Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment.

Aaron J Saathoff,Elaine K Chow,Bruce S Dien,Christian M Tobias,Gautam Sarath

doi:10.1371/journal.pone.0016416

Abstract

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. “Alamo” with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity of one line indicated greater incorporation of hydroxycinnamyl aldehydes in the lignin.

Highlights

Recalcitrance of biomass to enzymatic digestion reduces the efficiencies of processes that utilize fermentable sugars from lignocellulosic material for the production of ethanol and other biofuels
The silencing construct was derived from pWBVec8 and contained two copies of a 575 bp fragment of the PviCAD2 coding region in inverted orientation separated by a spacer region derived from the barley Cre intron
A subset of these lines was chosen for Cinnamyl alcohol dehydrogenase (CAD) activity screening by measuring reduction of coniferaldehyde in stem tissue extracts in a colorimetric assay (Figure 1b)

Summary

Introduction

Recalcitrance of biomass to enzymatic digestion reduces the efficiencies of processes that utilize fermentable sugars from lignocellulosic material for the production of ethanol and other biofuels. Though grasses are less recalcitrant than woody perennials, slow digestion rates and requirements for substantial quantities of cell wall degrading enzymes add significant process costs and inefficiencies [1]. Barriers to cell wall deconstruction include the heterogeneous, cross-linked, nature of the cell wall, the crystalline nature of the cellulose that prevents enzyme access, the variety of covalent and hydrogen bonded linkages that must be cleaved, feedback inhibition of the products of hydrolysis [2], and the presence of enzymatic inhibitors including furans produced by thermochemical breakdown of sugars, phenolic compounds, and carboxylic acids. The composition of biomass feedstocks themselves is variable and may be bioengineered for altered cell wall architecture. Improving biomass quality parameters is a key focus for the development of plants with enhanced conversion properties for use in future biorefineries [3,4]

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Conclusion