Immunogold labelling of xylans and arabinoxylans in the plant cell walls of maize stem

Summary— Polyclonal antibodies against 4-O-methyl-glucuronoxylan and α L-1-3 arabinofuranosyl poly-β-d-1-4-xylopyranosyl were raised from rabbits. An immunocytochemical technique was used to localize xylans and arabinoxylans in the plant cell walls of the apical internode of two maize lines of different digestibility. The sclerenchyma, fibres and xylem (lignified tissues) and the parenchyma (non-lignified tissue) were studied. The arabinoxylans were more heavily labelled than the xylans in the lignified tissues of the less digestible maize whereas in the more digestible line the labelling of the two polysaccharides was similar. The xylans and arabinoxylans were localized in the secondary cell wall. In both maize lines, labelling increased from the base upwards of the apical internode, reflecting the changes in growth stage.     

Impact of fine litter chemistry on lignocellulolytic enzyme efficiency during decomposition of maize leaf and root in soil

Residue recalcitrance controls decomposition and soil organic matter turnover. We hypothesized that the complexity of the cell wall network regulates enzyme production, activity and access to polysaccharides. Enzyme efficiency, defined as the relationship between cumulative litter decomposition and enzyme activities over time, was used to relate these concepts. The impact of two contrasting types of cell walls on xylanase, cellulase and laccase efficiencies was assessed in relation to the corresponding changes in residue chemical composition (xylan, glucan, lignin) during a 43-day incubation period. The selected residues were maize roots, which are rich in secondary cell walls that contain lignin and covalent bridges between heteroxylans and lignin, and maize leaves having mostly non-lignified primary cell walls thus making the cellulose and hemicelluloses less resistant to enzymes. Relationships between C mineralization and change in residue quality through decomposition indicated that the level of substitution of arabinoxylans (arabinan to xylan ratio) provides a good explanation of the decomposition process. In leaves enriched in primary cell walls, arabinose substitution of xylan controlled C mineralization rate but hampered polysaccharide decomposition, but to a lesser extent than in roots in which arabinoxylans were mostly cross-linked with lignin. Enzyme activity was higher in leaf than root amended soils while enzyme efficiency was systematically higher in the presence of roots. This apparent paradox suggests that residue quality could preselect the microbial community. Indeed, we found that microorganisms exhibited an initial rapid growth in the presence of a high quality litter and produced enzymes that are not efficient in degrading recalcitrant cell walls while, in the presence of the more recalcitrant maize roots, microbial biomass grew more slowly but produced enzymes of higher efficiency. This high enzyme efficiency could be explained by the synergistic action of hydrolytic and oxidative enzymes even in the early stage of decomposition.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls I : Purification of Novel Enzymes Capable of Dissociating Feraxan Fragments from Zea mays Coleoptile Cell Wall

Three novel β-xylan xylanohydrolases capable of dissociating ferulated arabinoxylan (Feraxan) from maize (Zea mays L. hybrid B73 × Mo17) coleoptile sections and two conventional β-xylan xylanohydrolases (xylanases) were purified from a Bacillus subtilis industrial enzyme preparation (Novo Ban L-120). The Feraxan-dissociating enzymes (designated as feraxanases) exhibit optimum activities between pH 6.5 and 7.0 and have common molecular weights of 45 kilodaltons as studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two xylanases exhibit their optimum activities between pH 4.5 and 6.0 and have common molecular weights of 27 kilodaltons. Feraxanases liberate oligomeric fragments, which accounted for the following percentages of walls of Zea mays coleoptile sections that had been pretreated by boiling in 80% ethanol: 76% of the ferulic acid, 96% of the arabinose, 71% of the xylose, 27% of the galactose, 50% of the uronic acid, and 4% of the glucose. Monomers, dimers, trimers, or tetramers were not found among enzyme digestion products. The enzymes hydrolyzed both Feraxan in intact cell wall and maize arabinoxylans extracted from walls by alkaline solutions but did not degrade other substrates including larch arabinoxylan and Rhodymenia xylan. Structural analyses of the fragments released by the enzymes from the maize cell wall indicated the presence of 2,4/3,4-linked-xylopyranosyl, terminal-arabinofuranosyl, 5-linked-arabinofuranosyl, 4-linked-xylopyranosyl, terminal-glucuronopyranosyl, and ferulic acid as major components. This result is consistent with the idea that most of the fragments were derived from Feraxan. Because of high enzyme specificity and substantial recovery of digestion products from maize cell walls, these new enzymes offer opportunities not only for enhanced structural analyses of cell walls but also for assistance in protoplast preparation from cereals.     

