Expression of a fungal ferulic acid esterase in suspension cultures of tall fescue (Festuca arundinacea) decreases cell wall feruloylation and increases rates of cell wall digestion

In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase (faeA) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11240-017-1168-9) contains supplementary material, which is available to authorized users

Manipulating the Phenolic Acid Content and Digestibility of Forage Grasses by Targeted Expression of Fungal Cell Wall Degrading Enzymes

Grass cell walls constitute 30-80% of forage dry matter, representing a major source of energy for ruminants. Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid) and other hydroxycinnamic acids are ester linked to arabinosyl residues in arabinoxylans of grass cell walls and undergo oxidative coupling reactions resulting in the formation of a variety of dehydrodiferulate dimers which cross-link cell wall polymers. Although such cross-links have a number of important roles in the cell wall, they also hinder the rate and extent of cell wall degradation by ruminant microbial and fungal enzymes. We have shown previously the expression of a ferulic acid esterase gene from Aspergillus niger in Festuca arundinacea and the potential of the expressed FAE to break phenolic cross-links and release monomeric and dimeric ferulic acids on cell death in vacuole targeted FAE plants. This was enhanced several fold by the addition of exogenous recombinant xylanase (Buanafina et al., 2002). We propose to decrease the level of phenolic cross-linking of cell wall carbohydrate by inducible expression of FAE to the apoplast, ER and golgi and by co-expressing FAE and endo-ß-1,4-xylanase from Trichoderma reesei to the apoplast and vacuole

A Comparison of Hygromycin and Paromomycin Selection Strategies in the Genetic Transformation of Seven \u3ci\u3eLolium, Festuca, Poa\u3c/i\u3e, and \u3ci\u3eAgrostis\u3c/i\u3e Species

Hygromycin selection for the hpt gene, expressed from the CaMV-35S promoter, has been successful in transgenesis of a limited number of grass species. As an alternative to hpt selection Altpeter et al., (2000) reported successful transformation using paromomycin selection for the nptII gene expressed by the maize ubiquitin promoter. We have tested the utility of a number of selection cassettes using previously sporadically transformable species which nevertheless had very good tissue culture and regeneration protocols

Nylon Mesh as an Improved Support for Bombarded Calli or Cell Suspensions

Using cell suspensions to transform some grass species by particle bombardment has a number of disadvantages including increased somoclonal variation in liquid cell culture and poor performance due to polysaccharide production. The use of calli avoids these problems, but the manipulation of calli through numerous media changes is laborious and time-consuming. We investigated a possible mechanism to facilitate the use of calli in transformation by immobilising calli on mesh

Linkage Mapping of Stem Saccharification Digestibility in Rice

Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties

A Genome Wide Association Study of arabinoxylan content in 2-row spring barley grain

In barley endosperm arabinoxylan (AX) is the second most abundant cell wall polysaccharide and in wheat it is the most abundant polysaccharide in the starchy endosperm walls of the grain. AX is one of the main contributors to grain dietary fibre content providing several health benefits including cholesterol and glucose lowering effects, and antioxidant activities. Due to its complex structural features, AX might also affect the downstream applications of barley grain in malting and brewing. Using a high pressure liquid chromatography (HPLC) method we quantified AX amounts in mature grain in 128 spring 2-row barley accessions. Amounts ranged from ~ 5.2 μg/g to ~ 9 μg/g. We used this data for a Genome Wide Association Study (GWAS) that revealed three significant quantitative trait loci (QTL) associated with grain AX levels which passed a false discovery threshold (FDR) and are located on two of the seven barley chromosomes. Regions underlying the QTLs were scanned for genes likely to be involved in AX biosynthesis or turnover, and strong candidates, including glycosyltransferases from the GT43 and GT61 families and glycoside hydrolases from the GH10 family, were identified. Phylogenetic trees of selected gene families were built based on protein translations and were used to examine the relationship of the barley candidate genes to those in other species. Our data reaffirms the roles of existing genes thought to contribute to AX content, and identifies novel QTL (and candidate genes associated with them) potentially influencing the AX content of barley grain. One potential outcome of this work is the deployment of highly associated single nucleotide polymorphisms markers in breeding programs to guide the modification of AX abundance in barley grain