[Show abstract][Hide abstract] ABSTRACT: Predictions about future supply of crude oil, increase in greenhouse gases and burgeoning human population have recently necessitated a search for sustainable and environmentally responsible sources of alternative energy. Bioconversion of organic biomass, largely derived from plant resi-dues, to biofuels has been viewed as one of the most attractive avenues of research. More than half of the plant biomass produced on earth consists of cell walls that are enriched in carbohydrate polymers such as cellulose and hemicellulose. Breakdown of these carbohydrate polymers by pre-treatments and cellulolytic enzymes to monomer sugars (saccharification) which is followed by fermentation of sugars to bioethanol is one of the favoured ways of producing biofuels. Presence of phenolic compounds in form of lignin hinders access of cell wall degrading enzymes to carbohydrates, and genetic engineering of genes and metabolic pathways involved in cell wall formation could dramatically alter the outcome for biofuel production. In recent years, several reports have described such approaches that improve saccharification efficiency and this review discusses some of those advances. Another approach is to use quantitative trait locus (QTL) mapping and/or association mapping to identify genes/molecular markers that are associated with cell wall polymer synthesis. These candidate genes can be used for marker-assisted breeding and/or could be manipulated using transgenic approaches. A combination of these techniques is likely to provide novel tools in producing feedstocks for efficient use of selected plant biomass in biofuel production.
Review Methodology: Published articles related to the topic of this review were searched using keywords " Biofuel, Biomass, Cell wall, Genetic modification, and QTL " through databases such as Pubmed, Google scholar and Web of science. Information was also obtained from book chapters and online resources such as 'Biopact' for data pertaining to the topic of interest.
CAB Reviews Perspectives in Agriculture Veterinary Science Nutrition and Natural Resources 06/2014; 9:1-10. DOI:10.1079/PAVSNNR20149017
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We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes. The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the "food versus fuel" issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and leafy cotyledon2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80% increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.
[Show abstract][Hide abstract] ABSTRACT: Economical production of bioethanol from lignocellulosic biomass still faces many technical limitations. Cost-effective production of fermentable sugars is still not practical for large-scale production of bioethanol due to high costs of lignocellulolytic enzymes. Therefore, plant molecular farming, where plants are used as bioreactors, was developed for the mass production of cell wall degrading enzymes that will help reduce costs. Subcellular targeting is also potentially more suitable for the accumulation of recombinant cellulases. Herein, we generated transgenic tobacco plants (Nicotiana tabacum cv. SR1) that accumulated Thermotoga maritima BglB cellulase, which was driven by the alfalfa RbcsK-1A promoter and contained a small subunit of the rubisco complex transit peptide. The generated transformants possessed high specific BglB activity and did not show any abnormal phenotypes. Furthermore, we genetically engineered the RbcsK-1A promoter (MRbcsK-1A) and fused the amplification promoting sequence (aps) to MRbcsK-1A promoter to obtain high expression of BglB in transgenic plants. AMRsB plant lines with aps-MRbcsK-1A promoter showed the highest specific activity of BglB, and the accumulated BglB protein represented up to 9.3 % of total soluble protein. When BglB was expressed in Arabidopsis and tobacco plants, the maximal production capacity of recombinant BglB was 0.59 and 1.42 mg/g wet weight, respectively. These results suggests that suitable recombinant expression of cellulases in subcellular compartments such as chloroplasts will contribute to the cost-effective production of enzymes, and will serve as the solid foundation for the future commercialization of bioethanol production via plant molecular farming.
[Show abstract][Hide abstract] ABSTRACT: All known orthologs of a secondary wall-associated cellulose synthase (CesA) gene from Arabidopsis, AtCesA8, encode CesA proteins with two consecutive methionines at their N-termini (MM or 2M). Here, we report that these 2Ms in an aspen ortholog of AtCesA8, PtdCesA8A, are important for maintaining normal wood cellulose biosynthesis in aspen trees. Overexpression of an altered PtdCesA8A cDNA encoding a PtdCesA8A protein missing one methionine at the N-terminus (1M) in aspen resulted in substantial decrease in cellulose content and caused negative effects on wood strength, suggesting that both methionines are essential for proper CesA expression and function in developing xylem tissues. Transcripts from a pair of paralogous native PtdCesA8 genes, as well as introduced PtdCesA8A:1M transgenes were significantly reduced in developing xylem tissues of transgenic aspen plants, suggestive of a co-suppression event. Overexpression of a native PtdCesA8A cDNA encoding a CesA protein with 2Ms at the N-terminus did not cause any such phenotypic changes. These results suggest the importance of 2Ms present at the N-terminus of PtdCesA8A protein during cellulose synthesis in aspen.
