Lisa M. Baird

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.

Senescence, dormancy and tillering in perennial C4 grasses.

Perennial, temperate, C4 grasses, such as switchgrass and miscanthus have been tabbed as sources of herbaceous biomass for the production of green fuels and chemicals based on a number of positive agronomic traits. Although there is important literature on the management of these species for biomass production on marginal lands, numerous aspects of their biology are as yet unexplored at the molecular level. Perenniality, a key agronomic trait, is a function of plant dormancy and winter survival of the below-ground parts of the plants. These include the crowns, rhizomes and meristems that will produce tillers. Maintaining meristem viability is critical for the continued survival of the plants. Plant tillers emerge from the dormant crown and rhizome meristems at the start of the growing period in the spring, progress through a phase of vegetative growth, followed by flowering and eventually undergo senescence. There is nutrient mobilization from the aerial portions of the plant to the crowns and rhizomes during tiller senescence. Signals arising from the shoots and from the environment can be expected to be integrated as the plants enter into dormancy. Plant senescence and dormancy have been well studied in several dicot species and offer a potential framework to understand these processes in temperate C4 perennial grasses. The availability of latitudinally adapted populations for switchgrass presents an opportunity to dissect molecular mechanisms that can impact senescence, dormancy and winter survival. Given the large increase in genomic and other resources for switchgrass, it is anticipated that projected molecular studies with switchgrass will have a broader impact on related species.

Chinch Bug (Hemiptera: Blissidae) Mouthpart Morphology, Probing Frequencies, and Locations on Resistant and Susceptible Germplasm

Abstract Chinch bugs are common pests of many agronomic and horticulturally important crops and turfgrasses. Previous research has indicated that some grasses exhibit resistance to multiple chinch bug species, whereas others are resistant to only one species. The objectives of this research were to document differences in the probing frequencies and locations among Blissus species as well as differences in mouthpart morphology as a first step in understanding the differential responses of grasses to chinch bug feeding. Scanning electron microscopy detected differences in the total lengths of proboscises as well as individual mouthpart segments among the four species studied. Blissus occiduus Barber probed significantly more often on buffalograss, Buchloë dactyloides (Nuttall) Engelmann, than any other plant material. Probing locations of B. occiduus and Blissus leucopterus leucopterus (Say) were similar on both B. occiduus-resistant and susceptible buffalograsses and KS94 sorghum, Sorghum bicolor (L.) Moench (B. occiduus-resistant, B. l. leucopterus-resistant). However, on ‘Wheatland’ sorghum (B. occiduus-resistant, B. l. leucopterus-susceptible), stylet tracts of B. l. leucopterus most often terminated in the bundle sheath cells, whereas those of B. occiduus generally terminated in the vascular tissues.

P39, a novel soybean protein allergen, belongs to a plant-specific protein family and is present in protein storage vacuoles

Soybean lecithins are seeing increasing use in industry as an emulsifier and food additive. They are also a growing source of human food allergies, which arise principally from the proteins fractionating with the lecithin fraction during manufacture. A previous study (Gu, X.; Beardslee, T.; Zeece, M.; Sarath, G.; Markwwell, J. Int Arch. Allergy Immunol. 2001, 126, 218-225) identified several allergenic proteins in soybean lecithins and a soybean IgE-binding protein termed P39 was discovered. However, very little was known about this protein except that it was coded by the soybean genome. This paper investigates key biological and immunological properties of this potential soybean lecithin allergen. P39 is encoded by a multigene family in soybeans and in several other higher plants. The soybean P39-1 protein and its essentially indistinguishable homologue, P39-2, have been cloned and studied. These proteins and their homologues belong to a family of plant-specific proteins of unknown function. In soybeans, P39-1 is seed specific, and its transcript levels are highest in developing seeds and decline during seed maturation. In contrast, P39 protein was detectable only in the fully mature, dry seed. Subcellular fractionation revealed that P39 protein was strongly associated with oil bodies; however, immunolocalization indicated P39 was distributed in the matrix of the protein storage vacuoles, suggesting that association with oil bodies was an artifact arising from the extraction procedure. By the use of recombinant techniques it has also been documented that IgE-binding epitopes are present on several different portions of the P39-1 polypeptide.

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.

Morphogenesis of Effective and Ineffective Root Nodules in Phaseolus vulgaris L.

Seedlings of Phaseolus vulgaris, inoculated with an effective strain of Rhizobium phaseoli (CIAT 73) or an ineffective strain (14c), were maintained on an N-free nutrient solution in sterilized sand. Acetylene reduction was measured periodically, and nodules were harvested for light and electron microscopy. In plant host cells infected with CIAT 73, there was an increase in cell volume, nuclear size, and mitochondrial length as bacteria multiplied and became bacteroidal. Golgi activity and rough endoplasmic reticulum (RER) were sparse at nodule maturity, but close associations of amyloplasts and elongated, energized mitochondria were observed. Nitrogen fixation peaked at 5 wk and dropped sharply coincident with flowering. Plant host cells infected with 14c typically contained numerous mitochondria; Golgi bodies and RER were conspicuous throughout nodule development. By the time the bacteria became bacteroidal, plant shoots were chlorotic, and under nitrogen stress the apex had made the transition to flowering. Data suggest that such ineffective nodulation may accelerate the process of normal nodule senescence.

