The maize Brown midrib1 locus affects cell wall composition and plant development in a dose-dependent manner

The four brown midrib (bm) mutants of maize have a reduced content and altered subunit composition of the cell wall polymer lignin. The bm mutations have traditionally been considered completely recessive, because the brown midrib phenotype is only apparent in plants homozygous for the mutation. In addition to an effect on cell wall composition, some bm mutations have been shown to affect flowering time. We had preliminary evidence for a dosage effect of the Bm1 locus on flowering time, which prompted this detailed study on the Bm1 locus. In this study, near-isogenic lines (in an A619 background) with zero, one or two bm1 mutant alleles were compared. The bm1 heterozygotes flowered significantly earlier than both the wild-type plants and bm1 mutants. This difference can at least be partly attributed to an accelerated growth rate in the later stages of plant development. Furthermore, Fourier transform infrared spectroscopy revealed that the cell wall composition of the bm1 heterozygous plants is distinct from both the bm1 and wild-type homozygotes. The combination of the data on flowering time and the data on cell wall composition provide evidence for a dosage effect at the Bm1 locus.     

Genotypic covariations of traits underlying sorghum stem biomass production and quality and their regulations by water availability: Insight from studies at organ and tissue levels

Sweet and biomass sorghum are expected to contribute increasingly to bioenergy production. Better understanding the impacts of the genotypic and environmental variabilities on biomass component traits and their properties is essential to optimize energy yields. This study aimed to evaluate whether traits contributing to stem biomass growth and biochemical composition at different biological scales (co)vary with the genotype and the water status in sorghum. Height genotypes were studied over two years in field conditions in southern France under two water treatments (well watered vs. 25 days’ dry down during stem elongation). Main stem internode number, size, (non)structural carbohydrate, and lignin contents were measured at the end of the stress period and/or at final harvest, together with biochemical and histological analyses of the youngest expanded internode. The tallest genotypes showed the highest stem dry weights and lignin contents. Stem (structural) biomass density was positively correlated with lignin content, particularly in internode parenchyma. Stem soluble sugar and lignin contents were inversely proportional across genotypes and water conditions. Genotypes contrasted for drought sensitivity and recovery capacity of stem growth and biochemical composition. The length and cell wall deposition of internodes expanding under water deficit were reduced and did not recover, these responses being weakly correlated. Genotypic variability was pointed out in the growth recovery of internodes expanding under re‐watered conditions. According to the observed genotypic variability and the absence of antagonistic correlations between the responses of the different traits to water availability, it is suggested that biomass sorghum varieties optimizing their responses to water availability in terms of growth and cell wall deposition can be developed for different bioenergy targets.     

Expression of cell wall related genes in basal and ear internodes of silking <Emphasis Type="Italic">brown-midrib-3</Emphasis>, caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase (COMT) down-regulated,and normal maize plants

Background  

Silage maize is a major forage and energy resource for cattle feeding, and several studies have shown that lignin content and structure are the determining factors in forage maize feeding value. In maize, four natural brown-midrib mutants have modified lignin content, lignin structure and cell wall digestibility. The greatest lignin reduction and the highest cell wall digestibility were observed in the brown-midrib-3 (bm3) mutant, which is disrupted in the caffeic acid O-methyltransferase (COMT) gene.     

Colocalizations Between Several QTLs for Cell Wall Degradability and Composition in the F288 × F271 Early Maize RIL Progeny Raise the Question of the Nature of the Possible Underlying Determinants and Breeding Targets for Biofuel Capacity

