Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+ß barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

Monoxia obesula Blake, 1939, a species native to the U.S.A. and adventive to Sardinia, Italy (Coleoptera: Chrysomelidae: Galerucinae: Galerucini).

Monoxia obesula Blake, 1939, a species native to North America, is newly found in Sardinia, Italy. This discovery constitutes the first report of the species from Europe, as well as the first report from outside of the U.S.A. The species is redescribed and illustrated, and errors in the original description are corrected. Host plants are species of Atriplex and Chenopodium occurring in saline habitats.

Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus).

BACKGROUND: Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves. RESULTS: Transcripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial) clades. CONCLUSIONS: Our analysis of the hop transcriptome significantly expands the genomic resources available for this agriculturally-important crop. This study provides evidence for the lupulin gland-specific biosynthesis of BCAAs and prenyl diphosphates to provide precursors for the production of bitter acids. The biosynthetic pathway leading to BCAAs in lupulin glands involves the plastidial enzyme, HlBCAT2. The mitochondrial enzyme HlBCAT1 degrades BCAAs as the first step in the catabolic pathway leading to branched chain-acyl-CoAs.

Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in apple fruit.

BACKGROUND: The ubiquitous, non-proteinaceous amino acid GABA (γ-aminobutyrate) accumulates in plants subjected to abiotic stresses such as chilling, O2 deficiency and elevated CO2. Recent evidence indicates that controlled atmosphere storage causes the accumulation of GABA in apple (Malus x domestica Borkh.) fruit, and now there is increasing interest in the biochemical mechanisms responsible for this phenomenon. Here, we investigated whether this phenomenon could be mediated via Ca(2+)/calmodulin (CaM) activation of glutamate decarboxylase (GAD) activity. RESULTS: GAD activity in cell-free extracts of apple fruit was stimulated by Ca(2+)/CaM at physiological pH, but not at the acidic pH optimum. Based on bioinformatics analysis of the apple genome, three apple GAD genes were identified and their expression determined in various apple organs, including fruit. Like recombinant Arabidopsis GAD1, the activity and spectral properties of recombinant MdGAD1 and MdGAD2 were regulated by Ca(2+)/CaM at physiological pH and both enzymes possessed a highly conserved CaM-binding domain that was autoinhibitory. In contrast, the activity and spectral properties of recombinant MdGAD3 were not affected by Ca(2+)/CaM and they were much less sensitive to pH than MdGAD1, MdGAD2 and Arabidopsis GAD1; furthermore, the C-terminal region neither bound CaM nor functioned as an autoinhibitory domain. CONCLUSIONS: Plant GADs typically differ from microbial and animal GAD enzymes in possessing a C-terminal 30-50 amino acid residue CaM-binding domain. To date, rice GAD2 is the only exception to this generalization; notably, the C-terminal region of this enzyme still functions as an autoinhibitory domain. In the present study, apple fruit were found to contain two CaM-dependent GADs, as well as a novel CaM-independent GAD that does not possess a C-terminal autoinhibitory domain.

Seasonal and microclimatic effects on leaf beetles (Coleoptera, Chrysomelidae) in a tropical forest fragment in northeastern Mexico.

