Allelic Association, Chemical Characterization and Saccharification Properties of brown midrib Mutants of Sorghum (Sorghum bicolor (L.) Moench)

Genetic improvement of biomass crops can significantly reduce the overall cost of biomass-to-ethanol conversion. The conversion of cellulose to monomeric sugar units is affected by lignin content and composition. Sorghum has attracted the attention of the scientific and industrial community as a promising source of biomass for bioenergy due to its great yield potential and tolerance to stresses. The brown midrib (bmr) mutants of sorghum are characterized by brown vascular tissue associated with altered lignin content. Twenty-eight bmr mutants have been identified since the late 1970s, but the allelic relationships have not been fully established, and the function of only one of the Bmr loci has been unequivocally established. In this study, we combined genetic and chemical approaches to establish that there are mutations at least four independent bmr loci, represented by the bmr2, bmr6, bmr12 and bmr19 groups. Since each allelic group presents unique staining characteristics, rapid classification of emerging bmr lines into the existing groups can be achieved using phloroglucinol-HCl as a histochemical stain. In addition, pyrolysis-gas chromatography-mass spectrometry, enabled the characterization of changes in subunit lignin composition in each of the allelic groups, to help predict the genes underlying the mutations. Enzymatic saccharification of stover from plants representing each allelic bmr group demonstrated that lignin changes in lines belonging to the bmr2, bmr6 and bmr12 groups can increase glucose yields, up to 25% compared to wild-type isolines. In order to expedite the selection of the bmr mutant alleles in breeding populations, we have developed molecular markers specific for bmr7 and bmr25, two novel mutant alleles of the gene encoding caffeic acid O-methyl transferase. Based on the results from this study, we propose to rename the bmr mutants in a manner that reflects the number of independent loci.     

Identification and Characterization of Four Missense Mutations in Brown midrib 12 (Bmr12), the Caffeic O-Methyltranferase (COMT) of Sorghum

Modifying lignin content and composition are targets to improve bioenergy crops for cellulosic conversion to biofuels. In sorghum and other C4 grasses, the brown midrib mutants have been shown to reduce lignin content and alter its composition. Bmr12 encodes the sorghum caffeic O-methyltransferase, which catalyzes the penultimate step in monolignol biosynthesis. From an EMS-mutagenized TILLING population, four bmr12 mutants were isolated. DNA sequencing identified the four missense mutations in the Bmr12 coding region, which changed evolutionarily conserved amino acids Ala71Val, Pro150Leu, Gly225Asp, and Gly325Ser. The previously characterized bmr12 mutants all contain premature stop codons. These newly identified mutants, along with the previously characterized bmr12-ref, represent the first allelic series of bmr12 mutants available in the same genetic background. The impacts of these newly identified mutations on protein accumulation, enzyme activity, Klason lignin content, lignin subunit composition, and saccharification yield were determined. Gly225Asp mutant greatly reduced protein accumulation, and Pro150Leu and Gly325Ser greatly impaired enzyme activity compared to wild type (WT). All four mutants significantly reduced Klason lignin content and altered lignin composition resulting in a significantly reduced S/G ratio relative to WT, but the overall impact of these mutations was less severe than bmr12-ref. Except for Gly325Ser, which is a hypomorphic mutant, all mutants increased the saccharification yield relative to WT. These mutants represent new tools to decrease lignin content and S/G ratio, possibly leading toward the ability to tailor lignin content and composition in the bioenergy grass sorghum.     

A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.     

Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents

Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.     

Biomass and Cellulosic Ethanol Production of Forage Sorghum Under Limited Water Conditions

This study presents results from a 2-year evaluation of biomass and cellulosic ethanol (EtOH) production potential of forage sorghum (Sorghum bicolor L. Moench) cultivars differing in brown midrib trait (i.e., bmr12) under dryland (no irrigation) and limited irrigation (2.88 mm?day^?1; subsurface drip) in the semiarid Southern High Plains of the USA. Commercial cultivar Sorghum Partners 1990 (SP 1990, conventional non-bmr) produced significantly more biomass (29–62 %) than a bmr12 cultivar PaceSetter bmr (PS bmr) under irrigated and dryland conditions during both years of this study. However, PS bmr biomass had higher cellulosic EtOH conversion efficiency than SP 1990 in both years according to simultaneous saccharification and fermentation analysis. Irrigation resulted in 26–49 % more biomass and 28–72 % more cellulosic EtOH production during both growing seasons, indicating that limited irrigation had favorable effects on both biomass and biofuel production. In the first year, when precipitation was below average, both cultivars produced similar amounts of cellulosic EtOH. During the second year, when precipitation was above average, higher biomass production of SP 1990 resulted in 28 % higher cellulosic EtOH production than PS bmr when averaged across both irrigated and dryland conditions. The large range of cellulosic EtOH production (1,600 to 3,380 L?ha^?1) during the 2 years of this study was primarily driven by differences in water availability that resulted from precipitation and irrigation. Our findings indicates that chemical composition and biomass yield potential of sorghum cultivars are critical factors that affect biomass and biofuel production under limited water conditions.     