Hemicellulosic polymers from the cell walls of beeswing wheat bran: Part I,polymers solubilised by alkali at 2°

Cell-wall material from beeswing wheat bran was sequentially extracted with 0.05m NaOH at 2°, m KOH at 2° and 20°, and 4m KOH at 20° followed by delignification and further extraction with m and 4m KOH at 20°, to leave the α-cellulose residue which contained a significant amount of arabinoxylan. The hemicellulosic polymers solubilised by m KOH at 2°, which represented ∼20% of the dry weight of the cell walls, were fractionated by graded precipitation with alcohol prior to anion-exchange chromatography and then subjected to methylation analysis. The major polymers were closely related, highly branched arabinoxylans, slightly branched arabinoxylans, and arabinoxylans in close association with xylogucans (arabinoxylan-xyloglucan complexes); the arabinoxylans were acidic and were associated with various amounts of phenolics. The various polymers exhibit heterogeneity, and phenolic ester and phenolic ether cross-links play a major role in the architecture of the cell walls.     

Factors affecting xylanase functionality in the degradation of arabinoxylans

Endo-beta-1,4-xylanases are key enzymes in the degradation of arabinoxylans, the main non-starch polysaccharides from grain cell walls. Due to the heterogeneity of arabinoxylans, xylanases with different characteristics are required in industrial applications but the choice of the enzyme is still largely empirical. Although the classification into glycoside hydrolase families greatly helped to derive mechanistic information on the catalytic and substrate specificity of xylanases, other factors e.g. their sensitivity to endogenous inhibitors, the presence of carbohydrate-binding module(s) and their degree of selectivity towards soluble versus insoluble substrate may play a role in determining the functionality of these enzymes in the degradation of arabinoxylans.     

Extracellular cross-linking of maize arabinoxylans by oxidation of feruloyl esters to form oligoferuloyl esters and ether-like bonds

Primary cell walls of grasses and cereals contain arabinoxylans with esterified ferulate side chains, which are proposed to cross‐link the polysaccharides during maturation by undergoing oxidative coupling. However, the mechanisms and control of arabinoxylan cross‐linking in vivo are unclear. Non‐lignifying maize (Zea mays L.) cell cultures were incubated with l‐ [1‐³H]arabinose or (E)‐[U‐¹⁴C]cinnamate (radiolabelling the pentosyl and feruloyl groups of endogenous arabinoxylans, respectively), or with exogenous feruloyl‐[³H]arabinoxylans. The cross‐linking rate of soluble extracellular arabinoxylans, monitored on Sepharose CL‐2B, peaked suddenly and transiently, typically at ～9 days after subculture. This peak was not associated with appreciable changes in peroxidase activity, and was probably governed by fluctuations in H₂O₂ and/or inhibitors. De‐esterified arabinoxylans failed to cross‐link, supporting a role for the feruloyl ester groups. The cross‐links were stable in vivo. Some of them also withstood mild alkaline conditions, indicating that they were not (only) based on ester bonds; however, most were cleaved by 6 m NaOH, which is a property of p‐hydroxybenzyl–sugar ether bonds. Cross‐linking of [¹⁴C]feruloyl‐arabinoxylans also occurred in vitro, in the presence of endogenous peroxidases plus exogenous H₂O₂. During cross‐linking, the feruloyl groups were oxidized, as shown by ultraviolet spectra and thin‐layer chromatography. Esterified diferulates were minor oxidation products; major products were: (i) esterified oligoferulates, released by treatment with mild alkali; and (ii) phenolic components attached to polysaccharides via relatively alkali‐stable (ether‐like) bonds. Thus, feruloyl esters participate in polysaccharide cross‐linking, but mainly by oligomerization rather than by dimerization. We propose that, after the oxidative coupling, strong p‐hydroxybenzyl–polysaccharide ether bonds are formed via quinone‐methide intermediates.     