Leaf beetles (Coleoptera: Chrysomelidae) constitute a family of abundant, diverse, and ecologically important herbivorous insects, due to their high specificity with host plants, a close association with vegetation and a great sensitivity to microclimatic variation (factors that are modified gradually during the rainy and dry seasons). Therefore, the effects of seasonality (rainy and dry seasons) and microclimate on the community attributes of chrysomelids were evaluated in a semideciduous tropical forest fragment of northeastern Mexico. Monthly sampling was conducted, between March 2016 and February 2017, with an entomological sweep net in 18 plots of 20 × 20 m, randomly distributed from 320 to 480 m a.s.l. Seven microclimatic variables were simultaneously recorded during each of the samplings, using a portable weather station. In total, 216 samples were collected at the end of the study, of which 2,103 specimens, six subfamilies, 46 genera, and 71 species were obtained. The subfamily Galerucinae had the highest number of specimens and species in the study area, followed by Cassidinae. Seasonality caused significant changes in the abundance and number of leaf beetle species: highest richness was recorded in the rainy season, with 60 species, while the highest diversity (lowest dominance and highest H' index) was obtained in the dry season. Seasonal inventory completeness of leaf beetles approached (rainy season) or was higher (dry season) than 70%, while the faunistic similarity between seasons was 0.63%. The outlying mean index was significant in both seasons; of the seven microclimatic variables analyzed, only temperature, heat index, evapotranspiration and wind speed were significantly related to changes in abundance of Chrysomelidae. Association between microclimate and leaf beetles was higher in the dry season, with a difference in the value of importance of the abiotic variables. The results indicated that each species exhibited a different response pattern to the microclimate, depending on the season, which suggests that the species may exhibit modifications in their niche requirements according to abiotic conditions. However, the investigations must be replicated in other regions, in order to obtain a better characterization of the seasonal and microclimatic influence on the family Chrysomelidae.

Role of plant glyoxylate reductases during stress: a hypothesis.

Molecular modelling suggests that a group of proteins in plants known as the beta-hydroxyacid dehydrogenases, or the hydroxyisobutyrate dehydrogenase superfamily, includes enzymes that reduce succinic semialdehyde and glyoxylate to gamma-hydroxybutyrate and glycolate respectively. Recent biochemical and expression studies reveal that NADPH-dependent cytosolic (termed GLYR1) and plastidial (termed GLYR2) isoforms of succinic semialdehyde/glyoxylate reductase exist in Arabidopsis. Succinic semialdehyde and glyoxylate are typically generated in leaves via two distinct metabolic pathways, gamma-aminobutyrate and glycolate respectively. In the present review, it is proposed that the GLYRs function in the detoxification of both aldehydes during stress and contribute to redox balance. Outstanding questions are highlighted in a scheme for the subcellular organization of the detoxification mechanism in Arabidopsis.

Prathapanius fortis, a new genus and new species of Galerucinae from Ecuador (Coleoptera, Chrysomelidae).

Prathapanius Viswajyothi & Clark, gen. nov., is described and illustrated. The genus is placed in the chrysomelid section Diabroticites Chapuis (subtribe Diabroticina Chapuis, tribe Luperini Chapuis, subfamily Galerucinae Latreille). It is monobasic, containing Prathapanius fortis Viswajyothi & Clark, sp. nov., from Ecuador. The new genus is briefly compared with Acalymma Barber, Isotes Weise, and Zischkaita Bechyné.

Biochemical characterization, mitochondrial localization, expression, and potential functions for an Arabidopsis gamma-aminobutyrate transaminase that utilizes both pyruvate and glyoxylate.

Gamma-aminobutyrate transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, the previously identified Arabidopsis thaliana (L.) Heyhn GABA-T (AtGABA-T) was characterized in more detail. Full-length AtGABA-T contains an N-terminal 36 amino acid long targeting pre-sequence (36 amino acids) that is both sufficient and necessary for targeting the enzyme to mitochondria. Removal of the pre-sequence encoding this N-terminal targeting domain and co-expression of the resulting truncated AtGABA-T cDNA with the GroES/EL molecular chaperone complex in Escherichia coli yielded good recovery of the soluble recombinant proteins. Activity assays indicated that purified recombinant GABA-T has both pyruvate- and glyoxylate-dependent activities, but cannot utilize 2-oxoglutarate as amino acceptor. Kinetic parameters for glyoxylate- and pyruvate-dependent GABA-T activities were similar, with physiologically relevant affinities. Assays of GABA-T activity in cell-free leaf extracts from wild-type Arabidopsis and two knockout mutants in different genetic backgrounds confirmed that the native enzyme possesses both pyruvate- and glyoxylate-dependent activities. The GABA-T transcript was present throughout the plant, but its expression was highest in roots and increased as a function of leaf development. A GABA-T with dual functions suggests the potential for interaction between GABA metabolism and photorespiratory glyoxylate production.