Tree Physiology 10/2012; 32(11). DOI:10.1093/treephys/tps096 · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dwindling supply and unpredictable price fluctuations of fossil fuels has necessitated a renewed worldwide search for alternative fuels. Plant cell walls, rich in cellulose and hemicellulose, are actively pursued for their use in production of second-generation biofuels. It has been envisioned that degradation of these carbohydrate polymers to their sugar monomer subunits (saccharification) will provide necessary substrates for their yeast-mediated fermentation to ethanol. But the major component of plant cell walls, cellulose, is not easily amenable to this type of deconstruction and is tightly interlinked with hemicelluloses and lignin, making cell walls highly recalcitrant to saccharification. Thus, altering cell wall properties through manipulation of cell wall genes is one of the ways for improving saccharification of cell walls for bioethanol production. In recent years, efforts have been made via genetic engineering approaches for improving saccharification, yet we are still far away from replacing fossil fuels with biofuels. This review focuses on recent progress made in the alteration of cell wall properties through manipulation of native cell wall genes for enhanced saccharification and bioethanol production.
[Show abstract][Hide abstract] ABSTRACT: Plants are attractive expression systems for large-scale, low-cost production of high-value proteins. The xylanase 2 gene (Xyn2), encoding an endo-β-1,4-xylanase from Trichoderma reesei, was cloned and expressed in Escherichia coli and the poplar (Populus spp.). The optimal temperature and pH of the recombinant xylanase were 50 °C and 5.0 respectively when expressed in E. coli. The purpose of this study was to produce recombinant xylanase in poplar. The Xyn2 gene was transferred into poplars by Agrobacterium-mediated transformation. The transgenic status and transgene expression of the transformed poplar were confirmed by polymerase chain reaction (PCR) genotyping and reverse transcription (RT)-PCR analysis. The poplar-expressed xylanase was biologically active, with an expression level of up to 14.4% of total leaf soluble protein. In the leaves, the average xylanase content was 1.016 mg per g of leaf fresh weight in the transgenic poplar. We found that the poplar might make possible the large-scale production of commercially important recombinant proteins.
[Show abstract][Hide abstract] ABSTRACT: Genetic manipulation of cellulose biosynthesis in trees may provide novel insights into the growth and development of trees. To explore this possibility, the overexpression of an aspen secondary wall-associated cellulose synthase (PtdCesA8) gene was attempted in transgenic aspen (Populus tremuloides L.) and unexpectedly resulted in silencing of the transgene as well as its endogenous counterparts. The main axis of the transgenic aspen plants quickly stopped growing, and weak branches adopted a weeping growth habit. Furthermore, transgenic plants initially developed smaller leaves and a less extensive root system. Secondary xylem (wood) of transgenic aspen plants contained as little as 10% cellulose normalized to dry weight compared to 41% cellulose typically found in normal aspen wood. This massive reduction in cellulose was accompanied by proportional increases in lignin (35%) and non-cellulosic polysaccharides (55%) compared to the 22% lignin and 36% non-cellulosic polysaccharides in control plants. The transgenic stems produced typical collapsed or 'irregular' xylem vessels that had altered secondary wall morphology and contained greatly reduced amounts of crystalline cellulose. These results demonstrate the fundamental role of secondary wall cellulose within the secondary xylem in maintaining the strength and structural integrity required to establish the vertical growth habit in trees.
[Show abstract][Hide abstract] ABSTRACT: The genus Populus has emerged as one of the premier systems for studying multiple aspects of tree biology, combining diverse ecological characteristics, a suite of hybridization complexes in natural systems, an extensive toolbox of genetic and genomic tools, and biological characteristics that facilitate experimental manipulation. Here we review some of the salient biological characteristics that have made this genus such a popular object of study. We begin with the taxonomic status of Populus, which is now a subject of ongoing debate, though it is becoming increasingly clear that molecular phylogenies are accumulating. We also cover some of the life history traits that characterize the genus, including the pioneer habit, long-distance pollen and seed dispersal, and extensive vegetative propagation. In keeping with the focus of this book, we highlight the genetic diversity of the genus, including patterns of differentiation among populations, inbreeding, nucleotide diversity, and linkage disequilibrium for species from the major commercially-important sections of the genus. We conclude with an overview of the extent and rapid spread of global Populus culture, which is a testimony to the growing economic importance of this fascinating genus.
