Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass (Panicum virgatum L.) in the field.

BACKGROUND: Switchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass 'Alamo' plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic 'Alamo 2' switchgrass clones were planted in pairs adjacent to pollen traps. RESULTS: In the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m. CONCLUSIONS: In these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.

Chromosome stickiness impairs meiosis and influences reproductive success in Panicum maximum (Poaceae) hybrid plants.

Chromosome stickiness has been studied in several species of higher plants and is characterized by sticky clumps of chromatin resulting in sterility. Chromosome stickiness was recorded in Panicum maximum hybrid plants that were cultivated in the field. In the meiocytes affected, chromosomes clumped into amorphous masses that did not orient themselves on the equatorial plate, and anaphase I disjunction failed to occur. After a normal cytokinesis, the masses of chromatin were divided between both daughter cells. Metaphase and anaphase of the second division also did not occur, and after the second cytokinesis, polyads were formed. This abnormality arose spontaneously. Abnormalities that cause male sterility are an important tool for obtaining hybrid seeds in plant breeding. This is the first report of an abnormality affecting pollen viability in P. maximum. This finding can open a new opportunity in the breeding program of this species that is devoted to hybridization where manual cross-pollination is difficult and time consuming.

Phytochemical and antiproliferative activity of proso millet.

The phytochemical content, antioxidant activity and antiproliferative properties of three diverse varieties of proso millet are reported. The free phenolic content ranged from 27.48 (Gumi 20) to 151.14 (Mi2504-6) mg gallic acid equiv/100 g DW. The bound phenolic content ranged from 55.95 (Gumi20) to 305.81 (Mi2504-6) mg gallic acid equiv/100 g DW. The percentage contribution of bound phenolic to the total phenolic content of genotype samples analyzed ranged between 62.08% and 67.05%. Ferulic acid and chlorogenic acid are the predominant phenolic acid found in bound fraction. Caffeic acid and p-coumaric acid were also detected. Syringic acid was detected only in the free fraction. The antioxidant activity was assessed using the hydrophilic peroxyl radical scavenging capacity (PSC) assay. The PSC antioxidant activity of the free fraction ranged from 57.68 (Mi2504-6) to 147.32 (Gumi20) µmol of vitamin C equiv/100 g DW. The PSC antioxidant activity of the bound fraction ranged from 95.38 (Mizao 52) to 136.48 (Gumi 20) µmol of vitamin C equiv/100 g DW. The cellular antioxidant activity (CAA) of the extract was assessed using the HepG2 model. CAA value ranged from 2.51 to 6.10 µmol equiv quercetin/100 g DW. Antiproliferative activities were also studied in vitro against MDA human breast cancer and HepG2 human liver cancer cells. Results exhibited a differential and possible selective antiproliferative property of the proso millet. These results may be used to direct the consumption of proso millet with improved health properties.

Transgene autoexcision in switchgrass pollen mediated by the Bxb1 recombinase.

BACKGROUND: Switchgrass (Panicum virgatum L.) has a great potential as a platform for the production of biobased plastics, chemicals and energy mainly because of its high biomass yield on marginal land and low agricultural inputs. During the last decade, there has been increased interest in the genetic improvement of this crop through transgenic approaches. Since switchgrass, like most perennial grasses, is exclusively cross pollinating and poorly domesticated, preventing the dispersal of transgenic pollen into the environment is a critical requisite for the commercial deployment of this important biomass crop. In this study, the feasibility of controlling pollen-mediated gene flow in transgenic switchgrass using the large serine site-specific recombinase Bxb1 has been investigated. RESULTS: A novel approach utilizing co-transformation of two separate vectors was used to test the functionality of the Bxb1/att recombination system in switchgrass. In addition, two promoters with high pollen-specific activity were identified and thoroughly characterized prior to their introduction into a test vector explicitly designed for both autoexcision and quantitative analyses of recombination events. Our strategy for developmentally programmed precise excision of the recombinase and marker genes in switchgrass pollen resulted in the generation of transgene-excised progeny. The autoexcision efficiencies were in the range of 22-42% depending on the transformation event and assay used. CONCLUSION: The results presented here mark an important milestone towards the establishment of a reliable biocontainment system for switchgrass which will facilitate the development of this crop as a biorefinery feedstock through advanced biotechnological approaches.