Subcellular localization and expression of multiple tomato gamma-aminobutyrate transaminases that utilize both pyruvate and glyoxylate.

Gamma-aminobutyric acid transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, three GABA-T isoforms were identified in the tomato (Solanum lycopersicum L.) plant. The deduced amino acid sequences of the three isoforms are highly similar over most of their coding regions with the exception of their N-terminal regions. Transient expression of the individual full-length GABA-T isoforms fused to the green fluorescent protein in tobacco suspension-cultured cells revealed their distinct subcellular localizations to the mitochondrion, plastid or cytosol, and that the specific targeting of the mitochondrion- and plastid-localized isoforms is mediated by their predicted N-terminal presequences. Removal of the N-terminal targeting presequences from the mitochondrion and plastid GABA-T isoforms yielded good recovery of the soluble recombinant proteins in Escherichia coli when they were co-expressed with the GroES/EL molecular chaperone complex. Activity assays indicated that all three recombinant isoforms possess both pyruvate- and glyoxylate-dependent GABA-T activities, although the mitochondrial enzyme has a specific activity that is significantly higher than that of its plastid and cytosolic counterparts. Finally, differential expression patterns of the three GABA-T isoforms in reproductive tissues, but not vegetative tissues, suggest unique roles for each enzyme in developmental processes. Overall, these findings, together with recent information about rice and pepper GABA-Ts, indicate that the subcellular distribution of GABA-T in the plant kingdom is highly variable.

Successional and seasonal changes of leaf beetles and their indicator value in a fragmented low thorn forest of northeastern Mexico (Coleoptera, Chrysomelidae).

Leaf beetles (Chrysomelidae: Coleoptera) constitute a highly diverse family of phytophagous insects with high ecological relevance, due to their host plant specificity and their close association to vegetation variables. Therefore, secondary succession and seasonal changes after loss of vegetal cover will have a significant influence on their community patterns. Accordingly, responses of leaf beetles to such environmental heterogeneity make them a suitable taxon for monitoring disturbance, which is more important for endangered habitats such as the low thorn forests (LTF) in northeastern Mexico. We conducted a study in a LTF fragment in order to assess the effects of secondary succession and seasonality on leaf beetle communities, as well as to quantify the importance of Chrysomelidae as an indicator taxon. Landsat scenes were used for delimiting a successional gradient, in which four succession categories were selected: four years, 17 years, and 31 years since loss of vegetal cover, and conserved areas. Eight plots of 100 m2 were randomly delimited in each category; plots were sampled monthly, using an entomological sweep net, from May 2016 to April 2017. In total, 384 samples were collected by the end of study, from which 6978 specimens, six subfamilies, 57 genera, and 85 species were obtained. Species richness was higher in early succession areas. Abundance declined significantly from early successional to conserved areas, but the conserved areas had the higher diversity. Furthermore, differences in abundance were significant between rainy and dry seasons in areas of four, 17, and 31 years of succession, but not in conserved areas; also, all categories had a similar abundance during the dry season. Intermediate (17 and 31 years) and conserved areas differed in the season of higher diversity. Regarding inventory completeness, it was close to or above 70 % for all comparisons, although it was very low for the 17-year category during the rainy season. Faunistic similarity was higher between intermediate categories. A total of 24 species had a significant indicator value. Effects of succession time and seasonality on leaf beetle communities are here quantified for first time in LTF forests. Influences of environmental heterogeneity and intermediate disturbance are discussed as main drivers of the results obtained. Several leaf beetle species are proposed that could be useful for monitoring succession time and secondary LTF vegetation in northeastern Mexico. However, studies must be replicated at other regions, in order to obtain a better characterization of disturbance influence on leaf beetles.

