening of mechanosensitive channels¹³ and generation of signals for the altered cell wall structure. To test this hypothesis, we are now analyzing the immediate and early responses of tobacco BY-2 cells to B deprivation, and preliminary results do indicate the involvement of Ca²⁺ influx in the responses. Identification of the mechanism by which cells sense the external B status will greatly contribute to our understanding of the cell wall-symplast interaction in plants.¹⁴     

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly.

Abbreviations: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II     

The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently cross-linked dimer

The location of the 1:2 borate-diol ester cross-link in the dimer of the plant cell wall polysaccharide rhamnogalacturonan II (RG-II) has been determined. The ester cross-links the apiofuranosyl residue of the 2-O-methyl-D-xylose-containing side chains in each of the subunits of the dimer. The apiofuranosyl residue in each of the two aceric acid-containing side chains is not esterified. The site of borate esterification is identical in naturally occurring and in in vitro synthesized dimer. Pb2+, La3+, and Ca2+ increase dimer formation in vitro in a concentration- and pH-dependent manner. Pb2+ is the most effective cation. The dimer accounts for 55% of the RG-II when the monomer (0.5 mM) is treated for 5 min at pH 3.5 with boric acid (1 mM) and Pb2+ (0.5 mM); at pH 5 the rate of conversion is somewhat slower. Hg2+ does not increase the rate of dimer formation. A cation's charge density and its ability to form a coordination complex with RG-II, in addition to steric factors, may regulate the rate and stability of dimer formation in vitro. Our data provide evidence that the structure of RG-II itself determines which apiofuranosyl residues are esterified with borate and that in the presence of boric acid and certain cations, two RG-II monomers self-assemble to form a dimer.     

Boron nutrition of cultured tobacco BY-2 cells. III. Characterization of the boron-rhamnogalacturonan II complex in cells acclimated to low levels of boron

Cultured cells of tobacco (Nicotiana tabacum L.) BY-2 which could propagate at the same rate as the parent cells (1 mg B liter(-1)) under a lower level of boron (0.01 mg B liter(-1)) were obtained. The selected cells had swollen cell walls. In the parent cells, all the RG-II occurred as a B-RG-II complex, however, two-thirds of the RG-II occurred in a monomeric form in the selected cells.     

2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis.     

Changes in Cell Wall Polysaccharides of Green Bean Pods during Development

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, beta-galactosidase, and alpha-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.     

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up ∼90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of α-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.     

Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp

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)-beta-galactan epitope was absent from cell walls in regions of pit fields. A (1-->5)-alpha-arabinan epitope occurred most abundantly at the inner face of cell walls in regions surrounding the pit fields.     

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.     

Depletion of UDP-D-apiose/UDP-D-xylose synthases results in rhamnogalacturonan-II deficiency, cell wall thickening, and cell death in higher plants

D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D-apiose as well as 2-O-methyl-L-fucose and 2-O-methyl-D-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a D-apiose deficiency.