Characterization of Peroxidase Changes in Tolerant and Susceptible Soybeans Challenged by Soybean Aphid (Hemiptera: Aphididae)

ABSTRACT Changes in protein content, peroxidase activity, and isozyme profiles in response to soybean aphid feeding were documented at V1 (fully developed leaves at unifoliate node, first trifoliate leaf unrolled) and V3 (fully developed leaf at second trifoliate node, third trifoliate leaf unrolled) stages of soybean aphid-tolerant (KS4202) and -susceptible (SD76R) soybeans. Protein content was similar between infested and control V1 and V3 stage plants for both KS4202 and SD76R at 6, 16, and 22 d after aphid introduction. Enzyme kinetics studies documented that control and aphid-infested KS4202 V1 stage and SD76R V1 and V3 stages had similar levels of peroxidase activity at the three time points evaluated. In contrast, KS4202 aphid-infested plants at the V3 stage had significantly higher peroxidase activity levels than control plants at 6 and 22 d after aphid introduction. The differences in peroxidase activity observed between infested and control V3 stage KS4202 plants at these two time points suggest that peroxidases may be playing multiple roles in the tolerant plant. Native gels stained for peroxidase were able to detect differences in the isozyme profiles of aphidinfested and control plants for both KS4202 and SD76R.

Expression Profiling of Four Defense-Related Buffalograss Transcripts in Response to Chinch Bug (Hemiptera: Blissidae) Feeding

ABSTRACT Oxidative enzymes are one of many key players in plant tolerance responses and defense signaling pathways. This study evaluated gene expression of four buffalograss transcripts (two peroxidases, a catalase, and a GRAS (gibberellic acid insensitive [GAI], repressor of GAI, and scarecrow) and total peroxidase activity in response to western chinch bug (Blissus occiduus Barber) feeding in susceptible and resistant buffalograsses (Buchloë dactyloides (Nuttall) Engelmann). Basal levels of all four transcripts were consistently higher in the resistant buffalograss when compared with the susceptible genotype, which suggests important physiological differences exist between the two buffalograsses. The four defense-related transcripts also showed differential expression between infested and control plants for both the resistant and susceptible buffalograsses. Differences in total peroxidase activity were also detected between control and infested plants, and basal peroxidase activity was higher in the resistant genotype. Overall, this study indicates that elevated basal levels of specific peroxidases, catalases, and GRAS may be an effective buffalograss defense strategy against chinch bug feeding and other similar biotic stresses.

Transcriptome analysis of two buffalograss cultivars

BackgroundBuffalograss [Buchloë dactyloides (Nutt.) Engel. syn. Bouteloua dactyloides (Nutt.) Columbus] is a United States native turfgrass species that requires less irrigation, fungicides and pesticides compared to more commonly used turfgrass species. In areas where water is limited, interest in this grass species for lawns is increasing. While several buffalograss cultivars have been developed through buffalograss breeding, the timeframe for new cultivar development is long and is limited by a lack of useful genetic resources. Two high throughput next-generation sequencing techniques were used to increase the genomic resources available for buffalograss.ResultsTotal RNA was extracted and purified from leaf samples of two buffalograss cultivars. ‘378’ and ‘Prestige’ cDNA libraries were subjected to high throughput sequencing on the Illumina GA and Roche 454 Titanium FLX sequencing platforms. The 454 platform (3 samples) produced 1,300,885 reads and the Illumina platform (12 samples) generated approximately 332 million reads. The multiple k-mer technique for de novo assembly using Velvet and Oases was applied. A total of 121,288 contigs were assembled that were similar to previously reported Ensembl commelinid sequences. Original Illumina reads were also mapped to the high quality assembly to estimate expression levels of buffalograss transcripts. There were a total of 325 differentially expressed genes between the two buffalograss cultivars. A glycosyl transferase, serine threonine kinase, and nb-arc domain containing transcripts were among those differentially expressed between the two cultivars. These genes have been previously implicated in defense response pathways and may in part explain some of the performance differences between ‘Prestige’ and ‘378’.ConclusionsTo date, this is the first high throughput sequencing experiment conducted on buffalograss. In total, 121,288 high quality transcripts were assembled, significantly expanding the limited genetic resources available for buffalograss genetic studies. Additionally, 325 differentially expressed sequences were identified which may contribute to performance or morphological differences between ‘Prestige’ and ‘378’ buffalograss cultivars.

ABA, ROS and NO are Key Players During Switchgrass Seed Germination

Seed dormancy and germination are complex physiological processes usually under hormonal control. Germination of seeds from many plants including switchgrass, are inhibited by ABA and promoted by NO or ROS. However, ABA apparently requires both ROS and NO as intermediates in its action, with ROS produced by membrane-bound NADPH-oxidases responsive to ABA. In switchgrass seeds, externally supplied hydrogen peroxide (ROS), but not NO will overcome ABA-imposed inhibition of germination. Stimulation of germination by external ROS can be partially blocked by NO-scavengers, suggesting that NO is required for seed germination in switchgrass as well as for ABA-induced inhibition of germination. Collectively, these data suggest that multiple mechanisms might be required to sense and respond to varying levels of ABA, NO and ROS in switchgrass seeds.