R. Neil Reese

Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside as primary phenolic antioxidants in black raspberry.

Anthocyanin constituents in black raspberries (Rubus occidentalis L.) were investigated by HPLC-DAD, and their involvement as potent, significant antioxidants in black raspberries was demonstrated by three common antioxidant assays (FRAP, DPPH, ABTS) in this study. Five anthocyanins were present in black raspberries: cyanidin 3-sambubioside, cyanidin 3-glucoside, cyanidin 3-xylosylrutinoside, cyanidin 3-rutinoside, and pelargonidin 3-rutinoside. Their identities and structures, with particular emphasis on cyanidin 3-xylosylrutinoside, were confirmed by NMR spectroscopy. Two of these anthocyanins, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside, predominated, comprising 24-40 and 49-58%, respectively, of the total anthocyanins in black raspberries. On the basis of both potency and concentration, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were found to be the significant contributors to the antioxidant systems of black raspberries. These findings indicate that these two anthocyanin compounds may function as the primary phenolic antioxidants in black raspberries. These two compounds exhibit potential biological activities that may be exploited in conjunction with other naturally occurring bioactive compounds in black raspberry fruit-based products used in clinical trials for the treatment of various types of cancer.

The WRKY transcription factor family in Brachypodium distachyon

BackgroundA complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement.ResultsWe have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors.ConclusionsThe description of the WRKY transcription factor family in Brachypodium that we report here provides a framework for functional genomics studies in an important model system. Our database is a resource for both Brachypodium and wheat studies and ultimately projects aimed at improving wheat through manipulation of WRKY transcription factors.

Total phenolic, anthocyanin contents and antioxidant capacity of selected elderberry (Sambucus canadensis L.) accessions.

Fourteen purple-black American elderberry accessions (Sambucus canadensis L.) obtained from various sites in midwestern USA and then grown at a single Ohio production site in USA were analyzed for their total phenolic (TP) and total monomeric anthocyanin (TMA) contents and for their antioxidant capacity by the ferric reducing antioxidant power (FRAP) and DPPH radical scavenging assays. Total phenolic and anthocyanin contents were measured using the Folin-Ciocalteu reagent and the pH differential methods, respectively. Overall, the phytonutrient contents and antioxidant capacity of our elderberry accessions were similar to those typically reported for black raspberries, blackberries and other dark-fleshed small fruits. Variability among accessions was greatest for TMA content (CV 37.5%); individuals ranged nearly threefold from 1308 to 4004 μg cy3-GE/g on a fresh weight basis. Variation among accessions was also evident for TP, FRAP and DPPH values (CV 14.4, 21.7 and 26.8%, respectively). TP and TMA values were very highly correlated (r = 0.93), although individuals differed in the estimated proportion of total phenolics attributable to anthocyanins. Both TP and TMA also highly correlated to antioxidant capacity values (r = 0.70–0.85). Within this limited study of 14 accessions, variability for phytonutrient content and antioxidant capacity suggested the employment of wild germplasm within an elderberry improvement program to incorporate an array of superior horticultural, post-harvest or processing traits into new or existing cultivars with superior phytonutrient profiles.

Tobacco drought stress responses reveal new targets for Solanaceae crop improvement.

BackgroundThe Solanaceae are an economically important family of plants that include tobacco (Nicotiana tabacum L.), tomato, and potato. Drought is a major cause of crop losses.ResultsWe have identified major changes in physiology, metabolites, mRNA levels, and promoter activities during the tobacco response to drought. We have classified these as potential components of core responses that may be common to many plant species or responses that may be family/species-specific features of the drought stress response in tobacco or the Solanaceae. In tobacco the largest increase in any metabolite was a striking 70-fold increase in 4-hydroxy-2-oxoglutaric acid (KHG) in roots that appears to be tobacco/Solanaceae specific. KHG is poorly characterized in plants but is broken down to pyruvate and glyoxylate after the E. coli SOS response to facilitate the resumption of respiration. A similar process in tobacco would represent a mechanism to restart respiration upon water availability after drought. At the mRNA level, transcription factor gene induction by drought also showed both core and species/family specific responses. Many Group IX Subgroup 3 AP2/ERF transcription factors in tobacco appear to play roles in nicotine biosynthesis as a response to herbivory, whereas their counterparts in legume species appear to play roles in drought responses. We observed apparent Solanaceae-specific drought induction of several Group IId WRKY genes. One of these, NtWRKY69, showed ABA-independent drought stress-inducible promoter activity that moved into the leaf through the vascular tissue and then eventually into the surrounding leaf cells.ConclusionsWe propose components of a core metabolic response to drought stress in plants and also show that some major responses to drought stress at the metabolome and transcriptome levels are family specific. We therefore propose that the observed family-specific changes in metabolism are regulated, at least in part, by family-specific changes in transcription factor activity. We also present a list of potential targets for the improvement of Solanaceae drought responses.

