A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Proteome profile of the endomembrane of developing coleoptiles from switchgrass (Panicum virgatum)

The cost‐effective production of biofuels from lignocellulosic material will likely require manipulation of plant biomass, specifically cell walls. The North American native prairie grass Panicum virgatum (switchgrass) is seen as a potential biofuel crop with an array of genetic resources currently being developed. We have characterized the endomembrane proteome of switchgrass coleoptiles to provide additional information to the switchgrass community. In total, we identified 1750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD001351 ( http://proteomecentral.proteomexchange.org/dataset/PXD001351 ).     

Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpression or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was functionally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.     

Protoplast isolation and transient gene expression in different petunia cultivars

Protoplasma - The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White)....     

利用SCoT分析柳枝稷遗传资源的多样性

本研究利用SCoT标记对96份柳枝稷种质的亲缘关系和遗传变异进行了研究。筛选出20条引物对96份供试材料进行PCR扩增,共获得445条带,其中多态性条带402条,平均多态性条带比率(PPB)达90.31%,多态性信息含量(PIC)为0.166~0.410,平均值为0.332,标记指数(MI)为10.20。遗传相似系数(GS)为0.498~0.912,平均值为0.688。表明SCoT标记能够揭示柳枝稷种质间的遗传变异。通过UPGMA分析表明,96份种质资源聚为高地型和低地型两大类。经POPGENE1.32软件分析结果显示:96份柳枝稷基因多样性指数(H)为0.285,Shannon指数(I)为0.431,表明供试的种质间遗传多样性丰富,遗传多样性水平高。经AMOVA 1.55方差分析揭示:96份柳枝稷生态型内的遗传变异占总变异的72.85%,生态型间遗传变异占总变异的27.15%,结果表明ScoT可用于柳枝稷遗传多样性研究,该研究结果可为柳枝稷种质资源的进一步开发利用提供重要信息。     

Genic microsatellite markers derived from EST sequences of switchgrass (Panicum virgatum L.)

Switchgrass is a large, North American, perennial grass that is being evaluated as a potential energy crop. There is a need to assess genetic diversity in stored accessions and in remaining native stands to assist breeding and conservation efforts. Marker development will also be necessary for genetic linkage mapping. Toward this end, 32 switchgrass genic di‐, tri‐ and tetranucleotide repeat microsatellites were identified from expressed sequence tags (ESTs). These microsatellites were used to screen individuals from two different named cultivars. The markers displayed a high level of polymorphism consistent with the tetraploid, allogamous behaviour of the cultivars tested.     

Investigation of genomic organization in switchgrass (Panicum virgatum L.) using DNA markers

We report an early investigation into genomic organization and chromosomal transmission in switchgrass based on restriction fragment length polymorphism (RFLP) markers. The segregation of 224 single dose restriction fragments (SDRF) in 85 full-sib progeny of a cross between the genotypes Alamo (AP13) and Summer (VS16) was used to determine linkage associations in each parent. In the seed parent AP13, 11 cosegregation groups were identified by 45 SDRF markers with a cumulative recombination length of 412.4 cM. In the pollen parent VS16, 57 SDRF markers were assigned to 16 cosegregation groups covering a length of 466.5 cM. SDRF markers identified by the same probes and mapping to different cosegregation groups were used to combine the two maps and identify homology groups. Eight homology groups were identified among the nine haploid linkage groups expected in switchgrass. The high incidence of repulsion phase associations indicates that preferential pairing between homologous chromosomes is predominant in switchgrass. Based on marker distribution in the paternal map (VS16), we estimated the recombinational length of switchgrass genome to be 4,617 cM. In order to link 95% of the genome to a marker at a 15-cM distance, a minimum of 459 markers will be required. Using information from the ratio of repulsion to coupling linkages, we infer that switchgrass is an autotetraploid with a high degree of preferential pairing. The information presented in this study establishes a foundation for extending genetic mapping in this crop and constitutes a framework for basic and applied genetic studies.Electronic Supplementary Material Supplementary material is available for this article at     

Effects of ecotypes and morphotypes in feedstock composition of switchgrass (Panicum virgatum L.)

