Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : III. Structural Changes in the Feraxan during Coleoptile Elongation

Changes in structural features of feraxan (feruloylated arabinoxylans) in cell walls during development of maize (Zea mays L.) coleoptiles were investigated by analysis of fragments released by feraxanase, a specific enzyme purified from Bacillus subtilis. The following patterns were identified: (a) The total quantity of carbohydrate dissociated from a given dry weight of cell wall by feraxanase remained rather constant throughout the initial 10 days of coleoptile development. However, during the same period the proportion of ferulic acid in the fraction increased 12-fold. The absolute amount of ferulic acid per coleoptile also increased rapidly during this developmental phase. (b) Fragments dissociated by the enzyme were resolved into feruloylated and nonferuloylated components by reversed phase chromatography. While the quantity of feruloylated fractions represented a small portion of the total arabinoxylan during the phase of maximum coleoptile elongation (days 2-4) this component increased in abundance to reach a plateau (after 8-10 days). In contrast, nonferuloylated fractions were most abundant during the stage of maximum elongation but declined to a constant level by day 6. (c) Glycosidic linkage analysis of carbohydrate reveals that substitution of the xylan backbone of feraxan by arabinosyl residues decreased during coleoptile growth. We conclude that significant incorporation of ferulic acid occurs and/or more feruloyated domains are added to the arabinoxylan during development. This augmentation in phenolic acids is accompanied by a concerted displacement of arabinosyl residues and/or a reduction in the incorporation of regions enriched in arabinosyl sidechains.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls : II. Fractionation and Partial Characterization of Feraxan Fragments Dissociated by a Bacillus subtilis Enzyme (Feraxanase)

Structural features of feruloylated arabinoxylan (feraxan) present in Zea mays L. (hybrid B 73 × Mo 17) coleoptile cell walls have been studied using a purified feraxan-dissociating enzyme (feraxanase) and an α-arabinofuranosidase. This experimental approach has demonstrated the following. (a) Feraxanase dissociated ca. 20% (dry weight basis) of the maize wall preparation. The predominant oligosaccharides enzymically liberated were allocated into seven major subfractions designated A-1 (0.8%), B-1 (1.6%), B-2 (2.4%), B-3 (4.6%), C-1 (1.0%), C-2 (4.2%), and C-3 (0.3%). Values in parentheses reflect the percentage of the wall associated with each subfraction. Subfractions represent samples enriched in different degrees of polymerization, sugar composition, linkage arrangements, and phenolic acid content. (b) B-1, B-2, and B-3 fractions are not feruloylated and have smaller molecular mass (less than 10⁴ kilodaltons) and consist chiefly of t-arabinosyl-5-arabinosyl, 4-xylosyl, 2,4/3,4-xylosyl, and glucuronosyl residues, suggesting that these fragments constitute nonferuloylated regions of arabinoxylan. (c) C-2 and C-3 fractions contain ferulic acid (6.2% and 12.1%, respectively) and are similar to the B series in their sugar linkage arrangements but were derived from feruloylated regions. (d) Alkali treatment of the C-2 fraction decreases the molecular size of the fragment and liberates phenolic acids. The results suggest the presence of alkaline-labile links, probably diferulate bridges. (e) A-1 and C-1 fractions are larger (more than 5 × 10⁵ kilodalton) and contain t-galactosyl-, 4-galactosyl, 2,4-rhamnosyl-residues, galacturonic acid, and the sugar linkage arrangements common to other fractions. The A-1 fraction is not feruloylated, whereas C-1 fraction contains 0.5% ferulic acid. The presence of galactose, rhamnose, and galacturonic acid suggests that pectic polymers, probably homopolygalacturonans and rhamnogalacturonans, are linked to nonferuloylated and feruloylated segments of arabinoxylans.     

