Substantial Decrease In Lignin Content Biology Essay

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Cell wall digestibility is considered to be regulated with differences in G and S ratios where lignin abundant in G-unit has more crosslinked areas than lignin augmented with S-unit (Boerjan et al. 2003; Abramson et al. 2010). Since F5H is the chief enzyme in producing S-lignin, its over-expression led to an increase in S-lignin while its down-regulation resulted in lignin essentially composed of G-units (Stewart et al. 2009). S/G ratio could be enhanced by the down-regulation of HCT or C3H genes as they reduced the synthesis of G-lignin (Ralph et al. 2006; Wagner et al. 2007; Hoffmann et al. 2004; Coleman et al. 2008). On the other hand, enhanced S/G ratio could be obtained by COMT down-regulation as it leads to the assimilation of 5-hydroxyconiferyl alcohol (Vanholme et al. 2008; Fu et al. 2011). In transgenic alfalfa, down-regulation of C4H, HCT, C3H, CCoAOMT and COMT reduced the lignin content while down-regulation of F5H and CAD did not have any effect on the lignin content (Baucher et al. 1999; Guo et al. 2001a,b; Reddy et al. 2005; Shadle et al. 2007). Down-regulation of CCoAOMT and COMT, in Poplar, led to a reduction in lignin content while the down-regulation of CAD and up-regulation of F5H did not change the lignin content (Petit-Conil et al. 1999; Jouanin et al. 2000; Huntley et al. 2003; Lapierre et al. 2004). Transgenic tobacco lines independently modified for CCR or CAD were crossed and those hybrids that included only one allele of each antisense transgene displayed a normal phenotype with a remarkable decrease in lignin content (Chabannes et al. 2001). To understand the role of laccases in lignifications, Berthet et al. (2011) down-regulated LAC4 and LAC17 in Arabidopsis and observed that the resulting plants had lower levels of lignin leading to enhanced saccharification efficiency. They also observed that the deposition of G lignin could be influenced by disturbing LAC17 (Berthet et al. 2011).

Manipulation of transcription factors Genes in the lignin pathway are also regulated by R2R3-type transcription factors which have the MYB DNA binding domain (Stracke et al. 2001; Rogers and Campbell 2004). Manipulation of these transcription factors has significant effect on lignin content and composition. In transgenic tobacco, overexpression of Antirrhinum majus, AmMYB308 and AmMYB330 led to the down-regulation of 4CL, CAD and C4H (Tamagnone et al. 1998) while overexpression of Eucalyptus, EgMYB2 down-regulated the transcription of CCR and CAD and regulated both biosynthesis of lignin and secondary cell wall synthesis (Goicoechea et al. 2005; Legay et al., 2007). On the other hand, EgMYB1 was found to be a negative controller of CCR and CAD in the lignin pathway (Legay et al., 2007). Pinus taeda MYB4 (PtMYB4) was also overexpressed in transgenic tobacco and augmented the deposition of lignin by modifying the expression of lignin biosynthesis genes (Patzlaff et al. 2003).

In maize, ZmMYB31 and ZmMYB42 were identified as down regulators of COMT (Fornale et al. 2006; Sonbol et al. 2009). A.thaliana plants overexpressing ZmMYB42 had a reduced growth rate and decreased fresh weight (Fornale et al. 2006). Its overexpression also reduced the total lignin content, and modified the lignin composition by increasing the H-lignin and G-lignin while decreasing the S-lignin leading to the overall decrease in the S/G ratio (Sonbol et al. 2009). Transcription factors like SND1, SND2, NST1 and their homologs (Zhong et al. 2006, 2008; Hussey et al. 2011) act as master switches by regulating genes early in the lignin pathway and affect growth severely making them unsuitable for genetic manipulation of lignocellulosic biomass. The overexpression of PvMYB4 transcription factor, a lignin repressor which binds to the AC elements, in genetically modified switchgrass reduced its recalcitrance (Shen et al., 2012). Arabidopsis AtMYB4 and AtMYB32 also acted as repressors of the lignin biosynthesis pathway (Jin et al., 2000; Preston et al., 2004). The most desirable phenotype for lignocellulosic biomass has been demonstrated in transgenic rice by the overexpression of Arabidopsis transcription factor SHINE which increased the cellulose content, decreased the lignin content and changed the lignin composition without altering the biomass of the transformed plants (Ambavaram et al. 2011). Therefore, genetic manipulation of genes and transcription factors can be used to develop varieties with the desired phenotype for the production of cellulosic biomass.