Switchgrass (Panicum virgatum L.) is a C4 grass with high biomass yield potential and is now a model species for the Bioenergy Feedstock Development Program. Two distinct ecotypes (e.g., upland and lowland) and a range of plant morphotypes (e.g., leafy and stemmy) have been observed in switchgrass. The objective of this study was to determine the influence of ecotype and morphotype on biomass feedstock quality. Leaf and stem tissues of leafy and stemmy morphotypes from both lowland and upland ecotypes were analyzed for key feedstock traits. The leaf : stem ratio of leafy morphotype was more than 40% higher than the stemmy morphotype in both upland and lowland ecotypes. Therefore, the stemmy morphotype has significant advantages over leafy morphotype during harvesting, storage, transportation and finally the feedstock quality. Remarkable differences in feedstock quality and mineral composition were observed in switchgrass genotypes with distinct ecotypic origins and variable plant morphotypes. Lignin, hemicelluloses and cellulose concentrations were higher in stems than in the leaves, while ash content was notably high in leaves. A higher concentration of potassium was found in the stems compared to the leaves. In contrast, calcium was higher and magnesium was generally higher in the leaves compared to stems. The upland genotypes demonstrated considerably higher lignin (14.4%) compared with lowland genotypes (12.4%), while hemicellulose was higher in lowland compared with upland. The stemmy type demonstrated slightly higher lignin compared with leafy types (P < 0.1). Differences between the ecotypes and morphotypes for key quality traits demonstrated the potential for improving feedstock composition of switchgrass through selection in breeding programs.     

Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9

The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self‐infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar ‘Alamo’ switchgrass. We first developed a transient assay by which a non‐functional green‐fluorescent protein gene containing a 1‐bp frameshift insertion in its 5′ coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium‐mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9‐induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1‐bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding.     

Nanoparticle titanium dioxide affects the growth and microRNA expression of switchgrass (Panicum virgatum)

Nanoparticle TiO₂ is a common chemical used in daily life. As increasing usage of TiO₂, it is becoming a potentially dangerous contaminant to the environment. However, the impact of TiO₂ is not well understood. In this paper, switchgrass was employed to investigate the impacts of nanoparticle TiO₂ on plant growth and development as well as the potential impact on the expression of microRNAs (miRNAs). TiO₂ significantly affected switchgrass seed generation as well as plant growth and development in a dose-dependent manner. Particularly, TiO₂ significantly inhibited root development. miRNA expressions were also significantly altered. Nanoparticle TiO₂ may regulate plant development through controlling the expression of certain miRNAs. Among the 16 tested miRNAs, the expression of some miRNAs, such as miR390 and miR399 was increased with increasing TiO₂ concentrations; the expression of some miRNAs, such as miR169 was decreased with increasing TiO₂ concentrations; the other miRNAs show different expression patterns.     

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.     

Five nuclear loci resolve the polyploid history of switchgrass (Panicum virgatum L.) and relatives

Polyploidy poses challenges for phylogenetic reconstruction because of the need to identify and distinguish between homoeologous loci. This can be addressed by use of low copy nuclear markers. Panicum s.s. is a genus of about 100 species in the grass tribe Paniceae, subfamily Panicoideae, and is divided into five sections. Many of the species are known to be polyploids. The most well-known of the Panicum polyploids are switchgrass (Panicum virgatum) and common or Proso millet (P. miliaceum). Switchgrass is in section Virgata, along with P. tricholaenoides, P. amarum, and P. amarulum, whereas P. miliaceum is in sect. Panicum. We have generated sequence data from five low copy nuclear loci and two chloroplast loci and have clarified the origin of P. virgatum. We find that all members of sects. Virgata and Urvilleana are the result of diversification after a single allopolyploidy event. The closest diploid relatives of switchgrass are in sect. Rudgeana, native to Central and South America. Within sections Virgata and Urvilleana, P. tricholaenoides is sister to the remaining species. Panicum racemosum and P. urvilleanum form a clade, which may be sister to P. chloroleucum. Panicum amarum, P. amarulum, and the lowland and upland ecotypes of P. virgatum together form a clade, within which relationships are complex. Hexaploid and octoploid plants are likely allopolyploids, with P. amarum and P. amarulum sharing genomes with P. virgatum. Octoploid P. virgatum plants are formed via hybridization between disparate tetraploids. We show that polyploidy precedes diversification in a complex set of polyploids; our data thus suggest that polyploidy could provide the raw material for diversification. In addition, we show two rounds of allopolyploidization in the ancestry of switchgrass, and identify additional species that may be part of its broader gene pool. This may be relevant for development of the crop for biofuels.     

