Analytical implications of different methods for preparing plant cell wall material.

Almost all plant cells are surrounded by a wall constructed of co-extensive networks of polysaccharides and proteoglycans. The capability to analyse cell wall components is essential for both understanding their complex biology and to fully exploit their numerous practical applications. Several biochemical and immunological techniques are used to analyse cell walls and in almost all cases the first step is the preparation of an alcohol insoluble residue (AIR). There is significant variation in the protocols used for AIR preparation, which can have a notable impact on the downstream extractability and detection of cell wall components. To explore these effects, we have formally compared ten AIR preparation methods and analysed polysaccharides subsequently extracted using high-performance anion exchange chromatography (HPAEC-PAD) and Micro Array Polymer Profiling (MAPP). Our results reveal the impact that AIR preparation has on downstream detection of cell wall components and the need for optimisation and consistency when preparing AIR.

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

Oxidative stability and chemical safety of mayonnaise enriched with grape seed extract.

Grape seed extract (GSE), a by-product from the wine industry, was explored for its use as enrichment to mayonnaise, due to its potential health effects. Mayonnaises were enriched with 0 mg GSE per mL, 0.5 mg GSE per mL (~0.050%), 0.9 mg GSE per mL (~0.10%) and 1.4 mg GSE per mL (~0.15%) during preparation and stored in the dark at room temperature for 8 weeks. The antioxidative capacity of the mayonnaises was evaluated by their ability to scavenge the stable radical TEMPO by electron spin resonance (ESR) spectroscopy. The oxidative stability of the mayonnaises was determined by the content of lipid hydroperoxides (peroxide value, POV), the content of conjugated diene hydroperoxides and the content of thiobarbituric acid reactive substances (TBARS). The highest antioxidative capacity and the lowest content of lipid hydroperoxides and TBARS were found in the mayonnaise with the highest percentage of GSE (0.15%). Therefore, the oxidative stability of the mayonnaises enriched with GSE was slightly improved through storage. However, mayonnaise without GSE had the highest sensorial acceptability compared to mayonnaise enriched with GSE. In the Artemia salina assay, a fast screening method for overall toxicity, the death rate of brine shrimps larvae was found to increase for increasing percentage of GSE. A level of 0.05% GSE in mayonnaise is concluded not to constitute any toxicological risks, but to provide significant protection against oxidation during storage.

Palatability and chemical safety of apple juice fortified with pomegranate peel extract.

Pomegranate peel extract (PPE), a by-product of the pomegranate juice industry with potential health effects, was explored for use to fortify reconstituted apple juice in the concentration range 0.5 to 2.0% (w/w). Radical scavenging and antioxidative capacities of the fortified apple juices were evaluated using (i) electron spin resonance (ESR) to quantify their ability to scavenge the stable radical Fremy's salt and (ii) the Trolox equivalent antioxidant capacity (TEAC) assay and compared to apple juice without fortification as control. The highest antioxidative capacity was found in the apple juice fortified with the highest percentage of pomegranate peel extract, while the optimal sensory quality was found by addition of 0.5 g PPE per 100 mL. The Artemia salina assay was used as a fast screening method for evaluating overall toxicity, and showed little toxicity with up to 1.0 g per 100 mL addition of PPE, but increasing toxicity at higher concentrations. Accordingly, it is important to balance addition of PPE, when used for enrichment of apple juice in order to obtain a healthier product, without compromising the sensorial quality or toxicological safety of the apple juice. Concentrations between 0.5 and 1.0 g PPE per 100 mL seem to be acceptable.

ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta.

Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.

Characterisation of the arabinose-rich carbohydrate composition of immature and mature marama beans (Tylosema esculentum).

Marama bean (Tylosema esculentum) is an important component of the diet around the Kalahari Desert in Southern Africa where this drought resistant plant can grow. The marama bean contains roughly 1/3 proteins, 1/3 lipids and 1/3 carbohydrates, but despite its potential as dietary supplement little is known about the carbohydrate fraction. In this study the carbohydrate fraction of "immature" and "mature" marama seeds are characterised. The study shows that the marama bean contains negligible amounts of starch and soluble sugars, both far less than 1%. The cell wall is characterised by a high arabinose content and a high resistance to extraction as even a 6M NaOH extraction was insufficient to extract considerable amounts of the arabinose. The arabinose fraction was characterised by arabinan-like linkages and recognised by the arabinan antibody LM6 and LM13 indicating that it is pectic arabinan. Two pools of pectin could be detected; a regular CDTA (1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid) or enzymatically extractable pectin fraction and a recalcitrant pectin fraction containing the majority of the arabinans, of which about 40% was unextractable using 6M NaOH. Additionally, a high content of mannose was observed, possibly from mannosylated storage proteins.

Simultaneous in vivo truncation of pectic side chains.

