Dynamic Reconfiguration of Switchgrass Proteomes in Response to Rust (Puccinia novopanici) Infection.

Switchgrass (Panicum virgatum L.) can be infected by the rust pathogen (Puccinia novopanici) and results in lowering biomass yields and quality. Label-free quantitative proteomics was conducted on leaf extracts harvested from non-infected and infected plants from a susceptible cultivar (Summer) at 7, 11, and 18 days after inoculation (DAI) to follow the progression of disease and evaluate any plant compensatory mechanisms to infection. Some pustules were evident at 7 DAI, and their numbers increased with time. However, fungal DNA loads did not appreciably change over the course of this experiment in the infected plants. In total, 3830 proteins were identified at 1% false discovery rate, with 3632 mapped to the switchgrass proteome and 198 proteins mapped to different Puccinia proteomes. Across all comparisons, 1825 differentially accumulated switchgrass proteins were identified and subjected to a STRING analysis using Arabidopsis (A. thaliana L.) orthologs to deduce switchgrass cellular pathways impacted by rust infection. Proteins associated with plastid functions and primary metabolism were diminished in infected Summer plants at all harvest dates, whereas proteins associated with immunity, chaperone functions, and phenylpropanoid biosynthesis were significantly enriched. At 18 DAI, 1105 and 151 proteins were significantly enriched or diminished, respectively. Many of the enriched proteins were associated with mitigation of cellular stress and defense.

Divergent Metabolic Changes in Rhizomes of Lowland and Upland Switchgrass (Panicum virgatum) from Early Season through Dormancy Onset.

High-biomass-yielding southerly adapted switchgrasses (Panicum virgatum L.) frequently suffer from unpredictable winter hardiness at more northerly sites arising from damage to rhizomes that prevent effective spring regrowth. Previously, changes occurring over the growing season in rhizomes sampled from a cold-adapted tetraploid upland cultivar, Summer, demonstrated a role for abscisic acid (ABA), starch accumulation, and transcriptional reprogramming as drivers of dormancy onset and potential keys to rhizome health during winter dormancy. Here, rhizome metabolism of a high-yielding southerly adapted tetraploid switchgrass cultivar, Kanlow-which is a significant source of genetics for yield improvement-was studied over a growing season at a northern site. Metabolite levels and transcript abundances were combined to develop physiological profiles accompanying greening through the onset of dormancy in Kanlow rhizomes. Next, comparisons of the data to rhizome metabolism occurring in the adapted upland cultivar Summer were performed. These data revealed both similarities as well as numerous differences in rhizome metabolism that were indicative of physiological adaptations unique to each cultivar. Similarities included elevated ABA levels and accumulation of starch in rhizomes during dormancy onset. Notable differences were observed in the accumulation of specific metabolites, the expression of genes encoding transcription factors, and several enzymes linked to primary metabolism.

Effect of cultivar and temperature on the synergistic interaction between panicum mosaic virus and satellite panicum mosaic virus in switchgrass.

Panicum mosaic virus (PMV), the type member of the genus Panicovirus in the family Tombusviridae, naturally infects switchgrass (Panicum virgatum L.). PMV and its molecular partner, satellite panicum mosaic virus (SPMV), interact synergistically in coinfected millets to exacerbate the disease phenotype and increase the accumulation of PMV compared to plants infected with PMV alone. In this study, we examined the reaction of switchgrass cvs. Summer and Kanlow to PMV and PMV+SPMV infections at 24°C and 32°C. Switchgrass cv. Summer was susceptible to PMV at both temperatures. In contrast, cv. Kanlow was tolerant to PMV at 24°C, but not at 32°C, suggesting that Kanlow harbors temperature-sensitive resistance to PMV. At 24°C, PMV was readily detected in inoculated leaves, but not in upper uninoculated leaves of Kanlow, suggesting that resistance to PMV was likely mediated by abrogation of long-distance virus transport. Coinfection by PMV and SPMV at 24°C and 32°C in cv. Summer, but not in Kanlow, caused increased symptomatic systemic infection and mild disease synergism with slightly increased PMV accumulation compared to plants infected with PMV alone. These data suggest that the interaction between PMV and SPMV in switchgrass is cultivar-dependent, manifested in Summer but not in Kanlow. However, co-inoculation of cv. Kanlow with PMV+SPMV caused an enhanced asymptomatic infection, suggesting a role of SPMV in enhancement of symptomless infection in a tolerant cultivar. These data suggest that enhanced asymptomatic infections in a virus-tolerant switchgrass cultivar could serve as a source of virus spread and play an important role in panicum mosaic disease epidemiology under field conditions. Our data reveal that the cultivar, coinfection with SPMV, and temperature influence the severity of symptoms elicited by PMV in switchgrass.

