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 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

Results of nodule detection along AUV track SO239_115-1_AUV9 (Abyss_175) during SONNE cruise SO239

Images were acquired by the DeepSurvey Camera on board GEOMAR's AUV Abyss. Nodules were delineated by the CoMoNoD algorithm [see related to references]. Result files are computed per AUV dive. Nodule detections below 5cm^2 are neglected as are detections above 707cm^2. Abundance statistics are computed per m^2 and gridded per m^2 as well. For overlapping images, max-pooling has been applied to select the values reported in the result files. Pixel values in the rendered maps correspond to the units reported in the ASCI files (median-nodule-size: cm^2, nodule-number: m^-2, percent-coverage: %, sorting, skewness and pixel-contributions are unit-free)

Comparison of growth of newly emerging third leaves of control and apoplast (T27 and T27R) or Golgi (T28 and T29) FAE expressing plants.

<p>Growth as increase in leaf length (A), distribution of growth within the elongation zone of leaf blades, determined as the relative segmental elongation rate (B), maximum daily extension rate (C), and leaf length at maximum extension rate (D). Mean ± SEM (n = 20–30 from each plant). Third leaves from tillers of 2–3 plants per line were measured until leaf length was constant. Letters indicate significant difference (Tukey’s, α = 0.05) among mean values.</p

Chlorophyll content of leaves of control and an FAE expressing plant (T27R) during leaf senescence.

<p>Chlorophyll content of leaves of control and an FAE expressing plant (T27R) during leaf senescence.</p