Structure of the Threonine-Rich Extensin from Zea mays

Chymotryptic digestion of a threonine-rich hydroxyproline-rich glycoprotein (THRGP) purified from the cell surface of a Zea mays cell suspension culture gave a peptide map dominated by the hexadecapeptide TC5: Thr-Hyp-Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys-Pro-Thr-Hyp-Hyp-Thr-Tyr, in which the repetitive motif Ser-Hyp-Lys-Pro-Hyp-Thr-Pro-Lys is homologous with the dominant decamer of P1-type dicot extensins: Ser-Hyp-Hyp-Hyp-Hyp-Thr-Hyp-Val-Tyr-Lys, modified by a Lys for Hyp substitution at residue 3, a Val-Tyr deletion at residues 8 and 9, and incomplete post-translational modification of proline residues. One of the minor peptides (TC1) contained the 8-residue sequence: Thr-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Tyr corresponding to the C-terminal tail (judging from the recently isolated maize cDNA clone MC56) which is homologous with the major repetitive motif of the `P3' class of dicot extensins. Direct peptide sequencing defined potential glycosylated regions on the THRGP corresponding to clone MC56 and showing that glycosylated and nonglycosylated domains alternate with high regularity. The THRGP is not in the polyproline-II conformation, judging from circular dichroic spectra, but nevertheless is an extended rod, from electron microscopic data. HF-solvolysis of cell walls from maize coleoptile, root, and root tip released deglycosylated THRGP detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots with high titer rabbit polyclonal antibodies raised against the intact THRGP. In a quantitative enzyme-linked immunosorbent assay, these antibodies cross-reacted 20% with tomato P1 extensin, and 18% with anhydrous hydrogen fluoride-deglycosylated P1. These results, together with other previously published data, show that maize THRGP is homologous with the dicot P1 extensins and, as such, is the first extensin isolated from a graminaceous monocot.     

Uptake and Metabolic Fate of Glucose, Arabinose, and Xylose by Zea mays Coleoptiles in Relation to Cell Wall Synthesis

According to the acid-growth hypothesis, auxin-induced secretion of hydrogen ions activate “wall loosening” enzymes that change the rheological properties of the cell wall. The wall loosening process may yield monosaccharides by the enzymic cleavage of load-bearing polysaccharides. Our study was initiated to determine the metabolic fate of such sugars when released from the major hemicellulosic polysaccharides of the cell walls of Zea mays coleoptiles.     

Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach

Two major α-glucan phosphorylases (I and II) from leaves of the C₄ plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C₃ plant spinach. The two α-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K_m = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C₃ plant spinach with mesophyll cell phosphorylase of the C₄ plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.     

Heat Shock Protein Synthesis Induced by Methomyl in Maize (Zea mays L.) Seedlings

Exposure of plumules of intact maize seedlings (Zea mays L.) to S-methyl-N-[(methylcarbamoyl)-oxy]thioacetimidate (methomyl) represses synthesis of several polypeptides normally made under control conditions and induces synthesis of polypeptides similar to maize heat shock polypeptides (HSPs). Three of the methomyl-induced polypeptides (18 kilodaltons) are recognized by antibodies raised against 18 kilodalton maize heat shock polypeptides.     

Flavonoids from Zea mays pollen

The flavonol glycosides of quercetin, isorhamnetin and kaempferol were isolated from Zea mays pollen. The most prominent flavonols were diglycosides of quercetin and isorhamnetin. Flavonol 3-O-glucosides of quercetin, isorhamnetin and kaempferol, and triglucosides of quercetin and isorhamnetin, were minor components. The flavonoid pattern of maize pollen is characterized by the accumulation of quercetin and isorhamnetin diglycosides and by the absence of flavones, which are common in other maize tissues.     

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

Cell wall development in maize coleoptiles

The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.

The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages.

