Apoplastic pH and monolignol addition rate effects on lignin formation and cell wall degradability in maize

Monolignol polymerization rate and apoplastic pH and may influence the formation of lignin and its interactions in cell walls. Primary maize walls were artificially lignified by gradual "end-wise" or rapid "bulk" polymerization of coniferyl alcohol at pH 4 or 5.5. Lignification efficiency was greatest for end-wise polymers at pH 5.5 (90-98%), intermediate for bulk polymers formed at either pH (54-82%), and lowest for end-wise polymers at pH 4 (41-53%). End-wise polymers had about 2.2-fold more ether inter-unit linkages and 70% fewer end-groups than bulk polymers. Low pH enhanced the formation of ether linkages in end-wise but not in bulk polymers. Differences in lignin structure did not influence the enzymatic degradability of cell walls, but lowering apoplastic pH from 5.5 to 4.0 during lignification reduced cell wall degradability by 25%. Further studies indicated this pH-dependent depression in degradability was related to cell wall cross-links formed via lignin quinone methide intermediates.     

In search of a maize ideotype for cell wall enzymatic degradability using histological and biochemical lignin characterization

Grass cell wall degradability is conventionally related to the lignin content and to the ferulic-mediated cross-linking of lignins to polysaccharides. To better understand the variations in degradability, 22 maize inbred lines were subjected to image analyses of Fasga- and M?ule-stained stem sections and to chemical analyses of lignins and p-hydroxycinnamic acids. For the first time, the nearness of biochemical and histological estimates of lignin levels was established. Combination of histological and biochemical traits could explain 89% of the variations for cell wall degradability and define a maize ideotype for cell wall degradability. In addition to a reduced lignin level, such an ideotype would contain lignins richer in syringyl than in guaiacyl units and preferentially localized in the cortical region rather than in the pith. Such enrichment in syringyl units would favor wall degradability in grasses, contrary to dicots, and could be related to the fact that grass syringyl units are noticeably p-coumaroylated. This might affect the interaction capabilities of lignins and polysaccharides.     

Impact of plant cell wall network on biodegradation in soil: Role of lignin composition and phenolic acids in roots from 16 maize genotypes

Residue quality is a key factor governing biodegradation and the fate of C in soil. Most investigations of relationships existing between crop residue quality and soil decomposition have been based on determining the relative proportions of soluble, cellulose, hemicellulose and lignin components. However, cell wall cohesion is increased by tight interconnections between polysaccharides and lignin that involve cross-linking agents (phenolic acids). The aim of this study was to determine the role of lignin composition and phenolic acids on short- to medium-term decomposition of maize roots in soil. Sixteen maize genotypes, presenting a range of chemical characteristics related to root lignin and phenolic acids, were used. The main components were characterized by Van Soest (VS) extraction and cell wall acid hydrolysis, and the non-condensed Syringyl and Guaicyl lignin monomers, esterified phenolic acids and etherified phenolic acids were determined. Maize roots were then incubated in soil under controlled conditions (15 °C, −80 kPa moisture) for 796 days. Results showed that VS extraction over-estimated the structural hemicellulose content and that VS lignin was more recalcitrant than Klason lignin. The tremendous effect of cell wall chemical characteristics was shown by marked variations (almost two-fold differences in C mineralization), between the 16 maize roots. Decomposition was controlled by soluble residue components in the short term whereas lignin and the interconnections between cell wall polymers were important in the long-term. Notably the cell wall domain rich in non-condensed lignin and esterified phenolic acids was prone to decomposition whereas the presence of etherified ferulic acids seemed to hamper cell wall decomposition.     

