Background: Legumes are important to humans by providing food, feed and raw materials for industrial utilizations. Some legumes, such as alfalfa, are potential bioenergy crops due to their high biomass productivity. Global transcriptional profiling has been successfully used to identify genes and regulatory pathways in secondary cell wall thickening in Arabidopsis, but such transcriptome data is lacking in legumes.Results: A systematic microarray assay and high through-put real time PCR analysis of secondary cell wall development were performed along stem maturation in Medicago truncatula. More than 11,000 genes were differentially expressed during stem maturation, and were categorized into 10 expression clusters. Among these, 279 transcription factor genes were correlated with lignin/cellulose biosynthesis, therefore representing putative regulators of secondary wall development. The b-ZIP, NAC, WRKY, C2H2 zinc finger (ZF), homeobox, and HSF gene families were over-represented. Gene co-expression network analysis was employed to identify transcription factors that may regulate the biosynthesis of lignin, cellulose and hemicellulose. As a complementary approach to microarray, real-time PCR analysis was used to characterize the expression of 1,045 transcription factors in the stem samples, and 64 of these were upregulated more than 5-fold during stem maturation. Reverse genetics characterization of a cellulose synthase gene in cluster 10 confirmed its function in xylem development.Conclusions: This study provides a useful transcriptome and expression resource for understanding cell wall development, which is pivotal to enhance biomass production in legumes.

The evolution of lignin biosynthesis was critical in the transition of plants from an aquatic to an upright terrestrial lifestyle. Lignin is assembled by oxidative polymerization of two major monomers, coniferyl alcohol and sinapyl alcohol. Although two recently discovered laccases, LAC4 and LAC17, have been shown to play a role in lignin polymerization in Arabidopsis thaliana, disruption of both genes only leads to a relatively small change in lignin content and only under continuous illumination. Simultaneous disruption of LAC11 along with LAC4 and LAC17 causes severe plant growth arrest, narrower root diameter, indehiscent anthers, and vascular development arrest with lack of lignification. Genome-wide transcript analysis revealed that all the putative lignin peroxidase genes are expressed at normal levels or even higher in the laccase triple mutant, suggesting that lignin laccase activity is necessary and nonredundant with peroxidase activity for monolignol polymerization during plant vascular development. Interestingly, even though lignin deposition in roots is almost completely abolished in the lac11 lac4 lac17 triple mutant, the Casparian strip, which is lignified through the activity of peroxidase, is still functional. Phylogenetic analysis revealed that lignin laccase genes have no orthologs in lower plant species, suggesting that the monolignol laccase genes diverged after the evolution of seed plants.

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.

Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial plant secondary cell walls. We recently reported evidence of an unanticipated catechyl lignin homopolymer (C lignin) derived solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. We previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins, but not both. Here, we identified several dicot plants (Euphorbiaceae and Cleomaceae) that produce C lignin together with traditional G/S lignins in their seed coats. Solution-state NMR analyses, along with an in vitro lignin polymerization study, determined that there is, however, no copolymerization detectable (i.e., that the synthesis and polymerization of caffeyl alcohol and conventional monolignols in vivo is spatially and/or temporally separated). In particular, the deposition of G and C lignins in Cleome hassleriana seed coats is developmentally regulated during seed maturation; C lignin appears successively after G lignin within the same testa layers, concurrently with apparent loss of the functionality of O-methyltransferases, which are key enzymes for the conversion of C to G lignin precursors. This study exemplifies the flexible biosynthesis of different types of lignin polymers in plants dictated by substantial, but poorly understood, control of monomer supply by the cells.

Lignins are complex phenylpropanoid polymers mostly associated with plant secondary cell walls. Lignins arise primarily via oxidative polymerization of the three monolignols, p-coumaryl, coniferyl, and sinapyl alcohols. Of the two hydroxycinnamyl alcohols that represent incompletely methylated biosynthetic products (and are not usually considered to be monolignols), 5-hydroxyconiferyl alcohol is now well established as incorporating into angiosperm lignins, but incorporation of caffeyl alcohol has not been shown. We report here the presence of a homopolymer of caffeyl alcohol in the seed coats of both monocot and dicot plants. This polymer (C-lignin) is deposited to high concentrations in the seed coat during the early stages of seed development in the vanilla orchid (Vanilla planifolia), and in several members of the Cactaceae. The lignin in other parts of the Vanilla plant is conventionally biosynthesized from coniferyl and sinapyl alcohols. Some species of cacti contain only C-lignin in their seeds, whereas others contain only classical guaiacyl/syringyl lignin (derived from coniferyl and sinapyl alcohols). NMR spectroscopic analysis revealed that the Vanilla seed-coat polymer was massively comprised of benzodioxane units and was structurally similar to the polymer synthesized in vitro by peroxidase-catalyzed polymerization of caffeyl alcohol. CD spectroscopy did not detect any optical activity in the seed polymer. These data support the contention that the C-lignin polymer is produced in vivo via combinatorial oxidative radical coupling that is under simple chemical control, a mechanism analogous to that theorized for classical lignin biosynthesis.