     

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of ß(1–3),(1–4)-D-glucanand acid pH-induced extension of isolated Zea mays cell wallshas been investigated using a constant-load extension technique.Acidic buffer (4.5) was able to induce an additional extension(E_a) on cell walls already extended at pH 6.8 buffer under a20 g-mass load, indicating that the additional extension (E_a)was the parameter that better represented the effect of thedifferent treatments on the mechanical properties of maize coleoptilecell walls. The additional extension in response to acidic pHwas higher when cell walls had been previously autolysed for24 h at pH 5.5. Furthermore, the acid-pH effect was dependenton the presence during the constant load extension of some thermo-labilefactors, suggesting the participation of expansins. Acid pHincreased E_a of native cell walls through an increase in theplastic extension (E_p) in agreement with a one step mechanismleading directly to irreversible (plastic) wall extension assuggested by Cosgrove (1977). The autolytic degradation of ß(1–3),(1–4)-D-glucan was also able to modify the mechanicalproperties of maize coleoptile cell walls increasing its elasticextension (E_e) in response to pH 4.5 buffer but that modificationonly leads to an increase in wall extension when expansins areactive, suggesting a cooperation between ß-glucanturnover and expansin action. (Received August 5, 1998; Accepted March 16, 1999)     

5-hydroxyferulic acid in Zea mays and Hordeum vulgare cell walls

Considerable amounts of esterified E-5-hydroxyferulic acid and very small amounts of esterified E-sinapic acid were detected and identified in cell walls of young Zea mays and Hordeum vulgare, in addition to known E-p-coumaric and ferulic acids. Their relative amounts were determined by peak areas using GC. The ratios of E-p-coumaric-5-hydroxyferulic-sinapic-ferulic acid were 440:46:2:100 in corn, and 37:26:3:100 in barley, respectively.     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity     

Partial Purification and Reconstitution of the alpha-Ketoglutarate Carrier from Corn (Zea mays L.) Mitochondria

The α-ketoglutarate carrier from corn shoot mitochondria (Zea mays L., B 73) was solubilized in Triton X-114 and partially purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. On SDS-gel electrophoresis, the hydroxyapatite/celite eluate showed various protein bands between 12 and 70 kilodaltons. When reconstituted into liposomes, the α-ketoglutarate transport protein catalyzed a phthalonate-sensitive α-ketoglutarate/α-ketoglutarate exchange. The protein was purified 60-fold with a recovery of 88% with respect to the mitochondrial extract. The protein yield was 0.6%. The properties of the reconstituted carrier, i.e. requirement for a counter-anion, substrate specificity, and inhibitor sensitivity, were similar to those of the α-ketoglutarate transport system as characterized in plant and animal mitochondria.     

Oxygen fixation in the biosynthesis of ferulic acid by green Zea mays

The results of experiments in which seedlings of Zea mays were grown in the light in an atmosphere enriched with oxygen-18 indicate that the hydroxyl and methoxyl oxygen atoms in ferulic acid are derived from molecular oxygen.     

Hydrolytic Activity and Substrate Specificity of an Endoglucanase from Zea mays Seedling Cell Walls

An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1→3),(1→4)-β-d-glucosyl sequences. Substrates with (1→4)-β-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1→3)-β-glucans such as laminarin were not hydrolyzed. When (1→3),(1→4)-β-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 × 10⁴). Activity against a water soluble (1→3),(1→4)-β-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1→3),(1→4)-β-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1→3),(1→4)-β-d-glucan that contained blocks of (1→4) linkages and/or more than a single contiguous (1→3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1→3),(1→4)-β-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase.     

Investigating the roles of phenylpropanoids in the growth and development of Zea mays L.