Litter decomposition of three lignin-deficient mutants of Sorghum bicolor during spring thaw

Decomposition of plant litter during the freeze-thaw season has recently gained attention as having a significant role in nutrient cycling in many cold ecosystems. However, few studies have examined decomposition of crop remnants during the freeze-thaw season in an agronomic setting when microbial activity is presumably low. We examined decomposition of four cultivars of sorghum (Sorghum bicolor) leaves in a field in Southern Minnesota, USA using the litterbag method. Three of the four cultivars we examined expressed the brown midrib (bmr) mutation which have altered/reduced levels of lignin in their secondary cell walls compared to the wild-type (WT). Litter was buried in the fall and harvested during the spring thaw. After 160 d the bmr mutants lost 57–62% of their initial mass, compared to 51% in the WT. Mass loss agreed with presumed initial litter quality, as the bmr litter had higher initial N, and holocellulose:lignin and lower lignin, C:N and lignin:N values compared to the WT. The increased decomposition of the bmr cultivars appears to be related to increased loss of hemicellulose and holocellulose (cellulose+hemicellulose) or higher initial N concentrations. Alterations in cell-wall deposition in the bmr cultivars may increase accessibility of microbial cell-wall degrading enzymes that accelerate mass loss. Our results demonstrate that alterations in initial lignin chemistry may influence decomposition of sorghum litter in an agronomic setting.     

A candidate-gene approach to clone the sorghum <Emphasis Type="Italic">Brown midrib </Emphasis>gene encoding caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The brown midrib (bmr) mutants of sorghum have brown vascular tissue in the leaves and stem as a result of changes in lignin composition. The bmr mutants were generated via chemical mutagenesis with diethyl sulfate (DES) and resemble the brown midrib (bm) mutants of maize. The maize and sorghum brown midrib mutants are of particular value for the comparison of lignin biosynthesis across different, yet evolutionarily related, species. Although the sorghum brown midrib mutants were first described in 1978, none of the Brown midrib genes have been cloned. We have used a candidate-gene approach to clone the first Brown midrib gene from sorghum. Based on chemical analyses of the allelic mutants bmr12, bmr18 and bmr26, we hypothesized that these mutants had reduced activity of the lignin biosynthetic enzyme caffeic acid O-methyltransferase (COMT). After a northern analysis revealed strongly reduced expression of the COMT gene, the gene was cloned from the mutants and the corresponding wild types using PCR. In all three mutants, point mutations resulting in premature stop codons were identified: bmr12, bmr18 and bmr26 are therefore mutant alleles of the gene encoding COMT. RT-PCR indicated that all three mutants express the mutant allele, but at much lower levels relative to the wild-type controls. Molecular markers were developed for each of the three mutant alleles to facilitate the use of these mutant alleles in genetic studies and breeding programs.     

Improved sugar yields from biomass sorghum feedstocks: comparing low-lignin mutants and pretreatment chemistries

Background

For biofuel production processes to be economically efficient, it is essential to maximize the production of monomeric carbohydrates from the structural carbohydrates of feedstocks. One strategy for maximizing carbohydrate production is to identify less recalcitrant feedstock cultivars by performing some type of experimental screening on a large and diverse set of candidate materials, or by identifying genetic modifications (random or directed mutations or transgenic plants) that provide decreased recalcitrance. Economic efficiency can also be increased using additional pretreatment processes such as deacetylation, which uses dilute NaOH to remove the acetyl groups of hemicellulose prior to dilute acid pretreatment. In this work, we used a laboratory-scale screening tool that mimics relevant thermochemical pretreatment conditions to compare the total sugar yield of three near-isogenic brown midrib (bmr) mutant lines and the wild-type (WT) sorghum cultivar. We then compared results obtained from the laboratory-scale screening pretreatment assay to a large-scale pretreatment system.

