Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.     

Development of a Quantitative Assay for Mycobacterial Endogenous Arabinase and Ensuing Studies of Arabinase Levels and Arabinan Metabolism in Mycobacterium smegmatis

Treatment of either Mycobacterium tuberculosis or M. smegmatis with ethambutol results both in inhibition of arabinan synthesis and in copious loss of previously formed arabinan from the cell wall. The loss of arabinan has been shown to be due to the action of an endogenous arabinase. To better understand this phenomenon, a quantitative assay for endogenous arabinase was developed. Using the assay it was determined that various subcellular fractions of M. smegmatis showed significant amounts of endogenous arabinase activity. Surprisingly, treatment with ethambutol yielded only minor changes in the amounts of endogenous arabinase activities. Endogenous arabinase was present in the cell wall, and consistently, incubation of the M. smegmatis cell wall in only buffer resulted in the release of arabinan, mimicking the effect of ethambutol on whole cells. To determine if cell wall arabinan is rapidly turned over, the arabinan was labeled in the early log phase of culture by feeding [¹⁴C]glucose, followed by a “chase” with nonradioactive glucose. Most of the labeled arabinan remained in the cell wall after the culture was grown to late log phase. Thus, there is active arabinase in the cell wall, but arabinan is not rapidly removed unless ethambutol is present. Purification of the endogenous arabinase, using the assay described, is ongoing to help further discern its biological function.     

In muro fragmentation of the rhamnogalacturonan I backbone in potato (Solanum tuberosum L.) results in a reduction and altered location of the galactan and arabinan side-chains and abnormal periderm development

Rhamnogalacturonan (RG) I is a branched pectic polysaccharide in plant cell walls. Rhamnogalacturonan lyase (eRGL) from Aspergillus aculeatus is able to cleave the RG I backbone at specific sites. Transgenic potato (Solanum tuberosum L.) plants were made by the introduction of the gene encoding eRGL, under the control of the granule-bound starch synthase promoter. The eRGL protein was successfully expressed and translated into an active form, demonstrated by eRGL activity in the tuber extracts. The transgenic plants produced tubers with clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis of the isolated cell walls showed a large reduction in galactosyl and arabinosyl residues in transgenic tubers. Immunocytochemical studies using the LM5 (galactan) and LM6 (arabinan) antibodies also showed a large reduction in galactan and arabinan side-chains of RG I. Most of the remaining LM5 epitopes were located in the expanded middle lamella at cell corners of eRGL tubers, which is in contrast to their normal location in the primary wall of wild type tubers. These data suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm.     

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.     

Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development

Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure–function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.     

A gas chromatographic analysis of the release of arabinose from coleoptile cell walls under the influence of auxin and dextranase preparations

An analysis by gas chromatography of the products of digestion of cell wall pellets of Avena sativa L., coleoptiles shows that after short term autolysis of pellets from auxin-rich plants, arabinose is released. Addition of arabinan as a substrate increases the yield of arabinose considerably, suggesting that a similar substance is broken down in the walls.In pellets from auxin-poor coleoptiles much less arabinose is released and addition of arabinan has no effect. Dextranase preparations stimulate this release and can also break down arabinan. This suggests that auxin and a dextranase-like enzyme are involved in the process.     

Oligosaccharides as inhibitors of mycobacterial arabinosyltransferases. Di- and trisaccharides containing C-3 modified arabinofuranosyl residues

The assembly of the arabinan portions of cell wall polysaccharides in mycobacteria involves a family of arabinosyltransferases (AraT's) that promote the polymerization of decaprenolphosphoarabinose. Mycobacterial viability depends upon the ability of the organism to synthesize an intact arabinan and thus compounds that inhibit these AraT's are both useful biochemical tools as well as potential lead compounds for new anti-tuberculosis agents. We describe here the preparation of oligosaccharide fragments of mycobacterial arabinan that contain arabinofuranosyl residues modified at C-3 by the replacement of the hydroxyl group with an amino, azido or methoxy functionality. Subsequent testing of these oligosaccharides as inhibitors of mycobacterial AraT's revealed that all inhibited the enzymes, but to varying degrees. In further studies, each compound was shown to have only low activity as an inhibitor of mycobacterial growth.     

Deletion of Cg-emb in corynebacterianeae leads to a novel truncated cell wall arabinogalactan, whereas inactivation of Cg-ubiA results in an arabinan-deficient mutant with a cell wall galactan core

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for "priming" the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide.     