Identification, Nomenclature, and Evolutionary Relationships of Mitogen-Activated Protein Kinase (MAPK) Genes in Soybean

Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution.

Nonanthocyanin secondary metabolites of black raspberry (Rubus occidentalis L.) fruits: identification by HPLC-DAD, NMR, HPLC-ESI-MS, and ESI-MS/MS analyses.

Nonanthocyanin secondary metabolites potentially contributing to the antiproliferative bioactivity of black raspberry ( Rubus occidentalis L.) fruits were extracted in ethyl acetate and isolated by semipreparative and analytical HPLC and analyzed by NMR, HPLC-ESI-MS, and ESI-MS/MS techniques. Here we present complete and partial structures of a variety of the chemical entities such as quercetin 3-glucoside, quercetin 3-rutinoside, myricetin glucoside, dihydrokaempferol glucoside, benzoic acid β-d-glucopyranosyl ester, 3,4-dihydroxybenzoic acid, epicatechin, caffeic acid, p-coumaric acid, p-coumaryl glucoside, p-coumaryl sugar ester, ellagic acid, methyl ellagic acid acetylpentose, methyl ellagic acid valerylpentose, trans-piceid, phloretin glucoside (phloridzin), dihydrosinapic acid, salicylic acid β-d-glucopyranosyl ester, a salicylic acid derivative without attached sugar, p-alkylphenyl glucoside, and a citric acid derivative. To our knowledge, 15 of these compounds were not previously reported in black raspberry fruits.

NMR-based metabolomic investigation of bioactivity of chemical constituents in black raspberry (Rubus occidentalis L.) fruit extracts.

Black raspberry (Rubus occidentalis L.) (BR) fruit extracts with differing compound profiles have shown variable antiproliferative activities against HT-29 colon cancer cell lines. This study used partial least-squares (PLS) regression analysis to develop a high-resolution (1)H NMR-based multivariate statistical model for discerning the biological activity of BR constituents. This model identified specific bioactive compounds and ascertained their relative contribution against cancer cell proliferation. Cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside were the predominant contributors to the extract bioactivity, but salicylic acid derivatives (e.g., salicylic acid glucosyl ester), quercetin 3-glucoside, quercetin 3-rutinoside, p-coumaric acid, epicatechin, methyl ellagic acid derivatives (e.g., methyl ellagic acetyl pentose), and citric acid derivatives also contributed significantly to the antiproliferative activity of the berry extracts. This approach enabled the identification of new bioactive components in BR fruits and demonstrates the utility of the method for assessing chemopreventive compounds in foods and food products.

In Vitro Screening of Forty Medicinal Plant Extracts from the United States Northern Great Plains for Anthelmintic Activity Against Haemonchus Contortus