Understanding the genetic basis of the different traits which contribute to a given value of cell wall degradability is a key issue towards the breeding of grasses with higher feeding value or higher capability for bioenergy production. A quantitative trait loci (QTL) investigation for cell wall degradability and several cell wall component traits including lignin content, p-hydroxycinnamic acid content, and lignin monomeric structure was thus done with a simultaneous search for underlying candidate genes in the F288?×?F271 recombinant inbred line progeny. Genotype effects were highly significant for all cell wall investigated traits (P?<?0.001) and much higher than genotype?×?environment interaction effects. Out of 42 QTLs mapped, 11 and 23 QTLs explained more than 20 and 15 % of the observed trait phenotypic variation, respectively. Twenty-three QTLs were gathered into four large clusters shown in bins 3.06, 4.09, 6.05, and 6.07. Colocalizations of cell wall degradability QTLs occurred with lignin content QTLs and lignin structure QTLs. Moreover, for two positions, there were also colocalizations with etherified ferulic acid QTLs. Such simultaneous colocalizations between QTLs for cell wall degradability and both lignin- and ferulate-related traits led to questioning the possible underlying genetic determinant(s). A cluster of (linked) genes involved in the different mechanisms of cell wall biosynthesis and/or assembly is likely the simplest situation to consider. However, a single “master” regulation factor located upstream in the pathway of cell wall biosynthesis and assembly cannot be definitely ruled out. Candidate genes putatively involved in cell wall degradability variations highlighted especially the presence of ZmMYB Hv5/EgMYB1-like and COV-like genes in any of the clusters. Moreover, besides potential regulatory candidates, there are a number of candidates of still unknown functions. The question of the nature of the possible QTL underlying determinants is still partly unanswered, even if the results obtained strongly suggested that, in this progeny, genes involved in monolignol biosynthesis and important Arabidopsis NAC are not likely candidates. In addition, the positions of candidate genes suggested that ghost QTL positions should also be considered.     

Simultaneously disrupting AtPrx2,AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem

Plant class III heme peroxidases catalyze lignin polymerization. Previous reports have shown that at least three Arabidopsis thaliana peroxidases, At Prx2, At Prx25 and At Prx71,are involved in stem lignification using T-DNA insertion mutants,atprx2, atprx25, and atprx71. Here, we generated three double mutants, atprx2/atprx25, atprx2/atprx71, and atprx25/atprx71,and investigated the impact of the simultaneous de ficiency of these peroxidases on lignins and plant growth. Stem tissue analysis using the acetyl bromide method and derivatization followed by reductive cleavage revealed improved lignin characteristics, such as lowered lignin content and increased arylglycerolb-aryl(b-O-4) linkage type, especially b-O-4 linked syringyl units, in lignin, supporting the roles of these genes in lignin polymerization. In addition, none of the double mutants oexhibited severe growth defects, such as shorter plant stature, dwar fing, or sterility, and their stems had improved cell wall degradability. This study will contribute to progress in lignin bioengineering to improve lignocellulosic biomass.     

Genetic and physical fine mapping of the novel brown midrib gene bm6 in maize (Zea mays L.) to a 180?kb region on chromosome 2

Brown midrib mutants in maize are known to be associated with reduced lignin content and increased cell wall digestibility, which leads to better forage quality and higher efficiency of cellulosic biomass conversion into ethanol. Four well known brown midrib (bm) mutants, named bm1-4, were identified several decades ago. Additional recessive brown midrib mutants have been identified by allelism tests and designated as bm5 and bm6. In this study, we determined that bm6 increases cell wall digestibility and decreases plant height. bm6 was confirmed onto the short arm of chromosome 2 by a small mapping set with 181 plants from a F(2) segregating population, derived from crossing B73 and a bm6 mutant line. Subsequently, 960 brown midrib individuals were selected from the same but larger F(2) population for genetic and physical mapping. With newly developed markers in the target region, the bm6 gene was assigned to a 180?kb interval flanked by markers SSR_308337 and SSR_488638. In this region, ten gene models are predicted in the maize B73 sequence. Analysis of these ten genes as well as genes in the syntenic rice region revealed that four of them are promising candidate genes for bm6. Our study will facilitate isolation of the underlying gene of bm6 and advance our understanding of brown midrib gene functions.     

Effect of pretreatment and enzymatic hydrolysis of wheat straw on cell wall composition, hydrophobicity and cellulase adsorption

The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20-50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.     

