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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 ([less-than or equal to]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.

Panicum miliaceum (broomcorn millet) is a tetraploid cereal, which was among the first domesticated crops, but is now a minor crop despite its high water use efficiency. The ancestors of this species have not been determined; we aimed to identify likely candidates within the genus, where phylogenies are poorly resolved. Nuclear and chloroplast DNA sequences from P. miliaceum and a range of diploid and tetraploid relatives were used to develop phylogenies of the diploid and tetraploid species. Chromosomal in situ hybridization with genomic DNA as a probe was used to characterize the genomes in the tetraploid P. miliaceum and a tetraploid accession of P. repens. In situ hybridization showed that half the chromosomes of P. miliaceum hybridized more strongly with labelled genomic DNA from P. capillare, and half with labelled DNA from P. repens. Genomic DNA probes differentiated two sets of 18 chromosomes in the tetraploid P. repens. Our phylogenetic data support the allotetraploid origin of P. miliaceum, with the maternal ancestor being P. capillare (or a close relative) and the other genome being shared with P. repens. Our P. repens accession was also an allotetraploid with two dissimilar but closely related genomes, the maternal genome being similar to P. sumatrense. Further collection of Panicum species, particularly from the Old World, is required. It is important to identify why the water-efficient P. miliaceum is now of minimal importance in agriculture, and it may be valuable to exploit the diversity in this species and its ancestors.

Long-term climate change and periodic environmental extremes threaten food and fuel security 1 and global crop productivity 2 – 4 . Although molecular and adaptive breeding strategies can buffer the effects of climatic stress and improve crop resilience 5 , these approaches require sufficient knowledge of the genes that underlie productivity and adaptation 6 —knowledge that has been limited to a small number of well-studied model systems. Here we present the assembly and annotation of the large and complex genome of the polyploid bioenergy crop switchgrass ( Panicum virgatum ). Analysis of biomass and survival among 732 resequenced genotypes, which were grown across 10 common gardens that span 1,800 km of latitude, jointly revealed extensive genomic evidence of climate adaptation. Climate–gene–biomass associations were abundant but varied considerably among deeply diverged gene pools. Furthermore, we found that gene flow accelerated climate adaptation during the postglacial colonization of northern habitats through introgression of alleles from a pre-adapted northern gene pool. The polyploid nature of switchgrass also enhanced adaptive potential through the fractionation of gene function, as there was an increased level of heritable genetic diversity on the nondominant subgenome. In addition to investigating patterns of climate adaptation, the genome resources and gene–trait associations developed here provide breeders with the necessary tools to increase switchgrass yield for the sustainable production of bioenergy. The genome of the biofuel crop switchgrass ( Panicum virgatum ) reveals climate–gene–biomass associations that underlie adaptation in nature and will facilitate improvements of the yield of this crop for bioenergy production.

• The lignin content of feedstock has been proposed as one key agronomic trait impacting biofuel production from lignocellulosic biomass. 4‐Coumarate:coenzyme A ligase (4CL) is one of the key enzymes involved in the monolignol biosynthethic pathway. • Two homologous 4CL genes, Pv4CL1 and Pv4CL2, were identified in switchgrass (Panicum virgatum) through phylogenetic analysis. Gene expression patterns and enzymatic activity assays suggested that Pv4CL1 is involved in monolignol biosynthesis. Stable transgenic plants were obtained with Pv4CL1 down‐regulated. • RNA interference of Pv4CL1 reduced extractable 4CL activity by 80%, leading to a reduction in lignin content with decreased guaiacyl unit composition. Altered lignification patterns in the stems of RNAi transgenic plants were observed with phloroglucinol‐HCl staining. The transgenic plants also had uncompromised biomass yields. After dilute acid pretreatment, the low lignin transgenic biomass had significantly increased cellulose hydrolysis (saccharification) efficiency. • The results demonstrate that Pv4CL1, but not Pv4CL2, is the key 4CL isozyme involved in lignin biosynthesis, and reducing lignin content in switchgrass biomass by silencing Pv4CL1 can remarkably increase the efficiency of fermentable sugar release for biofuel production.