Manipulation of regulatory factors and developmental genes that regulate lignin A role of microRNAs (miRNAs) in the developmental biology of plants has been established (Aukerman and Sakai 2003; Palatnik et al. 2003). miRNAs regulate transcription factors and may possibly enhance biomass yield, modify lignin structure and composition, reduce recalcitrance and other such characteristics important for biofuel production (Fu et al., 2011a; Zhang et al., 2006). Being less lignified, the immature plant material demonstrates variation in accumulation of biomass and may be able to decrease the recalcitrance (Chuck et al., 2011; Poethig, 1990). miRNA such as the one encoded by the maize Corngrass1 (Cg1) gene, that belong to the miR156 class, target the SQUAMOSA PROMOTER BINDING LIKE (SPL) family of transcription factors and support the development of juvenile morphology with reduced lignifications in the cell wall (Rhoades et al., 2002). This Cg1 gene has been constitutively expressed in poplar and switchgrass (Rubinelli et al. 2012; Chuck et al., 2011). In both cases, it modified the content and composition of lignin along and also had a severe effects on the plant structure. Similar results were also obtained when PvmiR156 was over-expressed by the introduction of the fragment of the OsmiR156b precursor in switchgrass (Fu et al., 2012).

Gibberellins have been associated with diverse growth and developmental processes ranging from seed development to flowering (Cowling and Harberd 1999). Overexpression of Gibberellin 20-oxidase (GA20ox) in tobacco resulted in an increase in biomass with higher levels of lignin (Biemelt et al. 2004). This increase in lignin might be the result of up-regulation of genes in the lignin pathway (Israelsson et al. 2003). To change the lignin content of the biomass, dwarfing might also be of use as it shifts the biomass allocation from the stem to the leaves (Gressel, 2008). Mutant GA20ox, responsible for the dwarf and high yielding varieties of food crops in the green revolution (Ashikari et al. 2002; Spielmeyer et al. 2002) did not have any pleiotropic defects other than semi-dwarf stature. Taken together down-regulation of GA20ox genes will decrease lignin along with moderate decrease in biomass. Similarly, repressors of gibberellin synthesis can also be manipulated to modify lignin with slight reduction in biomass (Zhao et al. 2010). Overexpression or down-regulation of homeobox genes like ARK1 affect lignin content but these genes are important for the development of the plant and have deleterious effect on the survival of the plant and are therefore not suitable for genetic manipulation.

Consequences of lignin modification The consequences of the lignin reduction or modification are dependent on which gene in the lignin biosynthesis pathway has been manipulated. A decrease in the amount of lignin or alteration of the lignin configuration will significantly increase the accessibility and digestion of the cell-wall carbohydrates, cellulose and hemicelluloses during fermentation leading to more proficient production of biofuels (Casler, 2012; Vogel and Jung, 2001; Bouton, 2008). Reduction in lignin also reduces the severity of the pretreatment and enzyme requirements, and increase the energy that is available to microorganisms that conduct fermentation eventually leading to significant reduction in the costs of biofuel production (Fu et al., 2011). The reduction of CAD activity in transgenic CAD-RNAi maize plants led to higher accessibility and more efficient breakdown of cell-wall carbohydrates resulting in an increased biofuel production (Fornale et al. 2012). In transgenic switchgrass, silencing of CAD improved the release of glucose after cellulase treatment (Saathoff et al., 2011) while down-regulation of COMT enhanced ethanol production, required less harsher pretreatment and lesser dosages of cellulose (Fu et al., 2011). Increase in the number of tillers and enhanced saccharification efficiency was observed in transgenic switchgrass overexpressing the PvMYB4 (Shen et al., 2012). Down-regulation of 4CL activity decreased the lignin content, led to vessel cell wall collapse and stunted growth in transgenic tobacco plants (Kajita et al. 1997). Silencing of 4CL influenced the carbohydrate metabolism leading to enhanced galactose content in Pinus radiata (Wagner et al. 2009) and enhanced the availability of carbohydrate release for biofuel production in switchgrass (Xu et al. 2011).