Gamma-hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms.

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol are probably crucial in maintaining plant health during stress. Succinic semialdehyde (SSA) is a mitochondrially-generated intermediate in the metabolism of gamma-aminobutyrate (GABA), which accumulates in response to a variety of biotic and abiotic stresses. SSA can be reduced to gamma-hydroxybutyrate (GHB) under oxygen deficiency and high light conditions. Recent evidence indicates that distinct cytosolic and plastidial glyoxylate reductase isoforms from Arabidopsis (designated herein after as AtGR1 and AtGR2, respectively) catalyse the in vitro conversion of SSA to GHB, as well as glyoxylate to glycolate, via NADPH-dependent reactions. In the present report, the responses of GHB and related amino acids, as well as NADP(+) and NADPH, were monitored in leaves from Arabidopsis or tobacco plants subjected to various abiotic stresses (i.e. Arabidopsis during exposure to salinity, drought, submergence, cold, or heat; tobacco during exposure to, and recovery from, submergence). Time-course experiments revealed that GHB accumulated in both Arabidopsis and tobacco plants subjected to stress, and that this accumulation was generally accompanied by higher GABA and alanine levels, higher NADPH/NADP(+) ratio, and lower glutamate levels. Furthermore, the analysis of gene expression in Arabidopsis revealed that the relative abundance of GR1 (salinity, drought, submergence, cold, and heat) and GR2 (cold and heat) transcripts was enhanced by the stress tested. Thus, GHB accumulation in plants is a general response to abiotic stress and appears to be regulated by both biochemical and transcriptional processes.

Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification.

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (K(m) glyoxylate=34 microM), and SSA to gamma-hydroxybutyrate (K(m) SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (k(cat)/K(m)). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response.

Identification of an attenuated barley stripe mosaic virus for the virus-induced gene silencing of pathogenesis-related wheat genes.

BACKGROUND: Virus-induced gene silencing (VIGS) has become an emerging technology for the rapid, efficient functional genomic screening of monocot and dicot species. The barley stripe mosaic virus (BSMV) has been described as an effective VIGS vehicle for the evaluation of genes involved in wheat and barley phytopathogenesis; however, these studies have been obscured by BSMV-induced phenotypes and defense responses. The utility of BSMV VIGS may be improved using a BSMV genetic background which is more tolerable to the host plant especially upon secondary infection of highly aggressive, necrotrophic pathogens such as Fusarium graminearum. RESULTS: BSMV-induced VIGS in Triticum aestivum (bread wheat) cv. 'Fielder' was assessed for the study of wheat genes putatively related to Fusarium Head Blight (FHB), the necrotrophism of wheat and other cereals by F. graminearum. Due to the lack of 'Fielder' spike viability and increased accumulation of Fusarium-derived deoxynivalenol contamination upon co-infection of BSMV and FHB, an attenuated BSMV construct was generated by the addition of a glycine-rich, C-terminal peptide to the BSMV γ b protein. This attenuated BSMV effectively silenced target wheat genes while limiting disease severity, deoxynivalenol contamination, and yield loss upon Fusarium co-infection compared to the original BSMV construct. The attenuated BSMV-infected tissue exhibited reduced abscisic, jasmonic, and salicylic acid defense phytohormone accumulation upon secondary Fusarium infection. Finally, the attenuated BSMV was used to investigate the role of the salicylic acid-responsive pathogenesis-related 1 in response to FHB. CONCLUSIONS: The use of an attenuated BSMV may be advantageous in characterizing wheat genes involved in phytopathogenesis, including Fusarium necrotrophism, where minimal viral background effects on defense are required. Additionally, the attenuated BSMV elicits reduced defense hormone accumulation, suggesting that this genotype may have applications for the investigation of phytohormone-related signaling, developmental responses, and pathogen defense.