Location of the mycolyl ester substituents in the cell walls of mycobacteria

The question of the precise location of mycolic acids, the single most distinctive cell wall entity of members of the Mycobacterium genus, has now been addressed. The free hydroxyl functions of the arabinogalactan component of the mycobacterial cell wall were O-methylated under conditions in which the mycolyl esters were not cleaved. Subsequent replacement of the mycolyl functions with O-ethyl groups resulted in an acid- and base-stable differentially O-alkylated surrogate polysaccharide, more amenable to analysis. Complete hydrolysis, reduction, acetylation, and gas chromatography/mass spectrometry revealed the unexpected finding that the mycolyl substituents were selectively and equally distributed on the 5-hydroxyl functions of terminal- and 2-linked arabinofuranosyl (Araf) residues. Further analysis of the O-alkylated cell wall through partial acid hydrolysis, NaB[2H]4 reduction, pentadeuterioethylation, and gas chromatography/mass spectrometry demonstrated that the mycolyl units are clustered in groups of four on the previously recognized nonreducing terminal pentaarabinosyl unit [beta-Araf-(1----2)-alpha-Araf)2-3, 5-alpha-Araf. However, only about two-thirds of the available pentasaccharide units are so substituted. Thus, the antigenicity of the arabinan component of mycobacterial cell walls may be explained by the fact that about one-third of the pentaarabinosyl units are not mycolyated and are available for interaction with the immune system. On the other hand, the extreme hydrophobicity and impenetrability of the mycobacterial cell may be explained by the same motif also acting as the fulerum for massive esterified paraffin residues. New fundamental information on the structure of mycobacterial cell walls will aid in our comprehension of its impenetrability to antibiotics and role in immunopathogenesis and persistence of disease.     

Predominant structural features of the cell wall arabinogalactan of Mycobacterium tuberculosis as revealed through characterization of oligoglycosyl alditol fragments by gas chromatography/mass spectrometry and by 1H and 13C NMR analyses

The peptidoglycan-bound arabinogalactan of a virulent strain of Mycobacterium tuberculosis was per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides reduced and O-pentadeute-rioethylated. The per-O-alkylated oligoglycosyl alditol fragments were separated by high pressure liquid chromatography and the structures of 43 of these constituents determined by 1H NMR and gas chromatography/mass spectrometry. The arabinogalactan was shown to consist of a galactan containing alternating 5-linked beta-D-galactofuranosyl (Galf) and 6-linked beta-D-Galf residues. The arabinan chains are attached to C-5 of some of the 6-linked Galf residues. The arabinan is comprised of at least three major structural domains. One is composed of linear 5-linked alpha-D-arabinofuranosyl (Araf) residues; a second consists of branched 3,5-linked alpha-D-Araf units substituted with 5-linked alpha-D-Araf residues at both branched positions. The non-reducing terminal region of the arabinan was characterized by a 3,5-linked alpha-D-Araf residue substituted at both branched positions with the disaccharide beta-D-Araf-(1----2)-alpha-D-Araf. 13C NMR of intact soluble arabinogalactan established the presence of both alpha- and beta-Araf residues in this domain. This non-reducing terminal motif apparently provides the structural basis of the dominant immunogenicity of arabinogalactan within mycobacteria. A rhamnosyl residue occupies the reducing terminus of the galactan core and may link the arabinogalactan to the peptidoglycan. Evidence is also presented for the presence of minor structural features involving terminal mannopyranosyl units. Models for most of the heteropolysaccharide are proposed which should increase our understanding of a molecule responsible for much of the immunogenicity, pathogenicity, and peculiar physical properties of the mycobacterial cell.     

glmU基因敲除后对分枝杆菌细胞壁中聚糖影响的初步分析

应用已构建的glmU基因敲除的耻垢分枝杆菌作为实验模型,对细胞壁中的聚糖的组成成份和结构进行分析。气相色谱与高效液相Dionex的结果共同说明了在mc2155 glmU KOT菌株的细胞壁内,当缺失活性GlmU时,阿拉伯糖含量增加,且其增加是来自于具有分支的阿拉伯糖末端的增多。此结果将能更进一步地认识GlmU的功能以及当GlmU功能异常时对细菌造成的影响,这些都将为研究以GlmU为靶位点的药物对细菌的影响提供实验支持。     

The structure and function of an arabinan-specific alpha-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases

Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.     