In most cases, the modification of lignin biosynthetic genes not only impacts the lignin content and composition but also affects plant growth and development significantly (Jones et al., 2001; Nakashima et al., 2008; Vermerris et al., 2010) while others did not exhibit such negative consequences (Chabannes et al., 2001; Jackson et al., 2008). Plants lacking lignin tend to be dwarfs, sterile, more susceptible to infections, and incapable of standing upright (Bonawitz and Chapple 2010). Down-regulation of ZmMYB31 in transgenic Arabidopsis produced dwarf plants which exhibited decreased lignin content without any change in its composition (Fornale et al. 2010). Overexpression of PvMYB4 in transgenic switchgrass also displayed a similar decrease in plant stature (Shen et al., 2012). Plant with reduced activity of C4H,C3H, HCT, or CCR commonly demonstrate a modest to extreme dwarf phenotype as compared to plants lacking CAD and COMT (Franke et al. 2002, Hoffman et al. 2004; Reddy et al. 2005; Bonawitz and Chapple 2010). The difficulty in water transportation, or absence of a necessary phenylpropanoid-derived compound or buildup of lethal pathway intermediate may be possible causes of dwarfing in these plants. The association between disturbance of monolignol biosynthesis and dwarfing will be imperative for justifying the negative effects of genetic manipulation of lignin biosynthetic pathway for the production of biofuels (Bonawitz and Chapple 2010). When linked with a significant enhancement in saccharification efficiency, moderate alterations in the growth pattern may be acceptable but extreme dwarfism may not be agronomically feasible (Chapple et al. 2007; Chen and Dixon 2007; Bonawitz and Chapple 2010).

Genes encoding CAD are up-regulated in response to pathogen infection (Tronchet et al. 2010) indicating a role of lignin genes in disease resistance. On the other hand, down-regulation of gene encoding HCT had increased tolerance to fungal infection and drought, and exhibited dwarfism (Gallego-Giraldo et al. 2011). To minimize the effect of lignin modification on plant development and biomass, control of lignin level and the expression of transgene(s) in specific tissues only is necessary. Modifying the composition of lignin or incorporating novel genes that are able to sustain the many important functions of lignin along with enhancing its degradation and digestibility during fermentation or similar complex strategies may be required to avoid biomass reduction (Grabber et al. 2008). Hence, detailed and comprehensive knowledge of lignin biosynthetic enzymes is necessary to improve the quality of the feedstock without compromising plant fitness. Although a number of genes in the lignin biosynthetic pathway have been manipulated and transgenic plants and mutants obtained, they haven’t been fully characterized warranting a need to further study them to better understand the effects of these modifications on the assembly and regulation of the lignin biosynthetic pathway (Anterola and Lewis 2002). Solving this puzzle will not only be critical for completely exploiting plant-derived renewable resources but also in recognizing the role played by lignin in vivo (Bonawitz and Chapple 2010).

Perspectives

The existence of lignin and the complexity of cell walls makes the degradation of lignocellulosic biomass more complex than starch. Lignocellulosic biomass can become economically viable only if the production costs are below the cost of using fossil fuels. Though biomass are available at low cost, steps such as pretreatment and processing increase the cost of biofuels making them unprofitable. The ongoing research on lignin modification and manipulation has demonstrated that lignin content can be reduced, degradability of lignin can be increased by improving the accessibility of cellulases for digestion of cellulose, and the lignin composition can be altered in a way that pretreatment can be decreased or even excluded.

It has also been shown that lignin can be tailored to a certain extent without considerable loss of biomass. However, most of these studies have been limited to greenhouses and the actual test of these plants with altered lignin production mechanism under field conditions has not been investigated. Field trials will bring a whole new aspect to the growth of these plants as will their exposure to various biotic and abiotic stresses, weeds and pathogens. Even as more research is conducted to sort out the issues with negative effects of lignin manipulation, the indications are that this is the right track and lignin manipulation is the key to successful adoption of lignocellulosic biofuels. Similarly, along with lignin manipulation, efforts towards the manipulation of cellulose and hemicellulose may be anticipated in the future for more synergistic impacts. To achieve this, plant biologists, microbiologists, biochemists, agronomists and breeders need to make collaborative efforts to formulate the most favorable solution to improve the conversion efficiency and sustainability without compromising the quality and yield of the biomass (Chapple et al. 2007).

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