| Home |
| People |
| Research |
| Publications |
| Lab Tour |
| Lab Photos |
| Collaborators |
| Meetings / Seminars |
| Contact Us |
Research
Plant cells produce an extracellular matrix, the cell wall, that determines and maintains the shape of cells and serves a protective barrier. Plant cell walls are highly complex structures composed predominantly of a diverse set of polysaccharides that vary in structure and abundance. Cellulose is the most abundant polysaccharide on earth, followed by xylan, a hemicellulosic component of the plant cell wall. Characterisation of the different polysaccharides synthesised in plant species dates back to the early 19th century. However, apart from cellulose, enzymes catalysing the synthesis of the majority of other cell wall polysaccharides are still uncharacterised or even undiscovered. Recently, plant polysaccharides edge ever closer to the spotlight, as they provide a sustainable resource for energy production. They are also essential for nutritional value and other beneficial traits of food.
Figure 1: A model of polysaccharide biosynthesis in the Golgi
The biosynthesis of polysaccharides involves the action of hundreds of different glycosyltransferases (GTs),
the enzymes that catalyse the specific transfer of sugar moieties from activated nucleotide sugar donors to acceptor molecules,
forming glycosidic bonds. Specific nucleotide-sugar transporters located in the Golgi membrane are also involved as suppliers of
the monosaccharide (Figure 1). Our knowledge about the specific function of the glycosyltranferases and the requirement of specific sugar
transporters for biosynthesis is, however, limited.
At the moment we are predominantly working on understanding the biosynthesis of diverse variants of xylan and glucomannan
in different plants. Both xylan and glucomannan are hemicellulosic components of the plant cell wall
that are synthesized in the Golgi apparatus. In general, our research focuses on understanding how the polysaccharides are synthesized,
what their structure is and how the structure relates to function.
Recently published work on xylan biosynthesis and glucomannan biosynthesis exemplifies our approach of combining
reverse genetics, cell biology and biochemical methods to analyse protein function and polysaccharide structure. The complexity of
polysaccharide structures and their analysis are exemplified by our work on the structure of arabinogalactans
Glucuronoxylan biosynthesis
Xylans are based on a linear β-1,4-linked xylose polymer that can be substituted by arabinosyl (Ara) and/or glucuronyl (GlcA) side chains with the degree and nature of substitution varying between tissues and species (Figure 2). Xylans can be furthermore modified by additional sugar side chains, by methylation, acetylation or feruloylation.
Figure 2: Simple structure of glucuronoarabinoxylan
Our recent work on the biosynthesis of glucuronoxylan in the model plant Arabidopsis thaliana identified novel players in
xylan biosynthesis, GUX1 and GUX2, that are required for the addition of glucuronyl acid side chains to the xylan backbone.
Candidate glucuronyl acid transferases were identified on the basis of co-expression with known xylan biosynthesis genes and
their localisation confirmed to the Golgi apparatus. The analysis of knock-out plants revealed no apparent growth phenotype. Biochemical analysis of the cell wall,
however, showed that the double mutant gux1/gux2 lacks glucuronic acid substitutions on xylan (Figure 3A). In addition, microsomal material from
gux1/gux2 mutant plants - unlike the wildtype (wt) - showed no Glucuronic acid transfer activity in biochemical assays (Figure 3B).
The absence of glucuronic acid substitutions also seems to change the properties of xylan that is now easier chemically extractable from
cell wall material, demonstrating the potential of manipulating xylan structure in context of industrial applications, such as renewable materials and
energy from plants, and altered dietary fibre.
Figure 3: Analysis of gux1/gux2 mutants
Glucomannan biosynthesis
Glucomannans are based on a linear β-1,4-linked glucosyl (Glc) and mannosyl (Man) polymer that can be substituted by galactosyl (Gal) side chains (Figure 4). The composition of the glucosyl and mannosyl polymer backbone as well as the side chain substitution varies between tissues and species.
Figure 4: Structure of Galacto(gluco)mannan
Our recent work on the biosynthesis of glucomannan in Arabidopsis thaliana characterises a group of genes of the CSLA family that are required for glucomannan biosynthesis. There are nine members of the CSLA family in Arabidopsis, which are differentially expressed throughout development. Analysis of knock-out mutants reveals that CSLA2/CSLA3/CSLA9 are the major components required for glucomannan in stems, whereas CSLA7 seems to synthesise glucomannan in the embryo. Even though there is a lack of glucomannan in stems in the CSLA2/CSLA3/CSLA9 triple mutant (Figure 5A), no effect on stem development and strength can be detected. In contrast, altering the glucomannan levels by mutating CSLA7 or by expressing CSLAs using a strong and ubiquitous promotor (35Spro) interferes with embryo development (Figure 5B), suggesting a role of glucomannan in signalling processes.
Figure 5: Immunofluorescence labelling of Arabidopsis stem sections and analysis of embryo lethality in csla mutants and CSLA overexpressing lines
Structure of Arabinogalactan
Arabinogalatctans are structurally highly complex polysaccharides consisting predominantly of Galacose (Gal) and Arabinose (Ara) sugars. Arabinogalactans are the major component of gums. They are attached to diverse cell wall proteins via a hydroxyproline in arabinogalactan proteins (AGPs). The polysaccharide component, however, can account for more than 90% of the AGP, suggesting that the arabinogalactan is sometimes the functional part of the molecule.
We used a set of specific carbohydrate-active enzymes to reduce the complexity of the structure, analysed the oligosaccharides by mass spectrometry (MS). The structure of the oligosaccharides revealed the complexity and highly branched nature of arabinogalactan of wheat endosperm AGPs. The cleavage sites of different carbohydrate-active enzymes (arrowheads) are depicted in the structure of arabinogalactan in Figure 6
Figure 6: Structure of wheat arabinogalactan
Specific techniques:
Our group is developing diverse techniques in order to identify and characterise candidate genes involved in polysaccharide synthesis,
and in order to characterise and quantify the structure of cell wall polysaccharides. Many of the techniques are developed in
collaboration with other research laboratories. For a list of collaborators please click here.
Localization of Organelle Proteins by Isotope Tagging LOPIT:
Quantitative proteomics to identify the main polysaccharide synthase enzymes in the plant Golgi apparatus
(Dunkley et al., 2004;
Dunkley et al., 2006).
This technique was developed in collaboration with Dr Kathryn Lilley.
Proteomic Complex Detection using Sedimentation ProCoDeS:
Quantitative proteomics to detect endogenous protein complexes
(Hartman et al., 2007;
Segura at el., 2010).
This technique was developed in collaboration with Dr Kathryn Lilley.
Oligosaccharide relative Quantitation Using Isotope Tagging OliQuiT:
Relative quantification of oligosaccharides using normal-phase liquid chromatography/mass spectrometry
(Ridlova et al., 2008).
This technique was developed in collaboration with Dr. Elaine Stephens.
Polysaccharide Analysis by Carbohydrate gel Electrophoreses PACE:
Quantitative method to characterize enzymatically digested polysaccharides
(Goubet et al., 2003).
This method is currently established to become a high-throughput technique.