Methyl esterification divergently affects the degradability of pectic uronosyls in nonlignified and lignified maize cell walls

Nonlignified cell walls from Zea mays (L.) cell suspensions were incubated with and without pectin methylesterase (PME) and a portion were artificially lignified to assess how methyl esters influence the release of pectic uronosyls and total sugars from cell walls by fungal enzymes. Treatment with PME reduced uronosyl concentrations from 97 to 92 mg/g, reduced uronosyl methylation from 57% to 21%, and increased Klason lignin concentrations in artificially lignified cell walls from 99 to 116 mg/g. Although PME treatment slightly enhanced uronosyl release from nonlignified cell walls, it reduced uronosyl release from artificially lignified cell walls by 55% after 4 h and by 7% after 72 h of enzymatic hydrolysis. Pectin hydrolysis in PME treated cell walls was probably impaired by enhanced benzyl ester cross-linking of uronosyls to lignin via quinone methide intermediates. Variations in uronosyl methylation had little effect on the overall release of total sugars from cell walls.     

Variations in the cell wall composition of maize brown midrib mutants

Most studies published thus far on the four brown midrib (bm) mutants (bm1, bm2, bm3, and bm4) in maize (Zea mays L.) have focused on one or two individual mutants, and comparisons between studies have been difficult because of variation in genetic backgrounds, maturity, and source of tissue. Detailed analyses of the stalks of the four bm single mutants and a bm1-bm2 double mutant in a common genetic background (inbred A619) revealed structural and compositional changes in their isolated cell walls and lignins compared to the wild-type inbred. 2D-NMR revealed a significant presence of benzodioxane units in the bm3 isolated lignin. 1D (13)C NMR revealed increased aldehyde levels in the bm1 and bm1-bm2 mutants compared to the wild-type inbred. The bm3 and bm1-bm2 mutants contained less Klason lignin in the isolated cell walls. The bm1, bm3, and bm1-bm2 mutants contained approximately 50% less esterified p-coumaric acid with noticeably elevated levels of ferulate in the bm3 mutant. A difference among bm mutants in the solubility of p-coumaric acid-lignin complexes during cellulase enzyme treatment was also discovered, suggesting that the bm mutations might also differ in the structural organization of lignin.     

Impact of artificial root exudates on the bacterial community structure in bulk soil and maize rhizosphere

Bacterial densities, metabolic signatures and genetic structures were evaluated to measure the impact of soil enrichment of soluble organic carbon on the bacterial community structures. The exudates chosen were detected in natural maize exudates (glucose, fructose, saccharose, citric acid, lactic acid, succinic acid, alanine, serine and glutamic acid) and were used at a rate of 100 μg C g⁻¹ day⁻¹ for 14 days. Moreover two synthetic solutions with distinct carbon/nitrogen ratios (20.5 and 40.1), obtained by varying carboxylic and amino acids concentrations, were compared in order to evaluate the potential role of organic N availability. The in vitro experiment consisted of applying exudate solutions to bulk soil. In the case of the control, only distilled water was added. Both solutions significantly increased bacterial densities and modified the oxidation pattern of Biolog® GN2 plates with no effect of the C/N ratio on these two parameters. Genetic structure, measured by means of ribosomal intergenic spacer analysis (RISA), was also consistently modified by the organic amendments. N availability levels led to distinct genetic structures. In a second experiment, one of the previous exudate solutions (C/N 20.5) was applied to 15-day-old maize plants to determine the structural influence of exudates on the rhizosphere microbial community (in situ experiment). Bacterial densities were significantly increased, but to a lesser extent than had been found in the in vitro experiment. Metabolic potentials and RISA profiles were also significantly modified by the organic enrichment.     

Cross-linking of maize walls by ferulate dimerization and incorporation into lignin

Cross-linking of xylans and lignin by ferulates was investigated with primary maize walls acylated with 2% ferulate and with ferulate ethyl esters. Peroxidase-mediated coupling of wall ferulate and ethyl ferulate yielded mostly 8-coupled products, including three new dehydrodimers. Significant quantities of 5-5-coupled diferulate formed only within walls, suggesting that matrix effects influence dimer formation. Over 60% of wall ferulate dimerized upon H2O2 addition, suggesting that xylan feruloylation is highly regulated during wall biosynthesis to permit extensive dimer formation at the onset of lignification. During lignification, ferulate and 5-5-coupled diferulate copolymerized more rapidly and formed fewer ether-linked structures with coniferyl alcohol than 8-5-, 8-O-4-, and 8-8-coupled diferulates. The potential incorporation of most ferulates and diferulates into lignin exceeded 90%. As a result, xylans become extensively cross-linked by ferulate dimerization and incorporation to lignin, but only a small and variable proportion of these cross-links is measurable by solvolysis of lignified walls.     

