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Background The crosslinking of maize cell wall components, particularly mediated by the formation of ferulic acid dimers or diferulates, has been associated with important crop valorization traits such as increased pest resistance, lower forage digestibility, or reduced bioethanol production. However, these relationships were based on studies performed using diverse unrelated inbred lines and/or populations, so genetic background could interfere on these associations. Results In the present research, the success of a pedigree selection program aimed to obtain inbred lines from a common antecessor with contrasting diferulate concentration was evaluated. From the 10 inbreds lines developed we could validate the success of the breeding program, obtaining 4 inbred lines with significant contrating values of total diferulate content in the pith tissues (two of each group): high (X̅= 0.69 mg/g of DW) and low (X̅= 0.35 mg/g). Ferulate changes in the same way were also observed: high (X̅= 3.09 mg/g of DW) and low (X̅= 1.62 mg/g). On the other hand, we found strong and positive correlations between DFAT and individual dimers, and moderate negative correlations between total DFAT and a main cell wall component such as cellulose. However, we did not find a significant effect of DFAT on maize valorization traits, except of a negative effect of DFAT on the concentration of sugars released after the enzimatic hydrolysis of the pith tissues. Interestingly, increasing DFAT in the pith does not seem to affect the digestibility of the forage or the saccharification of the stover residue, highlighting that changes in a specific tissue do not encompass correlated changes in other resources. Conclusions Overall, we have obtained contrasting inbred lines with diferulates concentration, which could be uselful in further studies focussing in the identification of regions/genes predominantly involved in the hydroxycinnamate biosynthesis pathway and cell wall crosslinking network.

Background The development of bioenergy crops with reduced recalcitrance to enzymatic degradation represents an important challenge to enable the sustainable production of advanced biofuels and bioproducts. Biomass recalcitrance is partly attributed to the complex structure of plant cell walls inside which cellulose microfibrils are protected by a network of hemicellulosic xylan chains that crosslink with each other or with lignin via ferulate (FA) bridges. Overexpression of the rice acyltransferase OsAT10 is an effective bioengineering strategy to lower the amount of FA involved in the formation of cell wall crosslinks and thereby reduce cell wall recalcitrance. The annual crop sorghum represents an attractive feedstock for bioenergy purposes considering its high biomass yields and low input requirements. Although we previously validated the OsAT10 engineering approach in the perennial bioenergy crop switchgrass, the effect of OsAT10 expression on biomass composition and digestibility in sorghum remains to be explored. Results We obtained eight independent sorghum (Sorghum bicolor (L.) Moench) transgenic lines with a single copy of a construct designed for OsAT10 expression. Consistent with the proposed role of OsAT10 in acylating arabinosyl residues on xylan with p-coumarate (pCA), a higher amount of p-coumaroyl-arabinose was released from the cell walls of these lines upon hydrolysis with trifluoroacetic acid. However, no major changes were observed regarding the total amount of pCA or FA esters released from cell walls upon mild alkaline hydrolysis. Certain diferulate (diFA) isomers identified in alkaline hydrolysates were increased in some transgenic lines. The amount of the main cell wall monosaccharides glucose, xylose, and arabinose was unaffected. The transgenic lines showed reduced lignin content and their biomass released higher yields of sugars after ionic liquid pretreatment followed by enzymatic saccharification. Conclusions Expression of OsAT10 in sorghum leads to an increase of xylan-bound pCA without reducing the overall content of cell wall FA esters. Nevertheless, the amount of total cell wall pCA remains unchanged indicating that most pCA is ester-linked to lignin. Unlike other engineered plants overexpressing OsAT10 or a phylogenetically related acyltransferase with similar putative function, the improvements of biomass saccharification efficiency in sorghum OsAT10 lines are likely the result of lignin reductions rather than reductions of cell wall-bound FA. These results also suggest a relationship between xylan-bound pCA and lignification in cell walls.

The aim of this work was to investigate the role of pericarp phenylpropanoids as resistance factors to F. verticillioides in eleven maize genotypes. Disease severity and kernel fumonisin accumulation were measured after inoculation with F. verticillioides and related to contents of pericarp phenylpropanoids in field trials conducted during 2 years. Grain fumonisin concentrations were highly dependant on disease severity of the genotypes ( r  = 0.88). A detailed analysis of pericarp phenylpropanoids indicated the presence of trans -ferulic acid (tFA), cis -ferulic acid (cFA), p-coumaric acid (pCA), and five diferulates (DFAs). The most prominent diferulates were 8,5′-diferulic acid benzofuram form (8,5′-DFAbz), followed by 8,5′-DFA and 8,8′-DFA. Except for cFA, the most resistant genotypes exhibited high levels of phenylpropanoids which were related to low levels of disease severity and grain fumonisin concentration (−0.61 > r  > −0.90). A stepwise regression analysis revealed that total diferulates was the best explanatory parameter for variability of disease severity ( r 2  = 0.71). Grain fumonisin concentration was well depicted by contents of total diferulates, 8,5′DFAbz and pCA ( r 2  = 0.82). Our findings suggest that high level of phenylpropanoids in the kernel pericarp is a trait associated to less disease severity and fumonisin accumulation caused by F. verticillioides . Further research is in progress to map quantitative trait loci for these cell wall components in bi-parental populations derived by crossing resistant and susceptible genotypes included in this study.