Faunistic patterns of leaf beetles (Coleoptera, Chrysomelidae) within elevational and temporal gradients in Sierra de San Carlos, Mexico.

The study of biodiversity of Chrysomelidae in Mexico and its variation within ecological gradients has increased recently, although important areas in the country remain to be explored. We conducted a faunistic inventory and analyzed the elevational and temporal variation of leaf beetle communities in the Sierra de San Carlos, in the state of Tamaulipas, in northeastern Mexico. This is an area with high to extreme priority for conservation, and due to its insular geographical position and to the vegetational communities present, it must be considered as a sky island. We selected seven sample sites distributed in different elevations within three localities, and comprising different vegetational communities. At each site, we randomly delimited 12 sample plots of 400 m(2) where sampling was conducted by entomological sweep netting and collecting directly by hand. Sampling was conducted monthly at each plot, for a total of 1,008 samples between February 2013 and January 2014. By the end of the study, we had obtained a total of 3,081 specimens belonging to six subfamilies, 65 genera, and 113 species, with Trichaltica scabricula (Crotch, 1873) being recorded for first time in Mexico. Species richness was less than the values observed at other studies conducted in the same region, which is attributed to differences in the number of plant species and to the insular location of Sierra de San Carlos; however, the higher diversity values suggest a higher quality of natural resources and vegetational communities. No consistent pattern of leaf beetle communities was correlated with elevation, although higher values of species richness and diversity were obtained at the highest elevation site. The seasonal gradient showed that the rainy season is most favorable for leaf beetle communities. We found that species composition was different between sites and months, and also that there exists a significant association between the abundance obtained at each site and particular months. These results highlight the importance of different microhabitats for species distribution, and suggest that each species of Chrysomelidae has a differential response to environmental factors that vary within the elevational gradient and according to seasons. Also, we confirm and emphasize the important status of Sierra de San Carlos as a key natural area for biological conservation.

Checklist of leaf beetles (Coleoptera: Chrysomelidae) from the state of Morelos, Mexico.

We record 116 genera and 366 species of Chrysomelidae from the state of Morelos, Mexico. This represents an increase of 9.3% in the species richness of these beetles for the state. Also, Morelos is currently the third most diverse state in leaf beetles within Mexico, with 16.78% of total species recorded for the country. The most diverse genera were Calligrapha, Disonycha, Blepharida, Leptinotarsa, Cryptocephalus, Systena, Alagoasa, Diabrotica and Pachybrachis, each with more than eight species. Most of these genera contain large, showy beetles. When the chrysomelid fauna is more fully understood, some of the genera of tiny beetles will likely prove to be more diverse.

Atlas of Ohio Aquatic Insects: Volume II, Plecoptera.