MicroRNA expression analysis in the cellulosic biofuel crop switchgrass (Panicum virgatum) under abiotic stress

Switchgrass has increasingly been recognized as a dedicated biofuel crop for its broad adaptation to marginal lands and high biomass. However, little is known about the basic biology and the regulatory mechanisms of gene expression in switchgrass, particularly under stress conditions. In this study, we investigated the effect of salt and drought stress on switchgrass germination, growth and the expression of small regulatory RNAs. The results indicate that salt stress had a gradual but significant negative effect on switchgrass growth and development. The germination rate was significantly decreased from 82% for control to 36% under 1% NaCl treatment. However, drought stress had little effect on the germination rate but had a significant effect on the growth of switchgrass under the severest salinity stress. Both salt and drought stresses altered the expression pattern of miRNAs in a dose-dependent manner. However, each miRNA responded to drought stress in a different pattern. Salt and drought stress changed the expression level of miRNAs mainly from 0.9-fold up-regulation to 0.7-fold down-regulation. miRNAs were less sensitive to drought treatment than salinity treatment, as evidenced by the narrow fold change in expression levels. Although the range of change in expression level of miRNAs was similar under salt and drought stress, no miRNAs displayed significant change in expression level under all tested salt conditions. Two miRNAs, miR156 and miR162, showed significantly change in expression level under high drought stress. This suggests that miR156 and miR162 may attribute to the adaption of switchgrass to drought stress and are good candidates for improving switchgrass as a biofuel crop by transgenic technology.     

Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C₄ species, despite their potential of serving as model systems for their extraordinary C₄ photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C₄ species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C₄ photosynthetic mechanism are discussed.     

The effects of promoter on transient expression in conifer cell lines

Summary Protoplasts from suspension cultures of somatic embryos of white spruce (Picea glauca Moench Voss) were electroporated with plasmids containing the chimeric genes for chloramphenicol acetyl transferase (CAT) or -glucuronidase (GUS), under control of one of three promoters. Transient CAT gene expression of approximately equal magnitude resulted when the CAT gene was fused to either the cauliflower mosaic virus (CaMV) 35S promoter or the nopaline synthase (NOS) promoter. When the CAT gene was fused to a tandem repeat CaMV 35S promoter (pPBI-363), CAT enzyme activity compared to NOS or 35S promoters increased up to eightfold (cell line WS-34), and were up to 100-fold greater than control (electroporated without plasmid). Comparatively, protoplasts of black spruce (Picea mariana Mill) and jack pine (Pinus banksiana Lamb.), electroporated with pPBI-363, produced increases in CAT activity compared to control of 90-fold and 70-fold, respectively. White spruce (WS-34) protoplasts were subsequently electroporated with the GUS gene fused to the tandem repeat CaMV 35S promoter. Comparatively, GUS enzyme activity increased up to tenfold compared to GUS fused to a CaMV 35S promoter. The results indicated that transient expression of the CAT and GUS genes was influenced by the type of promoter and cell line used, as well as by electroporation conditions.NRCC No. 30498     

Physiological and growth responses of switchgrass (Panicum virgatum L.) in native stands under passive air temperature manipulation

In the central Great Plains of North America, climate change predictions include increases in mean annual temperature of 1.5–5.5 °C by 2100. Ecosystem responses to increased temperatures are likely to be regulated by dominant plant species, such as the potential biofuel species Panicum virgatum (switchgrass) in the tallgrass prairie. To describe the potential physiological and whole‐plant responses of this species to future changes in air temperatures, we used louvered open‐sided chambers (louvered OSC; 1 × 1 m, adjustable height) to passively alter canopy temperature in native stands of P. virgatum growing in tallgrass prairie at varying topographic positions (upland/lowland). The altered temperature treatment decreased daily mean temperatures by 1 °C and maximum temperatures by 4 °C in May and June, lowered daytime stomatal conductance and transpiration, decreased tiller density, increased specific leaf area, and delayed flowering. Among topographic contrasts, aboveground biomass, flowering tiller density, and tiller weight were greater in lowland sites compared to upland sites, with no temperature treatment interactions. Differences in biomass production responded more to topography than the altered temperature treatment, as soil water status varied considerably between topographic positions. These results indicate that while water availability as a function of topography was a strong driver of plant biomass, many leaf‐level physiological processes were responsive to the small decreases in daily mean and maximum temperature, irrespective of landscape position. The varying responses of leaf‐level gas exchange and whole‐plant growth of P. virgatum in native stands to altered air temperature or topographic position illustrate that accurately forecasting yields for P. virgatum in mixed communities will require greater integration of physiological responses to simulated climate change (increased temperature) and resource availability over natural environmental gradients (soil moisture).