Feruloyl esterases (FAEs) are a key group of enzymes that hydrolyze ferulic acids ester-linked to plant polysaccharides. The cow’s rumen is a highly evolved ecosystem of complex microbial microflora capable of converting fibrous substances to energy. From direct cloning of the rumen microbial metagenome, we identified seven active phagemids conferring feruloyl esterase activity. The genomic inserts ranged from 1633 to 4143 bp, and the ORFs from 681 to 1359 bp. BLAST search reveals sequence homology to feruloyl esterases and esterases/lipases identified in anaerobes. The seven genes were expressed in Escherichia coli , and the proteins were purified to homogeneity. The FAEs were found to cover types B, C, and D in the feruloyl esterase classification system using model hydroxycinnamic acid esters. The release of ferulic acid (FA) catalyzed by these enzymes was established using natural substrates corn fiber (CF) and wheat insoluble arabinoxylan (WIA). Three of the enzymes were demonstrated to cleave diferulates and hence the capability to break down Araf-FA-FA-Araf cross-links. The wide variation in the sequence, activity, and substrate specificity observed in the FAEs discovered in this study is a confirming evidence that combined actions of a full range of FAE enzymes contribute to the high-efficiency fiber digestion in the rumen microbial ecosystem.

Glyceryl diferulate (DFG) is a water-soluble ester of ferulic acid. A novel ionic liquid (IL) system for enzymatic transesterification of ethyl ferulate (EF) with glycerol to produce DFG was developed. Of three ILs with different anions (BF₄ ⁻, PF₆ ⁻ and TF₂N⁻) and cations (BDMIM, OMIM, HMIM, BMIM, and EMIM), EMIMTF₂N proved the best using a commercial lipase. It had a significant protective effect against thermal inactivation of the enzyme. High EF conversion (~100 %) and DFG yield (45 %) were achieved using 45 mg enzyme/ml; temperature, 70 °C; reaction time, 12 h.

The relationship between the primary cell wall phenolic acids, dehydrodimers of ferulic acid, and maize grain resistance to Fusarium graminearum, the causal agent of gibberella ear rot, was investigated. Concentrations of dehydrodimers of ferulic acid were determined in the pericarp and aleurone tissues of five inbreds and two hybrids of varying susceptibility and in a segregating population from a cross between a resistant and susceptible inbred. Significant negative correlations were found between disease severity and diferulic acid content. Even stronger correlations were observed between diferulic acid and the fungal steroid ergosterol, which is an indicator of fungal biomass in infected plant tissue. These results were consistent over two consecutive field seasons, which differed significantly for temperature and rainfall during pollination, the most susceptible stage of ear development. No correlation was found between the levels of these phenolics and deoxynivalenol levels. This is the first report of in vivo evidence that the dehydrodimers of ferulic acid content in pericarp and aleurone tissues may play a role in genotypic resistance of maize to gibberella ear rot.

Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5—20 min, the CoA pool had lost ist 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse—chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1—3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised < 1 min after their attachment to polysaccharides. For at least the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides < 1 min after the biosynthesis of the latter, and > 10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and largr products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed.

We examined relationships among cell wall feruloylation, diferulate cross-linking, p-coumarate deposition, and apoplastic peroxidase (EC 1.11.1.7) activity with changes in the elongation rate of leaf blades of slow and rapid elongating genotypes of tall fescue (Festuca arundinacea Schreb.). Growth was not directly influenced by ferulic acid deposition but leaf elongation decelerated as 8—5-, 8—O—4-, 8—8-, and 5—5-coupled diferulic acids accumulated in cell walls. Growth rapidly slowed and stopped with the deposition of p-coumarate, which is primarily associated with lignification in grass cell walls. Accretion of ferulate, diferulates and p-coumarate continued after growth ended, into the later stages of secondary wall formation. The concentration of 8-coupled diferulates dwarfed that of the more commonly measured 5—5-coupled isomer, suggesting that the latter dimer is a poor indicator of diferulate cross-linking in cell walls. Further work is required to clearly demonstrate the role of diferulate cross-linking and p-coumaroylated lignins in the cessation of leaf growth in grasses.