Abstract
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.
Highlights
Interest in the relationship between bound cell wall phenolics and their effects on the digestibility of plant material has interested plant scientists for many years
Ferulic acid residues are attached to cell wall arabinoxylans of grasses via the C5-hydroxyl of α-l-arabinose side-chains on the xylan polymers (Grabber et al 1998), and estimates have suggested the levels of ferulic acid in grass cell walls can be as high as 3% dry weight (Kroon et al 1999)
We have previously reported the effects of expressing ferulic acid esterase (faeA) from A. niger in transgenic plants of Festuca arundinacea with FAE constitutively or inducibly targeted to the vacuole
Summary
Interest in the relationship between bound cell wall phenolics and their effects on the digestibility of plant material has interested plant scientists for many years. Forage quality has been improved by reducing the lignin content and lignin composition of tall fescue plants by down regulating expression of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase, resulting in increased in-vitro dry matter digestibility without significant changes in cellulose, hemicellulose or neutral sugar composition, or in p-coumaric acid or ferulic acid levels (Chen et al 2003, 2004). In this work we report the effect of expression of an A. niger FAE gene targeted to the vacuole, apoplast or ER on the levels of cell wall ferulates of cell suspension cultures of the forage grass Festuca arundinacea, and subsequent effects on cell wall digestion in the absence of the complication of lignin affecting cell wall digestibility, as well as attempts to further increase the levels of FAEA expression. The work describes studies at the cell culture level aimed at establishing functionality and testing different strategies for achieving targeted expression
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