[Show abstract][Hide abstract] ABSTRACT: Virus-induced gene silencing (VIGS) is a powerful genetic tool for rapid assessment of plant gene functions in the post-genomic era. Here, we successfully implemented a Tobacco Rattle Virus (TRV)-based VIGS system to study functions of genes involved in either primary or secondary cell wall formation in Nicotiana benthamiana plants. A 3-week post-VIGS time frame is sufficient to observe phenotypic alterations in the anatomical structure of stems and chemical composition of the primary and secondary cell walls. We used cell wall glycan-directed monoclonal antibodies to demonstrate that alteration of cell wall polymer synthesis during the secondary growth phase of VIGS plants has profound effects on the extractability of components from woody stem cell walls. Therefore, TRV-based VIGS together with cell wall component profiling methods provide a high-throughput gene discovery platform for studying plant cell wall formation from a bioenergy perspective.
[Show abstract][Hide abstract] ABSTRACT: In plants, sucrose synthase (SUS) enzymes catalyze conversion of sucrose into fructose and UDP-glucose in the presence of UDP. To investigate the impact of overexpression of heterologous SUS on the growth and development of Arabidopsis, we transformed Arabidopsis plants with an overexpression vector containing an aspen SUS gene (PtrSUS1). The ge-nomic PCR confirmed the successful integration of PtrSUS1 transgene in the Arabidopsis genome. PtrSUS1 expression in transgenic Arabidopsis plants was confirmed by RT-PCR. The SUS activity was dramatically increased in all transgenic lines exam-ined. The three selected transgenic PtrSUS1 lines ex-hibited faster growth and flowered about 10 days earlier compared to untransformed controls, and also possessed 133%, 139%, and 143% SUS activity compared to controls. Both fresh weights and dry biomass yields of the whole plants from these three selected transgenic lines were significantly increased to 125% of the controls. Transgenic PtrSUS1 lines also had a higher tolerance to higher concentration of sucrose which was reflective of the increased SUS activity in transgenic versus wild-type plants. The growth differences between wild-type and transgenic plants, either in root and hypocotyl length or in fresh and dry weight of whole plant, became more pro-nounced on the media containing higher sucrose concentrations. Taken together, these results showed that the early flowering, faster growth and increased tolerance to higher sucrose in transgenic lines were caused by the genome integration and constitutive expression of the aspen PtrSUS1 gene in transgenic Arabidopsis.
Advances in Bioscience and Biotechnology 01/2010; 1(05):426-438. DOI:10.4236/abb.2010.15056
[Show abstract][Hide abstract] ABSTRACT: Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible inte-ractions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were de-tected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant spe-cies could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
Advances in Bioscience and Biotechnology 01/2010; 1(03):152-161. DOI:10.4236/abb.2010.13021
[Show abstract][Hide abstract] ABSTRACT: Cellulose synthase (CesA) is a central catalyst in the generation of the plant cell wall biomass and is, therefore, the focus of intense research. Characterization of individual CesA genes from Populus species has led to the publication of several different naming conventions for CesA gene family members in this model tree. To help reduce the resulting confusion, we propose here a new phylogeny-based CesA nomenclature that aligns the Populus CesA gene family with the established Arabidopsis thaliana CesA family structure.
[Show abstract][Hide abstract] ABSTRACT: Trees constitute the majority of lignocellulosic biomass existing on our planet. Trees also serve as important feedstock materials
for various industrial products. However, little is known about the regulatory mechanisms of cellulose synthase (CesA) genes of trees. Here, the cloning and characterization of three CesA genes (EgraCesA1, EgraCesA2, and EgraCesA3) from an economically important tree species, Eucalyptus grandis, are reported. All three genes were specifically expressed in xylem cells of eucalyptus undergoing secondary cell wall biosynthesis.
The GUS gene, expressed under the control of the EgraCesA2 or EgraCesA3 promoter, was also localized in the secondary xylem in transgenic tobacco stems. However, the EgraCesA1 promoter alone or along with its 5′-UTR introns was insufficient to direct appropriate GUS expression. EgraCesA2 and EgraCesA3 gene expression was up-regulated in tension-stressed eucalyptus xylem cells. Accordingly, GUS expression directed by the EgraCesA2 or EgraCesA3 promoter was also up-regulated. EgraCesA1 had no such response. Thus, it is most unlikely that EgraCesA1 is a subunit of the EgraCesA2–EgraCesA3 complex. The presence of at least two types of cellulose biosynthesis machinery in wood formation is an important clue in
deciphering the underpinnings of the perennial growth of trees in various environmental conditions. By analysing GUS gene expression directed by the EgraCesA3 promoter or its deletions, several negative and positive regulatory regions controlling gene expression in xylem or phloem
were identified. Also a region which is likely to contain mechanical stress-responsive elements was deduced. These results
will guide further studies on identifying cis-regulatory elements directing CesA gene transcription and wood formation regulatory networks.
