3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

A novel copy number variant in the murine Cdh23 gene gives rise to profound deafness and vestibular dysfunction.

Hearing loss is the most common congenital sensory deficit worldwide and exhibits high genetic heterogeneity, making molecular diagnoses elusive for most individuals. Detecting novel mutations that contribute to hearing loss is crucial to providing accurate personalized diagnoses, tailored interventions, and improving prognosis. Copy number variants (CNVs) are structural mutations that are understudied, potential contributors to hearing loss. Here, we present the Abnormal Wobbly Gait (AWG) mouse, the first documented mutant exhibiting waltzer-like locomotor dysfunction, hyperactivity, circling behaviour, and profound deafness caused by a spontaneous CNV deletion in cadherin 23 (Cdh23). We were unable to identify the causative mutation through a conventional whole-genome sequencing (WGS) and variant detection pipeline, but instead found a linked variant in hexokinase 1 (Hk1) that was insufficient to recapitulate the AWG phenotype when introduced into C57BL/6J mice using CRISPR-Cas9. Investigating nearby deafness-associated genes revealed a pronounced downregulation of Cdh23 mRNA and a complete absence of full-length CDH23 protein, which is critical for the development and maintenance of inner ear hair cells, in whole head extracts from AWG neonates. Manual inspection of WGS read depth plots of the Cdh23 locus revealed a putative 10.4 kb genomic deletion of exons 11 and 12 that was validated by PCR and Sanger sequencing. This study underscores the imperative to refine variant detection strategies to permit identification of pathogenic CNVs easily missed by conventional variant calling to enhance diagnostic precision and ultimately improve clinical outcomes for individuals with genetically heterogenous disorders such as hearing loss.

Negative regulation of plastidial isoprenoid pathway by herbivore-induced ß-cyclocitral in Arabidopsis thaliana.

Insect damage to plants is known to up-regulate defense and down-regulate growth processes. While there are frequent reports about up-regulation of defense signaling and production of defense metabolites in response to herbivory, much less is understood about the mechanisms by which growth and carbon assimilation are down-regulated. Here we demonstrate that insect herbivory down-regulates the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in Arabidopsis (Arabidopsis thaliana), a pathway making primarily metabolites for use in photosynthesis. Simulated feeding by the generalist herbivore Spodoptera littoralis suppressed flux through the MEP pathway and decreased steady-state levels of the intermediate 1-deoxy-D-xylulose 5-phosphate (DXP). Simulated herbivory also increased reactive oxygen species content which caused the conversion of ß-carotene to ß-cyclocitral (ßCC). This volatile oxidation product affected the MEP pathway by directly inhibiting DXP synthase (DXS), the rate-controlling enzyme of the MEP pathway in Arabidopsis and inducing plant resistance against S. littoralis ßCC inhibited both DXS transcript accumulation and DXS activity. Molecular models suggested that ßCC binds to DXS at the binding site for the thymine pyrophosphate cofactor and blocks catalysis, which was confirmed by direct assays of ßCC with the purified DXS protein in vitro. Another intermediate of the MEP pathway, 2-C-methyl-D-erythritol-2, 4-cyclodiphosphate, which is known to stimulate salicylate defense signaling, showed greater accumulation and enhanced export out of the plastid in response to simulated herbivory. Together, our work implicates ßCC as a signal of herbivore damage in Arabidopsis that increases defense and decreases flux through the MEP pathway, a pathway involved in growth and carbon assimilation.

An Arabidopsis GCMS chemical ionization technique to quantify adaptive responses in central metabolism.

We present a methodology to survey central metabolism in 13CO2-labeled Arabidopsis (Arabidopsis thaliana) rosettes by ammonia positive chemical ionization-gas chromatography-mass spectrometry. This technique preserves the molecular ion cluster of methyloxime/trimethylsilyl-derivatized analytes up to 1 kDa, providing unambiguous nominal mass assignment of >200 central metabolites and 13C incorporation rates into a subset of 111 from the tricarboxylic acid (TCA) cycle, photorespiratory pathway, amino acid metabolism, shikimate pathway, and lipid and sugar metabolism. In short-term labeling assays, we observed plateau labeling of ∼35% for intermediates of the photorespiratory cycle except for glyoxylate, which reached only ∼4% labeling and was also present at molar concentrations several fold lower than other photorespiratory intermediates. This suggests photorespiratory flux may involve alternate intermediate pools besides the generally accepted route through glyoxylate. Untargeted scans showed that in illuminated leaves, noncyclic TCA cycle flux and citrate export to the cytosol revert to a cyclic flux mode following methyl jasmonate (MJ) treatment. MJ also caused a block in the photorespiratory transamination of glyoxylate to glycine. Salicylic acid treatment induced the opposite effects in both cases, indicating the antagonistic relationship of these defense signaling hormones is preserved at the metabolome level. We provide complete chemical ionization spectra for 203 Arabidopsis metabolites from central metabolism, which uniformly feature the unfragmented pseudomolecular ion as the base peak. This unbiased, soft ionization technique is a powerful screening tool to identify adaptive metabolic trends in photosynthetic tissue and represents an important advance in methodology to measure plant metabolic flux.