Identifying new lignin bioengineering targets: impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and fermentation of maize cell walls

Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, primary maize cell walls were artificially lignified with normal monolignols plus various epicatechin, quercetin glycoside, and gallate derivatives added as 0 or 45% by weight of the precursor mixture. The flavonoids and gallates had variable effects on peroxidase activity, but all dropped lignification pH. Epigallocatechin gallate, epicatechin gallate, epicatechin vanillate, epigallocatechin, galloylhyperin, and pentagalloylglucose formed wall-bound lignin at moderate to high concentrations, and their incorporation increased 48 h in vitro ruminal fiber fermentability by 20-33% relative to lignified controls. By contrast, ethyl gallate and corilagin severely depressed lignification and increased 48 h fermentability by about 50%. The results suggest several flavonoid and gallate derivatives are promising lignin bioengineering targets for improving the inherent fermentability of nonpretreated cell walls.     

The maize primary cell wall microfibril: a new model derived from direct visualization

Understanding the molecular architecture of the plant cell wall is critical to reducing the biomass recalcitrance problem, which currently impedes economic bioconversion processing. The parenchyma cell walls from field senesced, maize stem pith have been directly visualized without extraction processes using high-resolution atomic force microscopy (AFM). By imaging the cell wall inner surfaces from different cells and different faces of the same cell, we were able to map the native primary cell wall ultrastructures. Depending on the thickness of non-cellulosic deposition, the parallel-microfibrils appear in various morphologies ranging from clearly defined to completely embedded in the wall matrixes forming cell wall lamella. Macrofibrils were found to exist only on the uppermost layer of the native primary cell wall and appeared to be bundles of elementary fibrils. This novel observation led us to a new hypothesis for the cell wall fibrillar network and biosynthesis processes. Put concisely, a number of elementary fibrils are synthesized at one locus, that of the cellulose synthase complex (CelS), and coalesce into much larger macrofibrils. These macrofibrils eventually split at the ends to form parallel microfibrils with deposition of other cell wall components (i.e. hemicelluloses, pectin, etc.) also evident. On the basis of these AFM surface measurements and current supportive evidence from cell wall biophysics, biosynthesis, and genomics, we propose a new molecular model consisting of a 36-glucan-chain elementary fibril, in which the 36-glucan chains form both crystalline and subcrystalline structures. We also propose a modified model of CelS based on recently reported experimental evidence from plant cell wall biosynthesis.     

不同种类精饲料中磷的瘤胃降解特性研究

测定了谷物籽实、豆类籽实及根茎三类共计15种饲料的磷含量,并应用尼龙袋法测定其在山羊瘤胃内的降解率。结果表明：三类饲料的磷含量存在明显差异,以豆类籽实最高,谷物籽实其次,根茎类最低。饲料种类间磷的瘤胃降解率差异极显著（P〈0.001）,谷物类、豆类和根茎类饲料中快速降解磷（A）分别以玉米（88.82%）、豌豆（92.66%）和红薯（94.69%）最高,而以大米、赤豆和木薯为最低。除大麦、大米和小米外,其他饲料的快速降解磷均在60%以上。除小麦外,慢速降解的磷（B）都低于40%。磷在瘤胃中的降解速率（C）都超过10%/h以上,仅大米、小米和土豆的慢速降解部分的降解速率低于10%/h。不同种类精饲料总的可降解磷（A＋B）都在80%以上。不同种类饲料磷的有效降解率都随外流速率的增大而下降,除大米和小米外,其他饲料磷的有效降解率都超过了80%。饲料中磷的含量与磷的有效降解率呈显著正相关关系,磷的降解速率与粗脂肪含量呈极显著正相关关系,淀粉含量则与磷的有效降解率呈显著负相关关系,磷的含量与粗蛋白含量间呈极显著正相关关系。上述结果显示,饲料中磷在瘤胃中绝大部分都可释放出来,故配制反刍动物日粮时必须考虑饲料原料中磷的含量及其有效降解率。     