An egg hatch assay (EHA) and a larval migration assay (LMA) involving Haemonchus contortus was used to evaluate the anthelmintic activity of methanol extracts from 40 plants that are native or naturalized within the U.S.A. Northern Great Plains. Only one of these 40 plants (i.e. Lotus corniculatus) had been previously evaluated for activity against any gastrointestinal nematode. The various extracts were initially screened at 50mg/ml diluted either in 0.5% dimethyl sulphoxide (DMSO) or 3-(N-morpholino) propanesulfonic acid (MOPS buffer), and plants showing 100% inhibition at 50mg/ml, were further evaluated at 8 other concentrations (25-0.19 mg/ml). Extracts with 100% activity with the EHA were again screened with the LMA (50mg/ml). Two extracts with the highest LMA inhibition were also evaluated at lower concentrations (25-3.1mg/ml). Of the 40 methanolic extracts screened, 7 (Chrysothamnus viscidiflorus, Ericameria nauseosa, Liatris punctata, Melilotus alba, Melilotus officinalis, Perideridia gairdneri, and Sanguinaria canadensis) showed significant egg-hatch inhibition in DMSO and MOPS buffer. Three extracts (Geranium viscosissimum, L. corniculatus, and Rhus aromatica) only showed significant inhibition in DMSO. The 8 extracts showing 100% efficacy at 50mg/ml exhibited dose-dependent effects at the 8 lower concentrations, and R. aromatica and E. nauseosa extracts had the lowest ED50 values. Similarly, when these 8 plant extracts were further evaluated with the LMA, the extracts of E. nauseosa and R. aromatica again exhibited the highest activity (p<0.001), with ED50 values of 4.0mg/ml and 10.43 mg/ml respectively. Three other extracts (C. viscidiflorus, M. alba and M. officinalis) also showed inhibitory activity in the LMA. These results support the need for additional evaluations of the nematocidal properties for at least these 5 plants.

Spatio temporal influence of isoflavonoids on bacterial diversity in the soybean rhizosphere

High bacterial density and diversity near plant roots has been attributed to rhizodeposit compounds that serve as both energy sources and signal molecules. However, it is unclear if and how specific rhizodeposit compounds influence bacterial diversity. We silenced the biosynthesis of isoflavonoids, a major component of soybean rhizodeposits, using RNA interference in hairy-root composite plants, and examined changes in rhizosphere bacteriome diversity. We used successive sonication to isolate soil fractions from different rhizosphere zones at two different time points and analyzed denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA gene amplicons. Extensive diversity analysis of the resulting spatio temporal profiles of soybean bacterial communities indicated that, indeed, isoflavonoids significantly influenced soybean rhizosphere bacterial diversity. Our results also suggested a temporal gradient effect of rhizodeposit isoflavonoids on the rhizosphere. However, the hairy-root transformation process itself significantly altered rhizosphere bacterial diversity, necessitating appropriate additional controls. Gene silencing in hairy-root composite plants combined with successive sonication is a useful tool to determine the spatio temporal effect of specific rhizodeposit compounds on rhizosphere microbial communities.

Diversity and Evolution of Disease Resistance Genes in Barley (Hordeum vulgare L.)

Plant disease resistance genes (R-genes) play a critical role in the defense response to pathogens. Barley is one of the most important cereal crops, having a genome recently made available, for which the diversity and evolution of R-genes are not well understood. The main objectives of this research were to conduct a genome-wide identification of barley Coiled-coil, Nucleotide-binding site, Leucine-rich repeat (CNL) genes and elucidate their evolutionary history. We employed a Hidden Markov Model using 52 Arabidopsis thaliana CNL reference sequences and analyzed for phylogenetic relationships, structural variation, and gene clustering. We identified 175 barley CNL genes nested into three clades, showing (a) evidence of an expansion of the CNL-C clade, primarily due to tandem duplications; (b) very few members of clade CNL-A and CNL-B; and (c) a complete absence of clade CNL-D. Our results also showed that several of the previously identified mildew locus A (MLA) genes may be allelic variants of two barley CNL genes, MLOC_66581 and MLOC_10425, which respond to powdery mildew. Approximately 23% of the barley CNL genes formed 15 gene clusters located in the extra-pericentromeric regions on six of the seven chromosomes; more than half of the clustered genes were located on chromosomes 1H and 7H. Higher average numbers of exons and multiple splice variants in barley relative to those in Arabidopsis and rice may have contributed to a diversification of the CNL-C members. These results will help us understand the evolution of R-genes with potential implications for developing durable resistance in barley cultivars.