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...

Summary• Independent antisense down-regulation of 10 individual enzymes in the monolignol pathway has generated a series of otherwise isogenic alfalfa (Medicago sativa) lines with varying lignin content and composition. These plants show various visible growth phenotypes, and possess significant differences in vascular cell size and number.• To better understand the phenotypic consequences of lignin modification, the distributions of lignin content and composition in stems of the various alfalfa lines at the cellular level were studied by confocal microscopy after staining for specific lignin components, and by chemical analysis of laser capture dissected tissue types.• Although all antisense transgenes were driven by the same promoter with specificity for vascular, fiber and parenchyma tissues, the impact of down-regulating a specific transgene varied in the different tissue types. For example, reducing expression of ferulate 5-hydroxylase reduced accumulation of syringyl lignin in fiber and parenchyma cells, but not in vascular elements.• The results support a model for cell type-specific regulation of lignin content and composition at the level of the monolignol pathway, and illustrate the use of laser capture microdissection as a new approach to spatially resolved lignin compositional analysis.

Lignin is a major component of plant secondary cell walls and is derived from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monolignols. Among higher plants, S lignin is generally considered to be restricted to angiosperms, which contain the S lignin-specific cytochrome P450-dependent monooxygenase, ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydoxylase (F5H). The transcription factor MYB58 directly regulates expression of monolignol pathway genes except for F5H. Here we show that F5H expression is directly regulated by the secondary cell wall master switch NST1/SND1, which is known to regulate expression of MYB58. Deletion of NST1 expression in Medicago truncatula leads to a loss of S lignin associated with a more than 25-fold reduction of F5H expression but only around a 2-fold reduction in expression of other lignin pathway genes. A detailed phylogenetic analysis showed that gymnosperms lack both F5H and orthologs of NST1/SND1. We propose that both F5H and NST1 appeared at a similar time after the divergence of angiosperms and gymnosperms, with F5H possibly originating as a component of a defense mechanism that was recruited to cell wall biosynthesis through the evolution of NST1-binding elements in its promoter.ferulate 5-hydroxylase | NAC transcription factor | pathway evolution L ignin is an aromatic heteropolymer derived primarily from the hydroxycinnamyl alcohols p-coumaryl, coniferyl, and sinapyl alcohol, which give rise to p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) subunits, respectively (1). Lignin is one of the three main components of plant secondary cell walls and plays important roles in vascular plant growth by contributing to structural rigidity, facilitating water transport, and providing a defensive barrier against pathogens.Lignin monomer composition varies among plant species. Although S lignin has arisen more than once during evolution (2), it does not appear to exist in the majority of gymnosperms or in ferns, whereas angiosperms deposit S units in lignin polymers in secondary cell walls. A cytochrome P450 (CYP)-dependent monooxygenase, ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydoxylase (F5H, CYP84A1) is specifically required for S lignin biosynthesis (3, 4) and diverts G monolignol intermediates into the S lignin branch pathway.The Arabidopsis lignin-specific transcription factor MYB58 can directly activate most lignin biosynthesis genes except F5H (5). This activation is through binding to AC elements, which are present in most of the promoters of lignin biosynthetic genes (6-9), although no AC element has been detected in the F5H gene (9). Two NAC transcription factors, NST1 and SND1 (NST3), act together as key regulators of the entire secondary cell wall biosynthesis program in Arabidopsis (10, 11) and activate a cascade of downstream transcription factors, which in turn activates the biosynthetic pathways for the individual secondary cell wall components cellulose, xylan, and lignin (12). MYB46 is a direct target of SND1 in Arabidopsis. Although downstream ...

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Background: Calsyntenin-3 (Cstn3) promotes synapse development, controversially interacting with neurexin 1␣ (n1␣). Results: Cstn3 binds n1␣ directly, and its structure adopts multiple forms. Conclusion: Cstn3 interacts with n1␣ via a novel mechanism and can produce distinct trans-synaptic bridges with n1␣. Significance: A complex portfolio of molecular interactions between proteins implicated in autism spectrum disorder and schizophrenia guide synapse development.