Lignocellulosic biofuels has received much attention as a replacement for US reliance on foreign petroleum fuels. Energy demands of the 21st century, increasing oil prices and environmental concerns prompted the US Congress to enact the Energy Independence and Security Act (EISA), which requires the use of lignocellulosic biofuels from 2010 and to achieve the goal of producing lignocellulosic biofuels up to 16 billion gallons per year by 2022 under baseline assumptions .
Lignocellulosic biomass has long been heralded as the prospective low cost substitute for the production of biofuels by fermentation of sugars. Major sources of lignocellulosic biomass are dedicated biofuel crops such as switchgrass, Miscanthus, and poplar, forest biomass and different types of waste products and residuals from crops, wood processing, which could provide enough biomass to replace nearly 30% of petroleum use . Lignocellulosic biomass has considerable environmental and resource advantages over non-renewable fossil fuels. For example, a five year field study estimates that lignocellulosic biofuels derived from switchgrass produce significantly lower greenhouse gases than fossil fuels. Switchgrass grown as lignocellulosic biofuel crop (Figure 1) also produced a greater amount of replenishable energy, considerably more than the energy needed to produce it .
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The carbohydrate-rich plant cell walls store the major energy in plant biomass . Compilation of the biomass is the first step in lignocellulosic biofuel production followed by pretreatment to loosen/breakdown the cell wall, its saccharification into sugars and fermentation of these sugars to biofuels . Though lignocellulosic biofuels assure energy savings and reduction in greenhouse gases, they are not as yet considered economically viable due to the high costs involved in pretreatment, saccharification and fermentation [3,5]. Biotechnology offers the promise of dramatically increasing ethanol production from lignocellulosic biomass by developing crop varieties with reduced lignin content.
What is lignin? Lignins, celluloses and hemicelluloses are the major constituents of lignocelluloses . Lignins are the second most abundant polymers, produced during the secondary cell wall synthesis of all vascular plants and account for around quarter percent of plant biomass . They function mainly as inter- and intramolecular adhesive, providing rigidity to the embedded plant cell, thus, giving structure and strength to the cell wall and the plant . Their hydrophobic nature makes the cell walls impermeable to water [8,9]. They also protect the plant by resisting microbe and pathogen entry, thereby curbing infections and degradation, and can be synthesized in response to biotic and abiotic stress . They are formed via oxidative coupling of dihydroxycinnamyl alcohols (or monolignols), hydroxycinnamaldehydes (coniferaldehyde and sinapaldehyde), ferulates and acylated monolignols [11-14]. They are synthesized from p-coumaryl, coniferyl and sinapyl alcohols which produce p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. The relative quantity of each lignin component differs with the species, part and maturity of a plant . G and S units are the main components of the dicot lignin and a combination of G, S, and H units form the monocot lignin, while only G-units form the gymnosperm lignin .
The adaptation of vascular plants to inhabit the terrestrial environments was made possible by the evolution of lignin (Figure 2) as it provided structural support required for an erect growth habit, supplied water and protection from dehydration and desiccation [16-18]. The detection of lignin in Marchantia polymorpha, a bryophyte, spreads its distribution in non-vascular plants . Initially, lignin was believed to be absent in green algae. Gene network studies have shown the earliest appearance of lignin biosynthetic pathway in moss Physcomitrella , however, studies by Martone et al.  have identified the existence of secondary walls and lignin in marine red alga Calliarthron cheilosporioides. The relevant pathways may have advanced more than one billion years ago prior to the divergence of red and green algae or have evolved convergently in C. cheilosporioides and land plants [21,22]. The identification of S-lignin, which is thought to be distinctive to angiosperms, in some lycophytes (Selaginella), ferns (Dennstaedtia bipinnata) and gymnosperms (Podocarpus macrophyllus, Tetraclinis articulate) recommended its creation several times in diverse ancestry of tracheophytes [19,21,23-26. In Selaginella, the detection of a novel ferulate 5-hydroxylase (F5H), which plays a role in biosynthesis of S-lignin even though it is structurally distinct from the F5H, revealed its co-evolution [18,27]. Another interesting observation has been made in Ginkgo biloba whose cell suspension cultures have the ability to synthesize S-lignin while its woody tissues are not able to synthesize it . Though lignin has been studied for few decades now, many phases of its biosynthesis, especially how and when phenylpropanoid metabolism in general and lignifications in particular arose and evolved, still remain unanswered even though many advances have been made in biochemical studies [15,18,20,29].
