SPL7 and SPL8 represent a novel flowering regulation mechanism in switchgrass.

The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses. Inflorescence development in switchgrass was characterized using scanning electron microscopy (SEM). Microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation (ChIP)-PCR and EMSA were used to identify regulators of phase transition and flowering. Gene function was characterized by downregulation and overexpression of the target genes. Overexpression of SPL7 and SPL8 promotes flowering, whereas downregulation of individual genes moderately delays flowering. Simultaneous downregulation of SPL7/SPL8 results in extremely delayed or nonflowering plants. Furthermore, downregulation of both genes leads to a vegetative-to-reproductive reversion in the inflorescence, a phenomenon that has not been reported in any other grasses. Detailed analyses demonstrate that SPL7 and SPL8 induce phase transition and flowering in grasses by directly upregulating SEPALLATA3 (SEP3) and MADS32. Thus, the SPL7/8 pathway represents a novel regulatory mechanism in grasses that is largely different from that in Arabidopsis. Additionally, genetic modification of SPL7 and SPL8 results in much taller plants with significantly increased biomass yield and sugar release.

Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass.

The switchgrass (SG) samples pretreated by cellulose solvent- and organic solvent-based lignocellulose fractionation were characterized by enzymatic hydrolysis, substrate accessibility assay, scanning electron microscopy, X-ray diffraction (XRD), cross polarization/magic angle spinning (CP/MAS) (13)C nuclear magnetic resonance (NMR), and Fourier transform infrared spectroscopy (FTIR). Glucan digestibility of the pretreated SG was 89% at hour 36 at one filter paper unit of cellulase per gram of glucan. Crystallinity index (CrI) of pure cellulosic materials and SG before and after cellulose solvent-based pretreatment were determined by XRD and NMR. CrI values varied greatly depending on measurement techniques, calculation approaches, and sample drying conditions, suggesting that the effects of CrI data obtained from dried samples on enzymatic hydrolysis of hydrated cellulosic materials should be interpreted with caution. Fast hydrolysis rates and high glucan digestibilities for pretreated SG were mainly attributed to a 16.3-fold increase in cellulose accessibility to cellulase from 0.49 to 8.0 m(2)/g biomass, because the highly ordered hydrogen-bonding networks in cellulose fibers of biomass were broken through cellulose dissolution in a cellulose solvent, as evidenced by CP/MAS (13)C-NMR and FTIR.

Ultrastructure and Enzymatic Hydrolysis of Deuterated Switchgrass.

Neutron scattering of deuterated plants can provide fundamental insight into the structure of lignocellulosics in plant cell walls and its deconstruction by pretreatment and enzymes. Such plants need to be characterized for any alterations to lignocellulosic structure caused by growth in deuterated media. Here we show that glucose yields from enzymatic hydrolysis at lower enzyme loading were 35% and 30% for untreated deuterated and protiated switchgrass, respectively. Lignin content was 4% higher in deuterated switchgrass but there were no significant lignin structural differences. Transmission electron microscopy showed differences in lignin distribution and packing of fibers in the cell walls that apparently increased surface area of cellulose in deuterated switchgrass, increasing cellulose accessibility and lowering its recalcitrance. These differences in lignification were likely caused by abiotic stress due to growth in deuterated media.

Internode structure and cell wall composition in maturing tillers of switchgrass (Panicum virgatum. L).

This work examined cell composition gradients in maturing tillers of switchgrass (Panicum virgatum L.) with the aim of developing baseline information on this important forage and biomass crop. Flowering tillers were collected from plants raised from seeds in a greenhouse and field, harvested at soil level and separated into internodes beginning with the node subtending the peduncle. Internodes were analyzed using microscopy, by fiber digestion, high-performance liquid chromatography and by gas chromatography-mass spectrometry to obtain anatomical and compositional data. Microscopy demonstrated the development and maturation of cortical fibers which eventually became confluent with the fiber sheath surrounding vascular bundles in the lower internodes. Detergent fiber analysis indicated increasing cellulose and lignin contents and decreases in cell solubles and hemicelluloses with increasing distance of the internodes from the top of the plant. Soluble phenolics were greatest in amounts and complexity in top internodes. The lower internodes contained greater levels of wall-bound phenolic acids, principally as 4-coumarate and ferulate.

Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells.

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3'-diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed-transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H2 O2 staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H2 O2 staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration.

Surface and ultrastructural characterization of raw and pretreated switchgrass.

The US Department of Energy-funded Biomass Refining CAFI (Consortium for Applied Fundamentals and Innovation) project has developed leading pretreatment technologies for application to switchgrass and has evaluated their effectiveness in recovering sugars from the coupled operations of pretreatment and enzymatic hydrolysis. Key chemical and physical characteristics have been determined for pretreated switchgrass samples. Several analytical microscopy approaches utilizing instruments in the Biomass Surface Characterization Laboratory (BSCL) at the National Renewable Energy Laboratory (NREL) have been applied to untreated and CAFI-pretreated switchgrass samples. The results of this work have shown that each of the CAFI pretreatment approaches on switchgrass result in different structural impacts at the plant tissue, cellular, and cell wall levels. Some of these structural changes can be related to changes in chemical composition upon pretreatment. There are also apparently different structural mechanisms that are responsible for achieving the highest enzymatic hydrolysis sugar yields.

Comparative data on effects of leading pretreatments and enzyme loadings and formulations on sugar yields from different switchgrass sources.

Dilute sulfuric acid (DA), sulfur dioxide (SO(2)), liquid hot water (LHW), soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), and lime pretreatments were applied to Alamo, Dacotah, and Shawnee switchgrass. Application of the same analytical methods and material balance approaches facilitated meaningful comparisons of glucose and xylose yields from combined pretreatment and enzymatic hydrolysis. Use of a common supply of cellulase, beta-glucosidase, and xylanase also eased comparisons. All pretreatments enhanced sugar recovery from pretreatment and subsequent enzymatic hydrolysis substantially compared to untreated switchgrass. Adding beta-glucosidase was effective early in enzymatic hydrolysis while cellobiose levels were high but had limited effect on longer term yields at the enzyme loadings applied. Adding xylanase improved yields most for higher pH pretreatments where more xylan was left in the solids. Harvest time had more impact on performance than switchgrass variety, and microscopy showed changes in different features could impact performance by different pretreatments.

Opportunities and roadblocks in utilizing forages and small grains for liquid fuels.

This review focuses on the potential advantages and disadvantages of forages such as switchgrass (Panicum virgatum), and two small grains: sorghum (Sorghum bicolor), and wheat (Triticum aesitvum), as feedstocks for biofuels. It highlights the synergy provided by applying what is known from forage digestibility and wheat and sorghum starch properties studies to the biofuels sector. Opportunities therefore, exist to improve biofuel qualities in these crops via genetics and agronomics. In contrast to cereal crops, switchgrass still retains tremendous exploitable genetic diversity, and can be specifically improved to fit a particular agronomic, management, and conversion platform. Combined with emerging studies on switchgrass genomics, conversion properties and management, the future for genetic modification of this species through conventional and molecular breeding strategies appear to be bright. The presence of brown-midrib mutations in sorghum that alter cell wall composition by reducing lignin and other attributes indicate that sorghum could serve as an important model species for C(4)-grasses. Utilization of the brown-midrib traits could lead to the development of forage and sweet sorghums as novel biomass crops. Additionally, wheat crop residue, and wheat and sorghum with improved starch content and composition represent alternate biofuel sources. However, the use of wheat starch as a biofuel is unlikely but its value as a model to study starch properties on biofuel yields holds significant promise.