The Poaceae includes some of the most important food, fiber, and bio-fuel crops. While there have been many studies investigating the function of phenylpropanoids in this family, most of our understanding is based on correlative data rather than experimental evidence. The current study was conducted to evaluate the roles of phenylpropanoids in the growth and development of Zea mays and to develop an experimental model for further investigations. Z. mays seedlings were grown in vitro with various concentrations of the competitive phenylalanine ammonia lyase inhibitor, 2-aminoindane-2-phosphonic acid (AIP). Ferulic acid, a downstream biosynthetic product, was added to determine if it could rescue the induced phenotypes. At lower concentrations of AIP, plants exhibited elongated roots and shoots, but at higher concentrations, growth was extremely stunted. At the cellular level, the epidermal cells of roots cultured with AIP exhibited a loss of intercellular adhesion and organization, and their cell walls were more readily degraded by enzymatic digestion. These characteristics were accompanied by significant reductions in primary cell wall autofluorescence, indicating that less ferulic acid and other phenolics were incorporated in the cell wall. The majority of these symptoms could be partially or entirely rescued by ferulic acid, providing further evidence that these differences were due to the inhibition of phenylpropanoid biosynthesis. This study provides experimental evidence supporting and expanding upon hypothesized functions of phenylpropanoids in the growth and development of Z. mays and provides an experimental system for further investigations in the Poaceae and other taxonomic groups.     

1-Naphthyl Acetate-Dependent Medium Acidification by Zea mays L. Coleoptile Segments

Zea mays L. cv INRA 5a coleoptile segments ecidify the incubation medium on the addition of 1-naphthyl acetate (1-NA). The buffering capacity of the bathing solution increases during 1-NA stimulated medium acidification. The solution bathing the 1-NA treated coleoptile segment was analyzed by high performance liquid chromatography. A considerable amount of acetic acid was detected in the bathing solution used to measure 1-NA-dependent medium acidification. For the first time, the data demonstrate directly the release of acetic acid from 1-NA. The extent of medium acidification was proportional to 1-NA concentration. Simultaneous measurement of medium acidification and acetate content upon addition of 1-NA showed that both processes were temporally correlated. The stoichiometry of proton equivalents to acetate ion was 0.966. Addition of 50 micromolar N,N′-dicyclohexylcarbodiimide had little effect on 1-NA-dependent medium acidification. The results indicate that 1-NA is hydrolyzed in the extracellular space of coleoptile cells.     

The Structure of Plant Cell Walls: VII. Barley Aleurone Cells

The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls.     

Enzymic Dissociation of Zea Shoot Cell Wall Polysaccharides : IV. Dissociation of Glucuronoarabinoxylan by Purified Endo-(1 --> 4)-beta-Xylanase from Bacillus subtilis

The structure of a glucuronoarabinoxylan in Zea mays L. (hybrid B73 x Mo17) shoot cell walls has been studied. The water-insoluble fraction of Zea shoot cell walls, pretreated with purified Bacillus subtilis (1 --> 3), (1 --> 4)-beta-d-glucan 4-glucanohydrolase, was treated with purified B. subtilis endo-(1 --> 4)-beta-xylanase. Carbohydrates (2.6% of the waterinsoluble fraction of Zea shoot cells walls) derived from the enzyme treatment consisted of glucuronoarabinoxylan fragments with molecular weights which varied from a few hundred to over 2.0 x 10(5) daltons. Structural analyses of the fragments suggested that the glucuronoarabinoxylan had a xylan backbone which contained (1 --> 4)-beta-d-xylopyranosyl residues, with about 60 to 70% substitution at the C-2 or C-3 position with arabinose, glucuronic acid, and other substituents. Furthermore, the glucuronoarabinoxylan contained a phenolic component which appeared to be primarily ferulic acid bonded to carbohydrate, probably by an ester linkage. The amount of ferulic acid was approximately 3 micrograms per 100 micrograms of carbohydrate.     

Photosystem II Activity in Agranal Bundle Sheath Chloroplasts from Zea mays

The photochemical activities of chloroplasts isolated from bundle sheath and mesophyll cells of maize (Zea mays var. DS606A) have been measured. Bundle sheath chloroplasts are almost devoid of grana, except in very young leaves, while mesophyll chloroplasts contain grana at all stages of leaf development.