The lignin polymer can be composed of different ratios of S, G, and H, and can tolerate incorporation of other phenolic components. For example, in interfascicular fibers of Arabidopsis stems, lignin has a high proportion of G monolignols; however, in vascular bundles of Arabidopsis stems, lignin is primarily composed of S monolignols (Chapple et al. 1992). It has also been shown that lignin can comprise about 90 percent of the benzodioxane units in transgenic Arabidopsis with up-regulated Ferulate 5-Hydroxylase (F5H) and downregulated COMT (Vanholme et al. 2010; Weng et al. 2010). Notably, transgenic Arabidopsis has a dwarf stature but still produces viable seeds (Vanholme et al. 2010; Weng et al. 2010), echoing the flexibility of lignin polymers. Lignin polymers with different compositions often have different strengths of chemical bonds, which impacts lignin digestion and degradation. The frequency of resistant bonds (or condensed bonds) in lignin can be detected by the monolignol yield in thioacidolysis, such that a higher frequency of resistant bonds results in a lower thioacidolysis yield (Berthet et al. 2011). S-enriched lignin is thought to have fewer crosslinked bonds than G-enriched lignin, and thereby an increase in the S/G ratio could lead to easier lignin digestion and degradation (Abramson et al.

2010) . For example, transgenic Arabidopsis with S-enriched lignin has a higher enzymatic hydrolysis efficiency than wild type plants after hot-water pretreatment (Li et al. 2010). Although the correlation between the S/G ratio and the enzymatic hydrolysis efficiency has not yet been universally recognized (Chen and Dixon 2007), the hypothesis and current experimental results suggest that altering lignin composition may decrease the strength of lignin bonds, which would facilitate enzymatic hydrolysis of plant cell walls.

Some transcriptional factors directly regulating monolignol biosynthetic genes have been identified (Zhou et al. 2009; Zhao et al. 2010a, b; Ambavaram et al. 2011). In Arabidopsis, MYB Domain Protein 58 (MYB58) directly regulates expression of genes involved in monolignol biosynthesis, except for F5H; and the expression of MYB58 is regulated by a "master regulator of genes" for secondary cell wall formation — NST1 /NST2 / MYB46/VND6/VND7 (NST stands for NAC Secondary Wall Thickening Promoting Factor, VND for Vascular-related NAC-domain Protein) (Zhou et al. 2009). Interestingly, F5H is directly regulated by NST1 and Secondary Wall-associated NAC Domain Protein 1 (SND1) (Zhao et al. 2010a). On the other hand, since secondary cell wall structure is hallmarked by not only lignification but also higher cellulose and hemicellulose contents, other transcriptional factors downstream of NST1 /SND1 /VND6/VND7 should be involved in the activation of cellulose and hemicellulose biosynthesis. Recently, an Arabidopsis gene, SHN, when overexpressed in rice, caused increased cellulose but decreased lignin content by directly binding to promoters of rice MYB58/63, NST1 /2, SND1, VND4/5/6, and MYB20/43 to downregulate genes involved in monolignol biosynthesis and to upregulate genes in cellulose biosynthesis (Ambavaram et al. 2011).

Most monolignol biosynthetic genes (except F5H) have AC elements (ACCT/AAC/AC) in their promoter regions (Raes et al. 2003). The AC cis-element can be bound by some MYB proteins, such as transactivators MYB58/63/85, and transrepressors MYB4/32 (Goicoechea et al. 2005; Zhou et al. 2009; Zhao and Dixon 2011). Many MYB transcription factors are regulated by environmental cues and by plant hormones, which at least partially explains why cell wall lignification is largely influenced by plant growth conditions (see review by Zhao and Dixon 2011). Understanding the functions of these transcriptional factors may assist in engineering low lignin content switchgrass independent of field conditions.

Monolignols are synthesized inside the cytoplasm and then transported across the cell membrane to the cell wall where they are oxidized and polymerized into lignin polymers (Miao and Liu 2010). Monolignol transportation is mediated by ATP-dependent ATP-binding cassette-like transporters. The transporters in the plasma membrane preferentially transport monolignol aglycones, whereas transporters in the vacuolar membrane prefer gluco-conjugated monolignols for vacuole storage (Miao and Liu 2010). Genes encoding proteins for these transporters have not yet been identified and it is unclear whether different transporters have preferences for different monolignols. Nonetheless, it is possible to reduce lignin content or alter lignin composition by engineering these transporters in the future.

Laccases and guaiacol peroxidases (class III peroxidases) have been proposed to oxidize monolignols to form lignin polymers. In Arabidopsis, 73 peroxidases and 17 laccase-like genes have been identified (Berthet et al.

2011) . The high number of guaiacol peroxidase genes potentially involved in the oxidization of lignin polymers makes it difficult to assign a specific function to each gene (Mathe et al. 2010). Certain laccase genes are expressed exclusively in lignifying cells (Boerjan et al. 2003). Recently, a study showed that Laccase 4 (LAC4) and LAC17 are involved in the lignification of stems because the lac17 single mutant has a reduced G lignin deposition and the lac4/17 double mutant has approximately 40 percent less overall lignin content (Berthet et al. 2011). Another laccase gene, LAC15, is specifically involved in oxidative polymerization of flavonoids and monolignols in Arabidopsis seed coats (Liang et al. 2006). Notably, MYB58 and MYB63 can directly transactivate the expression of the LAC4 gene (Zhou et al. 2009). Interestingly, the double mutant lac4/17 has semi-dwarfed peduncles under long-day conditions but retains normal plant size under continuous light; however, lignin content is consistently reduced under both conditions (Berthet et al. 2011). This result further suggests that many genes involved in lignin synthesis are affected by environmental cues; however, reducing lignin content is not necessarily associated with reduced biomass yield.

Strategies for Promoting Switchgrass Vegetative Growth