Abolishing activity against ascorbate in a cytosolic ascorbate peroxidase from switchgrass.

Switchgrass (Panicum virgatum L.) is being developed as a bioenergy species. Recently an early version of its genome has been released permitting a route to the cloning and analysis of key proteins. Ascorbate peroxidases (APx) are an important part of the antioxidant defense system of plant cells and present a well studied model to understand structure-function relationships. Analysis of the genome indicates that switchgrass encodes several cytosolic ascorbate peroxidases with apparent varying levels of tissue expression. A major cytosolic ascorbate peroxidase was thus selected for further studies. This gene was cloned and expressed in Escherichia coli cells to obtain purified active protein. Full heme incorporation of the enzyme was achieved utilizing slow growth and supplementing the media with 5-aminolevulinic acid. The enzyme was observed to be monomeric in solution via size exclusion chromatography. Activity toward ascorbate was observed that was non-Michaelis-Menten in nature. A site-directed mutant, R172S, was made in an attempt to differentiate activity against ascorbate versus other substrates. The R172S protein exhibited negligible ascorbate peroxidase activity, but showed near wild type activity toward other aromatic substrates.

Gene flow matters in switchgrass (Panicum virgatum L.), a potential widespread biofuel feedstock.

There currently exists a large push for the use, improvement, and expansion via landscape modification of dedicated biofuel crops (feedstocks) in the United States and in many parts of the world. Ecological concerns have been voiced because many biofuel feedstocks exhibit characteristics associated with invasiveness, and due to potential negative consequences of agronomic genes in native wild populations. Seed purity concerns for biofuel feedstock cultivars whose seeds would be harvested in agronomic fields also exist from the agribusiness sector. The common thread underlying these concerns, which have regulatory implications, is gene flow; thus detailed knowledge of gene flow in biofuel crop plants is important in the formulation of environmental risk management plans. Here, we synthesize the current state of knowledge of gene flow in an exemplary biofuel crop, switchgrass (Panicum virgatum L.), which is native to eastern North America and is currently experiencing conventional and technological advances in biomass yields and ethanol production. Surprisingly little is known regarding aspects of switchgrass pollen flow and seed dispersal, and whether native populations of conspecific or congeneric relatives will readily cross with current agronomic switchgrass cultivars. We pose that filling these important gaps will be required to confront the sustainability challenges of widespread planting of biofuel feedstocks.

Genotypic variability in mineral composition of switchgrass.

Switchgrass (Panicum virgatum L.) is a warm season perennial grass with great potential as an energy crop in the USA. It is widely adapted to many regions of the country, produces large amounts of biomass, serves as a useful forage grass, and provides ecosystem services that benefit soil and water quality and wildlife. Biological and thermochemical technologies are being developed to convert herbaceous biomass, including switchgrass, to energy. The objective of this research was to determine the effect of genotype and production environment on the concentration of minerals that affect the suitability of switchgrass for thermochemical conversion and to quantify the amount of potassium (K) and phosphorus (P) removed from the production system by harvest of the aboveground biomass, a measure of the sustainability of the practice. Straw dry biomass contained from 1.3 to 6.4 kg Mg(-1) and from 6.2 to 15.8 kg Mg(-1) of P and K, respectively. Variability in aluminum (Al), calcium (Ca), chloride (Cl), K, P, silicon (Si), and sulfur (S) concentrations across locations was relatively high, ranging from twofold (Al) to eightfold (Cl). Location had a strong impact on mineral concentrations among switchgrass genotypes evaluated in this study. Latitude of origin impacted the Cl and Si concentrations measured in plant tissues, but none of the other minerals analyzed in this study. Upland and lowland cytotypes explained some of the observed differences, but populationxlocation interactions were the primary source of variability in the concentration of these minerals.