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Molecular Biology of Lignification in Grasses

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Abstract

As we transition into the twenty-first century, the need for renewable resources to address global energy and food demands has become a major concern. Around the world, scientists are interested in engineering dedicated biomass feedstocks particularly for improved cell wall composition by modifying the major wall components, cellulose and lignin. In this chapter, we review the current knowledge of plant engineering specifically in the area of lignin biosynthesis and composition towards the goal of generating plants optimized for bioethanol production and animal feed. Crops dedicated as biomass feedstocks, i.e., miscanthus, switchgrass, triticals, sorghum and maize, are grasses, which have unique characteristics of making their cell walls ideal sources for bioethanol production. Our understanding of the grass cell wall has significantly improved in the past two decades through studies carried out primarily in maize (. Zea mays). Here, we discuxss several aspects of lignin deposition into the cell wall including the cellular and molecular aspects of lignin biosynthesis. Significant effort was dedicated to identifying the molecular regulators of these processes and the developmental defects resulting from gene modifications. In addition, we demonstrate genetic correlations between genes of the lignin biosynthesis pathway to those involved in cell wall deposition using a gene expression network program. Together this work lays the foundation for future studies addressing the molecular regulation of lignification in the grasses in hopes to develop, through genetic engineering, ideal biomass feedstocks for biofuel production.

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... The structure is thin and flexible, suitable for elongating cells, but still sufficiently strong to withstand arising turgor pressure [9,10]. It consists primarily of cellulose and hemicellulose, with higher amounts of pectin and proteins in dicots compared to monocots [5,11]. SCWs are formed between the PCW and the plasma membrane in specialised cells such as sclerenchyma and xylem vessels after cell elongation has been completed. ...
... Lignin of grasses primarily consists of S-and G-units. Additionally, grasses also contain H-units and significantly larger amounts of ferulic acid (FA) and p-coumaric acid (pCA) [11,32]. The FA and pCA cross-link to the lignocellulosic matrix, providing structural integrity of the cell wall. ...
Article
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Biomass rich in lignocellulose from grasses is a major source for biofuel production and animal feed. However, the presence of lignin in cell walls limits its efficient utilisation such as in its bioconversion to biofuel. Reduction of the lignin content or alteration of its structure in crop plants have been pursued, either by regulating genes encoding enzymes in the lignin biosynthetic pathway using biotechnological techniques or by breeding naturally-occurring low lignin mutant lines. The aim of this review is to provide a summary of these studies, focusing on lignin (monolignol) biosynthesis and composition in grasses and, where possible, the impact on recalcitrance to bioconversion. An overview of transgenic crops of the grass family with regulated gene expression in lignin biosynthesis is presented, including the effect on lignin content and changes in the ratio of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. Furthermore, a survey is provided of low-lignin mutants in grasses, including cereals in particular, summarising their origin and phenotypic traits together with genetics and the molecular function of the various genes identified.
... The hydroxycinnamates p-coumaric acid and ferulic acid are also incorporated into secondary walls in linkages between lignin and hemicellulose polymers. The biochemistry and genetics of grass lignin are extensively reviewed in Harrington et al. (Harrington et al. 2012). Lignin biosynthesis has been defined largely in terms of monolignol biosynthesis, with extensive characterization of the enzymes and transcriptional regulators governing this branch of the phenylpropanoid pathway (Vanholme et al. 2010). ...
... Consistent with the reduction of S lignin, there was also a reduction in the p-coumaric acid levels in Bdcad1 mutants . P-coumaric acids are involved in the cross linking between hemicellulose arabinoxylans and lignin polymers, particularly through ester linkage to S lignin monomers (Ralph 2010;Vanholme et al. 2010;Harrington et al. 2012;Bouvier et al. 2013). Ferulic acid levels, which are ester-linked to hemicelluloses much like p-coumaric acid is, were not affected ). ...
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The utilization of Brachypodium distachyon as a model system has allowed for a synthesis of known aspects of grass cell wall biosynthesis and provided a platform to investigate new areas of the field. Compositional analysis of B. distachyon cell walls shows many similarities with the walls of major food and energy crop species. This chapter presents a summary of these comparisons, as well as a review of work done in B. distachyon characterizing genes involved with cell wall biogenesis. Aspects of lignin biosynthesis and polymerization, cellulose and hemicellulose synthesis, and transcriptional regulation of secondary walls have all been characterized in B. distachyon, with genetic, biochemical, and phenotypic data outlined herein. Finally, the use of B. distachyon in identifying saccharification and digestibility traits relatable to biofuel feedstock quality in grasses are discussed. Taken together, the reviewed material demonstrates the utility of B. distachyon as a model for grass cell wall research, highlighting known and novel facets of cell wall biosynthesis.
... In contrast, lignin may be deposited at later www.frontiersin.org stages in other tissues (Boerjan et al., 2003;Baghdady et al., 2006;Harrington et al., 2012). For instance, parenchyma cells present in the secondary xylem of Arabidopsis hypocotyls are devoid of lignin during the vegetative stage (Sibout et al., 2008). ...
... Second, it is possible that acylation may affect lignin structure during polymerization as well as subsequent degradation by chemical or biochemical processes such as saccharification and digestibility. In grasses, p-coumarates are linked to syringyl units and plants with low coumarate levels also generally show modifications in their S, or S and G content (Harrington et al., 2012). Importantly, coumaroylation of cell wall polysaccharides is most likely (or at least partly) catalyzed in the cytosol prior to export and polymerization in the cell wall strongly suggesting the existence of specific monolignol transferases and transporters (Withers et al., 2012;Molinari et al., 2013; Figure 1). ...