Divergent selection for ester-linked diferulates in maize pith stalk tissues. Effects on cell wall composition and degradability

Cross-linking of grass cell wall components through diferulates (DFAs) has a marked impact on cell wall properties. However, results of genetic selection for DFA concentration have not been reported for any grass species. We report here the results of direct selection for ester-linked DFA concentration in maize stalk pith tissues and the associated changes in cell wall composition and biodegradability. After two cycles of divergent selection, maize populations selected for higher total DFA (DFAT) content (CHs) had 16% higher DFAT concentrations than populations selected for lower DFAT content (CLs). These significant DFA concentration gains suggest that DFA deposition in maize pith parenchyma cell walls is a highly heritable trait that is genetically regulated and can be modified trough conventional breeding. Maize populations selected for higher DFAT had 13% less glucose and 10% lower total cell wall concentration than CLs, suggesting that increased cross-linking of feruloylated arabinoxylans results in repacking of the matrix and possibly in thinner and firmer cell walls. Divergent selection affected esterified DFAT and monomeric ferulate ether cross link concentrations differently, supporting the hypothesis that the biosynthesis of these cell wall components are separately regulated. As expected, a more higher DFA ester cross-coupled arabinoxylan network had an effect on rumen cell wall degradability (CLs showed 12% higher 24-h total polysaccharide degradability than CHs). Interestingly, 8–8-coupled DFAs, previously associated with cell wall strength, were the best predictors of pith cell wall degradability (negative impact). Thus, further research on the involvement of these specific DFA regioisomers in limiting cell wall biodegradability is encouraged.     

Early cell-wall modifications of maize cell cultures during habituation to dichlobenil

Studies involving the habituation of plant cell cultures to cellulose biosynthesis inhibitors have achieved significant progress as regards understanding the structural plasticity of cell walls. However, since habituation studies have typically used high concentrations of inhibitors and long-term habituation periods, information on initial changes associated with habituation has usually been lost. This study focuses on monitoring and characterizing the short-term habituation process of maize (Zea mays) cell suspensions to dichlobenil (DCB). Cellulose quantification and FTIR spectroscopy of cell walls from 20 cell lines obtained during an incipient DCB-habituation process showed a reduction in cellulose levels which tended to revert depending on the inhibitor concentration and the length of time that cells were in contact with it. Variations in the cellulose content were concomitant with changes in the expression of several ZmCesA genes, mainly involving overexpression of ZmCesA7 and ZmCesA8. In order to explore these changes in more depth, a cell line habituated to 1.5 μM DCB was identified as representative of incipient DCB habituation and selected for further analysis. The cells of this habituated cell line grew more slowly and formed larger clusters. Their cell walls were modified, showing a 33% reduction in cellulose content, that was mainly counteracted by an increase in arabinoxylans, which presented increased extractability. This result was confirmed by immunodot assays graphically plotted by heatmaps, since habituated cell walls had a more extensive presence of epitopes for arabinoxylans and xylans, but also for homogalacturonan with a low degree of esterification and for galactan side chains of rhamnogalacturonan I. Furthermore, a partial shift of xyloglucan epitopes toward more easily extractable fractions was found. However, other epitopes, such as these specific for arabinan side chains of rhamnogalacturonan I or homogalacturonan with a high degree of esterification, seemed to be not affected.     