Cytosolic geraniol and citronellol biosynthesis require a Nudix hydrolase in rose-scented geranium (Pelargonium graveolens).

Geraniol, citronellol and their esters are high-value acyclic monoterpenes used in food technology, perfumery and cosmetics. A major source of these compounds is the essential oil of rose-scented geraniums of the genus Pelargonium. We provide evidence that their biosynthesis mainly takes place in the cytosol of glandular trichomes via geranyl monophosphate (GP) through the action of a Nudix hydrolase. Protein preparations could convert geranyl diphosphate (GDP) to geraniol in in vitro assays, a process which could be blocked by inorganic phosphatase inhibitors, suggesting a two-step conversion of GDP to geraniol. Pelargonium graveolens chemotypes enriched in either geraniol or (-)-citronellol accumulate GP or citronellyl monophosphate (CP), respectively, the presumed precursors to their monoterpenoid end products. Geranyl monophosphate was highly enriched in isolated glandular trichomes of lines producing high amounts of geraniol. In contrast, (-)-isomenthone-rich lines are depleted in these prenyl monophosphates and monoterpene alcohols and instead feature high levels of GDP, the precursor to plastidic p-menthane biosynthesis. A Nudix hydrolase cDNA from Pelargonium glandular trichomes, dubbed PgNdx1, encoded a cytosolic protein capable of hydrolyzing GDP to GP with a KM of about 750 nm but is only weakly active towards farnesyl diphosphate. In citronellol-rich lines, GDP, GP and CP were detected in nearly equimolar amounts, while citronellyl diphosphate was absent, suggesting that citronellol biosynthesis may proceed by reduction of GP to CP in this species. These findings highlight the cytosol as a compartment that supports monoterpene biosynthesis and expands the roles of Nudix hydrolases in the biosynthesis of plant volatiles.

Distinct metabolic pathways drive monoterpenoid biosynthesis in a natural population of Pelargonium graveolens.

Pelargonium graveolens is a wild predecessor to rose-scented geranium hybrids prized for their essential oils used as fragrances and flavorings. However, little is known about their biosynthesis. Here we present metabolic evidence that at least two distinct monoterpene biosynthetic pathways contribute to their volatile profiles, namely, cyclic p-menthanes such as (-)-isomenthone and acyclic monoterpene alcohols such as geraniol and (-)-citronellol and their derivatives (referred to here as citronelloid monoterpenes). We established their common origin via the 2C-methyl-d-erythritol-4-phosphate pathway but found no indication these pathways share common intermediates beyond geranyl diphosphate. Untargeted volatile profiling of 22 seed-grown P. graveolens lines demonstrated distinct chemotypes that preferentially accumulate (-)-isomenthone, geraniol, or (-)-citronellol along with approximately 85 minor volatile products. Whole plant 13CO2 isotopic labeling performed under physiological conditions permitted us to measure the in vivo rates of monoterpenoid accumulation in these lines and quantify differences in metabolic modes between chemotypes. We further determined that p-menthane monoterpenoids in Pelargonium are likely synthesized from (+)-limonene via (+)-piperitone rather than (+)-pulegone. Exploitation of this natural population enabled a detailed dissection of the relative rates of competing p-menthane and citronelloid pathways in this species, providing real time rates of monoterpene accumulation in glandular trichomes.

The plastidial metabolite 2-C-methyl-D-erythritol-2,4-cyclodiphosphate modulates defence responses against aphids.

Feeding by insect herbivores such as caterpillars and aphids induces plant resistance mechanisms that are mediated by the phytohormones jasmonic acid (JA) and salicylic acid (SA). These phytohormonal pathways often crosstalk. Besides phytohormones, methyl-D-erythriol-2,4-cyclodiphosphate (MEcPP), the penultimate metabolite in the methyl-D-erythritol-4-phosphate pathway, has been speculated to regulate transcription of nuclear genes in response to biotic stressors such as aphids. Here, we show that MEcPP uniquely enhances the SA pathway without attenuating the JA pathway. Arabidopsis mutant plants that accumulate high levels of MEcPP (hds3) are highly resistant to the cabbage aphid (Brevicoryne brassicae), whereas resistance to the large cabbage white caterpillar (Pieris brassicae) remains unaltered. Thus, MEcPP is a distinct signalling molecule that acts beyond phytohormonal crosstalk to induce resistance against the cabbage aphid in Arabidopsis. We dissect the molecular mechanisms of MEcPP mediating plant resistance against the aphid B. brassicae. This shows that MEcPP induces the expression of genes encoding enzymes involved in the biosynthesis of several primary and secondary metabolic pathways contributing to enhanced resistance against this aphid species. A unique ability to regulate multifaceted molecular mechanisms makes MEcPP an attractive target for metabolic engineering in Brassica crop plants to increase resistance to cabbage aphids.