Article
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Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units – the monolignols – that are linked together by several covalent bonds. Numerous studies have shown that monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of monolignol metabolism and the relationship that exists between lignin and other monolignol-derived compounds thereby appears essential if we wish to improve biomass quality.
... In contrast, lignin may be deposited at later www.frontiersin.org stages in other tissues (Boerjan et al., 2003;Baghdady et al., 2006;Harrington et al., 2012). For instance, parenchyma cells present in the secondary xylem of Arabidopsis hypocotyls are devoid of lignin during the vegetative stage (Sibout et al., 2008). ...
... Second, it is possible that acylation may affect lignin structure during polymerization as well as subsequent degradation by chemical or biochemical processes such as saccharification and digestibility. In grasses, p-coumarates are linked to syringyl units and plants with low coumarate levels also generally show modifications in their S, or S and G content (Harrington et al., 2012). Importantly, coumaroylation of cell wall polysaccharides is most likely (or at least partly) catalyzed in the cytosol prior to export and polymerization in the cell wall strongly suggesting the existence of specific monolignol transferases and transporters (Withers et al., 2012;Molinari et al., 2013; Figure 1). ...
Data
Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units – the monolignols – that are linked together by several covalent bonds. Numerous studies have shown that monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of monolignol metabolism and the relationship that exists between lignin and other monolignol-derived compounds thereby appears essential if we wish to improve biomass quality.
... In contrast, lignin may be deposited at later www.frontiersin.org stages in other tissues (Boerjan et al., 2003;Baghdady et al., 2006;Harrington et al., 2012). For instance, parenchyma cells present in the secondary xylem of Arabidopsis hypocotyls are devoid of lignin during the vegetative stage (Sibout et al., 2008). ...
... Second, it is possible that acylation may affect lignin structure during polymerization as well as subsequent degradation by chemical or biochemical processes such as saccharification and digestibility. In grasses, p-coumarates are linked to syringyl units and plants with low coumarate levels also generally show modifications in their S, or S and G content (Harrington et al., 2012). Importantly, coumaroylation of cell wall polysaccharides is most likely (or at least partly) catalyzed in the cytosol prior to export and polymerization in the cell wall strongly suggesting the existence of specific monolignol transferases and transporters (Withers et al., 2012;Molinari et al., 2013; Figure 1). ...
Article
Full-text available
Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units - the monolignols - that are linked together by several covalent bonds. Numerous studies have shown that monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of monolignol metabolism and the relationship that exists between lignin and other monolignol-derived compounds thereby appears essential if we wish to improve biomass quality.
... In grasses, alteration of lignin content and/or composition improves both feed digestibility [51,52] and saccharification yields [53][54][55]. However, even changes in low-abundance components, such as pectin, can have a dramatic impact on the yields of glucose and xylose in saccharification assays with poplar wood [56,57]. ...
Article
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Background: The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. Results: High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. Conclusions: Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.
... The fall sunshine, appropriate temperature, and light conditions were suitable for radish fleshy root thickening and photosynthetic product accumulation in taproot, which may result in the relative decrease of lignin content. With parenchyma cells starting to reinforce their secondary cell walls, the phloem and xylem extensively expanded and xylem exhibited strong lignification at later stages (Harrington et al. 2012). The huge deposition of lignin at later stages in radish root would directly affect radish quality. ...
Article
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Radish is an important root vegetable crop with high nutritional, economic, and medicinal value. Lignin is an important secondary metabolite possessing a great effect on plant growth and product quality. To date, lignin biosynthesis-related genes have been identified in some important plant species. However, little information on characterization of critical genes involved in plant lignin biosynthesis is available in radish. In this study, a total of 71,148 transcripts sequences were obtained from radish root, of which 66 assembled unigenes and ten candidate genes were identified to be involved in lignin monolignol biosynthesis. Full-length cDNA sequences of seven randomly selected genes were isolated and sequenced from radish root, and the assembled unigenes covered more than 80% of their corresponding cDNA sequences. Moreover, the lignin content gradually accumulated in leaf during the developmental stages, and it increased from pre-cortex to cortex splitting stage, followed by a decrease at thickening stage and then increased at mature stage in root. RT-qPCR analysis revealed that all these genes except RsF5H exhibited relatively low expression level in root at thickening stage. The expression profiles of Rs4CL5, RsCCoAOMT1, and RsCOMT genes were consistent with the changes of root lignin content, implying that these candidate genes may play important roles in lignin formation in radish root. These findings would provide valuable information for identification of lignin biosynthesis-related genes and facilitate dissection of molecular mechanism underlying lignin biosynthesis in radish and other root vegetable crops.
... The prolonged time to emergence was thus not an artefact of ambient humidity. It is possible that the cortical cells of water deficitstressed culms surrounding the galled tissue had increased lignin content ( Harrington, Mutwil, Barrière, & Sibout, 2012); however, the effects of water deficit stress on lignin are not consistent across grasses or other plant species ( Frei, 2013). Decreased water content also may have led to increased silification of culms (O' Reagain & Mentis, 1989). ...
