Paul Twigg

Comparative Genomics in Switchgrass Using 61,585 High-Quality Expressed Sequence Tags

The development of genomic resources for switchgrass (Panicum virgatum L.), a perennial NAD+‐malic enzyme type C4 grass, is required to enable molecular breeding and biotechnological approaches for improving its value as a forage and bioenergy crop. Expressed sequence tag (EST) sequencing is one method that can quickly sample gene inventories and produce data suitable for marker development or analysis of tissue‐specific patterns of expression. Toward this goal, three cDNA libraries from callus, crown, and seedling tissues of ‘Kanlow’ switchgrass were end‐sequenced to generate a total of 61,585 high‐quality ESTs from 36,565 separate clones. Seventy‐three percent of the assembled consensus sequences could be aligned with the sorghum [Sorghum bicolor (L.) Moench] genome at a E‐value of <1 × 10−20, indicating a high degree of similarity. Sixty‐five percent of the ESTs matched with gene ontology molecular terms, and 3.3% of the sequences were matched with genes that play potential roles in cell‐wall biogenesis. The representation in the three libraries of gene families known to be associated with C4 photosynthesis, cellulose and β‐glucan synthesis, phenylpropanoid biosynthesis, and peroxidase activity indicated likely roles for individual family members. Pairwise comparisons of synonymous codon substitutions were used to assess genome sequence diversity and indicated an overall similarity between the two genome copies present in the tetraploid. Identification of EST–simple sequence repeat markers and amplification on two individual parents of a mapping population yielded an average of 2.18 amplicons per individual, and 35% of the markers produced fragment length polymorphisms.

Analysis of expressed sequence tags and the identification of associated short tandem repeats in switchgrass.

Switchgrass is a large, North American, perennial grass that is being evaluated as a potential energy crop. Expressed sequence tags (ESTs) were generated from four switchgrass cv. “Kanlow” cDNA libraries to create a gene inventory of 7,810 unique gene clusters from a total of 11,990 individual sequences. Blast similarity searches to SwissProt and GenBank non-redundant protein and nucleotide databases were performed and a total of 79% of these unique clusters were found to be similar to existing protein or nucleotide sequences. Tentative functional classification of 61% of the sequences was possible by association with appropriate gene ontology descriptors. Significant differential representation between genes in leaf, stem, crown, and callus libraries was observed for many highly expressed genes The unique gene clusters were screened for the presence of short tandem repeats for further development as microsatellite markers. A total of 334 gene clusters contained repeats representing 3.8% of the ESTs queried.

Gene expression profiling of tolerant barley in response to Diuraphis noxia (Hemiptera: Aphididae) feeding

Aphids are, arguably, the single most damaging group of agricultural insect pests throughout the world. Plant tolerance, which is a plant response to an insect pest, is viewed as an excellent management strategy. Developing testable hypotheses based on genome-wide and more focused methods will help in understanding the molecular underpinnings of plant tolerance to aphid herbivory. As a first step in this process, we undertook transcript profiling with Affymetrix GeneChip Barley Genome arrays using RNA extracted from tissues of tolerant and susceptible genotypes collected at three hours, three days and six days after Diuraphis noxia introduction. Acquired data were compared to identify changes unique to the tolerant barley at each harvest date. Transcript abundance of 4086 genes was differentially changed over the three harvest dates in tolerant and susceptible barley in response to D. noxia feeding. Across the three harvest dates, the greatest number of genes was differentially expressed in both barleys at three days after aphid introduction. A total of 909 genes showed significant levels of change in the tolerant barley in response to D. noxia feeding as compared to susceptible plants infested with aphids. Many of these genes could be assigned to specific metabolic categories, including several associated with plant defense and scavenging of reactive oxygen species (ROS). Interestingly, two peroxidase genes, designated HvPRXA1 and HvPRXA2, were up-regulated to a greater degree in response to D. noxia feeding on tolerant barley plants, indicating that specific peroxidases could be important for the tolerance process. These findings suggest that the ability to elevate and sustain levels of ROS-scavenging enzymes could play an important role in the tolerant response.

Switchgrass contains two cinnamyl alcohol dehydrogenases involved in lignin formation.

