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- Materials and Methods
- Supporting Information
Globally, ethanol is the most widely produced liquid biofuel. Currently, more than 10 billion gallons of ethanol are produced per year from starch [from maize (Zea mays)] or sugar [from sugarcane (Saccharum species) and sugar beet (Beta vulgaris)] through mature industrial procedures for the hydrolysis of starch and fermentation of sugar (Goldemberg, 2007; Rass-Hansen et al., 2007). Maize grain and other cereals, such as sorghum (Sorghum bicolor), are the primary feedstocks for US ethanol production. However, the use of these as feedstocks for bioethanol has been criticized as diverting material away from the human food chain, with resulting food shortages and price rises (Runge & Senauer, 2007).
An alternative to starch or sugar ethanol (classified as first-generation biofuels) is ethanol produced from lignocellulosic biomass via saccharification and fermentation (a second-generation biofuel) (Smeets & Faaij, 2007; Schmer et al., 2008; Yuan et al., 2008). Dedicated perennial energy crops, such as switchgrass (Panicum virgatum), crop residues and forestry biomass are major cellulosic ethanol sources that could potentially displace 30% of our current petroleum consumption (Schmer et al., 2008).
Switchgrass has been proposed as a major perennial bioenergy feedstock in the USA because it is widely adapted, has high biomass production, high C-4 photosynthetic efficiency and efficient use of water and nitrogen (Yuan et al., 2008). Switchgrass grown and managed as a bioenergy crop produces five times more renewable energy than the energy consumed in its production as a source of bioethanol, and has significant environmental benefits related to soil conservation and low net greenhouse gas emissions (Schmer et al., 2008). Although higher energy ratios would be obtainable from switchgrass through direct combustion than through saccharification and fermentation to ethanol (Powlson et al., 2005), most research efforts in the USA are directed towards the improvement of the efficiency of switchgrass processing for the generation of liquid transportation fuels.
The most critical biological obstacle to be overcome in the transition from starch-based to lignocellulosic biofuels is the recalcitrance of cell walls to chemical and/or enzymatic breakdown. The processing of lignocellulosic biomass requires pretreatment (usually with hot acid), saccharification (hydrolysis) and fermentation (Ragauskas et al., 2006). Reducing the recalcitrance of lignocellulosic biomass could eliminate or reduce pretreatment, reduce cellulase loading and, ultimately, facilitate consolidated bio-processing. These improvements would reduce production costs and increase the economic competitiveness of lignocellulosic bioethanol (Lynd et al., 2008).
Lignin is probably the major factor affecting the recalcitrance of lignocelluloses (Yuan et al., 2008). It is an aromatic polymer that fills the spaces in the secondary cell wall between the cellulose, hemicellulose and pectin components, especially in tracheids, sclereids and xylem. It is covalently linked to hemicellulose and thereby cross-links plant polysaccharides, conferring mechanical strength and hydrophobicity to the cell wall. Several studies have demonstrated the link between reduced lignin levels and decreased recalcitrance with improved saccharification efficiency (Grabber, 2005; Davison et al., 2006; Ralph et al., 2006; van der Rest et al., 2006; Talukder, 2006; Chen & Dixon, 2007; Jackson et al., 2008).
Lignin content and composition have been manipulated transgenically in a variety of plant species, mainly by targeting the monolignol biosynthesis pathway (Fig. S1, see Supporting Information) (Hoffmann et al., 2004; Chen et al., 2006; Ralph et al., 2006; Millar et al., 2007; Shadle et al., 2007; Wagner et al., 2007; Jackson et al., 2008). In the above studies, reduced lignin content and/or altered lignin composition were achieved by the down-regulation of the individual genes encoding phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-hydroxycinnamate:CoA ligase, hydroxycinnamoyl CoA shikimate hydroxycinnamoyl transferase (HCT), p-coumaroylshikimate 3-hydroxylase, caffeoyl CoA 3-O-methyltransferase, caffeic acid/5-hydroxyferulic acid 3-O-methyltransferase, cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) (Fig. S1). However, when lignin levels become too low, plant growth and development can be affected, and this has been attributed to either the dysfunction of the vascular system or altered hormone transport (van der Rest et al., 2006; Besseau et al., 2007; Do et al., 2007; Leple et al., 2007; Shadle et al., 2007; Mir Derikvand et al., 2008).
In general, the down-regulation of the later enzymes in monolignol biosynthesis has less effect on plant growth and yield than the down-regulation of the earlier enzymes. This may result from the avoidance of pleiotropic growth effects as a result of perturbations in metabolites that feed into other pathways. CCR and CAD catalyze steps devoted specifically to monolignol biosynthesis, and could therefore be good targets for down-regulation in bioenergy crops to improve saccharification efficiency without compromising biomass yields. The reduction in lignin content through the down-regulation of CCR has been observed in tobacco (Dauwe et al., 2007), tomato (van der Rest et al., 2006), poplar (Leple et al., 2007) and other species. In a recent study (Jackson et al., 2008), CCR and CAD were down-regulated individually in alfalfa; a comparison between lines with similar reductions in lignin level revealed greater saccharification efficiency in the case of the CCR-down-regulated lines.
It is important to determine whether the improvements in saccharification efficiency recorded for dicot species, such as alfalfa, can be reproduced through targeting the monolignol pathway in switchgrass. However, little is known at present concerning the genes and corresponding enzymes involved in monolignol biosynthesis in this species. Here, we identify four cDNAs similar to CCRs in the tetraploid lowland switchgrass variety Alamo, and show that two of them (PvCCR1 and PvCCR2) encode enzymes with CCR activity. A combination of biochemical and gene expression analyses indicate that PvCCR1 is the enzyme involved in lignification, and therefore a target for down-regulation to improve switchgrass as a bioenergy crop. PvCCR2 appears to be involved in plant defense. We also describe potential allelic variants of switchgrass CCRs.