Article
Water deficit stress can reduce the reproductive performance of galling insects, but have not previously been studied in the context of mass-rearing of a galling agent on a perennial grass. The effects of water deficit were examined for the wasp Tetramesa romana Walker (Hymenoptera: Eurytomidae), released in the Lower Rio Grande Basin of Texas and Mexico in 2009 and in northern California in 2010 for control of giant reed (Arundo donax (L)) (Poaceae). In one study, water deficit was imposed for 7 to 10 weeks during gall maturation, ending when adult progeny began to emerge. Above-ground water content was reduced by 1.7% and culm height by 41% in pots receiving 1/4th of normal watering (soil saturation), indicating that water deficit stress occurred. Water deficit did not affect proportion of culms successfully galled or number of progeny produced. However, time to first exit hole appearance was 2 to 4 days longer and adult wasp generation time 5 to 7 days longer on galls on water deficit-stressed compared to control culms, thereby reducing the wasp's intrinsic rate of increase. Water deficit imposed only during parent wasp oviposition had no effect on galling success or reproduction. Water deficit thus has a negative effect on rearing by delaying adult emergence and reducing the rate of population increase, even without affecting adult fertility. Mass-rearing should involve well-watered plants, and variable drought conditions in the field should be considered when evaluating T. romana establishment and impact.
... Co-expression analysis using PlaNet (http://aranet.mpimp-golm.mpg.de/index.html), a web-based platform that predicts transcriptomic co-expression networks based on temporal and spatial gene expression data (Mutwil et al., 2011; Harrington et al., 2012), revealed that BdPMT expression clustered with those of other predicted lignin biosynthetic genes including Phenylalanine Ammonia Lyase (PAL; Bradi3g49250 and Brad- i3g49260), Cinnamoyl-CoA Reductase (CCR; Bradi3g36887), and Caffeic acid O-Methyltransferase (COMT; Brad- i3g16530) (Table S1). To study how altered BdPMT expression and function affects cell wall composition and structure as well as plant growth, we generated Brachypodium BdPMT RNA interference (RNAi) as well as overexpression (OX) lines. ...
Article
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Grass lignins contain substantial amounts of p-coumarate (pCA) acylating the sidechains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages. However, p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was specifically involved in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD acyltransferases that specifically acylate monolignols with pCA, producing monolignol p-coumarate conjugates that are used for lignification in planta. This article is protected by copyright. All rights reserved.
... In conclusion, the three mutant lines that have an altered lignin content and/or composition share at least a mutation located in the vicinity of the SAM/SAH binding and catalytic domain. It is worth noting that the three COMT mutants do not show coloured leaf veins as observed in the brown-midrib mutants of maize, pearl millet or sorghum [56]. In addition, the levels of S units is still high in the mutants (as revealed by the 45 to 55% of S thioacidolysis monomers). ...
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The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called “BRACHYTIL”, for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6 , a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to asses
... Model plants are important tools for designing lignified cell walls with improved saccharification. Brachypodium distachyon (Brachypodium) has a range of features that make it an excellent model grass species in which to identify genes important for cereals and energy grasses, as described in recent reviews (Draper et al., 2001;Vogel et al., 2006;Opanowicz et al., 2008;Bevan et al., 2010;Brkljacic et al., 2011;Mur et al., 2011;Harrington et al., 2012). Importantly, the genomic sequence of the Bd21 accession has been released (International Brachypodium Initiative, 2010). ...
Article
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Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge about its lignins and no data about lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. The down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically-induced population of Brachypodium mutants (Bd21-3 background) for red culm coloration. Thus, we identified two mutants Bd4179 and Bd7591, with mutations in the BdCAD1 gene. The mature stems of these mutants displayed a reduced CAD activity and a lower lignin content. Their lignins were enriched in 8-O-4 and 4-O-5 coupled sinapaldehyde units as well as in resistant interunit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end-groups. Moreover, sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. The functional complementation of the Bd4179 mutant with the wild-type (WT) BdCAD1 allele restored a WT phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than WT plants. In this work, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the branching degree of lignins through the incorporation of sinapaldehyde. These changes make the saccharification of alkaline-pretreated cell walls easier without compromising plant growth. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
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Grass cell walls are typified by ferulic esters linked to polysaccharides. In past research, these feruloylated esters have been repeatedly speculated to be cross-linking agents with lignins, via ether bonds. Whereas this hypothesis is strongly supported by degradative studies, model experiments, and NMR data, diagnostic fragments associating ferulic acid and lignin precursors, through an ether bond, have never been isolated from grass walls. This paper reports the isolation of such products by saponification of wheat and oat straws. New dimers associating ferulic acid to the beta position of coniferyl alcohol are characterized by gas chromatography/mass spectrometry and authenticated by independently synthesized compounds. The biochemical implication is that ferulate esters are copolymerized with lignin precursors through oxidative coupling. These ferulate esters thereby provide points of growth for the polymer Lignin, via ether bonds that anchor Lignins to wall polysaccharides.
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Grass lignins are made of guaiacyl and syringyl units together with minor amounts of p-hydroxyphenyl units. The specific association of p-coumaric (pCA) and ferulic (FA) acids to grass lignins has suspected important consequences on wall properties which are however, poorly understood. Genetic variation for lignin content, lignin structure, and p-coumaric and ferulic acid contents has been shown in normal maize, after an earlier description in brown-midrib (bm) mutants. QTL analyses for lignin-related traits have established that nearly 40 genomic regions are involved in maize variation of lignin content. For most of these locations, no candidate genes have been validated, or have been still defined. Whereas all steps of lignin biosynthesis have been presumably identified, little is known about the number of gene members encoding each enzymatic step and the role of each member in organ, stage and/or tissue specificity. Moreover, even if the lignin pathway has often been displayed as a metabolic grid, available results, especially in maize bm and genetically engineered plants, suggest that the hydroxylation/methylation steps at the aromatic C-3 position have a key role in controlling the flux to lignins. Recent studies of cell wall-related gene expression in young and silking maize plants also illustrated an unexpected diversity of genes with differential expression profiles, especially in bm mutants. Breeding maize and other grasses for phenolic structures more suitable for animal nutrition or energy production could now be considered a realistic goal by integrating new genomic-based knowledge on maize lignification.