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Biosynthesis of monolignols followed by their polymerization are the two main steps in lignin biosynthesis (Figure 3). Though the biochemical pathways of monolignol biosynthesis are highly conserved throughout vascular plants, over the past two decades, it has undergone major revisions and the understanding of the primary intermediates in the lignin biosynthesis has evolved. The deamination of phenylalanine to produce cinnamic acid in the presence of the catalyst phenylalanine ammonia lyase (PAL) followed by the conversion of cinnamic acid to p-coumaric acid by cinnamate 4-hydroxylase (C4H) are the initial steps in the monolignol biosynthesis pathway [20,30,31]. Earlier researchers contemplated that hydrolation and methylation reactions took place at the cinnamic acid stage and the sequential action of 4-coumarate:CoA ligase (4CL), cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) led to the conversion of p-coumaric acid, ferulic acid and sinapic acid to the corresponding monolignols (.
Characterization of most of the enzymes in the monolignol biosynthesis pathway along with the identification of various intermediates and precursors has led to the discovery of various pathways for further hydrolation and methylation steps [31,32]. Most of the enzymes in the monolignol biosynthesis pathway have been identified and characterized. The downregulation of PAL, C4H, 4CL, p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyltrasnferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), CCR, caffeic acid-O-methyltransferase (COMT), F5H, sinapyl alcohol dehydrogenase (SAD) and CAD, also affect lignin biosynthesis and content[15,32-35]. The quantity of various monolignols produced in the earlier steps of lignin biosynthesis and the enzymes catalyzing these steps determine its structure [15,17].
Why lignin needs to be manipulated? Though plant biomass is the largest source of cellulose, plant cell walls have evolved to be recalcitrant to degradation and crystalline cellulose, embedded in a complex matrix of lignin and hemicelluloses, is resistant to hydrolysis. Lignin provides the resistance to degradation by shielding the cell wall from microbial decomposition [18,36-38]. Thus, lignin has received significant consideration due to its complex nature and difficulty in degrading it. This negatively impacts the production of biofuels by reducing the conversion efficiency of lignocellulosic biomass to fermentable sugars [18,39-41]. Besides total lignin content, the composition of lignin is also an important factor in the recalcitrance of cellulosic biomass. G-lignin is more resistant to chemical degradation, implying that modification of S/G ratio is also important for improving the digestibility of cellulosic biomass . Lignin will not be of concern if thermal process is used to convert biomass to fuels, but the presence of lignin hinders the access of hydrolytic enzymes to the cellulose polymer and, thus, ethanol production is directly related to the recalcitrance of the cell wall . Lignin can adsorb hydrolytic enzymes and prevent it from breaking down cellulose into monosaccharides, while lignin degradation products inhibit the activity of the enzymes for fermentation of monosaccharides . Derivatives of lignin are also toxic to the microorganisms . The lignin in biomass has to be either removed by pretreatment or decreased by genetic manipulation of genes in the lignin pathway. The elimination of lignin from plant biomass is an expensive procedure.
To attain superior yields and quick hydrolysis of the carbohydrates to monomeric sugars, the size, structure and chemical composition of the biomass needs to be changed by pretreatment . Pretreatment methods like acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis are currently being used to breakdown the lignocellulosic matrix  and to remove lignin from the cellulosic biomass before it is used for fermentation into ethanol. The digestibility of the biomass is also distinguished by the lignin content, and accessibility of cellulose to cellulase . Hence, pretreatment is also viewed as an important bottleneck in the processing of lignocellulosic biomass. Different pretreatment methods have been suggested depending on the source of biomass [45,46] which contributes the most to cost of production of cellulosic biofuel . Pretreatment also has immense prospective for enhancing the efficiency and reducing the cost through research and improvement [45,48]. It is improbable that any one pretreatment method will develop into an alternative for all types of biomass, even though some methods have shown advantages over others . Therefore, reduction of lignin content in lignocellulosic biomass will decrease the cost of pretreatment. Additionally, the cost of production of dedicated crops is greater compared to straw and stover which have negative value as a byproduct. Unless the lignin in lignocellulosic biofuel crops is modified, the available cellulose for simultaneous saccharification and fermentation will be low, thereby, increasing the cost of production .