Moderate ferulate and diferulate levels do not impede maize cell wall degradation by human intestinal microbiota

The degradation of plant fiber by human gut microbiota could be restricted by xylan substitution and cross-linking by ferulate and diferulates, for example, by hindering the association of enzymes such as xylanases with their substrates. To test the influence of feruloylation on cell wall degradability by human intestinal microbiota, nonlignified primary cell walls from maize cell suspensions, containing various degrees of ferulate substitution and diferulate cross-linking, were incubated in nylon bags in vitro with human fecal microbiota. Degradation rates were determined gravimetrically, and the cell walls were analyzed for carbohydrates, ferulate monomers, dehydrodiferulates, dehydrotriferulates, and other minor phenolic constituents. Shifting cell wall concentrations of total ferulates from 1.5 to 15.8 mg/g and those of diferulates from 0.8 to 2.6 mg/g did not alter the release of carbohydrates or the overall degradation of cell walls. After 24 h of fermentation, the degradation of xylans and pectins exceeded 90%, whereas cellulose remained undegraded. The results indicate that low to moderate levels of ferulates and diferulates do not interfere with hydrolysis of nonlignified cell walls by human gut microbiota.     

Impact of genetics and environment on nutritional and metabolite components of maize grain

The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnology-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnology-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly associated with a newly introduced trait. Therefore, steps have been initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against two different testers. These were grown at three different, but geographically similar, locations in the United States. In addition to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, organic acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant organic acid was citric acid, with only minor amounts of other organic acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and organic acids appeared to be markedly higher than that observed for the OECD analytes.     

Inhomogeneities in the chemical structure of sugarcane bagasse lignin

Sequential treatments of dewaxed bagasse with distilled water, 0.5 M NaOH, 0.5, 1.0, 1.5, 2.0, and 3.0% H(2)O(2) at pH 11.5, and 2 M NaOH at 55 degrees C for 2 h solubilized 2.8, 52.5, 14.9, 3.3, 5.5, 5.0, 2.8, and 2.2% of the original lignin, respectively. The eight isolated lignin fractions were subjected to a comprehensive structural characterization by UV, FT-IR, and (1)H and (13)C NMR spectroscopies and thermal analysis. The nitrobenzene oxidation method was also applied to the in situ lignins. The seven lignin fractions, isolated successively with alkali and alkaline peroxide, were all SGH-type lignins, with a small amount of esterified p-coumaric acid and mainly etherified ferulic acid. No significant differences were found in the weight-average molecular weights (1680-2220 g/mol) of the seven alkali and alkaline peroxide dissolved lignins. However, the first four lignin fractions, isolated with 0.5 M NaOH and 0.5, 1.0, and 1.5% H(2)O(2) at pH 11.5, were rich in syringyl units and contained large amounts of noncondensed ether structures, whereas the last three lignin fractions, isolated sequentially with 2.0 and 3.0% H(2)O(2) at pH 11.5 and 2 M NaOH at 55 degrees C for 2 h, had a higher degree on condensation and were rich in guaiacyl lignins.     

Studies on the effect of ball milling on lignin structure using a modified DFRC method.