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Lignin content and structure were examined in seven caffeic acid O-methyltransferase antisense (COMT-AS) maize progenies and their corresponding normal inbred lines in relation to cell wall digestibility. The seven parental inbreds were chosen for their highly divergent in vitro wall digestibility. Maize plants were grown under field conditions to determine (i) if the positive effect of COMT down-regulation on wall chemistry and digestibility was similar to that previously observed for COMT-AS maize grown in the greenhouse and (ii) to what extent the genetic background was a factor in determining the effect of the transgene. All␣COMT-AS progenies displayed a significant reduction in endogenous COMT activity (14–43% residual activity). In all but one genetic background (F4), the COMT-AS gene resulted in an expected increase in wall digestibility accompanied by changes in lignin composition. These effects varied greatly among parental lines, and independently of the inherent digestibility values in the corresponding non-transformed lines. Curiously, in the highly digestible F4 background, the typical decrease in syringyl (S) unit lignin and a higher frequency of 5-OH guaiacyl lignin resulting from the introduction of the COMT-AS transgene were not observed. Our results indicate that COMT down-regulation via an antisense strategy is an efficient tool for forage maize improvement in the field.
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The European corn borer (ECB, Ostrinia nubilalis Hübner) is a major pest of maize in central Europe. The objectives of our study were to (1) identify QTL for resistance to ECB, (2) estimate their genetic effects, and (3) investigate the consistency of QTL across two different populations. A total of 230 F 2:3 families derived from cross 1396A (resistant) × F478 (susceptible) were used for QTL analyses. Each F 2:3 family was evaluated for resistance traits tunnel length (TL), stalk damage ratings (SDR), and relative grain yield (RGY) using manual infestation with ECB larvae. The agronomic traits comprised grain yield under insecticide protection (GYP) and manual infestation (GYI), date of anthesis (ANT), dry matter content (DMC), and in vitro digestible organic matter (IV-DOM) of stover. The field experiment was performed with two replications in two environments in 1995. Two QTL for SDR and two QTL for TL were detected explaining 24.7% and 26.0% of the genotypic variance (σ g2), respectively. For agronomic traits one to three QTL were found, explaining between 2.0% and 11.8% of σ g2. No common QTL for resistance traits were found across population 1396A×F478 and a second population of 230 F 2:3 families derived from cross D06 (resistant) × D408 (susceptible). Two QTL for IVDOM and DMC were in common among both populations. Due to the low consistency of QTL across populations, marker-assisted selection (MAS) is not recommended for improving ECB resistance in early maturing dent germplasm.
Chapter
Ferulate polysaccharide esters in grasses enter into free-radical coupling reactions in the cell wall. By radical dimerization of ferulates, polysaccharide-polysaccharide cross-linking is effected. A range of diferulate isomers are produced, only one of which had been quantitated in the past. Diferulates have been underestimated by factors of up to 20, belittling their contribution to functions in the cell wall. Both ferulates and diferulates participate in lignification reactions and become intimately bound with lignin. Under-quantitation is significant since it is not possible to release ferulate or diferulates from some of the structures generated. Overall ferulates play a significant role in cell wall development and impact upon polysaccharide utilization in grasses.
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Plant mechanical strength is an important agronomic trait. To understand the molecular mechanism that controls the plant mechanical strength of crops, we characterized the classic rice mutant brittle culm1 (bc1) and isolated BC1 using a map-based cloning approach. BC1, which encodes a COBRA-like protein, is expressed mainly in developing sclerenchyma cells and in vascular bundles of rice. In these types of cells, mutations in BC1 cause not only a reduction in cell wall thickness and cellulose content but also an increase in lignin level, suggesting that BC1, a gene that controls the mechanical strength of monocots, plays an important role in the biosynthesis of the cell walls of mechanical tissues.
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Referee: Dr. E. Charles Brummer, Forage Breeding and Genetics, 1204 Agromonomy, Iowa State University, Ames, IA 50011 Much of the research on the genetic modification of herbaceous plant cell walls has been conducted to improve the utilization of forages by ruminant livestock. The rumen of these animals is basically an anaerobic fermentation vat in which the micro flora break down the complex polysaccharides of plant cell walls into simpler compounds that can be further digested and absorbed by the mammalian digestive system. Research on improving the forage digestibility of switchgrass, Panicum virgatum L., and other herbaceous species has demonstrated that genetic improvements can be made in forage quality that can have significant economic value. To meet future energy needs, herbaceous biomass will need to be converted into a liquid fuel, probably ethanol, via conversion technologies still under development. If feedstock quality can be genetically improved, the economics and efficiency of the conversion processes could be significantly enhanced. Improving an agricultural product for improved end product use via genetic modification requires knowledge of desired quality attributes, the relative economic value of the quality parameters in relation to yield, genetic variation for the desired traits, or for molecular breeding, knowledge of genes to suppress or add, and knowledge of any associated negative consequences of genetic manipulation. Because conversion technology is still under development, desirable plant feedstock characteristics have not been completely delineated. Some traits such as cellulose and lignin concentration will undoubtably be important. Once traits that affect biomass feedstock conversion are identified, it will be highly feasible to genetically modify the feedstock quality of herbaceous plants using both conventional and molecular breeding techniques. The use of molecular markers and transformation technology will greatly enhance the capability of breeders to modify the morphologic structure and cell walls of herbaceous species. It will be necessary to monitor gene flow to remnant wild populations of biomass plants and have strategies available to curtail gene flow if it becomes a potential problem. It will also be necessary to monitor plant survival and long-term productivity as affected by these genetic changes to herbaceous species.
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The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.
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Considerable effort has been expended toward the genetic characterization of native resistance to stalk tunneling by the European corn borer [ECB; Ostrinia nubilalis (Hübner)], indicative of the importance of this pest and the difficulty in obtaining conclusive results. In this study, 191 recombinant inbred lines (RILs) of B73 maize (Zea mays L.) (susceptible to stalk tunneling by ECB) x De811 (resistant) were evaluated for stalk tunneling, anthesis, and plant height in Iowa at two locations in 1998 and one location in 1999 with the objectives of (i) determining the genetic relationship between these traits and (ii) mapping quantitative trait loci (QTL) associated with resistance to stalk tunneling. The genotypic correlation between plant height and stalk tunneling (rg = 0.1) was negligible, but the correlation between stalk tunneling and anthesis was very high (rg = -0.8) necessitating the adjustment of the means of former with the latter. Ten QTL for stalk tunneling adjusted for anthesis associated with 42% of the phenotypic variation were observed in the mean across trials, only one of which was observed in each of the individual trials. The lack of consistent QTL detection across environments is a common characteristic among studies of ECB tunneling and underscores a major problem of breeding for resistance. QTL observed in F3 lines of the same cross and in RILs of B73 X B52 are linked to three QTL each for the mean across trials herein, providing further evidence of association between these genomic regions and resistance to stalk tunneling.
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Forage plants are the basis of ruminant nutrition. Among cereal forages, maize cropped for silage making is the most widely used. Much research in genetics, physiology, and molecular biology of cereal forages is thus devoted to maize, even if silage of sorghum or immature small-grain cereals and straws of small-grain cereals are also given to cattle. Cell wall digestibility is the limiting factor of forage feeding value and is, therefore, the first target for improving their feeding value. Large genetic variation for cell wall digestibility was proven from both in vivo and in vitro experiments in numerous species. Among the regular maize hybrids [excluding brown-midrib (bm) ones], the cell wall digestibility nearly doubled from 32.9% to 60.1%. Genetic variation has also been proven in cell wall digestibility of sorghum and wheat, barley or rice forage, or straw, with lower average values than in maize. Despite lignin content is well known as an important factor making cell wall indigestible, breeding for a higher digestibility of plant needs the use of specific traits estimating the plant cell wall digestibility. Quantitative trait loci (QTL) analysis, studies of single-nucleotide polymorphism (SNP) × feeding value traits relationships, studies of mutants and deregulated plants, and expression studies will contribute to the comprehensive knowledge of the lignin pathway and cell wall biogenesis. Plant breeders will then be able to choose the best genetic and genomic targets for the improvement of plant digestibility. Favorable alleles or favorable QTL for cereal cell wall digestibility will thus be introgressed in elite lines through marker-assisted introgression. Efficient breeding of maize and others annual forage plants demands a renewing of genetic resources because only a limited number of lines are actually known with a high cell wall digestibility. Among bm genes, the bm3 mutant in maize and the bmr12 (and possibly bmr18) mutant in sorghum, which are both altered in the caffeic acid O-methyltransferase (COMT) activity, appeared as the most efficient in cell wall digestibility improvement. Genetic engineering is both an inescapable tool in mechanism understanding and an efficient way in cereal breeding for improved feeding value. Moreover, gene mining and genetic engineering in model plant and systems (Arabidopsis, Zinnia, Brachypodium, …) are also essential complementary approaches for improvement of cell wall digestibility in grass and cereal forage crops.
Article
Improving forage quality is a major goal in maize breeding for cooler climates. In this study, we mapped and characterized quantitative trait loci (QTL) affecting testcross (TC) performance of important forage maize quality traits and investigated their consistency across testers. Two elite flint inbred lines were crossed to generate 380 F2 individuals, 345 of which were genotyped at 89 RFLP marker loci. The 380 F3 lines produced by selfing the F2 individuals were testcrossed to two dent inbred testers. Each TC series was evaluated in field trials with two replications in five environments. The following six traits were analyzed: in vitro digestible organic matter (IVDOM), acid detergent fiber (ADF), metabolizable energy concentration (MEC), and protein concentration (CPC), all determined by near infra-red reflectance spectroscopy (NIRS), as well as metabolizable energy yield (MEY) and protein yield (CPY). Genotypic variances (θ(g)/2) were mostly significant for these traits in both TC series. Heritabilities ranged from 0.24 to 0.69 and were low for IVDOM, ADF, and MEC. Genotypic correlations between testers exceeded 0.64 for each trait. Between four (CPY) and ten (CPC) QTL were detected in each TC experiment by composite interval mapping, explaining between 48.4% and 85.3% of θ(g)/2 in a simultaneous fit. QTL results were consistent across testers for CPC and CPY, but not for IVDOM, ADF, MEC, and MEY. Few of the detected QTL displayed significant digenic epistatic interactions. The digestibility traits IVDOM, ADF, and MEC were tightly correlated (|r(g)| > 0.88) with each other and displayed intermediate genotypic correlations (r(g)) with plant height and starch concentration, but low r(g) values with dry matter yield or dry matter concentration. In most cases, the magnitude of r(g) corresponded well with the number of common QTL regions affecting both traits.
