Ronald D. Hatfield

Lax leaf maize: cell wall composition and nutritional value

Forage nutritive value, which comprises traits such as digestibility, fibre, lignin and protein content, is an important criterion for maize (Zea mays L) harvested as silage. Lines with a characteristic phenotype (‘lax leaf’) could be useful sources of genes for improved nutritive value in maize. A study was conducted to characterise the cell wall composition of the lax leaf line. Lax leaf inbreds and inbreds representing ‘normal’ maize were evaluated for cell wall neutral sugars, uronic acids, Klason lignin and phenolic acids in five tissues from the ear node and the internode above it. Acid detergent fibre (ADF) and neutral detergent fibre (NDF) and 48 h in vitro true digestibility (IVTD) were predicted using near-infrared reflectance spectrophotometry (NIRS) calibrated with a subset of the scanned samples. Lax leaf inbred tissues had lower levels of ADF, NDF, lignin and xylose and were more digestible than tissues from the inbreds representing ‘normal’ maize. It was not known whether the lax leaf phenotype resulted from alterations in nutritive value traits or whether laxness and nutritive value traits are independent from one another. A second study was conducted to determine the nature of genetic control of the lax leaf character and to determine the genotypic relation between the lax leaf character and nutritive value. A recombinant inbred mapping population was developed from a cross between the lax leaf line and an inbred line with stiff upright leaves. Whole-plant samples from each recombinant inbred line were evaluated for ADF, NDF, acid detergent lignin (ADL) and IVTD of dry matter using NIRS. Laxness, measured by number of broken leaves, was associated with lower nutritive value in this population (genetic correlations 0.16–0.34), which was contrary to expectation. Amplified restriction fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were used to identify linkage groups associated with the lax leaf character, digestibility and fibre content. Several linkage groups were associated with both the lax leaf character and nutritive value. Where these characters were associated with the same linkage group, the lax leaf parent allele was associated with greater laxness but reduced nutritive value. The lax leaf parent allele was associated with increased nutritive value in linkage groups unassociated with the lax leaf character. While the lax leaf line may be a good source for alleles for improved nutritive value, selection for laxness will not likely be accompanied by improvement in forage quality. © 2000 Society of Chemical Industry

Lignin-ferulate cross-links in grasses: active incorporation of ferulate polysaccharide esters into ryegrass lignins

Abstract Active incorporation of ferulate polysaccharide esters into ryegrass lignins has been demonstrated by NMR spectroscopy of uniformly 13 C-labeled ryegrass. Observation, in the HMBC spectrum, of products of ferulate at its 8-position coupling with hydroxycinnamyl alcohols at the β-position (producing 8-β′-linked structures) is proof that ferulate-lignin radical cross-coupling reactions occur in vivo. Correlations of H-α′ (hydroxycinnamyl alcohol moiety) with guaiacyl and syringyl 1-, 2-, and 6-aromatic carbons in 8-β′ structures indicates that ferulates couple with both coniferyl and sinapyl alcohol monomers. As notable as the presence of this and other ferulate products is the absence of coupling of ferulate at its 8-position with the 5- and O -4-positions of lignin units. Such structures were significant when ferulate was biomimetically incorporated into a synthetic lignin. Since hydroxycinnamyl alcohols couple almost exclusively at their β-position in cross-coupling reactions, the 8-5′ and 8- O -4′ structures would only be formed by coupling with higher lignin oligomers (with no side-chain conjugation). Exclusive reaction of ferulates with lignin monomers is the first real evidence that ferulate polysaccharide esters in grasses are acting as initiation or nucleation sites for lignification and are critical entities in directing cell-wall cross-linking during plant growth and development.

Identification and synthesis of new ferulic acid dehydrodimers present in grass cell walls

Seven isomeric dehydrodimers of ferulic acid (4-hydroxy-3-methoxycinnamic acid) have been synthesized and identified in extracts of saponified cell walls of cocksfoot, switchgrass, and suspension-cultured corn. Dehydrodimers (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid, (Z)-β-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-3-methoxycinnamic acid, (E)-3-{4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy}-4-hydroxy-5-methoxycinnamic acid, (E,E)-4,4′-dihydroxy-3,5′-dimethoxy-β,3′-bicinnamic acid, 4,4′-dihydroxy-3,3′-dimethoxy-β,β′-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-6-methoxy-1,2-dihydronaphthalene-2,3-dicarboxylic acid, all arise from oxidative coupling of ferulate esters in cell walls and represent products of 8–5, 8–8, 8–O–4, 4–O–5, and 5–5 radical coupling. Prior literature has acknowledged only the presence of the 5–5-coupled dehydrodimer (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid. Consequently, by measuring only a single dehydrodimer and assuming inappropriate response factors, ferulate dehydrodimers have been underestimated by factors of up to 20. Synthetic routes to all seven isomers have been developed to provide structural authentication and determination of GC response factors.