Results

After pretreatment and enzymatic hydrolysis, the bmr mutants had higher total sugar yields than the WT sorghum cultivar. Increased pretreatment temperatures increased reactivity for all sorghum samples reducing the differences observed at lower reaction temperatures. Deacetylation prior to dilute acid pretreatment increased the total sugar yield for all four sorghum samples, and reduced the differences in total sugar yields among them, but solubilized a sizable fraction of the non-structural carbohydrates. The general trends of increased total sugar yield in the bmr mutant compared to the WT seen at the laboratory scale were observed at the large-scale system. However, in the larger reactor system, the measured total sugar yields were lower and the difference in total sugar yield between the WT and bmr sorghum was larger.

Conclusions

Sorghum bmr mutants, which have a reduced lignin content showed higher total sugar yields than the WT cultivar after dilute acid pretreatment and enzymatic hydrolysis. Deacetylation prior to dilute acid pretreatment increased the total sugar yield for all four sorghum samples. However, since deacetylation also solubilizes a large fraction of the non-structural carbohydrates, the ability to derive value from these solubilized sugars will depend greatly on the proposed conversion process.

     

Characterization of novel Brown midrib 6 mutations affecting lignin biosynthesis in sorghum

The presence of lignin reduces the quality of lignocellulosic biomass for forage materials and feedstock for biofuels. In C4 grasses,the brown midrib phenotype has been linked to mutations to genes in the monolignol biosynthesis pathway. For example,the Bmr6 gene in sorghum(Sorghum bicolor) has been previously shown to encode cinnamyl alcohol dehydrogenase(CAD),which catalyzes the final step of the monolignol biosynthesis pathway. Mutations in this gene have been shown to reduce the abundance of lignin,enhance digestibility,and improve saccharification efficiencies and ethanol yields. Nine sorghum lines harboring five different bmr6 alleles were identified in an EMS-mutagenized TILLING population. DNA sequencing of Bmr6 revealed that the majority of the mutations impacted evolutionarily conserved amino acids while three-dimensional structural modeling predicted that all of these alleles interfered with the enzyme's ability to bind with its NADPH cofactor. All of the new alleles reduced in vitro CAD activity levels and enhanced glucose yields following saccharification. Further,many of these lines were associated with higher reductions in acid detergent lignin compared to lines harboring the previously characterized bmr6-ref allele. These bmr6 lines represent new breeding tools for manipulating biomass composition to enhance forage and feedstock quality.     