Lignin content of switchgrass (Panicum virgatum L.), a bioenergy species, is a critical determinant of biomass quality since it can negatively impact conversion of biomass into liquid fuels via biochemical platforms. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. Here, we have shown that cv. Kanlow switchgrass contains at least two closely related CAD genes (PviCAD1 and PviCAD2) that code for proteins containing highly conserved domains and residues that identify them as bona fide CADs. Both recombinant proteins displayed substrate kinetics consistent with their presumed role in cell wall lignification. Proteomic and immunoblotting detected CAD containing spots in internode protein extracts, and proteomic analyses demonstrated that both CADs were expressed. In planta CAD activity, CAD protein levels were observed at all stages of tiller development. A real-time qPCR analysis of the two CADs and one CAD-like sequence indicated that transcripts coding for PviCAD1 were present in greater abundance than those coding for PviCAD2. Transcripts for a third CAD-like sequence (PviAroADH) were present at intermediate levels as compared to PviCAD1 and CAD2. The predicted protein sequence of PviAroADH indicated that it was an enzyme unrelated to lignification based on phylogenetic and protein modeling data.

Next-Generation Sequencing of Crown and Rhizome Transcriptome from an Upland, Tetraploid Switchgrass

The crown and rhizome transcriptome of an upland tetraploid switchgrass cultivar cv Summer well adapted to the upper Midwest was investigated using the Roche 454-FLX pyrosequencing platform. Overall, approximately one million reads consisting of 216 million bases were assembled into 27,687 contigs and 43,094 singletons. Analyses of these sequences revealed minor contamination with non-plant sequences (< 0.5%), indicating that a majority were for transcripts coded by the switchgrass genome. Blast2Gos comparisons resulted in the annotation of ~65% of the contig sequences and ~40% of the singleton sequences. Contig sequences were mostly homologous to other plant sequences, dominated by matches to Sorghum bicolor genome. Singleton sequences, while displaying significant matches to S. bicolor, also contained sequences matching non-plant species. Comparisons of the 454 dataset to existing EST collections resulted in the identification of 30,177 new sequences. These new sequences coded for a number of different proteins and a selective analysis of two categories, namely, peroxidases and transcription factors, resulted in the identification of specific peroxidases and a number of low-abundance transcription factors expected to be involved in chromatin remodeling. KEGG maps for glycolysis and sugar metabolism showed high levels of transcript coding for enzymes involved in primary metabolism. The assembly provided significant insights into the status of these tissues and broadly indicated that there was active metabolism taking place in the crown and rhizomes at post-anthesis, the seed maturation stage of plant development.

Cloning and characterization of a caesalpinoid (Chamaecrista fasciculata) hemoglobin: The structural transition from a nonsymbiotic hemoglobin to a leghemoglobin

Nonsymbiotic hemoglobins (nsHbs) and leghemoglobins (Lbs) are plant proteins that can reversibly bind O2 and other ligands. The nsHbs are hexacoordinate and appear to modulate cellular concentrations of NO and maintain energy levels under hypoxic conditions. The Lbs are pentacoordinate and facilitate the diffusion of O2 to symbiotic bacteroids within legume root nodules. Multiple lines of evidence suggest that all plant Hbs evolved from a common ancestor and that Lbs originated from nsHbs. However, little is known about the structural intermediates that occurred during the evolution of pentacoordinate Lbs from hexacoordinate nsHbs. We have cloned and characterized a Hb (ppHb) from the root nodules of the ancient caesalpinoid legume Chamaecrista fasciculata. Protein sequence, modeling data, and spectral analysis indicated that the properties of ppHb are intermediate between that of nsHb and Lb, suggesting that ppHb resembles a putative ancestral Lb. Predicted structural changes that appear to have occurred during the nsHb to Lb transition were a compaction of the CD‐loop and decreased mobility of the distal His inhibiting its ability to coordinate directly with the heme‐Fe, leading to a pentacoordinate protein. Other predicted changes include shortening of the N‐ and C‐termini, compaction of the protein into a globular structure, disappearance of positive charges outside the heme pocket and appearance of negative charges in an area located between the N‐ and C‐termini. A major consequence for some of these changes appears to be the decrease in O2‐affinity of ancestral nsHb, which resulted in the origin of the symbiotic function of Lbs. Proteins 2008.

