Summary• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks.• The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters.• PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues.• We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

SUMMARYBiochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species.

The lignin content of biomass can impact the ease and cost of biomass processing. Lignin reduction through breeding and genetic modification therefore has potential to reduce costs in biomass-processing industries (e.g. pulp and paper, forage, and lignocellulosic ethanol). We investigated compositional changes in two low-lignin alfalfa (Medicago sativa) lines with antisense down-regulation of p-coumarate 3-hydroxylase (C3H) or hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT). We investigated whether the difference in reactivity during lignification of 4-coumaryl alcohol (H) monomers versus the naturally dominant sinapyl alcohol and coniferyl alcohol lignin monomers alters the lignin structure. Sequential base extraction readily reduced the H monomer content of the transgenic lines, leaving a residual lignin greatly enriched in H subunits; the extraction profile highlighted the difference between the control and transgenic lines. Gel permeation chromatography of isolated ball-milled lignin indicated significant changes in the weight average molecular weight distribution of the control versus transgenic lines (CTR1a, 6000; C3H4a, 5500; C3H9a, 4000; and HCT30a, 4000).The advent of large-scale liquid fuel production from biomass has served to highlight how difficult it is to commercially process biomass effectively and efficiently. Much of the difficulty is due to the recalcitrant nature of lignocelluloses (1), a complex interlinking structure composed of cellulose, hemicelluloses, and lignin that makes up the bulk of terrestrial biomass. Accessibility to the cell wall is influenced by lignin, which provides structural integrity to the cell wall. Both total lignin content and lignin monomer composition may impact the ease with which biomass is processed. This study examines whether lignin molecular weight is altered by changing the lignin monomer composition and if these changes affect the ease with which lignin can be removed by chemical processing.Three monomers (Fig. 1), 4-coumaryl alcohol (H), 2 coniferyl alcohol (G), and sinapyl alcohol (S), polymerize in what is thought to be a combinatorial fashion to form the bulk of the lignin polymer (2, 3). The amount of each unit depends on the species, age, cell type, and tissue type (4, 5). The presence of each additional methoxy group on a lignin unit results in one less reactive site (S Ͻ G Ͻ H) and therefore fewer possible combinations during the polymerization reaction. For example, the S unit has no vacant 5-position; therefore 5,5Ј-cross-linking is unavailable for lignin S subunits. As a result, lignin rich in S subunits is more easily depolymerized than lignin rich in G subunits.The relative level of S and G lignin subunits is expressed as the S/G ratio, an important measurement used in the assessment of biomass. H lignin subunits are present in low levels in natural materials (4.9% of lignin in wild-type alfalfa (1) and 0.8% in Norway Spruce (6)); consequently, less is known about lignin high in H subunits and its impact on biomass processing. I...