We have recently described a hitherto unsuspected catechyl lignin polymer (C-lignin) in the seed coats of Vanilla orchid and in cacti of one genus, Melocactus (Chen et al., Proc. Natl. Acad. Sci. USA. 2012, 109, 1772-1777.). We have now determined the lignin types in the seed coats of 130 different cactus species. Lignin in the vegetative tissues of cacti is of the normal guaiacyl/syringyl (G/S) type, but members of most genera within the subfamily Cactoidae possess seed coat lignin of the novel C-type only, which we show is a homopolymer formed by endwise β-O-4-coupling of caffeyl alcohol monomers onto the growing polymer resulting in benzodioxane units. However, the species examined within the genera Coryphantha, Cumarinia, Escobaria and Mammillaria (Cactoideae) mostly had normal G/S lignin in their seeds, as did all six species in the subfamily Opuntioidae that were examined. Seed coat lignin composition is still evolving in the Cactaceae, as seeds of one Mammillaria species (M. lasiacantha) possess only C-lignin, three Escobaria species (E. dasyacantha, E. lloydii and E. zilziana) contain an unusual lignin composed of 5-hydroxyguaiacyl units, the first report of such a polymer that occurs naturally in plants, and seeds of some species contain no lignin at all. We discuss the implications of these findings for the mechanisms that underlie the biosynthesis of these newly discovered lignin types.

• 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.

NAC domain transcription factors act as master switches for secondary cell wall thickening, but how they exert their function and how their expression is regulated remains unclear. Here we identify a loss-of-function point mutation in the NST1 gene of Medicago truncatula. The nst1-3 mutant shows no lignification in interfascicular fibers, as previously seen in tnt1 transposon insertion alleles. However, the C→A transversion, which causes a T94K mutation in the NST1 protein, leads to increased NST1 expression. Introduction of the same mutation into the Arabidopsis homolog SND1 causes both protein mislocalization and loss of target DNA binding, with a resultant inability to trans-activate downstream secondary wall synthesis genes. Furthermore, trans-activation assays show that the expression of SND1 operates under positive feedback control from itself, and SND1 was shown to bind directly to a conserved motif in its own promoter, located within a recently described 19-bp secondary wall NAC binding element. Three MYB transcription factors downstream of SND1, one of which is directly regulated by SND1, exert negative regulation on SND1 promoter activity. Our results identify a conserved amino acid critical for NST1/SND1 function, and show that the expression of the NAC master switch itself is under both positive (autoregulatory) and negative control.

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

The temporal proteome response of Medicago truncatula suspension cell cultures to yeast elicitation (which mimics fungal infection) was investigated using two-dimensional polyacrylamide gel electrophoresis (2-DE) and nanoliquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Reproducibility of 2-DE was assessed using the number of the visualized protein spots and spot volume. Average coefficient of variation was determined to be less than 6% for the number of spots and around 50% for spot volume. About 4% of the total visualized proteins, that is, 34 out of 861, were differentially accumulated in the suspension cells 24 h after yeast elicitation, including isoflavononid biosynthetic enzymes and a putative laccase. The induction of the putative laccase was highly correlated with the polymerization of phenolics such as 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, and ferulic acid into cell walls. In contrast, lignin was only induced at the later stages of the temporal study, indicating that this specific laccase is primarily involved in cell wall modifications and/or fortifications rather than in lignification in response to yeast elicitation.

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.

To identify genes controlling secondary cell wall biosynthesis in the model legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant population for plants with altered patterns of lignin autofluorescence. From more than 9000 R1 plants screened, four independent lines were identified with a total lack of lignin in the interfascicular region. The mutants also showed loss of lignin in phloem fibers, reduced lignin in vascular elements, failure in anther dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls. Microarray and PCR analyses confirmed that the mutations were caused by the insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in different combinations to control cell wall composition in stems and anthers. Loss of MtNST1 function resulted in reduced lignin content, associated with reduced expression of most lignin biosynthetic genes, and a smaller reduction in cell wall polysaccharide content, associated with reduced expression of putative cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase digestion released significantly more sugars from cell walls of nst1 mutants compared with the wild type. We discuss the implications of these findings for the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.

Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase.

Stems of dicotyledonous plants consist of an outer epidermis, a cortex, a ring of secondarily thickened vascular bundles and interfascicular cells, and inner pith parenchyma cells with thin primary walls. It is unclear how the different cell layers attain and retain their identities. Here, we show that WRKY transcription factors are in part responsible for the parenchymatous nature of the pith cells in dicotyledonous plants. We isolated mutants of Medicago truncatula and Arabidopsis thaliana with secondary cell wall thickening in pith cells associated with ectopic deposition of lignin, xylan, and cellulose, leading to an ∼50% increase in biomass density in stem tissue of the Arabidopsis mutants. The mutations are caused by disruption of stem-expressed WRKY transcription factor (TF) genes, which consequently up-regulate downstream genes encoding the NAM, ATAF1/2, and CUC2 (NAC) and CCCH type (C3H) zinc finger TFs that activate secondary wall synthesis. Direct binding of WRKY to the NAC gene promoter and repression of three downstream TFs were confirmed by in vitro assays and in planta transgenic experiments. Secondary wall-bearing cells form lignocellulosic biomass that is the source for second generation biofuel production. The discovery of negative regulators of secondary wall formation in pith opens up the possibility of significantly increasing the mass of fermentable cell wall components in bioenergy crops.