The structures of milled wood lignin (MWL), cellulolytic enzyme lignin (CEL), and residual lignin (REL) from a loblolly pine were analyzed using a modified derivatization followed by reductive cleavage (DFRC) method developed to allow the quantitative determination of three different structural monomeric products originating in lignin: phenolic beta-O-4, alpha-O-4, and etherified beta-O-4 structures. Results show that MWL and CEL are structurally identical, with an increased phenolic beta-O-4 content compared to that of the original Wiley milled wood. These results indicate that the portion of lignin linked to carbohydrates and that not linked to carbohydrates are structurally the same. Modified DFRC analysis of the effect of ball milling on the structure of lignin in wood, MWL, CEL, and REL indicate that vibratory ball milling does not change the lignin structure provided certain precautions are taken. Specifically, dry vibratory ball milling under a nitrogen atmosphere causes substantial structural changes including condensation, whereas vibratory ball milling in toluene had little effect on the lignin structure. This indicates that the structural differences observed in MWL and CEL arise because of the extraction procedure, which preferentially extracts phenolic lignin structures. MWL and CEL are representative of the total lignin in wood; however, due primarily to the solvent extraction process, higher phenolic hydroxyl contents are observed. Nitrobenzene oxidation showed structural results similar to those from the modified DFRC method.     

Impact of VAM on phosphorus nutrition of maize with low soluble phosphate fertilization

In a greenhouse pot experiment, maize was grown inoculated with the spores of the VAM fungi Glomus mosseae and Glomas multicaulis or non‐inoculated. Low soluble ferrous phosphate (FePO₄.₄H₂O) was added to the mycorrhized and non‐mycorrhized maize. The fresh and dry weights of mycorrhized plants with added phosphate (P) were higher than in mycorrhized plants without added P or non‐mycorrhized plants with added P. The amount of P in the soil samples from pots with mycorrhizal plants fertilized with P was evidently smaller than those in samples also fertilized non‐mycorrhizal plants. The percentage of P was higher in tissues of fertilized mycorrhizal plants than in those mycorrhized plants without or non‐mycorrhized plants with added low‐ soluble P. These results indicated that plants in VAM symbiosis mobilize P better from low‐soluble P than non‐mycorrhized plants.     

Organosolv ethanol lignin from hybrid poplar as a radical scavenger: relationship between lignin structure, extraction conditions, and antioxidant activity

Twenty-one organosolv ethanol lignin samples were prepared from hybrid poplar (Populus nigra xP. maximowiczii) under varied conditions with an experimental matrix designed using response surface methodology (RSM). The lignin preparations were evaluated as potential antioxidants. Results indicated that the lignins with more phenolic hydroxyl groups, less aliphatic hydroxyl groups, low molecular weight, and narrow polydispersity showed high antioxidant activity. Processing conditions affected the functional groups and molecular weight of the extracted organosolv ethanol lignins, and consequently influenced the antioxidant activity of the lignins. In general, the lignins prepared at elevated temperature, longer reaction time, increased catalyst, and diluted ethanol showed high antioxidant activity. Regression models were developed to enable the quantitative prediction of lignin characteristics and antioxidant activity based on the processing conditions.     

Impact of inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the genetic structure of the rhizobacterial community of field-grown maize

The phytostimulatory PGPR Azospirillum lipoferum CRT1 was inoculated to maize seeds and the impact on the genetic structure of the rhizobacterial community in the field was determined during maize growth by Automated Ribosomal Intergenic Spacer Analysis (ARISA) of rhizosphere DNA extracts. ARISA fingerprints could differ from one plant to the next as well as from one sampling to the next. Inoculation with strain CRT1 enhanced plant-to-plant variability of the ARISA fingerprints and caused a statistically significant shift in the composition of the indigenous rhizobacterial community at the first two samplings. This is the first study on the ecological impact of Azospirillum inoculation on resident bacteria done in the field and showing that this impact can last at least one month.     

Individual contribution of grain outer layers and their cell wall structure to the mechanical properties of wheat bran