Ferulate cross-linking in cell walls isolated from maize cell suspensions

Abstract Cross-linking of arabinoxylans by ferulate dehydrodimers and incorporation of feruloylated arabinoxylans into lignin were modelled with maize walls (Zea mays cv Black Mexican) containing 5.3–18.0 mg g−1 of total ferulates. The proportion of dehydrodimers to total ferulates increased from ca 20 to 45% when dilute hydrogen peroxide was added to walls containing bound peroxidase. About 45% of the dehydrodimers were coupled by 8-5 linkages, with 8-8, 8-O-4 and 5-5 coupled dehydrodimers each comprising 10–25% of the total. The quantity of ferulates released by saponification were reduced by 83–95% when exogenously supplied hydroxycinnamyl alcohols were polymerized into walls by wall-bound peroxidase and in situ generated hydrogen peroxide. Only 40% of the ferulates incorporated into lignin were recovered following hydrolysis of ether linkages. These results indicate that primary walls in grasses become extensively cross-linked by ferulic and dehydrodiferulic acids during lignification, and that only a portion of ferulates in lignified tissues are measurable by current solvolytic methods.

Diferulate cross-links impede the enzymatic degradation of non-lignified maize walls

We assessed the e†ect of ferulate substitution and diferulate cross- linking of xylans on the degradation of cell walls by two fungal enzyme mixtures, one of which contained feruloyl esterase and high xylanase activities. Non- ligni-ed cell suspensions of maize (Zea mays) were grown with 0 or 40 lM 2- aminoindan-2-phosphonic acid to produce walls with normal (17E 2m g g~1 )o r reduced (5E 1m g g~1) ferulate concentrations. Walls were incubated with mercap- toethanol to inhibit diferulate formation or with hydrogen peroxide to stimulate diferulate formation by wall bound peroxidases. Varying the ferulate substitution of xylans did not a†ect cell wall hydrolysis. In contrast, increasing ferulate dimer- isation from 18 to 40% reduced carbohydrate release by 94E122 mg g~1 after 3 h and by 0E48 mg g~1 after 54 h of enzymatic hydrolysis. Diferulate cross- links impeded the release of xylans, cellulose and pectins from walls. These results provide compelling evidence that diferulate cross-links reduce the rate and, to a lesser degree, the extent of wall hydrolysis by fungal enzymes. Our results also suggest that enzyme mixtures containing high xylanase activity but not feruloyl esterase activity can partially overcome the inhibitory e†ects of diferulate cross-linking on wall hydrolysis. 1998 SCI. (

Unexpected variation in lignin

Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Pines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. These results argue in favor of an increased potential for genetic modification of lignin and indicate that our knowledge of the biosynthesis of lignin is far from complete.

Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls

AbstractPeroxidases are heavily implicated in plant cell wall cross-linking reactions, altering the properties of the wall and impacting its utilization. Polysaccharide-polysaccharide cross-linking in grasses is achieved by dehydrodimerization of hydroxycinnamate-polysaccharide esters; a complex array of hydroxycinnamic acid dehydrodimers are released by saponification. Ferulates are the major cross-linking agents, but sinapate-ferulate cross-products have been discovered implicating sinapates in a similar role. New dehydrodimers have been authenticated, expanding our knowledge of the chemistry, role, and extent of cross-linking reactions. Ferulate dehydrotrimers have been discovered; whether these trimers truly cross-link three independent polysaccharide chains or only two remains to be determined. Hydroxycinnamates and their dehydrodimers also undergo radical coupling reactions with lignin monomers and possibly oligomers, resulting in lignin-polysaccharide cross-linking in the wall. Both polysaccharide-polysaccharide and lignin-polysaccharide cross-links inhibit the enzymatic hydrolysis of cell walls. The cross-linking process has particular relevance to plant physiology, human and animal nutrition and health, and food technology. Abbreviations: CW – cell wall; DFA – dehydrodiferulic acid (or dehydrodiferulate in context); DSA – dehydrodisinapic acid; TFA – dehydrotriferulic acid; SA – sinapic acid (1S); TA – thomasidioic acid (5C3SS); IDF – insoluble dietary fiber; SDF – soluble dietary fiber; GC-MS – gas chromatography-mass spectrometry; NMR – nuclear magnetic resonance (spectroscopy).

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Lignin–feruloyl ester cross-links in grasses. Part 1. Incorporation of feruloyl esters into coniferyl alcohol dehydrogenation polymers

Methyl 5-O-(E)-[γ-13C]feruloyl-α-L-arabinofuranoside (FA-Ara) has been synthesized and incorporated into a synthetic lignin dehydrogenation polymer (DHP) of coniferyl alcohol. Inverse-detected long-range C–H correlation NMR experiments on the DHP lignin gave correlation peaks indicative of the copolymerization of the FA-Ara and coniferyl alcohol into the DHP polymer. The bonding sites and modes, as determined by analysis of the carbonyl region of the long-range C–H correlated 2D NMR experiment, are predictable from free-radical coupling mechanisms. In addition to the abundant 4-O-α′ and 4-O-β′ ether couplings, structures involving the β-position of the feruloyl moiety of FA-Ara in β-ether, phenylcoumaran and pinoresinolide structures were present. The incorporation of feruloyl esters into a lignin DHP results in some structures which would not release ferulic acid by solvolytic schemes currently used for quantitation of ferulic acid in plant materials. Thus the degree to which hydroxycinnamic acids are involved in the lignification of forages may be significantly underestimated.

Cloning and Characterization of Red Clover Polyphenol Oxidase cDNAs and Expression of Active Protein in Escherichia coli and Transgenic Alfalfa

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%–83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.