BACKGROUND: We provide volume II of a distributional atlas of aquatic insects for the eastern USA state of Ohio. This treatment of stoneflies (Plecoptera) is companion to Armitage et al. (2011) on caddisflies (Trichoptera). We build on a recent analysis of Ohio stonefly diversity patterns based on large drainages (DeWalt et al. 2012), but add 3717 new records to the data set. We base most analyses on the United States Geological Survey Hierarchical Unit Code eight (HUC8) drainage scale. In addition to distributional maps for each species, we provide analyses of species richness versus HUC8 drainage area and the number of unique locations in a HUC8 drainage, species richness versus Ohio counties, analyze adult presence phenology throughout the year, and demonstrate stream size range affiliation for each species. NEW INFORMATION: This work is based on a total of 7797 specimen records gathered from 21 regional museums, agency data, personal collections, and from the literature Table 1. To our knowledge this is the largest stonefly data set available for a similarly sized geopolitical area anywhere in the world. These data are made available as a Darwin Core Archive supported by the Pensoft Integrated Publishing Toolkit (DeWalt et al. 2016b). All known published papers reporting stoneflies from Ohio are detailed in Suppl. material 1. We recovered 102 species from Ohio, including all nine Nearctic families Table 2​. Two species were removed from the DeWalt et al. (2012) list and two new state records added. Perlidae (32 spp.) was most speciose, compared to the low diversity Pteronarcyidae (2 spp.) and Peltoperlidae (1 sp.). The richest HUC8 drainages occurred in northeastern, south-central, and southern regions of the state where drainages were heavily forested, had the highest slopes, and were contained within or adjacent to the unglaciated Allegheny and Appalachian Plateaus. Species poor drainages occurred mainly in the northwestern region where Wisconsinan aged lake plains climaxed to an expansive wooded wetland, the Black Swamp. The unglaciated Lower Scioto drainage (72 spp.) in south-central Ohio supported the greatest species richness. There was no relationship between species richness and HUC8 drainage size, but the number of unique locations in a drainage strongly related to species richness. All Ohio counties were represented in the data set with Hocking County (59 spp.) of the Lower Scioto drainage being the richest and most heavily sampled. Adult presence phenology was influenced by phylogenetic relationships such that the superfamily Nemouroidea (Capniidae, Leuctridae, Nemouridae, and Taeniopterygidae) generally emerged in winter and spring while the superfamilies Pteronarcyoidea (Pteronarcyidae, Peltoperlidae) and Perloidea (Chloroperlidae, Perlidae, Perlodidae) emerged later, some species continuing emergence through summer months. Species often occupied specific stream size ranges, while others were generalists. Two species once histrorically abundant in the western Lake Erie Bass Islands no longer reside there. Each of the 102 species is discussed in detail, including several that require additional collecting efforts to confirm their identities, presence, and distribution in Ohio.

Characterization of Triticum aestivum Abscisic Acid Receptors and a Possible Role for These in Mediating Fusairum Head Blight Susceptibility in Wheat.

Abscisic acid (ABA) is a well-characterized plant hormone, known to mediate developmental aspects as well as both abiotic and biotic stress responses. Notably, the exogenous application of ABA has recently been shown to increase susceptibility to the fungal pathogen Fusarium graminearum, the causative agent of Fusarium head blight (FHB) in wheat and other cereals. However roles and mechanisms associated with ABA's modulation of pathogen responses remain enigmatic. Here the identification of putative ABA receptors from available genomic databases for Triticum aestivum (bread wheat) and Brachypodium distachyon (a model cereal) are reported. A number of these were cloned for recombinant expression and their functionality as ABA receptors confirmed by in vitro assays against protein phosphatases Type 2Cs. Ligand selectivity profiling of one of the wheat receptors (Ta_PYL2DS_FL) highlighted unique activities compared to Arabidopsis AtPYL5. Mutagenic analysis showed Ta_PYL2DS_FL amino acid D180 as being a critical contributor to this selectivity. Subsequently, a virus induced gene silencing (VIGS) approach was used to knockdown wheat Ta_PYL4AS_A (and similar) in planta, yielding plants with increased early stage resistance to FHB progression and decreased mycotoxin accumulation. Together these results confirm the existence of a family of ABA receptors in wheat and Brachypodium and present insight into factors modulating receptor function at the molecular level. That knockdown of Ta_PYL4AS_A (and similar) leads to early stage FHB resistance highlights novel targets for investigation in the future development of disease resistant crops.

Review of the West Indian genus Monotalla Bechyné (Coleoptera, Chrysomelidae, Galerucinae, Alticini) with description of five new species.

The West Indian genus Monotalla Bechyné is reviewed, redescribed and illustrated. Five new species are added: Monotalladominica sp. n. (Dominica); Monotallalecticofolia sp. n. (St. Lucia); Monotallamaierae sp. n. (St. Lucia); Monotallaobrienorum sp. n. (Grenada); and Monotallaviridis sp. n. (St. Lucia). A key to Monotalla species is provided.

Separation of isomeric short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography coupled with tandem mass spectrometry.

Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants.