Modification of Cellulose Content via Cellulose Genes 631 Cell Wall Formation and Cellulose Synthesis

The biopolymer cellulose is the most abundant naturally occurring organic substance, being found as the principal component of cell walls in higher plants where it provides the main structural feature. Cellulose occurs in almost pure form in cotton (98%), and in lower percentages in flax (80%) and wood (40-50%). It is very insoluble, but drastic chemical disintegration reveals that cellulose is a long chain polymer, made up of repeating units of glucose, a simple sugar. Light scattering methods reveal that the length of cellulose chains range from 2000 glucose residues in cotton up to 14,000 in secondary walls of wood, and there is no branching (Haigler and Brown1985).

The special properties of cellulose result from the association of the long chains to form fibres called microfibrils (Brown 1996). The microfibrils associate to form larger fibrils or fibres, which in the secondary walls are then laid down in a criss-cross pattern (http://www-biol.paisley.ac.uk/courses/ stfun-mac/glossary/cellulose.html). The number of these glucan chains in each bundle varies from 36 in the elementary fibrils to more than 1200 in some algal species (Brown et al. 1996). In tree species the content of cellulose varies in the primary and secondary cell wall types. The elastic and expanding primary wall, containing largely pectins but also 2-15% cellulose and hemicelluloses, is 0.1-0.2 ^m thick and cellulose microfibrils are irregularly oriented. In contrast, the secondary wall has orderly oriented cellulose microfibrils, is rich in cellulose (50-75%), and also contains lignin (20-30%) and hemicellulose (10-15%). It consists of three layers, namely S1, S2 and S3. While the outer and inner layers (S1 and S3, respectively) are relatively thin (less than 0.3 ^m), the middle S2 layer is up to 5 ^m thick.

Until 1996, when the first report on the identification of bacterial cellulose synthase gene homologs in cotton was published (Pear et al. 1996), only little information was available on molecular aspects of cellulose biosynthesis in plants. Later, it was found that a large multigene family of encoding cellulose synthase (ces) genes exists in Arabidopsis and other plants (Joshi 2004). This fact has initially lowered the inspiration to modify cellulose content or quality via a genetic engineering approach.

Specific plant cellulose synthases (CesA) are necessary for secondary wall synthesis in vascular tissues, which is critical to wood production. In Arabidopsis a family of at least ten CesA isoforms exists which by mutant analyses have been shown to play distinct role/s in the cellulose synthesis or cell wall composition (Delmer 1999; Richmond and Somerville 2000; Joshi 2003; Joshi et al. 2004). The CesA mutants work indicates that three different CesA proteins interact as subunits within a cellulose synthase complex: CesA1, CesA3, and CesA6 form the complex in primary cell wall biosynthesis, whereas CesA4, CesA7, and CesA8 form the complex in secondary cell walls (Eckard 2003). Taylor et al. (2003) have shown that some CesA proteins interact with each other in the formation of cellulose synthesizing complexes which are located in the plasma membrane (Joshi 2004). The different cellulose content between primary and secondary cell walls may be attributed to different functional properties of individual CesA subunits. There is no evidence that other proteins interact directly with CesA subunits within the CesA complex, this though remains a possibility (Eckard 2003).

By direct screening of the monosaccharide composition of total cell wall hydrolysates, a number of different Arabidopsis mutants were isolated which show defects in cell wall composition (Reiter et al. 1997). These mutants, however, did not reveal significant changes in cellulose content. Other Arabidopsis mutants called irregular xylem (irx) are defective in secondary cell wall deposition (Turner and Somerville 1997). Examination of the cell walls of these mutants by using electron microscopy showed that cellulose content is decreased which resulted in an alteration of the spatial organization of cell wall material.

While immense progress has been made in the analyses of Arabidopsis cellulose biosynthesis pathway, it is, nevertheless, important to understand cellulose biosynthesis in trees with special emphasis to wood formation. Significant progress in cloning and isolation of CesA genes in a tree species has been obtained in the tree model Populus. A number of different CesA cDNAs have been isolated which show a very high similarity to respective Arabidopsis CesA genes (Wu et al. 2000; Joshi et al. 2004). Three of these Populus genes (PtrCesAl, PtrCesA2, PtrCesA3) are thought to be related to secondary cell wall CesAs mainly based on high amino acid similarity to the Arabidopsis secondary cell wall genes (Joshi et al. 2004).

To study whether cellulose content can be increased in plants by genetic engineering, it is first worthwhile to express the three CesA genes that make secondary cell wall cellulose in cells that do not normally have secondary wall thickening (Somerville, online 2004; Somerville et al. 2004). However, because it is likely to be deleterious to induce extra cellulose synthesis in cells that need to divide and expand to support normal growth and development, the genes must be placed under transcriptional control of a promoter that is active at a time that is compatible with normal development. This will allow production and propagation of the transgenic plants and will also facilitate studies of the consequences of induced expression of the CesA genes at specific time and places and to differencing degrees. The transgenic plants containing the ectopic CesA genes can be analysed for cell wall composition (e.g., cellulose and other polymers) and for effects on growth and development. If increased cellulose is obtained from chemical induction of the genes, the next step will be to test the feasibility of engineering enhanced cellulose under the control of developmental stage-specific promoters.

6.3.2 Cellulose Degradation

Constitutive expression of poplar cellulase in Arabidopsis revealed severe effects on plant growth (Park et al. 2003). Cellulase over-expressing transgenic plants showed increased size of the cellulase synthase complex which is due to both larger leaf blades and petioles. This result is in agreement with the observation that suppression of cellulases achieved by either expression of the antisense gene or co-suppression of Cellulase mRNA by overexpression of the sense gene resulted in reduced leaf growth (Ohmiya et al. 2003).

Determination of the sizes of parenchyma cells (palisade and epidermal) in leaves of cellulase-over-expressing and control plants revealed that these cells were larger in leaves of the transgenic plants (Park et al. 2003). These changes were accompanied with changes in mechanical properties. Chemical analyses of cellulose composition in transgenic plants did not show significant differences to wildtype - only the amount of xyloglucan present in the 4% KOH-insoluble fraction was slightly higher (Park et al. 2003). Studies on microfibril structure using nuclear magnetic resonance (NMR) revealed that the transgenic plants have an increased proportion of trans-gauche conformation (one of the three preferred ones) at the C6 carbon of 1,4-P-glucan. The consequence of this modification is that the transgenic plants have a greater proportion of crystalline cellulose.

Similarly to the compensatory relationship of "reducing lignin leads to increase of cellulose", the reversal statements also seems be true. Tension wood is formed in response to mechanical stress or gravitational stimuli on the upper side of non-vertically growing stems. A widely known feature during the formation of tension wood is the increase of cellulose associated with reduced lignin content (Wu et al. 2000). The increased cellulose formation is caused by the unique role of cellulose synthase A in cellulose biosynthesis (Wu et al. 2000). This relationship clearly indicates the existence of complex but well-defined signalling mechanisms which regulates both cellulose and lignin biosynthesis.

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