[Show abstract][Hide abstract] ABSTRACT: Cellulose is the most abundant biopolymer on earth. Despite its simple structure, omnipresence in the plant kingdom, and ever increasing global importance as industrial raw material, the genetic and biochemical regulation of cellulose biosynthesis continues to be unclear. Over the past ten years, the advances in functional genomics have significantly improved our understanding of the processes of cellulose biosynthesis in higher plants. However, for each question answered myriad new unanswered ones have arisen.
Current Opinion in Plant Biology 07/2007; 10(3):220-6. DOI:10.1016/j.pbi.2007.04.013 · 7.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anualmente, as plantas produzem aproximadamente 180 bilhões de toneladas de celulose, sendo o maior reservatório de carbono orgânico no planeta. A celulose é um homopolímero linear composto por resíduos de glicose unidos por meio de ligações b(1-4). A síntese coordenada das cadeias de glicose é orquestrada por complexos específicos ligados à membrana plasmática (CelS). Postula-se que o CelS é composto por aproximadamente 36 subunidades da sintase da celulose (CESA). Cada CelS sintetiza 36 cadeias de glicose dispostas lado a lado antes de serem organizadas em microfibrilas, que são, posteriormente, associadas com outros polímeros da parede celular. As 36 cadeias de glicose presentes em uma microfibrila são estabilizadas por pontes de hidrogênio intra e inter-cadeias, conferindo grande estabilidade às microfibrilas. As microfibrilas elementares são dispostas lado a lado, permitindo a formação das macrofibrilas. Várias isoformas da CESA podem estar envolvidas no processo de biossíntese de celulose e, no mínimo, três tipos de isoformas da CESA podem ser necessárias para a organização funcional de cada CelS em plantas superiores.
Brazilian Journal of Plant Physiology 03/2007; 19(1). DOI:10.1590/S1677-04202007000100001
[Show abstract][Hide abstract] ABSTRACT: In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.
[Show abstract][Hide abstract] ABSTRACT: We report the draft genome of the black cottonwood tree, Populus trichocarpa. Integration of shotgun sequence assembly with genetic mapping enabled chromosome-scale reconstruction of the genome. More
than 45,000 putative protein-coding genes were identified. Analysis of the assembled genome revealed a whole-genome duplication
event; about 8000 pairs of duplicated genes from that event survived in the Populus genome. A second, older duplication event is indistinguishably coincident with the divergence of the Populus and Arabidopsis lineages. Nucleotide substitution, tandem gene duplication, and gross chromosomal rearrangement appear to proceed substantially
more slowly in Populus than in Arabidopsis. Populus has more protein-coding genes than Arabidopsis, ranging on average from 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene. However, the relative frequency of protein domains in the two genomes is similar. Overrepresented exceptions in Populus include genes associated with lignocellulosic wall biosynthesis, meristem development, disease resistance, and metabolite
[Show abstract][Hide abstract] ABSTRACT: The quality and quantity of cellulose deposited in the primary and secondary cell walls of plants vary in accordance with their biological function. However, the molecular basis of such cellulose heterogeneity has so far remained unclear. Since enrichment of better-quality cellulose, in terms of increased degree of polymerization and crystallinity, is one of the goals of forest biotechnology, our main objective is to decipher the roles of distinct cellulose synthase (CesA) genes in tree development, with special reference to wood production. Here, we report two full-length CesA cDNAs, PtrCesA3 and PtrCesA4, from an economically important tree aspen (Populus tremuloides). PtrCesA3 is orthologous to the Arabidopsis AtCesA4 gene involved in secondary wall formation, whereas PtrCesA4 is orthologous to the Arabidopsis AtCesA1 gene involved in primary cell wall formation. To define the specific cell types expressing these CesA genes, we explored the natural distribution patterns of PtrCesA3 and PtrCesA4 transcripts in a variety of aspen organs, such as leaves, petiole, stem, and roots, using in situ hybridization with hypervariable region-specific antisense riboprobes. Such a side-by-side comparison suggested that PtrCesA3 is exclusively expressed in secondary-wall-forming cells of xylem and phloem fibers, whereas PtrCesA4 is predominantly expressed in primary-wall-forming expanding cells in all aspen organs examined. These findings suggest a functionally distinct role for each of these two types of PtrCesAs during primary and secondary wall biogenesis in aspen trees, and that such functional distinction appears to be conserved between annual herbaceous plants and perennial trees.