Branched arabino-oligosaccharides isolated from sugar beet arabinan

Sugar beet arabinan consists of an α-(1,5)-linked backbone of l-arabinosyl residues, which can be either single or double substituted with α-(1,2)- and/or α-(1,3)-linked l-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an α-(1,5)-linked backbone of l-arabinosyl residues. One series carries single substituted α-(1,3)-linked l-arabinosyl residues at the backbone, whereas the other series consists of a double substituted α-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.     

Changes in distribution of cell wall polysaccharides in floral and fruit abscission zones during fruit development in tomato (Solanum lycopersicum)

After fruit development has been triggered by pollination, the abscission zone (AZ) in the pedicel strengthens its adhesion to keep the fruit attached. Unpollinated flowers are shed at their respective AZs, whereas an enlargement of the same tissue is observed in pollinated flowers. After the fruit has developed and is fully ripened, shedding occurs easily at the AZ, indicating an acceleration of abscission. Cell wall degradation and synthesis may play important roles in these processes; however, little is understood. In this report, we have visualized changes in polysaccharide distribution in the AZs of pollinated versus unpollinated flowers and in the ripened fruits using immunohistochemistry. During floral abscission, a large increase was observed in LM15 labeling of xyloglucan specifically at the AZ in the abscising pedicel. LM5 and LM6 labeling of galactan and arabinan, respectively, also increased—LM5 throughout the pedicel and LM6 at the basal side of the AZ. The results suggest that xyloglucan, pectic galactan and arabinan play key roles in the abscission process. During fruit abscission, unlike in floral abscission, no AZ-specific cell wall polysaccharide deposition was observed; however, high autofluorescence was seen in the AZ of over-ripe fruit pedicels, suggesting secondary cell wall synthesis and lignification of the AZ prior to fruit abscission.     

A Single Arabinan Chain Is Attached to the Phosphatidylinositol Mannosyl Core of the Major Immunomodulatory Mycobacterial Cell Envelope Glycoconjugate,Lipoarabinomannan

Lipoarabinomannan (LAM) is composed of a phosphatidylinositol anchor followed by a mannan followed by an arabinan that may be capped with various motifs including oligosaccharides of mannose. A related polymer, lipomannan (LM), is composed of only the phosphatidylinositol and mannan core. Both the structure and the biosynthesis of LAM have been studied extensively. However, fundamental questions about the branching structure of LM and the number of arabinan chains on the mannan backbone in LAM remain. LM and LAM molecules produced by three different glycosyltransferase mutants of Mycobacterium smegmatis were used here to investigate these questions. Using an MSMEG_4241 mutant that lacks the α-(1,6)-mannosyltransferase used late in LM elongation, we showed that the reducing end region of the mannan that is attached to inositol has 5–7 unbranched α-6-linked-mannosyl residues followed by two or three α-6-linked mannosyl residues branched with single α-mannopyranose residues at O-2. After these branched mannosyl residues, the α-6-linked mannan chain is terminated with an α-mannopyranose at O-2 rather than O-6 of the penultimate residue. Analysis of the number of arabinans attached to the mannan core of LM in two other mutants (ΔembC and ΔMSMEG_4247) demonstrated exactly one arabinosyl substitution of the mannan core suggestive of the arabinosylation of a linear LM precursor with ∼10–12 mannosyl residues followed by additional mannosylation of the core and arabinosylation of a single arabinosyl “primer.” Thus, these studies suggest that only a single arabinan chain attached near the middle of the mannan core is present in mature LAM and allow for an updated working model of the biosynthetic pathway of LAM and LM.     

The presence of an endogenous endo-D-arabinase in Mycobacterium smegmatis and characterization of its oligoarabinoside product

Endogenous mycobacterial endo-D-arabinase activity, which degrades cell wall polysaccharide arabinogalactan, was found in Mycobacterium smegmatis. The arabinan product contains 20-30 arabinosyl residues but no galactofuranosyl residues. Recognition of this endogenous activity results in the possibility of developing antituberculosis drugs that do not require bacterial growth for activity.     