The mechanical properties of wheat bran and the contribution of each constitutive tissue on overall bran properties were determined on a hard wheat (cv. Baroudeur) and a soft wheat (cv. Scipion). Manual dissection allowed three different layers to be separated from wheat bran, according to radial and longitudinal grain orientations, which were identified by confocal laser scanning microscopy as outer pericarp, an intermediate strip (comprising inner pericarp, testa, and nucellar tissue), and aleurone layer, respectively. Tissue microstructure and cell wall composition were determined. Submitted to traction tests, whole bran, intermediate, and aleurone layers demonstrated elastoplastic behavior, whereas pericarp exhibited elastic behavior. By longitudinal orientation, pericarp governed 50% bran elasticity (elastic strength and rigidity), whereas, in the opposite orientation, bran elastic properties were mostly influenced by the other tissues. Regardless of test orientation, the linear force required to bran rupture corresponded to the sum of intermediate and aleurone layer strengths. According to radial orientation, the intermediate strip governed bran extensibility, but according to longitudinal orientation, all tissues contributed until bran disruption. Tissues from both wheat cultivars behaved similarly. A structural model of wheat bran layers illustrated the detachment of pericarp from intermediate layer within radial bran strips.     

Impact of pea growth and arbuscular mycorrhizal fungi on the decomposition of 15N-labeled maize residues

A pot experiment was carried out (1) to compare C and N yield of different plant parts, nutrient concentrations, and root colonization between the non-mycorrhizal mutant P2 (myc ^?) and the symbiotic isoline Frisson (myc ⁺), (2) to investigate the effects of arbuscular mycorrhizal fungi and growing pea plants on microbial decomposition of ¹⁵N-labeled maize residues, and (3) to follow the distribution of the added substrate over different soil fractions, such as particulate organic matter, soil microbial biomass, and microbial residues. Yields of C in straw, grain, and roots of myc ⁺ peas were significantly higher by 27%, 11%, and 92%, respectively, compared with those of myc ^? peas. The δ¹³ C values in the different plant parts were significantly higher in myc ⁺ than in myc ^? tissue with and without maize. Application of labeled maize residues generally resulted in ¹⁵N enrichment of pea plants. At the end of the experiment, the ergosterol concentration in roots of mature peas did not differ between the two isolines, indicating similar colonization by saprotrophic fungi. The decomposition of added maize residues was significantly reduced by the myc ^? peas, but especially by myc ⁺ peas. The formation of microbial residue C was increased and that of microbial residue N was reduced in the presence of plants. The insufficient N supply to soil microorganisms reduced decomposition of maize residues in the presence of peas, especially myc ⁺ peas.     

Cell wall composition in juvenile and adult leaves of maize (Zea mays L.)

Many leaf characteristics vary with position along the culm in maize (Zea mays L.) due to the existence of vegetative phase change and heteroblasty. The objective of this work was to determine if differences in cell wall composition exist among developmental phases and between Cg1, a developmental mutant, and wild-type maize. In one experiment, the middle third of fully elongated leaf blades from lower and upper regions of the shoot was harvested (midribs removed) and analyzed for several cell wall components. Averaged over five inbreds (De811, Ia5125, Mo17, P39, and Wh8584), lower leaf blades had higher levels of xylose and lower levels of total uronosyls, glucose, arabinose, and galactose (P < 0.05) than did upper leaf blades. With the exception of glucose, upper and lower leaves of Cg1 plants varied in the same manner as their near-isogenic siblings, except cell walls of Cg1 plants were more "juvenile" than cell walls of wild-type siblings at the same leaf stage. These data support the hypothesis that Cg1 delays but does not eliminate the transition from juvenile-vegetative to adult-vegetative phase. In a second experiment, juvenile (leaves 3 and 5), transition (leaf 7), and adult (leaves 9 and 11) leaves from inbreds B73 and De811 were harvested and analyzed as in the first experiment. As leaf number rose, total cell wall content of sample dry matter, total neutral sugars, glucose, xylose, and ester-linked monomers of p-coumaric acid and total ferulates including ferulate dimers increased linearly while total uronosyls acids, arabinose, and galactose declined linearly (P < 0.05). Glucose and xylose are major cell wall components released from cellulose and xylans after acid hydrolysis. Pectin, a minor component of grass cell walls, is composed of galacturonosyls, arabinose, and galactose. Secondary cell wall deposition increased between leaves 3 and 11 in a heteroblastic series, due to either increased cell wall content concomitant with decreased cell lumen size, changes in proportion of cell types (i.e., sclerenchyma), or a combination of these factors.