Despite the wide occurrence of pectin in nature only a few source materials have been used to produce commercial pectins. One of the reasons for this is that many plant species contain pectins with high levels of neutral sugar side chains or that are highly substituted with acetyl or other groups. These modifications often prevent gelation, which has been a major functional requirement of commercial pectins until recently. We have previously shown that modification of pectin is possible through heterologous expression of pectin degrading enzymes in planta. To test the effect of simultaneous modification of the two main neutral pectic side chains in pectic rhamnogalacturonan I (RGI), we constitutively expressed two different enzymes in Arabidopsis thaliana that would either modify the galactan or the arabinan side chains, or both side chains simultaneously. Our analysis showed that the simultaneous truncation of arabinan and galactan side chains is achievable and does not severely affect the growth of Arabidopsis thaliana.

Identification of a xylogalacturonan xylosyltransferase involved in pectin biosynthesis in Arabidopsis.

Xylogalacturonan (XGA) is a class of pectic polysaccharide found in plant cell walls. The Arabidopsis thaliana locus At5g33290 encodes a predicted Type II membrane protein, and insertion mutants of the At5g33290 locus had decreased cell wall xylose. Immunological studies, enzymatic extraction of polysaccharides, monosaccharide linkage analysis, and oligosaccharide mass profiling were employed to identify the affected cell wall polymer. Pectic XGA was reduced to much lower levels in mutant than in wild-type leaves, indicating a role of At5g33290 in XGA biosynthesis. The mutated gene was designated xylogalacturonan deficient1 (xgd1). Transformation of the xgd1-1 mutant with the wild-type gene restored XGA to wild-type levels. XGD1 protein heterologously expressed in Nicotiana benthamiana catalyzed the transfer of xylose from UDP-xylose onto oligogalacturonides and endogenous acceptors. The products formed could be hydrolyzed with an XGA-specific hydrolase. These results confirm that the XGD1 protein is a XGA xylosyltransferase. The protein was shown by expression of a fluorescent fusion protein in N. benthamiana to be localized in the Golgi vesicles as expected for a glycosyltransferase involved in pectin biosynthesis.

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis.

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

Xylogalacturonan exists in cell walls from various tissues of Arabidopsis thaliana.

Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH). Upon enzymatic treatment, XGA oligosaccharides were primarily produced from pectin extracts obtained from the young and mature leaves and to a lesser extent from those originating from the stem of A. thaliana. The oligosaccharide GalA(3)Xyl was predominantly formed from these pectin extracts. No XGA oligosaccharides were detected in digests of pectin extracts from the seeds and roots. A low number of XGA oligosaccharides was obtained from pectins of A. thaliana. This indicates a uniform distribution of xylose in XGA from A. thaliana. The predominant production of GalA(3)Xyl, as well as the release of linear GalA oligosaccharides pointed to a lower degree of xylose substitution in XGA from A. thaliana than in XGA from apple and potato. The estimated amount of XGA accounted for approximately 2.5%, 7% and 6% (w/w) of the total carbohydrate in the pectin fraction of the stem, young leaves and mature leaves, respectively.

QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects homogalacturonan and xylan biosynthesis.

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan alpha-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of beta-1-4-D-xylan synthase, beta-1-4-D-galactan synthase and beta-glucan synthase II activities were also measured in microsomal membranes. Of these, only beta-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on alpha-1,4-D-galacturonosyltransferase and beta-1,4-D-xylan synthase activities.

Rhamnogalacturonan I in Solanum tuberosum tubers contains complex arabinogalactan structures.

A rhamnogalacturonan I polysaccharide was isolated from potato (Solanum tuberosum cv. Posmo) tuber cell walls and characterised by enzymatic digestion with an endo-beta-1 --> 4-galactanase and an endo-alpha-1 --> 5-arabinanase, individually or in combination. The reaction products were separated using size-exclusion chromatography and further analysed for monosaccharide composition and presence of epitopes using the LM5 anti-beta-1 --> 4-galactan and LM6 anti-alpha-1 --> 5-arabinan monoclonal antibodies. The analyses point to distinct structural features of potato tuber rhamnogalacturonan I, such as the abundance of beta-1 --> 4-galactan side chains that are poorly substituted with short arabinose-containing side chains, the presence of alpha-1 --> 5-arabinan side chains substituted with beta-1 --> 4-galactan oligomers (degree of polymerisation > 4), and the presence of alpha-1 --> 5-arabinans that resist enzymatic degradation. A synergy between the enzymes was observed towards the degradation of arabinans but not towards the degradation of galactans. The effect of the enzymes on isolated RG I is discussed in relation to documented effects of enzymes heterologously expressed in potato tubers. In addition, a novel and rapid method for the determination of the monosaccharide and uronic acid composition of cell wall polysaccharides using high-performance anion exchange chromatography with pulsed amperometric detection is described.