Transcriptome divergence during leaf development in two contrasting switchgrass (Panicum virgatum L.) cultivars.

The genetics and responses to biotic stressors of tetraploid switchgrass (Panicum virgatum L.) lowland cultivar 'Kanlow' and upland cultivar Summer are distinct and can be exploited for trait improvement. In general, there is a paucity of data on the basal differences in transcription across tissue developmental times for switchgrass cultivars. Here, the changes in basal and temporal expression of genes related to leaf functions were evaluated for greenhouse grown 'Kanlow', and 'Summer' plants. Three biological replicates of the 4th leaf pooled from 15 plants per replicate were harvested at regular intervals beginning from leaf emergence through senescence. Increases and decreases in leaf chlorophyll and N content were similar for both cultivars. Likewise, multidimensional scaling (MDS) analysis indicated both cultivar-independent and cultivar-specific gene expression. Cultivar-independent genes and gene-networks included those associated with leaf function, such as growth/senescence, carbon/nitrogen assimilation, photosynthesis, chlorophyll biosynthesis, and chlorophyll degradation. However, many genes encoding nucleotide-binding leucine rich repeat (NB-LRRs) proteins and wall-bound kinases associated with detecting and responding to environmental signals were differentially expressed. Several of these belonged to unique cultivar-specific gene co-expression networks. Analysis of genomic resequencing data provided several examples of NB-LRRs genes that were not expressed and/or apparently absent in the genomes of Summer plants. It is plausible that cultivar (ecotype)-specific genes and gene-networks could be one of the drivers for the documented differences in responses to leaf-borne pathogens between these two cultivars. Incorporating broad resistance to plant pathogens in elite switchgrass germplasm could improve sustainability of biomass production under low-input conditions.

Genomic prediction accuracy for switchgrass traits related to bioenergy within differentiated populations.

BACKGROUND: Switchgrass breeders need to improve the rates of genetic gain in many bioenergy-related traits in order to create improved cultivars that are higher yielding and have optimal biomass composition. One way to achieve this is through genomic selection. However, the heritability of traits needs to be determined as well as the accuracy of prediction in order to determine if efficient selection is possible. RESULTS: Using five distinct switchgrass populations comprised of three lowland, one upland and one hybrid accession, the accuracy of genomic predictions under different cross-validation strategies and prediction methods was investigated. Individual genotypes were collected using GBS while kin-BLUP, partial least squares, sparse partial least squares, and BayesB methods were employed to predict yield, morphological, and NIRS-based compositional data collected in 2012-2013 from a replicated Nebraska field trial. Population structure was assessed by F statistics which ranged from 0.3952 between lowland and upland accessions to 0.0131 among the lowland accessions. Prediction accuracy ranged from 0.57-0.52 for cell wall soluble glucose and fructose respectively, to insignificant for traits with low repeatability. Ratios of heritability across to within-population ranged from 15 to 0.6. CONCLUSIONS: Accuracy was significantly affected by both cross-validation strategy and trait. Accounting for population structure with a cross-validation strategy constrained by accession resulted in accuracies that were 69% lower than apparent accuracies using unconstrained cross-validation. Less accurate genomic selection is anticipated when most of the phenotypic variation exists between populations such as with spring regreening and yield phenotypes.

Seasonal below-ground metabolism in switchgrass.

Switchgrass (Panicum virgatum), a perennial, polyploid, C4 warm-season grass is among the foremost herbaceous species being advanced as a source of biomass for biofuel end uses. At the end of every growing season, the aerial tissues senesce, and the below-ground rhizomes become dormant. Future growth is dependent on the successful over-wintering of the rhizomes. Although the importance of rhizome health to overall year-upon-year plant productivity has been long recognized, there is limited information on seasonal changes occurring during dormancy at both the transcriptome and metabolite levels. Here, global changes in transcriptomes and metabolites were investigated over two growing seasons in rhizomes harvested from field-grown plants. The objectives were: (a) synthesize information on cellular processes that lead to dormancy; and (b) provide models that could account for major metabolic pathways present in dormant switchgrass rhizomes. Overall, metabolism during dormancy appeared to involve discrete but interrelated events. One was a response to abscisic acid that resulted in dehydration, increases in osmolytes and upregulation of autophagic processes, likely through the target of rapamycin complex and sucrose non-fermentative-related kinase-based signaling cascades. Another was a recalibration of energy transduction through apparent reductions in mitochondrial oxidative phosphorylation, increases in substrate level generation of ATP and reducing equivalents, and recycling of N and possibly CO2 through refixation. Lastly, transcript abundances indicated that cold-related signaling was also occurring. Altogether, these data provide a detailed overview of rhizome metabolism, especially during dormancy, which can be exploited in the future to improve winter survival in switchgrass.