Expression of a fungal ferulic acid esterase increases cell wall digestibility of tall fescue (Festuca arundinacea)

In the cell walls of forage grasses, ferulic acid is esterified to arabinoxylans and participates with lignin monomers in oxidative coupling pathways to generate ferulate–polysaccharide–lignin complexes that cross-link the cell wall. Such cross-links hinder cell wall degradation by ruminant microbes, reducing plant digestibility. In this study, genetically modified Festuca arundinacea plants were produced expressing an Aspergillus niger ferulic acid esterase (FAEA) targeted to the vacuole. The rice actin promoter proved to be effective for FAEA expression, as did the cauliflower mosaic virus (CaMV) 35S and maize ubiquitin promoters. Higher levels of expression were, however, found with inducible heat-shock and senescence promoters. Following cell death and subsequent incubation, vacuole-targeted FAEA resulted in the release of both monomeric and dimeric ferulic acids from the cell walls, and this was enhanced several fold by the addition of exogenous endo-1,4-β-xylanase. Most of the FAEA-expressing plants showed increased digestibility and reduced levels of cell wall esterified phenolics relative to non-transformed plants. It is concluded that targeted FAEA expression is an effective strategy for improving wall digestibility in Festuca and, potentially, other grass species used for fodder or cellulosic ethanol production.     

Abscisic acid is a key inducer of hydrogen peroxide production in leaves of maize plants exposed to water stress

The histochemical and cytochemical localization of water stress-induced H(2)O(2) production in the leaves of ABA-deficient vp5 mutant and wild-type maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine and CeCl(3) staining, respectively, and the roles of endogenous ABA in the production of H(2)O(2) induced by water stress were assessed. Water stress induced by polyethylene glycol resulted in the accumulation of H(2)O(2) in mesophyll cells, bundle-sheath cells and vascular bundles of wild-type maize leaves, and the accumulation was substantially blocked in the mutant maize leaves exposed to water stress. Pre-treatments with several apoplastic H(2)O(2) manipulators abolished the majority of H(2)O(2) accumulation induced by water stress in the wild-type leaves. The subcellular localization of H(2)O(2) production was demonstrated in the cell walls, xylem vessels, chloroplasts, mitochondria and peroxisomes in the leaves of wild-type maize plants exposed to water stress, and the accumulation of H(2)O(2) induced by water stress in the cell walls and xylem vessels, but not in the chloroplasts, mitochondria and peroxisomes, was arrested in the leaves of the ABA mutant or the ABA biosynthesis inhibitor (tungstate)-pre-treated maize plants. Pre-treatments with the apoplastic H(2)O(2) manipulators also blocked the apoplastic but not the intracellular H(2)O(2) accumulation induced by water stress in the leaves of wild-type plants. These data indicate that under water stress, the apoplast is the major source of H(2)O(2) production and ABA is a key inducer of apoplastic H(2)O(2) production. These data also suggest that H(2)O(2) generated in the apoplast could not diffuse freely into subcellular compartments.     

Effects of cell wall components on the functionality of wheat gluten

Normal white wheat flours and especially whole meal flour contain solids from the inner endosperm cell walls, from germ, aleurone layer and the outer layers of cereal grains. These solids can prevent either gluten formation or gas cell structure. The addition of small amounts of pericarp layers (1–2%) to wheat flour had a marked detrimental effect on loaf volume. Microstructural studies indicated that in particular the epicarp hairs appeared to disturb the gas cell structure. The detrimental effects of insoluble cell walls can be prevented by using endoxylanases. It has been shown that some oxidative enzymes, naturally present in flour or added to the dough, will oxidise water-extractable arabinoxylans via ferulic acid bridges, and the resulting arabinoxylan gel will hinder gluten formation. The negative effects of water-unextractable arabinoxylans on gluten yield and rheological properties can be compensated by the addition of ferulic acid. Free ferulic acid can probably prevent arabinoxylan cross-linking via ferulic acid.     

Modification of cell wall architecture of wheat coleoptiles grown under hypergravity conditions.