Down-regulation of the<Emphasis Type="Italic"> AtCCR1</Emphasis> gene in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: effects on phenotype,lignins and cell wall degradability

Cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is the first enzyme specific to the biosynthetic pathway leading to monolignols. Arabidopsis thaliana (L.) Heynh. plants transformed with a vector containing a full-length AtCCR1 cDNA in an antisense orientation were obtained and characterized. The most severely down-regulated homozygous plants showed drastic alterations to their phenotypical features. These plants had a 50% decrease in lignin content accompanied by changes in lignin composition and structure, with incorporation of ferulic acid into the cell wall. Microscopic analyses coupled with immunolabelling revealed a decrease in lignin deposition in normally lignified tissues and a dramatic loosening of the secondary cell wall of interfascicular fibers and vessels. Evaluation of in vitro digestibility demonstrated an increase in the enzymatic degradability of these transgenic lines. In addition, culture conditions were shown to play a substantial role in lignin level and structure in the wild type and in the effects of AtCCR1 repression efficiency.     

Impact of drought stress on growth and quality of miscanthus for biofuel production

Miscanthus has a high potential as a biomass feedstock for biofuel production. Drought tolerance is an important breeding goal in miscanthus as water deficit is a common abiotic stress and crop irrigation is in most cases uneconomical. Drought may not only severely reduce biomass yields, but also affect biomass quality for biofuel production as cell wall remodeling is a common plant response to abiotic stresses. The quality and plant weight of 50 diverse miscanthus genotypes were evaluated under control and drought conditions (28 days no water) in a glasshouse experiment. Overall, drought treatment decreased plant weight by 45%. Drought tolerance – as defined by maintenance of plant weight – varied extensively among the tested miscanthus genotypes and ranged from 30% to 110%. Biomass composition was drastically altered due to drought stress, with large reductions in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides. Stress had only a small effect on lignin content. Cell wall structural rigidity was also affected by drought conditions; substantially higher cellulose conversion rates were observed upon enzymatic saccharification of drought‐treated samples with respect to controls. Both cell wall composition and the extent of cell wall plasticity under drought varied extensively among all genotypes, but only weak correlations were found with the level of drought tolerance, suggesting their independent genetic control. High drought tolerance and biomass quality can thus potentially be advanced simultaneously. The extensive genotypic variation found for most traits in the evaluated miscanthus germplasm provides ample scope for breeding of drought‐tolerant varieties that are able to produce substantial yields of high‐quality biomass under water deficit conditions. The higher degradability of drought‐treated samples makes miscanthus an interesting crop for the production of second‐generation biofuels in marginal soils.     

Genetic variations of cell wall digestibility related traits in floral stems of Arabidopsis thaliana accessions as a basis for the improvement of the feeding value in maize and forage plants

Floral stems of Arabidopsis thaliana accessions were used as a model system relative to forage plant stems in genetic variation studies of lignin content and cell wall digestibility related traits. Successive investigations were developed in a core collection of 24 Arabidopsis accessions and in a larger collection of 280 accessions. Significant genetic variation for lignin content in the cell wall, and for the two in vitro cell wall digestibility investigated traits, were found both in the core collection and in the large collection. Genotype × environment interactions, investigated in the core collection, were significant with a few genotypes contributing greatly to interactions, based on ecovalence value estimates. In the core collection, genotypes 42AV, 224AV, and 8AV had low cell wall digestibility values, whatever be the environmental conditions. Genotype 157AV, observed only in one environment, also appeared to have a low cell wall digestibility. Conversely, genotypes 236AV, 162AV, 70AV, 101AV, 83AV had high cell wall digestibility values, genotype 83AV having a slightly greater instability across differing environments than others. The well-known accession Col-0 (186AV) appeared with a medium level of cell wall digestibility and a weak to medium level of interaction between environments. The ranges of variation in cell wall digestibility traits were higher in the large collection than in the core collection of 24 accessions, these results needing confirmation due to the lower number of replicates. Accessions 295AV, 148AV, and 309AV could be models for low stem cell wall digestibility values, with variable lignin content. Similarly, accessions 83AV and 162AV, already identified from the study of the core collection, and five accessions (6AV, 20AV, 91AV, 114AV, and 223AV) could be models for high stem cell wall digestibility values. The large variations observed between Arabidopsis accessions for both lignin content and cell wall digestibility in floral stems have strengthened the use this species as a powerful tool for discovering genes involved in cell wall biosynthesis and lignification of dicotyledons forage plants. Investigations of this kind might also be applicable to monocotyledons forage plants due to the basic similarity of the genes involved in the lignin pathway of Angiosperms and the partial homology of the cell wall composition and organization of the mature vascular system in grasses and Arabidopsis.     