Summary Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

Broomcorn millet (Panicum miliaceum L.), one of the first domesticated crops, has been grown in Northern China for at least 10,000 years. The species is presently a minor crop, and evaluation of its genetic diversity has been very limited. In this study, we analyzed the genetic diversity of 88 accessions of broomcorn millet collected from various provinces of China. Amplification with 67 simple sequence repeat (SSR) primers revealed moderate levels of diversity in the investigated accessions. A total of 179 alleles were detected, with an average of 2.7 alleles per locus. Polymorphism information content and expected heterozygosity ranged from 0.043 to 0.729 (mean = 0.376) and 0.045 to 0.771 (mean = 0.445), respectively. Cluster analysis based on the unweighted pair group method of mathematical averages separated the 88 accessions into four groups at a genetic similarity level of 0.633. A genetic structure assay indicated a close correlation between geographical regions and genetic diversity. The uncovered information will be valuable for defining gene pools and developing breeding programs for broomcorn millet. Furthermore, the millet-specific SSR markers developed in this study should serve as useful tools for assessment of genetic diversity and elucidation of population structure in broomcorn millet.

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.

Premise of the study: The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia. Methods: We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed. Key results: Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1-2 d). Plants inoculated with M.robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. Conclusions: These results suggest that M. robertsii is not only rhizosphere competent but also displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs.

Potassium (K) is essential for the productivity of tropical grasses, but its optimal supply remains unclear. This study evaluated the effects of K on gas exchange, photosynthetic rate (A), forage production (FP), and root development (RP) in Panicum maximum cultivars. The experiment was conducted using a randomized block design in a 6 × 4 factorial scheme (six cultivars: Tanzânia, Quênia, Mombaça, Zuri, Massai and Tamani; and four K rates: 0, 205, 410 and 820 mg dm −3 ). There was interaction between cultivars and K rates for A and stomatal conductance (g s ), with linear increases in A for all cultivars. The g s response in the Zuri cultivar was quadratic, with a maximum of 5.32 mmol m −2  s −1 at the dose of 410 mg dm −3  K, and linear for the other cultivars. The CO 2 concentration (Ci) and leaf temperature (Tleaf) were not influenced by the K dose or by the cultivars, maintaining an average of 129.28 ppm and 29.32 °C, respectively. Transpiration (E) increased by 0.018 mmol m −2  s −1 with increasing K doses. The chlorophyll content fitted a quadratic model, with a maximum of 35 SPAD at the dose of 530 mg dm −3 . The FP increased linearly for all cultivars, with the highest FP in the cultivars Quênia, Mombaça and Zuri. Increasing K doses improved A and g s , reflecting in higher FP. The cultivars Quênia and Mombaça showed the greatest increases in FP up to 820 mg dm −3  K, while the maximum efficiency for RP occurred between 205 and 410 mg dm −3 , suggesting that excessive K supply may not proportionally increase root development.

The physiological implications of C sub(3)-C sub(4) photosynthesis were investigated using closely related Panicum species exposed to industrial-age climate change. Panicum bisulcatum (C sub(3)), P. milioides (C sub(3)-C sub(4)), and P. coloratum (C sub(4)) were grown in a glasshouse at three CO sub(2) concentrations ([CO sub(2)]: 280, 400, and 650 mu l l super(-1)) and two air temperatures [ambient (27/19 degree C day/night) and ambient + 4 degree C] for 12 weeks. Under current ambient [CO sub(2)] and temperature, the C sub(3)-C sub(4) species had higher photosynthetic rates and lower stomatal limitation and electron cost of photosynthesis relative to the C sub(3) species. These photosynthetic advantages did not improve leaf- or plant-level water (WUE) or nitrogen (NUE) use efficiencies of the C sub(3)-C sub(4) relative to the C sub(3) Panicum species. In contrast, the C sub(4) species had higher photosynthetic rates and WUE but similar NUE to the C sub(3) species. Increasing [CO sub(2)] mainly stimulated photosynthesis of the C sub(3) and C sub(3)-C sub(4) species, while high temperature had no or negative effects on photosynthesis of the Panicum species. Under ambient temperature, increasing [CO sub(2)] enhanced the biomass of the C sub(3) species only. Under high temperature, increasing [CO sub(2)] enhanced the biomass of the C sub(3) and C sub(3)-C sub(4) species to the same extent, indicating increased CO sub(2) limitation in the C sub(3)-C sub(4) intermediate at high temperature. Growth [CO sub(2)] and temperature had complex interactive effects, but did not alter the ranking of key physiological parameters amongst the Panicum species. In conclusion, the ability of C sub(3)-C sub(4) intermediate species partially to recycle photorespired CO sub(2) did not improve WUE or NUE relative to congeneric C sub(3) or C sub(4) species grown under varying [CO sub(2)] and temperature conditions.