A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype

brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele.Plant cell walls constitute a vast reserve of fixed carbon. Cellulose and lignin are the first and second most abundant polymers on the planet, respectively (Jung and Ni, 1998). The world community has started to look to biomass as substrates for plant-based biologically sustainable fuels, which would mitigate carbon dioxide emission and reduce petroleum dependence (Sarath et al., 2008; Schmer et al., 2008). In the current generation of biofuels, ethanol is being synthesized via the fermentation of grain starch or sugarcane juice. For the next generation of biofuels, research is being directed toward the conversion of lignocellulosic biomass into biofuels (Chang, 2007). As bioenergy technologies progress, the conversion of biomass to biofuels could involve a range of chemical, biochemical, and fermentation processes to produce biofuels; alternate biofuels, such as butanol or dimethylfuran, are also on the horizon (Ezeji et al., 2007; Roman-Leshkov et al., 2007). Most liquid biofuel production processes will likely rely on the conversion of the cell wall polysaccharides cellulose and hemicellulose into monomeric sugars.Plant cell walls consist of a complex polysaccharide moiety composed of cellulose microfibrils, composed of β-1,4-linked Glc polymers (Carpita and McCann, 2000). Connecting the cellulose microfibrils to each other is a hemicellulose network, whose structure and composition are species dependent, and which is mainly composed of glucuronoarabinoxylans in grasses (Carpita and McCann, 2000). Lignin, a nonlinear heterogeneous polymer derived from aromatic precursors, cross-links these polysaccharides, rigidifying and reinforcing the cell wall structure (Carpita and McCann, 2000). The addition of lignin polymers to the polysaccharide matrix creates a barrier that is chemically and microbially resistant.Lignin can block the liberation of sugars from the cell wall polysaccharide moieties, release compounds that can inhibit microbes used for fermenting sugars to fuels, and adhere to hydrolytic enzymes. Understanding lignin synthesis, structure, and function to increase cell wall digestibility has long been a goal for forage improvement and paper processing (Mackay et al., 1997; Jung and Ni, 1998). Recently, manipulating lignin has also become an important target for bioenergy feedstock improvement (Chen and Dixon, 2007; Li et al., 2008).Lignin is derived from the phenylpropanoid pathway and contains primarily three types of phenolic subunits: p-hydroxyphenyl, guaiacyl, and syringyl units (Dixon et al., 2001). The phenolic aldehyde precursors are reduced into their corresponding alcohols (monolignols) and subsequently transported to the cell wall (Fig. 1), where laccases and peroxidases catalyze lignin polymerization through the formation of monolignol radicals (Boerjan et al., 2003). Therefore, most research efforts to manipulate lignin have focused on biosynthesis of the monolignols. Most of the enzymes involved in monolignol synthesis have been cloned and characterized in Arabidopsis (Arabidopsis thaliana) and other dicot species, using both mutagenic and transgenic approaches to study the impact of these gene products on dicot cell walls (Anterola and Lewis, 2002). However, there are significant differences in the architecture, polysaccharide composition, and phenylpropanoid composition of grass cell walls compared with those of dicots (Carpita and McCann, 2000; Vogel and Jung, 2001). For example, grasses contain significant amounts of p-coumaric acid and ferulic acid that are cross-linked to cell wall polysaccharides through ester and ether linkages in addition to their presence in lignin (Grabber et al., 1991; Boerjan et al., 2003). Because many of the proposed dedicated bioenergy crops are grasses, there is a need to identify and understand the function of the gene products involved in lignin biosynthesis in these species (Vermerris et al., 2007; Li et al., 2008; Sarath et al., 2008).Open in a separate windowFigure 1.The CAD enzyme and its role in the monolignol biosynthetic pathway. A, CAD catalyzes the conversion of cinnamyl aldehydes to alcohols using NADPH as its cofactor. p-Coumaryl aldehyde and alcohol, R₁ and R₂ = H; caffeoyl aldehyde and alcohol, R₁ and R₂ = OH; coniferyl aldehyde and alcohol, R₁ = H and R₂ = OCH₃; sinapyl aldehyde and alcohol, R₁ and R₂ = OCH₃. B, A simplified model of the lignin biosynthetic pathway where CAD catalyzes the final step in monolignol biosynthesis.The brown midrib phenotype has been useful for identifying mutants affecting lignin synthesis in grasses because it is a visible phenotype. Spontaneous brown midrib mutants were first discovered in maize (Zea mays; Jorgenson, 1931) and were subsequently generated in sorghum (Sorghum bicolor) using diethyl sulfate mutagenesis (Porter et al., 1978). Brown midrib mutants in maize, sorghum, and pearl millet (Pennisetum glaucum) have increased forage digestibility for livestock (Cherney et al., 1990; Akin et al., 1993; Jung et al., 1998; Oliver et al., 2004). In maize and sorghum, there are at least four brown midrib loci in their respective genomes (Jorgenson, 1931; Porter et al., 1978; Gupta, 1995). The genes encoding bm3 in maize and bmr12 in sorghum are the only loci cloned to date, and both encode highly similar caffeic acid O-methyl transferases (Vignols et al., 1995; Bout and Vermerris, 2003). A second brown midrib locus associated with reduced cinnamyl alcohol dehydrogenase (CAD) activity has been identified both in maize (bm1; Halpin et al., 1998) and sorghum (bmr6; Bucholtz et al., 1980; Pillonel et al., 1991). CAD is a member of the alcohol dehydrogenase superfamily of proteins that catalyzes the conversion of the hydroxycinnamoyl aldehydes into alcohols prior to their incorporation into lignin polymers (Fig. 1). Reduced CAD activity results in increased digestibility on dry weight basis, altered cell wall architecture, reduced lignin level, and the incorporation of phenolic aldehydes into lignin in sorghum and maize (Pillonel et al., 1991; Provan et al., 1997; Halpin et al., 1998; Marita et al., 2003; Shi et al., 2006; Palmer et al., 2008). The reduced CAD activity in bm1 has been genetically mapped to a region of the maize genome that contained a CAD gene, ZmCAD2 (Halpin et al., 1998), but a mutation was not identified. However, it has recently been shown that bm1 down-regulated the expression of several lignin biosynthetic genes, suggesting its gene product may be a regulatory protein (Shi et al., 2006; Guillaumie et al., 2007).To identify the mutation responsible for the bmr6 phenotype and to characterize how bmr6 impacts the lignin biosynthetic pathway, a candidate gene approach was taken. Here, we describe the cloning and characterization of Bmr6 and a related protein, SbCAD4. The identification and characterization of Bmr6 has revealed the major monolignol CAD protein in the grasses, which is likely to aid the development of new strategies to increase conversion of sorghum and other grass feedstocks to biofuels.     

