Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis.

In the near future, grasses must provide most of the biomass for the production of renewable fuels. However, grass cell walls are characterized by a large quantity of hydroxycinnamic acids such as ferulic and p-coumaric acids, which are thought to reduce the biomass saccharification. Ferulic acid (FA) binds to lignin, polysaccharides and structural proteins of grass cell walls cross-linking these components. A controlled reduction of FA level or of FA cross-linkages in plants of industrial interest can improve the production of cellulosic ethanol. Here, we review the biosynthesis and roles of FA in cell wall architecture and in grass biomass recalcitrance to enzyme hydrolysis.

The allelochemical L-DOPA increases melanin production and reduces reactive oxygen species in soybean roots.

The non-protein amino acid, L-3,4-dihydroxyphenylalanine (L-DOPA), is the main allelochemical released from the roots of velvetbean and affects seed germination and root growth of several plant species. In the work presented here, we evaluated, in soybean roots, the effects of L-DOPA on the following: polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities; superoxide anion (O·-2), hydrogen peroxide (H(2)O(2)), and melanin contents; and lipid peroxidation. To this end, 3-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 0.1 to 1.0 mM L-DOPA in a growth chamber (at 25°C, with a light/dark photoperiod of 12/12 hr and a photon flux density of 280 µmol m(-2) s(-1)) for 24 hr. The results showed that L-DOPA increased the PPO activity and, further, the melanin content. The activities of SOD and POD increased, but CAT activity decreased after the chemical exposure. The contents of reactive oxygen species (ROS), such as O·-2 and H(2)O(2), and the levels of lipid peroxidation significantly decreased under all concentrations of L-DOPA tested. These results suggest that L-DOPA was absorbed by the soybean roots and metabolized to melanin. It was concluded that the reduction in the O·-2 and H(2)O(2) contents and lipid peroxidation in soybean roots was due to the enhanced SOD and POD activities and thus a possible antioxidant role of L-DOPA.

Cadmium uncouples mitochondrial oxidative phosphorylation and induces oxidative cellular stress in soybean roots.

Cadmium (Cd) inhibits soybean root growth, but its exact mode of action is still not completely understood. We evaluated the effects of Cd on growth, mitochondrial respiration, lipid peroxidation, total phenols, glutathione, and activities of lipoxygenase (LOX), superoxide dismutase (SOD), and catalase (CAT) in soybean roots. In primary roots, Cd stimulated KCN-insensitive respiration and KCN-SHAM-insensitive respiration, indicating the involvement of the alternative oxidase (AOX) pathway, while it decreased KCN-sensitive respiration, suggesting an inhibition of the cytochrome oxidase pathway (COX). In isolated mitochondria, Cd uncoupled the oxidative phosphorylation since it decreased state III respiration (coupled respiration) and ADP/O and respiratory control ratios, while it increased state IV respiration (depletion of exogenously added ADP). The uncoupling effect increased extramitochondrial LOX activity, lipid peroxidation, and oxidized and reduced glutathione, which induced an antioxidant response with enhanced SOD and CAT activities. In brief, our findings reveal that Cd acts as an uncoupler of the mitochondrial oxidative phosphorylation in soybean roots, disturbing cellular respiration and inducing oxidative cellular stress.

Cadmium-induced lignification restricts soybean root growth.

The effects of cadmium (Cd), a well-known environmental pollutant with high toxicity to plants, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) soluble plus cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H(2)O(2)) levels, and the content and monomeric composition of lignin in soybean (Glycine max) roots. Three-day-old seedlings were cultivated in half-strength Hoagland's solution (pH 6.0), with or without 25-100 µM CdCl(2) in a growth chamber (25°C, 12/12-h light/dark photoperiod, irradiance of 280 µmolm(-2)s(-1)) for 24h. In general, root length and the fresh and dry weights decreased followed by loss of cell viability after Cd treatment. PAL activity, soluble and cell wall-bound POD activities, and H(2)O(2) and lignin contents increased significantly after Cd exposure. The lignin monomeric composition of Cd-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and syringyl (S) units. These results suggest that the effects caused by Cd may be due to excessive production of monolignols forming lignin, which solidifies the cell wall and restricts root growth.

Exogenous application of rosmarinic acid improves saccharification without affecting growth and lignification of maize.

Biomimetically incorporated into the lignin structure, rosmarinic acid improves in vitro maize cell wall saccharification; however, no in planta studies have been performed. We hypothesized that rosmarinic acid, itself, could inducer saccharification without disturbing plant growth. Its effects on growth, enzymes of the phenylpropanoid pathway, lignin, monomeric composition, and saccharification of maize were evaluated. In a short-term (24 h) exposure, rosmarinic acid caused deleterious effects on maize roots, inhibiting the first enzymes of the phenylpropanoid pathway, phenylalanine ammonia-lyase and tyrosine ammonia-lyase, altering lignin composition and slightly increasing saccharification. In a long-term (14 d) exposure, rosmarinic acid increased saccharification of maize stems by about 50% without any deleterious effects on plant growth, the phenylpropanoid pathway and lignin formation. This demonstrated that exogenous application of rosmarinic acid on maize plants improved saccharification, and represented an interesting approach in facilitating enzymatic hydrolysis of biomass polysaccharides and increasing bioethanol production.

The acetyl bromide method is faster, simpler and presents best recovery of lignin in different herbaceous tissues than Klason and thioglycolic acid methods.

We compared the amount of lignin as determined by the three most traditional methods for lignin measurement in three tissues (sugarcane bagasse, soybean roots and soybean seed coat) contrasting for lignin amount and composition. Although all methods presented high reproducibility, major inconsistencies among them were found. The amount of lignin determined by thioglycolic acid method was severely lower than that provided by the other methods (up to 95%) in all tissues analyzed. Klason method was quite similar to acetyl bromide in tissues containing higher amounts of lignin, but presented lower recovery of lignin in the less lignified tissue. To investigate the causes of the inconsistencies observed, we determined the monomer composition of all plant materials, but found no correlation. We found that the low recovery of lignin presented by the thioglycolic acid method were due losses of lignin in the residues disposed throughout the procedures. The production of furfurals by acetyl bromide method does not explain the differences observed. The acetyl bromide method is the simplest and fastest among the methods evaluated presenting similar or best recovery of lignin in all the tissues assessed.

Enhanced lignin monomer production caused by cinnamic Acid and its hydroxylated derivatives inhibits soybean root growth.

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.