Plant Cell walls as renewable resource for Biofuels and other commodity chemicals

With an estimated annual production of 180 billion tons per year plant cell walls (lignocellulosics) present an enormous renewable resource, of which only 2% are utilized by humans. The major bottleneck in utilizing this carbon neutral resource for the sustainable production of second generation biofuels is the recalcitrance of the wall material to degradation. With our various research projects we aim at characterizing the structural features of the wall that lead to this recalcitrance. In the long run such knowledge will aid us in overcoming them.

The structure of the Plant Cell Wall

Higher plant cells are encased in cell walls that define their shape and contribute to the strength and structural integrity not only of individual cells, but also of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active. It plays crucial roles in diverse cell activities such as growth, differentiation, cell-to-cell communication and transport, senescence, abscission, and plant-pathogen interactions. Microcrystalline cellulose is embedded in a hydrated matrix consisting of coextensive networks of complex heteropolysaccharides and sometimes glycoproteins and polyphenols, such as lignin.

Our research entails the establishment of an analytical platform for the analysis of wall polysaccharides, a forward genetic approach to identify novel wall structures and reverse genetic approaches to gain insights into wall biosynthesis.

Analytical Platform for the Microanalysis of Wall Polysaccharides

Our lab uses mainly classical carbohydrate chemistry based methods to describe the structure of particular wall polysaccharides. These methods encompass solubilization of various wall polymers using sequential extraction procedures that make use of wall degrading enzymes and chemicals. The resulting fractions are then analysed using techniques such as monosaccharide composition and glycosidic linkage analysis see videos (Lignin analysis: http://www.jove.com/details.php?id=1745; Carbohydrate analysis http://www.jove.com/details.php?id=1837). We also determine the presence of ester substituents (such as O-acetyl-substituents). Since such an analysis is rather labor-intensive and time consuming, an oligosaccharide mass profiling method (OLIMP) using specific polysaccharide hydrolases in combination with mass spectrometry has been developed (Video: http://www.jove.com/details.php?id=2046). The sensitivity of OLIMP allows for the rapid assessment of even minute amount of tissue-materials. A profile can be obtained from preparations of as little as 500 Arabidopsis cells prepared by a laser-dissection catapulting instrument.

 

Forward Genetic Approach: Identification of Structural Wall Mutants

We identify wall mutants by screening of populations of chemically mutagenised seeds for novel structural wall mutants.

An Arabidopsis mutants population was screened using OLIMP. It has lead to the identification of 36 distinct mutants with altered xyloglucan structures including the abundance of ester-substituents. Map-based cloning of the mutated genes gave valuable insights into biosynthesis, metabolism and function of structural variations of xyloglucan. For example, it became evident that the microheterogeneity of wall polysaccharides is dependent not on its biosynthetic machinery but on apoplastic "trimming" of the sidechains by glycosidases.

A maize mutant population was screened for altered sugar content. Indeed, one of the mutants, termed "candy-leaf 1" or Cal-1, exhibited a 250% increase in glucan content. Cal-1 is currently tested in the field for enhanced yields of biofuel production.

Massive parallel sequencing: Cell Wall Biosynthesis

Although information about the structural components of cell walls has increased considerably in recent years, very little is known about the biosynthesis of individual wall components on a molecular level. We employed Ilumina sequencing of plant species that synthesize only a single polysaccharide. This approach lead to the identification of genes in whole biosynthetic pathways. A reverse genetic approach is then employed with the correspondent Arabidopsis genes/mutants to ascertain the function of the gene and its role in polysaccharide biosynthesis. Currently, numerous novel genes involved in the synthesis of nucleotide sugars, the substrates for polysaccharide synthesis, have been identified through bioinformatic means by comparison to gene-sequences of well-characterized bacterial enzymes. In addition, we have identified genes that are responisble for polysaccharide O-acetylation. This substitution is an impediment to enzymatic degradation in a biorefinery and once released presents an inhibitor to many fermenting organisms.

The overall goal of these projects is to be able to assemble and reconstruct whole plant polysaccharide pathways in yeast that would produce the particular hemicellulose.

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