Crosslinked Areas Than Lignin Augmented

Published Date: 02 Nov 2017

Manipulation of transcription factors

Genes in the lignin pathway are also regulated by R2R3-type transcription factors which have the MYB DNA binding domain [73,74]. 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 [75], while overexpression of Eucalyptus, EgMYB2 down-regulated the transcription of CCR and CAD and regulated both biosynthesis of lignin and secondary cell wall synthesis [76,77]. On the other hand, EgMYB1 was found to be a negative controller of CCR and CAD in the lignin pathway [77]. Pinus taeda MYB4 (PtMYB4) was also overexpressed in transgenic tobacco and augmented the deposition of lignin by modifying the expression of lignin biosynthesis genes [7].

In maize, ZmMYB31 and ZmMYB42 were identified as down regulators of COMT [78,79]. A.thaliana plants overexpressing ZmMYB42 had a reduced growth rate and decreased fresh weight [78]. 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 [79]. Transcription factors like SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), SND2, NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and their homologs [80-82] 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 [83]. Arabidopsis AtMYB4 and AtMYB32 also acted as repressors of the lignin biosynthesis pathway [84,85]. 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 [86]. 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

Role of microRNAs (miRNAs) in the developmental biology of plants has been established. 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. Being less lignified, the immature plant material demonstrates variation in accumulation of biomass and may be able to decrease the recalcitrance [87,88]. miRNA such as the one encoded by the maize Corngrass1 (Cg1) gene, that belongs 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 [89]. This Cg1 gene has been constitutively expressed in poplar and switchgrass [87,90]. In both the cases, it modified the content and composition of lignin and also had a severe effect 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 [91].

Gibberellins have been associated with diverse growth and developmental processes ranging from seed development to flowering [92]. Overexpression of Gibberellin 20-oxidase (GA20ox) in tobacco resulted in an increase in biomass with higher levels of lignin [93]. This increase in lignin might be the result of up-regulation of genes in the lignin pathway. 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. Mutant GA20ox, responsible for the dwarf and high yielding varieties of food crops in the green revolution [94,95] 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 [96]. Overexpression or down-regulation of homeobox genes like ARBORKNOX1 (ARK1) affects lignin content. However, these genes are important for the development of the plant and have deleterious effect on the survival of the plant making them unsuitable for genetic manipulation.

The above research efforts exploit the utilization of genetic engineering and emphasize its role in down-regulation or silencing of the genes that could modify the lignin composition and decrease the lignin content to improve the production of lignocellulosic biofuels. To test their viability, commercial pretreatments and fermentation procedures for ethanol production from these transgenics nned to be utilized. Though a number of genes, transcription and regulatory factors have been manipulated for the modification of lignin content and composition, no single method may be able to provide the much needed solution. Different gene(s) may give different results depending on the feedstock that is being modified. However, those genes which have minimal effect on the normal plant growth and development, and biomass yield would be preferential. Another effective strategy for improving the production of lignocellulosic biofuels may be provided by transgene pyramiding which can be achieved by breeding various transgenic lines showing higher saccharification efficiency.

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. 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. 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 [53]. In transgenic switchgrass, silencing of CAD improved the release of glucose after cellulase treatment [52] while down-regulation of COMT enhanced ethanol production, required less harsher pretreatment and lesser dosages of cellulose [60]. Increase in the number of tillers and enhanced saccharification efficiency was observed in transgenic switchgrass overexpressing the PvMYB4 [83]. Down-regulation of 4CL activity decreased the lignin content, led to vessel cell-wall collapse and stunted growth in transgenic tobacco plants [54]. Silencing of 4CL influenced the carbohydrate metabolism leading to enhanced galactose content in P. radiata [56] and enhanced the availability of carbohydrate release for biofuel production in switchgrass [58].

In most cases, the modification of lignin biosynthetic genes not only impacted the lignin content and composition but also affected plant growth and development significantly [97], while others did not exhibit such negative consequences [45,46]. Plants lacking lignin tend to be dwarfs, sterile, more susceptible to infections, and incapable of standing upright. Down-regulation of ZmMYB31 in transgenic Arabidopsis produced dwarf plants which exhibited decreased lignin content without any change in its composition [98]. Overexpression of PvMYB4 in transgenic switchgrass also displayed a similar decrease in plant stature [83]. 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 [13,44,65]. 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. 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.

Genes encoding CAD are up-regulated in response to pathogen infection [10] 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 [99]. 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 the biomass reduction. Hence, detailed and comprehensive knowledge of the 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. 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.

Concluding remarks The existence of lignin and the complexity of cell walls make 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. Approaches such as in planta expression of cellulases, genetic modification of the feedstock of interest to produce the enzymes that are either required or useful during fermentation, and developing strategies for recycling the enzymes may provide valuable solutions to reduce the cost of pretreatment. The recent advances in genome sequencing have led to the development of gene expression atlases which will augment the characterization and expression of more genes controlling the biofuel traits. The utilization of techniques such as TILLING, EcoTilling will also lead to the identification of traits such as biomass yield and saccharification efficiency which will further improve the potential feedstocks.

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 (Figure 5) 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 increasing the enzymatic hydrolysis rate and ready availability of cellulose, reducing the crystallinity of cellulose, manipulating cellulose and hemicellulose and improving the enzyme efficacies of cellulases and hemicellulases 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.