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.ignocellulosic biomass is an abundant, domestic, renewable feedstock source that can be converted to liquid transportation fuels and other chemicals by fermentation. Cellulosic ethanol is a promising near-term technological option to reduce transportation sector greenhouse gas emissions (1). Because lignocellulosic biomass is made up of the complex structures of cellulose, hemicellulose, and lignin, such feedstock is highly recalcitrant to bioconversion of its carbohydrates into ethanol compared with starch (2, 3). Current biomass fermentation processes for fuels and chemicals have a relatively high cost primarily because of this recalcitrance, which in turn has limited commercialization of biomass ethanol (4). To achieve sustainable energy production, it is necessary to overcome the chemical and structural properties of biomass that inhibit its deconstruction in dedicated bioenergy crops (5).The conversion of lignocellulosic biomass to ethanol is a threestep process that involves pretreatment followed by polysaccharide hydrolysis to simple sugars followed by sugar fermentation to ethanol (6). The presence of lignin in cell walls negatively impacts these conversion steps (7,8). Examination of natural variation in alfalfa, switchgrass, canarygrass, and sorghum has shown that decreased lignin levels improve in vitro enzyme hydrolysis (9, 10). Lignin pathway modification in alfalfa generated transgenic lines with increased enzymati...

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.

The root stem cell niche, which is composed of four mitotically inactive quiescent center (QC) cells and the surrounding actively divided stem cells in Arabidopsis, is critical for growth and root development. Here, we demonstrate that the Arabidopsis prohibitin protein PHB3 is required for the maintenance of root stem cell niche identity by both inhibiting proliferative processes in the QC and stimulating cell division in the proximal meristem (PM). PHB3 coordinates cell division and differentiation in the root apical meristem by restricting the spatial expression of ethylene response factor (ERF) transcription factors 115, 114, and 109. ERF115, ERF114, and ERF109 mediate ROS signaling, in a PLT-independent manner, to control root stem cell niche maintenance and root growth through phytosulfokine (PSK) peptide hormones in Arabidopsis.

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30°C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.

SummarySwitchgrass (Panicum virgatum L.) is a leading candidate for a dedicated lignocellulosic biofuel feedstock owing to its high biomass production, wide adaptation and low agronomic input requirements. Lignin in cell walls of switchgrass, and other lignocellulosic feedstocks, severely limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars and subsequently biofuels. Low-lignin transgenic switchgrass plants produced by the downregulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, were analysed in the field for two growing seasons. COMT transcript abundance, lignin content and the syringyl/guaiacyl lignin monomer ratio were consistently lower in the COMT-down-regulated plants throughout the duration of the field trial. In general, analyses with fully established plants harvested during the second growing season produced results that were similar to those observed in previous greenhouse studies with these plants. Sugar release was improved by up to 34% and ethanol yield by up to 28% in the transgenic lines relative to controls. Additionally, these results were obtained using senesced plant material harvested at the end of the growing season, compared with the young, green tissue that was used in the greenhouse experiments. Another important finding was that transgenic plants were not more susceptible to rust (Puccinia emaculata). The results of this study suggest that lignin down-regulation in switchgrass can confer real-world improvements in biofuel yield without negative consequences to biomass yield or disease susceptibility.

The switchgrass variety Alamo has been chosen for genome sequencing, genetic breeding, and genetic engineering by the US Department of Energy Joint Genome Institute (JGI) and the US Department of Energy BioEnergy Science Center. Lignin has been considered as a major obstacle for cellulosic biofuel production from switchgrass biomass. The purpose of this study was to provide baseline information on cell wall development in different parts of developing internodes of tillers of switchgrass cultivar Alamo and evaluate the effect of cell wall properties on biomass saccharification. Cell wall structure, soluble and wall-bound phenolics, and lignin content were analyzed from the top, middle, and bottom parts of internodes at different developmental stages using ultraviolet autofluorescence microscopy, histological staining methods, and high-performance liquid chromatography (HPLC). The examination of different parts of the developing internodes revealed differences in the stem structure during development, in the levels of free and well-bound phenolic compounds and lignin content, and in lignin pathwayrelated gene expression, indicating that the monolignol biosynthetic pathway in switchgrass is under complex spatial and temporal control. Our data clearly show that there was a strong negative correlation between overall lignin content and biomass saccharification efficiency. The ester-linked p-CA/FA ratio showed a positive correlation with lignin content and a negative correlation with sugar release. Our data provide baseline information to facilitate genetic modification of switchgrass recalcitrance traits for biofuel production.