Lignin is a complex aromatic heteropolymer deposited within the SCWs of all vas­cular plants, and accounts for approximately 30% of the terrestrial organic carbon fixed annually in the biosphere, placing it second to cellulose as the most abundant biopolymer on earth [12]. Lignification aids the plant by providing added strength to xylem fibers that give support for upright growth, by waterproofing tracheary elements that make up the vascular system and by helping increase the resistance of plants to pathogen attack [12] (Fig. 1). Lignin content can vary with environmental factors, but in general comprises around 13-19% of the biomass in switchgrass (Panicum virgatum) [108, 173], 22-25% in Miscanthus (Miscanthusxgiganteus) [18, 169], and around 20% in big bluestem (Andropogon gerardii) and eastern gamagrass (Tripsacum dactyloides) [173], all of which are C4 grass species that have potential as bioenergy feedstocks. In addition, lignin accounts for approxi­mately 25-30% of the dry weight of potential hardwood bioenergy tree crops like poplar and can be even higher in softwood species [134]. The prominence of lignin in a majority of plant tissues has been recognized by reference to the nonstarch or nonsugar components of the plant body as simply “lignocellulosic” biomass. Traditional research attention was given to lignin with respect to chemical pulping and forage digestibility, but recently interest has intensified concerning conversion processes to biofuels and biochemicals. Much of the focus has centered on the fact that the cellulose microfibrils of the SCWs are embedded in a meshwork of HCs and lignin that create a barrier for cellulase enzymes and decrease saccharification efficiency. However, from a thermochemical conversion prospective, the associa­tion of lignin with cellulose is not a major issue and more important is the fact that lignin contains structural units that are more chemically reduced and energy dense than any of the cell wall carbohydrates and thus could serve as a source of hydrocar­bon fuels and high-value chemicals, if means can be found to free those structural units from the polymer.

The ultimate source of lignin in the plant is the amino acid phenylalanine (Phe) which is derived from the shikimate biosynthesis pathway in the plastid ] 154]. Current evidence suggests that through the general phenylpropanoid and monolignol — specific pathways located on or near the cytosolic side of the ER membrane, Phe is deaminated to form cinnamic acid, followed by a series of ring hydroxylations,

O-methylations and side-chain modifications culminating in the production of the p-hydroxycinnamyl alcohol monomers (monolignols) coniferyl and sinapyl alcohol, and to a lesser extent p-coumaryl alcohol [105]. Upon incorporation into the lignin polymer, these monomers are referred to as guaiacyl (G), syringyl (S), or p-hydroxy — phenyl (H) units, respectively ] 149] , In general, angiosperm dicot lignins are composed of G — and S-units, while gymnosperms, with a few notable exceptions, are composed almost entirely of G-units with minor amounts of H-units [188]. Most, if not all, of the enzymes required for monolignol biosynthesis are known and include: phenylalanine ammonia lyase (PAL), the three ER membrane bound cytochrome P450 monooxygenases cinnamate 4 hydoxylase (C4H), coumarate 3-hydroxylase (C3’H) and ferulate 5-hydroxylase (F5H), the two methyltransferases caffeoyl-CoA

3- O-methyltransferase (CCoAOMT) and caffeic acid/5-hydroxyferulic acid

O-methyltransferase (COMT), the two oxidoreductases cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol reductase (CAD) as well as two enzymes 4-coumarate — CoA ligase (4CL) and shikimate hydroxycinnamoyl transferase (HCT) that are involved in the generation of pathway intermediates [37, 38, 70, 93, 109, 144, 194].

Although it is uncertain how the newly synthesized monolignols are translocated to the apoplast (cell wall), once there most evidence suggests that the single electron oxidation of the monolignol phenol by wall-bound peroxidases and/or laccases followed by combinatorial radical coupling commences formal lignin polymerization [12]. Presumably, the coupling of two monolignols with one another initiates polymer­ization. Most likely due to the lack of steric hindrance, coupling between monoli — gnols is favored at the central b carbon of their side chain, resulting in the most common b-b dimer, however b-O-4 and b-5 linked dimers can and do occur [188]. In order for polymerization to continue, the lignin dimer must be dehydrogenated once more to a phenolic radical before it can couple with the next monomer radical. Bond formation is again favored at the central b carbon of the monolignol side chain and depending on the bond configuration and the subunit composition of the dimer, the end-wise coupling process can produce either more b-O-4, when a mono­mer adds to S — and G-units (most common), or b-5 bonds that occur only when adding to G-units [13]. If only the three previously mentioned bonds contributed to lignin polymerization, then the lignin polymer would form a relatively straight lin­ear chain. However, two oligolignol radicals with G-unit ends can also react to form

4- O-5 or 5-5 couplings that generate a branch-like quality to the polymer structure. In fact, lignin containing a high proportion of G-units is more highly cross-linked than lignin rich in S-units, which may contribute to the more rigid and hydrophobic character of G-unit lignin [13]. Therefore, the relative proportion of a given lignin monomer dictates the relative abundance of the inter-unit linkage present in the lignin polymer. Interestingly, the b-O-4 linkage is not only the most common linkage found in plants [62], but it is also the easiest of all the linkages to chemically cleave and increasing this linkage could potentially enhance the efficiency of conversion processes [75]. It should also be noted that an alternative hypothesis with regards to lignin polymerization suggests that lignin monomers are coupled with absolute structural control by proteins in the cell wall bearing arrays of dirigent sites, how­ever there has been no genetic data yet produced to support this claim [ 188] . Additional evidence supporting the predominant radical coupling model of lignification has shown that all phenolic compounds, monolignols or otherwise, that enter into the region of the cell wall where oxidation and radical coupling occur have the potential to be radicalized and incorporated into the lignin polymer, sug­gesting a very flexible process not likely mediated by ligand-specific enzymes [188].

This phenomenon may also allow for a strategy of designing lignins for industrial applications, specifically by regulating the influx and species of monolignol or other phenolic compound into the cell wall [68]. And finally, regarding the global control of lignification, several transcription factors belonging to the MYB and NAC gene families, similar to those responsible for SCW biogenesis, have been shown to play a key role in regulating the expression of many of the genes in the monolignol biosynthesis pathway [204, 211].