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

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

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

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

Degradation of lignin β-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the beta-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized beta-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNAi silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in ~60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation by much smaller magnitude, possibly due to higher expression of functionally-redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, ~doubled arabinose acylated by p-coumarate, changes in 2D-NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production, and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for the key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining, and animal nutrition applications

The complex cell wall composition of syncytia induced by plant parasitic cyst nematodes reflects both function and host plant

Plant–parasitic cyst nematodes induce the formation of specialised feeding structures, syncytia, within their host roots. These unique plant organs serve as the sole nutrient resource for development and reproduction throughout the biotrophic interaction. The multinucleate syncytium, which arises through local dissolution of cell walls and protoplast fusion of multiple adjacent cells, has dense cytoplasm containing numerous organelles, surrounded by thickened outer cell walls that must withstand high turgor pressure. However, little is known about how the constituents of the syncytial cell wall and their conformation support its role during nematode parasitism. We used a set of monoclonal antibodies, targeted to a range of plant cell wall components, to reveal the microstructures of syncytial cell walls induced by four of the most economically important cyst nematode species, Globodera pallida, Heterodera glycines, Heterodera avenae and Heterodera filipjevi, in their respective potato, soybean and spring wheat host roots. In situ fluorescence analysis revealed highly similar cell wall composition of syncytia induced by G. pallida and H. glycines. Both consisted of abundant xyloglucan, methyl-esterified homogalacturonan and pectic arabinan. In contrast, the walls of syncytia induced in wheat roots by H. avenae and H. filipjevi contain little xyloglucan but are rich in feruloylated xylan and arabinan residues, with variable levels of mixed-linkage glucan. The overall chemical composition of syncytial cell walls reflected the general features of root cell walls of the different host plants. We relate specific components of syncytial cell walls, such as abundant arabinan, methyl-esterification status of pectic homogalacturonan and feruloylation of xylan, to their potential roles in forming a network to support both the strength and flexibility required for syncytium function

A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon

The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed. We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait. Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the BdGT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the BdGT43A gene in Brachypodium. The biomass of the silenced lines showed higher digestibility supporting a causative role of the BdGT43A gene, suggesting that it might form a good target for improving straw digestibility in crops