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The hypothesis is proposed that nodes of hollow plant stems act as spring-like joints by storing strain energy when stems are bent and releasing this energy to elastically restore the original postures of stems when bending forces are removed. This hypothesis was tested by subjecting stem segments consisting of four nodes and three intervening hollow internodes to axial compressive loads and by determining the natural frequencies of vibration of their nodes. Compression tests were used to determine the critical load required to produce elastically recoverable deformations for each of a total of 115 stem segments of the grassArundinaria técta(Walt.) Muhl. Each segment was observed to flex at or very near its nodes while internodes appeared to act as rigid bars. The natural (fundamental) frequencies of vibrations of the nodes of these stem segments were subsequently determined and equalled those predicted by engineering theory assuming that nodes behave as spring-like joints. The data from resonance frequency tests were then used to calculate the spring constants of stem segments (i.e. the force required to produce a unit deflection in stems). These constants were found to agree with those predicted by theory provided that nodes acted mechanically as spring-like joints. The transverse septa of the nodes of 20 randomly selected stem segments were perforated with a needle and the spring constants of the impaired nodes were remeasured and compared with those of the same stems before surgical manipulation. On average, nodal spring constants were reduced by 35%. This reduction agreed with the prediction that the perforation of septa would significantly reduce the ability of nodes to store strain energy. Collectively, these results are interpreted to support the hypothesis that septate nodes can store and release strain energy. The hypothesis is discussed further in light of the behaviour of a physical model which shows that nodal ‘diaphragms’ can substantially stiffen a hollow cylindrical structure, although they are neither essential for the storage of strain energy nor the subsequent elastic restoration of the model's shape once bending loads are removed.
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Dry matter digestibility is one of the most important characteristics of forage. The major constraint on ruminant digestion of forage cell walls is lignin. Sequences of cDNA encoding a key lignin biosynthetic enzyme, caffeic acid O-methyltransferase (COMT), was cloned from the widely grown monocot forage species tall fescue (Festuca arundinacea Schreb.). Enzymatic properties of recombinant COMT protein expressed in E. coli were determined using six substrates. The preferred substrates for tall fescue recombinant COMT were 5-hydroxyferulic acid and caffeoyl aldehyde. Transgenic tall fescue plants carrying either sense or antisense COMT gene constructs were obtained by microprojectile bombardment of single-genotype-derived embryogenic suspension cells. Consistent and closely related molecular and biochemical data demonstrated that two co-suppressed transgenic lines were down-regulated in their lignin biosynthesis. These COMT down-regulated transgenic tall fescue plants showed substantially reduced levels of transcripts, significantly reduced enzymatic activities, significantly decreased lignin content, apparently altered lignin composition and significantly increased (9.8-10.8%) digestibility.
Article
Lignin is a complex, aromatic polymer that limits plant cell wall degradation by ruminants and reduces the nutritional value of forages. Genetic engineering, using an antisense strategy, offers the potential to modulate enzymes hi the lignin biosynthetic pathway as a way to reduce lignin, thereby improving forage quality and animal performance. We investigated the effectiveness of expressing antisense sorghum O-methyltransferase gene (omt) to downregulate maize OMT and reduce lignin. Constructs contained a sorghum omt coding region hi the antisense orientation driven by the maize ubiquitin-1 (Ubi) promoter (with the first intron and exon) along with bar, that confers glufosmate herbicide resistance, driven by the CaMV 35S promoter. Twenty-eight T0 plants regenerated from 17 herbicide-resistant callus lines from 13 independent bombardments expressed the brown midrib phenotype. O-methyltransferase activity was significantly lower in T1 transgenics compared with controls, with some plants showing a 60% reduction. Those T1 transgenics with downregulated OMT averaged 20% less lignin in stems and 12% less lignin in leaves compared with controls. On a whole-plant basis, lignin was reduced by an average of 17% with the greatest reduction being 31%. Digestibility was significantly improved in transgenic plants by 2% in leaves and 7% in stems. Mean whole-plant digestibility increased from 72 to 76%. This research demonstrates that genetic engineering has the potential to improve forage grass digestibility. This could be important, especially in tropical forage species, which generally have lower quality than temperate species.
Article
Lignin distribution during formation of latewood tracheids in Pinus radiata, was determined by quantitative interference microscopy, and by potassium permanganate staining combined with transmission electron microscopy. Lignin distribution varied among trees sampled on the same date in late winter. In one tree, latewood tracheids were fully lignified up to the growth ring boundary. However in most trees sampled, latewood was only partially lignified. The extent of lignification varied from tree to tree but in all cases, at least some lignin was present in the middle lamella and primary wall at the growth ring boundary. Latewood was ideal for examining the lignification process because of the large number of different stages present in a single specimen.