Cell wall structure of wheat coleoptiles grown under continuous hypergravity (300 g) conditions was investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The amounts of cell wall polysaccharides substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. As a results, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. The major sugar components of the hemicellulose fraction, a polymer fraction extracted from cell walls with strong alkali, were arabinose (Ara), xylose (Xyl) and glucose (Glc). The molar ratios of Ara and Xyl to Glc in hypergravity-treated coleoptiles were higher than those in control coleoptiles. Furthermore, the fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. These results suggest that hypergravity stimuli bias the synthesis of hemicellulosic polysaccharides and increase the proportion of acidic polymers, such as arabinoxylans, in cell walls of wheat coleoptiles. These structural changes in cell walls may contribute to plant resistance to hypergravity stimuli.     

Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum

Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-β-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall.     

Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules.     

Comparative characterization of degraded and non-degradative hemicelluloses from barley straw and maize stems: Composition, structure, and thermal properties

Three organic solvents and one aqueous alkaline solution for fully fractional dissolving hemicelluloses from mild ball-milled cell wall of lignified barley straw and maize stems are described: 90% neutral dioxane, 80% dioxane containing 0.05 M HCl, dimethyl sulfoxide (DMSO), and 8% aqueous KOH. The four successive extractions resulted in dissolution of 94.6% and 96.4% of the original hemicelluloses and 93.7% and 95.3% of the original lignin from barley straw and maize stems, respectively. The structures of the hemicellulosic fractions released during the treatment with the neutral solvents of 90% dioxane and DMSO was found to remain intact, while the extractions with 80% acidic dioxane and 8% KOH under the conditions used resulted in a partial depolymerization of dissolved polysaccharides by cleavage of the glycosidic bonds and saponification of the ester groups in the polymers. The 90% neutral dioxane-soluble hemicellulosic fractions consisted mainly of the more branched arabinoxylans and mixed-linkage glucans such as β-glucans, whereas the hemicellulosic fractions solubilized during the sequential treatments with 80% acidic dioxane, DMSO, and 8% KOH are composed of arabino-(4-O-methyl-d-glucurono) xylans as the major hemicellulosic materials. In addition, the hemicellulosic polymers contained small amounts of ferulic and p-coumaric acids and lignins, revealing that the hemicelluloses removed are mostly unbound to the lignins in the cell walls of cereal straws. This non-degradative cell wall dissolution offers the potential to analyze polysaccharide components for the first time, and improve current hemicellulosic isolation method by using high concentration of aqueous alkali from the delignified cell walls.     

Cell wall remodeling under salt stress: Insights into changes in polysaccharides,feruloylation, lignification,and phenolic metabolism in maize

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.     

A maize homologue of the bacterial CMP-3-deoxy-D-manno-2-octulosonate (KDO) synthetases. Similar pathways operate in plants and bacteria for the activation of KDO prior to its incorporation into outer cellular envelopes

The eight-carbon acid sugar 3-deoxy-d-manno-2-octulosonate (KDO) is an essential component of Gram-negative bacterial cell walls and capsular polysaccharides. KDO is incorporated into these polymers as CMP-KDO, which is produced in an unusual activation step catalyzed by the enzyme CMP-KDO synthetase. CMP-KDO synthetase activity has traditionally been considered exclusive to Gram-negative bacteria. CMP-KDO synthetase inhibitors attract great interest owing to their potential as selective bactericides. The sugar KDO is also a component of the rhamnogalacturonan II pectin fraction of the primary cell walls of most higher plants and of the cell wall polysaccharides of some green algae. However, the metabolic pathway leading to its incorporation into the plant cell wall is unknown. This paper describes the isolation and characterization of a maize gene, which codes for a protein very similar in sequence and activity to prokaryotic CMP-KDO synthetases. Remarkably, the maize gene can complement a CMP-KDO synthetase (kdsB) Salmonella typhimurium mutant defective in cell wall synthesis. ZmCKS activity is novel in eukaryotes. The evolutionary origin of ZmCKS is discussed in relation to the high degree of conservation between the plant and bacterial genes and its atypical codon usage in maize.