EmbB 及 EmbC 功能结构域对分枝杆菌聚阿拉伯糖合成的影响

本研究通过构建精确保留 EmbB 的N端跨膜区和 EmbC 的C端结构域相拼接的杂化质粒,将该质粒导入到embB和embC 基因敲除的耻垢分枝杆菌中,观察杂合蛋白表达对这2种菌株细胞壁中阿拉伯糖合成的影响.结果表明,Embs 蛋白家族成员 N端完整的跨膜区能使其正确地定位于细胞膜,此为其发挥作用的必要前提;尽管 Emb 家族成员间同源性高,但相互间的定位作用不能替代;Embs 蛋白家族成员的 C 末端是其催化性的结构域,但在发挥作用时,需要其 N 末端的结构域能使其正确定位.本文通过研究Emb蛋白的功能性结构域,深入探讨Emb蛋白结构与功能间的关系,从而为研发有效的抗结核化合物,克服耐药性结核分枝杆菌提供理论依据.     

A role for pectin-associated arabinans in maintaining the flexibility of the plant cell wall during water deficit stress

One of the main components of pectin, a primary constituent of higher plant cell walls, is rhamnogalacturonan I. This polymer comprised of linked alternating rhamnose and galacturonic acid residues is decorated with side chains composed of arabinose and galactose residues. At present, the function of these side chains is not fully understood. Our research on Southern African resurrection plants, plants that are capable of surviving severe dehydration (desiccation), has revealed that their cell walls are capable of extreme flexibility in response to water loss. One species, Myrothamnus flabellifolia, has evolved a constitutively protected leaf cell wall, composed of an abundance of arabinose polymer side chains, suggested to be arabinans and/or arabinogalactans, associated with the pectin matrix. In this article, we propose a hypothetical model that explains how the arabinan rich pectin found in the leaves of this desiccation-tolerant plant permits almost complete water loss without deleterious consequences, such as irreversible polymer adhesion, from occurring. Recent evidence suggesting a role for pectin-associated arabinose polymers in relation to water dependent processes in other plant species is also discussed.Key words: arabinans, cell wall, desiccation, resurrection, rehydration, rhamnogalacturonan IThe flowering plant cell wall is a composite structure consisting of a skeletal framework of cellulose and hemicellulose embedded within a matrix of pectin polysaccharides and cell wall glycoproteins.^1,2 The pectin matrix, in turn, is composed of three primary types of polysaccharides, these being rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII) and homogalacturonan (HG).¹ RGII is a complex polysaccharide, consisting of many unusual sugar moieties, and is not present in large amounts in the wall.³ HG is effectively a linear homopolymer of galacturonic acid and is believed to facilitate the formation of tight junctions, ‘egg boxes’, by complexing with calcium ions present in the cell wall.¹ RGI is a polymer composed of a backbone of alternating glycosidically linked rhamnose and galacturonic acid residues.¹ Side chains, consisting of either arabinogalactan polymers or linear chains of arabinans and/or galactans, are then attached to the rhamnose residues of the RGI backbone.¹ The manner with which these polymers are attached or become entangled with each other and cellulosic polymers to form the pectin matrix has been a matter of debate. The classical theory is that the RGI and HG polymers alternate with each other as block polymers and that the side chains interact with neighbouring polysaccharide chains. Recently, this standard theory has been questioned and an argument whereby the HG polymers are actually side chains of a RGI backbone polymer has been advanced.⁴ Nevertheless, the complexity of pectin polysaccharides is such that ascribing definitive functions to this matrix of polysaccharides has proven quite difficult. The physical properties of the pectin matrix suggest a number of possible functions. The water binding properties of the galacturonic acid residues indicate that polymers containing these groups have the capacity to hydrate and swell and so possibly help maintain polymer separation in the wall.⁵ The side chains of RGI include arabinan and galactan polymers which have been shown to be highly mobile^6,7,8 with the potential to interact with each other forming a temporally entangled matrix.⁹ It is also believed that arabinan chains, which have been shown to contain ferulate residues attached to terminal arabinose groups, are able to oxidatively cross-link via the formation of diferulate bridges between arabinan chains that originate on separate RGI polysaccharides.¹⁰ The pectin matrix is now believed to contain sub-domains of RGI, HG and RGII which may interact with different polysaccharide components of the cell wall such as cellulose or xyloglucan.^11,12 Hence, it is possible that the pectin matrix may form these associations with other polysaccharides via covalent⁹ and/or non-covalent¹¹ (e.g., H-bonding) interactions and in so doing ensure the integrity of the wall and its polymer organisation. Although a number of general functions, such as hydration and ion binding, have been proposed for the pectin matrix, in particular the RGI polymer and its neutral side chains, there has been difficulty in elucidating specific functions for these polysaccharides. A number of molecular genetic studies have been performed with the aim of establishing specific functions for the RGI side chains. A recent study showed that genetic removal of the arabinan side chains in the cell walls of Nicotiana plumbaginfolia results in the formation of a non-organogenic callus culture with loosely attached cells.