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A phloroglucinol-HCl assay for lignin, which detects aldehyde groups, was performed on tissue of normal and brown midrib 6 (bmr-6) sorghum (Sorghum bicolor L. Moench). The color formed in this lignin assay was greater in the brown midrib mutant 6 than in the normal tissue. The mutant lignin contained ∼3 times more aldehyde groups than the normal lignin, but there was less lignin in the mutant than in the normal. Apparently, the mutation caused an accumulation of aldehyde lignin intermediates which were incorporated into the lignin polymer. The enzyme activity which catalyzed the reduction of aldehyde lignin intermediates, cinnamyl alcohol:NADPH oxidoreductase, was less in brown midrib mutant 6 than in normal tissue [i.e., 0.24 and 1.42 μmol min-1 (mg of protein)-1, respectively].
Article
Lignin, a cell wall component, limits digestibility of plant cell walls. Brown midrib (bmr) mutants of forages have lignin with altered chemical composition compared with their normal counterparts. The objectives of this study were to determine if bmr lignin is more inhibitory to digestion than is normal lignin and if bmr has a consistent effect on rate of digestion across species and environments. Extent and rate of in-vitro cell wall digestion of normal and bmr stems of sorghum (Sorghum bicolor (L) Moench, two comparisons), millet (Pennisetum americanum (L) Leeke) and maize (Zea mays L, two comparisons) were determined. Samples were incubated in rumen fluid, and data were fitted with a first-order, nonlinear model to estimate concentrations of potentially digestible neutral detergent fibre (PDNDF), digestion rate of PDNDF, concentration of indigestible residue (IR), and lag time before digestion. The NDF, acid-detergent fibre (ADF), and acid-detergent lignin (ADL) analyses were conducted sequentially on undigested samples. The IR: ADL ratio was 37% greater for bmr than for normal plants, which indicates that bmr lignin inhibits digestion more than normal lignin per unit of lignin. Digestion rate of PDNDF was faster in bmr than in normal counterparts in one of the two sorghum comparisons (difference of 59%) and in the millet comparison (difference of 27%), but in neither maize comparison. The bmr mutants were lower than normal genotypes in NDF (9%) and ADL (47%) concentrations. The PDNDF concentration was 19% greater for bmr than for normal lines. Thus, decreased lignin concentration in bmr mutants increased the extent of NDF digestion but did not consistently increase the rate of digestion.
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It is shown that a large proportion of the p-hydroxyphenylpropane moiety of grass lignin can be ascribed to p-coumaric acid esterified with the lignin. There may therefore be little fundamental difference between the polymeric systems of grass and hardwood lignins, although a small amount of p-hydroxyphenyl-glycerol-β-aryl ether structure is contained in the former. The results of incorporation of tyrosine-G-14C indicate that tyrosine is utilized for the synthesis of both the monomers of the grass lignin and the esterified p-coumaric acid by action of tyrosine-ammonia lyase.
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An enzyme was found which catalyzes the deamination of l-tyrosine, giving equimolar amounts of trans-p-coumaric acid and ammonia as the products. This enzyme (tyrase) was readily detected in sorghum, barley, rice, wheat, oat, corn and sugar cane plants; but not in pea, lupine, alfalfa or white sweet clover plants, or in yeast. Tyrase was concentrated in the stems of barley rather than the leaves, and reached its maximum concentration at about the time the heads were emerging. The crude, soluble protein extracted from an acetone powder of barley stems was purified about forty-fold with respect to tyrase. Tyrase preparations from this source were also found to convert dl-m-tyrosine to m-coumaric acid and ammonia, and have been shown by Koukol and Conn to contain an enzyme (phenylalanase) which can catalyze the conversion of l-phenylalanine to cinnamic acid and ammonia. The data suggest that tyrase is distinct from the enzymes (or enzyme) catalyzing the deaminations of phenylalanine and m-tyrosine.
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Gene co-expression analysis has emerged in the past 5 years as a powerful tool for gene function prediction. In essence, co-expression analysis asks the question ‘what are the genes that are co-expressed, that is, those that show similar expression profiles across many experiments, with my gene of interest?’. Genes that are highly co-expressed may be involved in the biological process or processes of the query gene. This review describes the tools that are available for performing such analyses, how each of these perform, and also discusses statistical issues including how normalization of gene expression data can influence co-expression results, calculation of co-expression scores and P values, and the influence of data sets used for co-expression analysis. Finally, examples from the literature will be presented, wherein co-expression has been used to corroborate and discover various aspects of plant biology.
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After a successful career at the University of São Paulo, Marcos Jank became the President and CEO of the Brazilian Sugarcane Industry Association (UNICA) in July 2007. He was hired with a mandate to establish ethanol as a global commodity and to open new markets for the industry's sugar, ethanol and bioelectricity output. But he faced complex challenges. The main challenge related to the role of UNICA in leading industry-wide sustainability initiatives. This required coordination of 70,000 sugarcane producers and 430 processors; engagement with outside stakeholders in Brazil and abroad; and implementing programs that balanced economic, social and environmental outcomes. A second set of challenges emanated from the rapid growth and dramatic structural changes occurring in the industry. This case study describes UNICA's unique approach to sustainability and how it is changing the industry, allowing the reader to analyze the effectiveness of this approach in delivering sustainability. © 2010 International Food and Agribusiness Management Association (IFAMA).
Article
The lignification process in different morphological regions of loblolly pine tracheids was studied by the SEM-EDXA technique. Prior to S2 layer formation, lignification was initiated in the cell corner middle lamella and compound middle lamella regions. Subsequently a rapid lignin deposition was observed in both regions, whereas secondary wall lignification was a more gradual process and initiated when the middle lamella lignin concentration was approximately 50% of maximum. Within the secondary wall, the S1 layer is lignified first. Then, lagging just behind cell wall formation, lignification of the S2 layer is initiated adjacent to the S1 layer and extends toward the lumen. Finally, the S3 layer lignified. Upon completion of lignification, the cell walls had a higher concentration of lignin in both the S1 and S3 layers than in the S2 layer.