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How lignin can be manipulated? Genes encoding the enzymes leading to the formation of monolignols have been identified [15,50]. Nearly 10 enzymes are thought to be involved in the production of monolignols, and polymerization of monolignols is carried out by peroxidases and laccases . Lignin synthesis and deposition is regulated by myriad factors besides gene encoding the enzymes in the pathway and many factors are unknown. Genes encoding the enzymes in the lignin pathway are themselves regulated by a series of transcription factors during secondary cell wall formation . Each step in the pathway can, therefore, be manipulated along with the transcription factors to produce the cellulosic biomass with desired lignin content. The regulation of lignin biosynthesis in lignocellulosic biomass crops needs to be studied in depth. This will further assist in manipulating the lignin content and composition in lignocellulosic biomass crops . Further, as lignin content is directly proportional to recalcitrance , by manipulating lignin content through genetic engineering, the biomass pretreatment and processing costs can be reduced . Lignocellulosic crops genetically engineered for biofuel production will not require expensive pretreatments thereby further decreasing the production costs .
Manipulation of genes in the lignin pathway
Manipulation of genes early in the lignin pathway has a significant effect on total lignin and lignin composition by restricting the metabolite flux to lignin synthesis . A number of genes have been over-expressed or down-regulated in planta to see their effect on lignin biosynthesis (Table 1). Genetic engineering of H/G/S composition of lignin has also been attempted . Down-regulation of PAL, C4H has significant effect on plant growth and biomass besides lignin content and S/G ratio [51,54]. In transgenic tobacco, down-regulation of PAL and C4H led to a reduction in lignin content and an increase in the S/G ratio, while down-regulation of CAD decreased the S/G ratio [55,56]. The negligible presence of detectable vascular elements in the cross-sections of petiole demonstrated the compromised vascular integrity of the PAL-silenced transgenic tobacco plants . The down-regulation of C4H also resulted in a gradual and measurable decrease in lignin content, thus, proving to be a main rate-limiting step . Down-regulation of C4H in alfalfa led to a reduction in both the lignin content and the S/G ratio . The final step in the biosynthesis of monolignols is catalyzed by CAD whose down-regulation directs the assimilation of hydroxycinnamaldehydes [15,59]. The involvement of CAD in lignin biosynthesis has been studied in tobacco, alfalfa, poplar, eucalyptus and Arabidopsis [34,60-64]. The absence of CAD, in grasses, reduces the lignin content, modifies the structure of lignin and leads to an enhanced saccharification efficiency [65,66]. Down-regulation of CAD in switchgrass and maize led to a considerable decrease in lignin content [67,68]. In maize, it was also accompanied by modifications in the composition of the cell wall and decrease in the S/G ratio .
Though a substantial decrease in lignin content was observed on down-regulation of these genes, it was also accompanied by a severe decline in biomass making them inappropriate for genetic modification. 4CL takes part in the regulation of monolignol precursors thereby controlling the lignin content and composition . Down-regulation of 4CL in transgenic plants of tobacco, Arabidopsis, rice, Populus, Pinus, switchgrass and sorghum has led to a decrease in lignin content [41,69-74]. However, the effect on the composition of lignin differed probably due to diverse levels of down-regulation . In poplar, down-regulation of CCR resulted in increased digestabilty and doubling of cell wall sugar release by Clostridium cellulolyticum as compared to wild-type plants . The important aspect of down-regulation of COMT is the lack of developmental abnormalities in the plant . In transgenic maize and switchgrass, COMT down-regulation modified its lignin content and composition (Figure 4), and also enhanced digestibility [75,76]. These studies signify the importance of down-regulation of lignin genes for the genetic improvement of germplasm of lignocellulosic crops and will play a vital role in biofuel production (Figure 5).