Nitrogen fertilizer application affects lodging resistance by altering secondary cell wall synthesis in japonica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

Stem mechanical strength is an important agricultural quantitative trait that is closely related to lodging resistance in rice, which is known to be reduced by fertilizer with higher levels of nitrogen. To understand the mechanism that regulates stem mechanical strength in response to nitrogen, we analysed stem morphology, anatomy, mechanical properties, cell wall components, and expression of cell wall-related genes, in two varieties of japonica rice, namely, Wuyunjing23 (lodging-resistant variety) and W3668 (lodging-susceptible variety). The results showed that higher nitrogen fertilizer increased the lodging index in both varieties due to a reduction in breaking strength and bending stress, and these changes were larger in W3668. Cellulose content decreased slightly under higher nitrogen fertilizer, whereas lignin content reduced remarkably. Histochemical staining revealed that high nitrogen application decreased lignin deposition in the secondary cell wall of the sclerenchyma cells and vascular bundle cells compared with the low nitrogen treatments, while it did not alter the pattern of cellulose deposition in these cells in both Wuyunjing23 and W3668. In addition, the expression of the genes involved in lignin biosynthesis, OsPAL, OsCoMT, Os4CL3, OsCCR, OsCAD2, OsCAD7, OsCesA4, and OsCesA7, were also down-regulated under higher nitrogen conditions at the early stage of culm growth. These results suggest that the genes involved in lignin biosynthesis are down-regulated by higher nitrogen fertilizer, which causes lignin deficiency in the secondary cell walls and the weakening of mechanical tissue structure. Subsequently, this results in these internodes with reduced mechanical strength and poor lodging resistance.     

On the role of cell wall lignin in determining the fineness of jute fibre

Though finer quality fibre is of great demand in the industry, a reasonable biological assessment of the factors controlling jute fibre fineness is lacking. Our aim was to relate lignin synthesis and accumulation in the secondary wall of the fibre cells with fibre fineness by anatomical and physiological evidences. Several jute genotypes including a low lignin mutant, dlpf (INGR No. 04107) and its lignin sufficient parent (JRC 212) were grown under different growth conditions. Their cell wall morphology and cellulose, hemicellulose and lignin content of fibre were estimated. The fineness of the extracted fibre was examined gravimetrically as well as by air-flow method on individual plant basis to relate it with their chemical constituents. Effect of incident light and some plant growth regulators on glucan and lignin biosynthetic enzymes as well as fibre fineness was determined. Positive relationship between cell wall thickness and lignin and negative relationship between fibre fineness and lignin of jute fibre were established. Application of the GA biosynthetic inhibitor helped to reduce lignin synthesis and to increase fibre fineness. Genotypes with thinner cell wall and lesser lignin may be utilized in breeding for improving the fibre fineness of jute. Field application of GA biosynthetic inhibitors, like daminozide, is recommended to reduce the cell wall thickness of lignin sufficient high yielding jute varieties.     

SRAP Polymorphisms Associated to Cell Wall Degradability in Lignified Stems of Alfalfa