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The European corn borer (ECB, Ostrinia nubilalis Hübner) is a major pest of maize in Central Europe. We mapped and characterized quantitative trait loci (QTLs) involved in resistance of maize against ECB damage, compared them with QTLs for agronomic traits, and evaluated the usefulness of marker-assisted selection (MAS) for improving ECB resistance in early maturing European maize germplasm. A total 226 F3 families from the cross D06 (resistant) × D408 (susceptible), together with 93 RFLP and two SSR markers were used for the QTL analyses. For each F3 family we measured the length of tunnels produced by larval stalk mining (TL), stalk damage ratings (SDR), and relative grain yield (RGY) in field experiments, with two replications in two environments in 1 year. The agronomic traits comprised grain yield under insecticide protection (GYP) and manual ECB larval infestation (GYI), the date of anthesis (ANT), and the in vitro digestibility of organic matter (IVDOM) of stover. Estimates of genotypic variance (σ2 g) were highly significant for all traits. Six QTLs for TL and five QTLs for SDR were detected, explaining about 50.0% of σ2 g. Most QTLs showed additive gene action for TL and dominance for SDR. No QTL was found for RGY. The number of QTLs detected for the agronomic traits ranged from two for GYI to 12 for ANT, explaining 12.5 to 57.3% of σ2 g, respectively. Only a single QTL was in common between the two resistance traits, as expected from the moderate trait correlation and the moderate proportions of σ2 g explained. Based on these results, MAS for improving ECB resistance can be competitive when cost-effective PCR-based marker systems are applied. However, it remains to be established whether the putative QTL regions for ECB resistance detected in the population D06 × D408 are consistent across other early maturing European maize germplasms.
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Lignin distribution in developing tracheids of Pinus radiata was studied throughout the growth' season using quantitative interference microscopy. The pattern of lignification remained constant although the number of lignifying cells varied reaching a maximum in summer. Lignification of the secondary wall of latewood tracheids was incomplete at the onset of winter. Each stage of lignification was preceded by deposition of carbohydrates with lignification of the middle lamella starting after S1 formation and lignification of the secondary wall starting after S3 formation. Lignification of the middle lamella was completed before the start of lignin deposition in the secondary wall. In one of the trees examined, the secondary wall lignified concurrently with the middle lamella and this was associated with a low lignin concentration in the middle lamella of mature cells. The secondary wall reached a mature lignin concentration of 21–22% v/v except in one specimen containing severe compression wood which reached 28% v/v. The cell corner middle lamella reached a mature lignin concentration of 74–87% v/v.
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Lignin biosynthesis via shikimate-cinnamate pathways in plants, and the biosynthetic differences of guaiacyl-and syringyl lignins between gymnosperms and angiosperms have been elucidated by tracer experiments using 14C labeled precursors and the following enzyme reactions. The formation of guaiacyl lignin but not syringyl lignin in gymnosperms was attributed to the following factors; absence of ferulate-5-hydroxylase, poor affinity of O-methyltransferase toward 5-hydroxyferulate, and lack of activation and/or reduction of sinapatc. A mechanism of lignin-carbohydrate complexes formation in wood cell walls was elucidated based on the reaction of the quinone methide of guaiacylglycerol--guaiacyl ether with sugars, and the analysis of DHP-polysaccharide complexes.The main cleavage mechanisms of side chains and aromatic rings of lignin model compounds and synthetic lignin (DHP) by white-rot fungi and their enzymes, lignin peroxidase and laccase have been elucidated using 2H, 13C and 18O-labeled lignin substructure dimcrs with 18O2 and H2 18O. Side chains and aromatic rings of these substrates were cleaved via aryl cation radical and phenoxy radical intermediates, in reaction mediated only by lignin peroxidase/H2O2 and laccase/O2.
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The caffeic acid O-methyltransferase (COMT) gene plays an important role in the synthesis of lignin. We have used the polymerase chain reaction in conjuction with genomic analysis to characterize deletion mutations of this gene in maize. In addition, we have analyzed and compared regions of the COMT gene from three distinct heterotic groups. Both PCR and Southern analysis indicate that the active wild-type COMT gene can be polymorphic. We suggest that the intron domain of at least one heterotic inbred can contribute to the alteration of the wild-type gene. In addition, multiple deletion mutations have occurred at this locus. We have found a previously uncharacterized deletion mutation in which segments of both the intron and exon have been deleted and replaced by other sequences. Precise knowledge of its sequence has allowed us to develop an assay by which we can follow this mutation in a breeding program.
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With 2 tables Brown midrib (bm) mutations are known to affect cell-wall digestibility by altering the quantity and composition of lignins in cell walls, resulting in higher ethanol yield and increased cell-wall digestibility. So far, four bm genes (bm1, bm2, bm3 and bm4) were identified and mapped in maize, the last one (bm4) in 1947. In this study, 13 spontaneous mutations (bm*A–M) resulting in the appearance of brown midribs were crossed with bm1–4 for tests of allelism. From these tests, we report two new bm mutants bm5 (bm*F) and bm6 (bm*J) while other bm* lines were either found allelic to bm1–4 or to one of the bm* lines.