¹³ Furthermore, it has been shown that ‘in muro’ fragmentation of the RG1 backbone in Solanum tuberosum results in abnormal development of the periderm.¹⁴ This suggests that these side chains may play at least some role in normal cell attachment and cell development. However, the real problem is that no obvious phenotypic differences between wild type and mutant plants (in which neutral side chains have been modified) have been observed.^15,16,17 It may be that the conditions under which phenotypic differences between wild type and mutant plants would arise have not yet been investigated. We believe the water binding and attachment properties of the pectin matrix are particularly important. This is especially so given the role pectin plays in the middle lamella ensuring attachment of cells to each other and in the formation of the apoplast where water mediated transport of solutes occurs.¹ Our research has focused on a group of Southern African plants termed ‘Resurrection plants’ because of their unique ability to survive severe dehydration (desiccation) to an almost air-dry state.¹⁸ We have been interested in how the cell walls of angiosperm resurrection plants such as Craterostigma wilmsii^19,20 and Myrothamnus flabellifolia^21,22 may have become adapted to survive this extreme water deficit stress (desiccation). We have shown that in the case of the Myrothamnus flabellifolia leaf cell wall, which becomes considerably folded when dried, does not undergo dramatic changes in composition or polymer location in response to desiccation.²¹ Rather we propose that this plant has evolved a constitutively protected cell wall which is able to undergo repeated cycles of desiccation and rehydration.^21,22 We have observed that the pectin component of the leaf cell wall in this species was unusually rich in arabinose polymers, most likely arabinan and arabinogalactan in nature, which we advanced was the reason that the cell wall of this species was able to tolerate desiccation.²¹ Here we provide a simple model (Fig. 1) whereby the arabinan side chains of the pectin polysaccharides are responsible for possibly buffering/replacing the lost water during desiccation and in so doing prevent the formation of tight junctions (e.g., egg boxes) or strong H-bonding interactions between the normally separate ‘skeletal’ polysaccharides (e.g., cellulose microfibrils and xyloglucan tethers) embedded in the pectin matrix. Our model is supported by the observation that cell wall arabinans play a crucial role in the response of guard cells to turgor pressure.²³ It was shown that removal of arabinans by enzymatic digestion of leaf strips of Commelina communis resulted in locking of the guard cell walls in either the open or closed position.²³ Additional roles for arabinan polymers in cell walls have recently been implied with respect to the salt tolerance of Mesembryanthemum crystallinum,²⁴ ensuring hydration of the seed endosperm of Gleditsia triacanthos during germination²⁵ and the tolerance of tropical legume seeds to dehydration.²⁶ We believe that the arabinan side chains of RGI play a critical role in the ability of cell walls to remain flexible during plant growth and may have important functions in relation to the water content of the cell. Further studies aimed at determining the relationship between wall water content, RGI side chains and cell wall flexibility may reveal hitherto unsuspected functions for these polysaccharides in the life of the plant.Open in a separate windowFigure 1A model proposing the role of arabinose rich pectin polymers in stabilising the cell wall against water loss. (A) Pectin consisting of short arabinan chains in the hydrated state, (B) pectin consisting of short arabinan chains in the dehydrated state; (C) pectin consisting of long arabinan chains in the hydrated state; (D) pectin consisting of long arabinan chains in the dehydrated state. The likelihood of irreversible tight junctions (e.g., egg boxes) forming in arabinan poor cell walls during dehydration is demonstrated in (B) while the reversible buffering effect of arabinan rich cell walls is proposed in (D) as would possibly occur in Myrothamnus flabellifolia. For simplicity arabinan chains not participating in the buffering interactions between the RGI backbone chains have been shortened to two arabinose residues in length. Note in (A) and (B) all arabinan chains are two arabinose residues in length.     

Structural investigation of an arabinan from cabbage (Brassica oleracea var. capitata)

An arabinan has been isolated from the hot water-soluble pectic substances of cabbage cell wall material. Methylation analysis involving GC-MS of methylated alditol acetates formed from the methylated arabinan has shown that the parent polysaccharide is highly branched and of the same structural type as other arabinans associated with seed pectins.     

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.