Overexpression of SbMyb60 impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in Sorghum bicolor.

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses.

Turnip crinkle virus coat protein inhibits the basal immune response to virus invasion in Arabidopsis by binding to the NAC transcription factor TIP

Turnip crinkle virus (TCV) has been shown to interact with a NAC transcription factor, TIP, of Arabidopsis thaliana, via its coat protein (CP). This interaction correlates with the resistance response manifested in TCV-resistant Arabidopsis ecotype Di-17. We report that failure of a mutated CP to interact with TIP triggered the corresponding TCV mutant (R6A) to cause more severe symptoms in the TCV-susceptible ecotype Col-0. We hypothesized that TCV regulates antiviral basal immunity through TIP-CP interaction. Consistent with this hypothesis, we found that the rate of accumulation of R6A was measurably slower than wild-type TCV over the course of an infection. Notably, R6A was able to accumulate at similar rates as wild-type TCV in mutant plants with defects in salicylic acid (SA) signaling. Finally, plants with altered TIP expression provided evidence R6As inability to evade the basal resistance response was likely associated with loss of ability for CP to bind TIP.

Towards uncovering the roles of switchgrass peroxidases in plant processes

Herbaceous perennial plants selected as potential biofuel feedstocks had been understudied at the genomic and functional genomic levels. Recent investments, primarily by the U.S. Department of Energy, have led to the development of a number of molecular resources for bioenergy grasses, such as the partially annotated genome for switchgrass (Panicum virgatum L.), and some related diploid species. In its current version, the switchgrass genome contains 65,878 gene models arising from the A and B genomes of this tetraploid grass. The availability of these gene sequences provides a framework to exploit transcriptomic data obtained from next-generation sequencing platforms to address questions of biological importance. One such question pertains to discovery of genes and proteins important for biotic and abiotic stress responses, and how these components might affect biomass quality and stress response in plants engineered for a specific end purpose. It can be expected that production of switchgrass on marginal lands will expose plants to diverse stresses, including herbivory by insects. Class III plant peroxidases have been implicated in many developmental responses such as lignification and in the adaptive responses of plants to insect feeding. Here, we have analyzed the class III peroxidases encoded by the switchgrass genome, and have mined available transcriptomic datasets to develop a first understanding of the expression profiles of the class III peroxidases in different plant tissues. Lastly, we have identified switchgrass peroxidases that appear to be orthologs of enzymes shown to play key roles in lignification and plant defense responses to hemipterans.

Contrasting Metabolism in Perenniating Structures of Upland and Lowland Switchgrass Plants Late in the Growing Season

Background Switchgrass (Panicum virgatum L.) is being developed as a bioenergy crop for many temperate regions of the world. One way to increase biomass yields is to move southern adapted lowland cultivars to more northern latitudes. However, many southerly adapted switchgrass germplasm can suffer significant winter kill in northerly climes. Materials and Methods Here, we have applied next-generation sequencing in combination with biochemical analyses to query the metabolism of crowns and rhizomes obtained from two contrasting switchgrass cultivars. Crowns and rhizomes from field-grown lowland (cv Kanlow) and upland (cv Summer) switchgrass cultivars were collected from three randomly selected post-flowering plants. Summer plants were senescing, whereas Kanlow plants were not at this harvest date. Results Principal component analysis (PCA) differentiated between both the Summer and Kanlow transcriptomes and metabolomes. Significant differences in transcript abundances were detected for 8,050 genes, including transcription factors such as WRKYs and those associated with phenylpropanoid biosynthesis. Gene-set enrichment analyses showed that a number of pathways were differentially up-regulated in the two populations. For both populations, protein levels and enzyme activities agreed well with transcript abundances for genes involved in the phenylpropanoid pathway that were up-regulated in Kanlow crowns and rhizomes. The combination of these datasets suggests that dormancy-related mechanisms had been triggered in the crowns and rhizomes of the Summer plants, whereas the crowns and rhizomes of Kanlow plants had yet to enter dormancy. Conclusions Delayed establishment of dormancy at more northerly latitudes could be one factor that reduces winter-survival in the high-yielding Kanlow plants. Understanding the cellular signatures that accompany the transition to dormancy can be used in the future to select plants with improved winter hardiness.