RNAi‐suppression of barley caffeic acid O‐methyltransferase modifies lignin despite redundancy in the gene family

Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.     

Genetic background impacts soluble and cell wall-bound aromatics in <Emphasis Type="Italic">brown midrib</Emphasis> mutants of sorghum

Sorghum (Sorghum bicolor (L.). Moench) BMR-6 and BMR-12 encode cinnamylalcohol dehydrogenase and caffeic acid-O-methyltransferase, respectively. We have evaluated the impact of two bmr alleles, bmr-6 and bmr-12, respectively, on soluble and wall-bound aromatics in near isogenic, wild-type (WT), bmr-6, bmr-12 and double-mutant (DM; bmr-6 and bmr-12) plants in two genetic backgrounds, RTx430 and Wheatland. Immunoblots confirmed that COMT protein was essentially absent in bmr-12 and DM plants, but was present in bmr-6 and WT plants. In contrast, although CAD activity was not detected in bmr-6 and DM plants, proteins crossreacting to anti-CAD sera were found in stem extracts from all genotypes. In both sorghum backgrounds, WT plants had lowest amounts of free aromatics, higher levels of cell wall-bound pCA and FA esters and guaiacyl (G), syringyl (S), and p-hydroxyphenyl (H) lignins. Soluble aromatics and cell wall phenolic ester content in Wheatland DM plants resembled that of Wheatland bmr-6 plants, whereas in the RTx430 background, levels of these components in the DM plants more closely resembled those observed in bmr-12 plants. In both backgrounds, bmr-6 plants exhibited reduced levels of G, S, and H lignins relative to WT, and increased incorporation of G-indene into lignin. In bmr-12 plants, there was greater incorporation of G- and 5-hydroxyguaiacyl (5-OHG) lignin into cell walls. Histochemical staining of internode sections from Wheatland plants indicated that apparent lignification of cortical sclerenchyma and vascular bundle fibers was greatest and most uniform in WT plants. Relative staining intensity of these tissues was decreased in bmr-6, followed by bmr-12 plants. DM plants exhibited poor staining of cortical sclerenchyma and vascular bundle fibers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.     

Produced by a Strain of Unidentified Actinomycetes sp.

The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.     

Nucleotide diversity and linkage disequilibrium in Populus nigra cinnamyl alcohol dehydrogenase (CAD4) gene

Cinnamyl alcohol dehydrogenase (CAD) is involved in the biosynthesis of lignin, a component of plant cell wall which negatively impacts paper pulp processing and biomass fermentation to ethanol. Transgenic poplars with depressed CAD activity show structural alterations of lignin. Natural CAD mutants have been identified in several plants; however, no natural CAD mutants have been identified in poplar. We surveyed the natural genetic variation in CAD4, a gene coding for CAD, in 360 poplar trees from Western Europe. We measured linkage disequilibrium (LD) between single-nucleotide polymorphisms (SNPs), performed neutrality tests and estimated diversity indexes, and investigated their dependence from sample size. We identified 45 SNPs, six of which caused an amino acid substitution. Our results suggest a short span of LD in Populus nigra CAD4 gene. We identified carriers of different nonsynonymous SNPs in CAD4; those subjects are candidate to be used in classical breeding programs to obtain carriers of different combinations of functional polymorphisms. We showed that use of small sample size might lead to biased estimates of LD, neutrality tests, and diversity indexes.     

Catechol-O-methyltransferase: Effects of the val108met polymorphism on protein turnover in human cells

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT^H and COMT^L, respectively. Tissues and cells with a COMT^LL genotype have decreased COMT activity compared to COMT^HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT^L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT^LL compared to COMT^HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT^H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT^L compared to S-COMT^H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT^LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT^L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.