The identification of DNA polymorphisms associated to increased cell wall (CW) degradability could accelerate the development of alfalfa (Medicago sativa L.) cultivars with superior ethanol conversion yields. Genotypes with high (D+) or low (D?) CW degradability were recently identified within a biomass-type and three winter-hardy-type populations using near-infrared reflectance spectroscopy (NIRS) prediction of CW glucose released by enzyme saccharification. In this report, we used sequence-related amplified polymorphism to search for DNA variations associated to differences in enzyme-released glucose. A bulk segregant analysis (BSA) of pooled DNA samples (20 plants/bulk) from D+, D?, and randomly chosen genotypes uncovered polymorphisms associated to CW degradability. Polymorphisms that increase or decrease in intensity between D+ and D? bulks indicated the presence of genomic regions with either positive or negative effects on CW degradability. A primer pair (Me4-R14) generated a fragment, which increased in intensity in the D+ bulk of the biomass-type population. Conversely, the amplification of that fragment declined in the D+ bulks of the winter-hardy-type populations. Interestingly, these populations differ in their degradability. Assessment of the genotypic occurrence of this fragment confirmed that polymorphism detected with BSA reflects changes in the frequency of occurrence within populations. Sequence analysis of the Me4-R14 fragment revealed homologies with sequences from Medicago truncatula, a model species for legumes with documented synteny with M. sativa. Our results show that genomic regions associated to CW degradability can be identified using the combination of BSA of genotypes with contrasted degradability and a PCR-based amplification technique.     

Chemical composition,cell wall features and degradability of stem,leaf blade and sheath in untreated and alkali-treated rice straw

Three dominant morphological fractions (i.e. leaf blade (LB), leaf sheath (LS) and stem) were analysed for chemical composition and ruminal degradability in three rice straw varieties. In one variety treated with alkali, cell wall features were also characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy. The highest concentrations of cell wall carbohydrates (hemicellulose and cellulose) were observed in LS, whereas the highest concentrations of non-fibre (silica, phenolic compounds and CP) and lignin were recorded for LB. The stem had the lowest silica and hemicellulose contents but intermediate levels of other components. In terms of ruminal degradability, stem ranked higher than LB, which was followed by LS. Hemicellulose was found to be less degradable than either dry matter or cellulose in all the three fractions investigated. FTIR results indicated that the highest levels of hydrogen bonding, esterification and crystallinity within the cell wall components belonged to LS. In the alkaline treatment, these indices decreased to a larger extent for leaf fractions and a greater improvement was achieved in the degradability of LB and LS compared with that of stem. In the 24-h ruminal incubation, the silicified layer of epidermis and the underlying cell walls showed a rigid structure in the control fractions, whereas the treatment with NaOH resulted in crimping of the silicified cuticle layer and the loss of integrity in cell structure. Despite the highest silica and lignin contents observed in LB, LS showed the lowest degradability, which might be due to its high level of hydrogen bonding, crystallinity and esterification within its cell wall components as well as its high hemicellulose content.     

Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO₄-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical woody plant lignin, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.     

Activity and isoforms of peroxidases, lignin and anatomy, during adventitious rooting in cuttings of Ebenus cretica L

Adventitious rooting of Ebenus cretica cuttings was studied in order to examine a) the rooting ability of different genotypes in relation to electrophoretic patterns of peroxidases. b) the activity and electrophoretic patterns of soluble and wall ionically bound peroxidases, the lignin content and anatomical changes in the control and IBA treated cuttings of and genotypes in the course of adventitious root formation. In addition, a fraction of soluble cationic peroxidases was separated by gel filtration chromatography from the total soluble peroxidases of a genotype. No rooting occurred in cuttings without IBA-treatment. In both genotypes, electrophoretic patterns of soluble anionic peroxidases revealed two common peroxidase isoforms, while a fast-migrating anionic peroxidase isoform (A3) appeared only in genotypes. Both genotypes showed similar patterns of soluble, as well as wall ionically bound cationic peroxidase isoforms. The number of isoforms was unchanged during the rooting process (induction, initiation and expression phase) but an increase in peroxidase activity (initiation phase) followed by decrease has been found in IBA-treated cuttings. During initiation phase the lignin content was almost similar to that on day 0 in genotype while it was reduced at by about 50% in genotype at the respective time. Microscopic observations revealed anatomical differences between genotypes. According to this study, the and genotypes display differences in anatomy, lignin content, activity of soluble peroxidases and the electrophoretic patterns of soluble anionic peroxidase isoforms. The A3-anionic peroxidase isoform could be used as biochemical marker to distinguish and genotypes of E. cretica and seems to be correlated to lignin synthesis in rooting process.