Medically Useful Plant Terpenoids: Biosynthesis, Occurrence, and Mechanism of Action.

Specialized plant terpenoids have found fortuitous uses in medicine due to their evolutionary and biochemical selection for biological activity in animals. However, these highly functionalized natural products are produced through complex biosynthetic pathways for which we have a complete understanding in only a few cases. Here we review some of the most effective and promising plant terpenoids that are currently used in medicine and medical research and provide updates on their biosynthesis, natural occurrence, and mechanism of action in the body. This includes pharmacologically useful plastidic terpenoids such as p-menthane monoterpenoids, cannabinoids, paclitaxel (taxol®), and ingenol mebutate which are derived from the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, as well as cytosolic terpenoids such as thapsigargin and artemisinin produced through the mevalonate (MVA) pathway. We further provide a review of the MEP and MVA precursor pathways which supply the carbon skeletons for the downstream transformations yielding these medically significant natural products.

Non-invasive quantification of ethylene in attached fruit headspace at 1 p.p.b. by gas chromatography-mass spectrometry.

Ethylene is a gaseous plant hormone involved in defense, adaptations to environmental stress and fruit ripening. Its relevance to the latter makes its detection highly useful for physiologists interested in the onset of ripening. Produced as a sharp peak during the respiratory burst, ethylene is biologically active at tens of nl L-1 . Reliable quantification at such concentrations generally requires specialized instrumentation. Here we present a rapid, high-sensitivity method for detecting ethylene in attached fruit using a conventional gas chromatography-mass spectrometry (GC-MS) system and in situ headspace collection chambers. We apply this method to melon (Cucumis melo L.), a unique species consisting of climacteric and non-climacteric varieties, with a high variation in the climacteric phenotype among climacteric types. Using a population of recombinant inbred lines (RILs) derived from highly climacteric ('Védrantais', cantalupensis type) and non-climacteric ('Piel de Sapo', inodorus type) parental lines, we observed a significant variation for the intensity, onset and duration of the ethylene burst during fruit ripening. Our method does not require concentration, sampling times over 1 h or fruit harvest. We achieved a limit of detection of 0.41 ± 0.04 nl L-1 and a limit of quantification of 1.37 ± 0.13 nl L-1 with an analysis time per sample of 2.6 min. Validation of the analytical method indicated that linearity (>98%), precision (coefficient of variation ≤2%) and sensitivity compared favorably with dedicated optical sensors. This study adds to evidence of the characteristic climacteric ethylene burst as a complex trait whose intensity in our RIL population lies along a continuum in addition to two extremes.

The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana.

2-C-Methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) is an intermediate of the plastid-localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co-factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds-3 mutant, defective in the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2-C-methyl-D-erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds-3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds-3 mutant also showed enhanced resistance to the phloem-feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP-mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds-3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.

Arabidopsis J-protein J20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control.

Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress.

Comparative transcriptional profiling analysis of developing melon (Cucumis melo L.) fruit from climacteric and non-climacteric varieties.

BACKGROUND: In climacteric fruit-bearing species, the onset of fruit ripening is marked by a transient rise in respiration rate and autocatalytic ethylene production, followed by rapid deterioration in fruit quality. In non-climacteric species, there is no increase in respiration or ethylene production at the beginning or during fruit ripening. Melon is unusual in having climacteric and non-climacteric varieties, providing an interesting model system to compare both ripening types. Transcriptomic analysis of developing melon fruits from Védrantais and Dulce (climacteric) and Piel de sapo and PI 161375 (non-climacteric) varieties was performed to understand the molecular mechanisms that differentiate the two fruit ripening types. RESULTS: Fruits were harvested at 15, 25, 35 days after pollination and at fruit maturity. Transcript profiling was performed using an oligo-based microarray with 75 K probes. Genes linked to characteristic traits of fruit ripening were differentially expressed between climacteric and non-climacteric types, as well as several transcription factor genes and genes encoding enzymes involved in sucrose catabolism. The expression patterns of some genes in PI 161375 fruits were either intermediate between. Piel de sapo and the climacteric varieties, or more similar to the latter. PI 161375 fruits also accumulated some carotenoids, a characteristic trait of climacteric varieties. CONCLUSIONS: Simultaneous changes in transcript abundance indicate that there is coordinated reprogramming of gene expression during fruit development and at the onset of ripening in both climacteric and non-climacteric fruits. The expression patterns of genes related to ethylene metabolism, carotenoid accumulation, cell wall integrity and transcriptional regulation varied between genotypes and was consistent with the differences in their fruit ripening characteristics. There were differences between climacteric and non-climacteric varieties in the expression of genes related to sugar metabolism suggesting that they may be potential determinants of sucrose content and post-harvest stability of sucrose levels in fruit. Several transcription factor genes were also identified that were differentially expressed in both types, implicating them in regulation of ripening behaviour. The intermediate nature of PI 161375 suggested that classification of melon fruit ripening behaviour into just two distinct types is an over-simplification, and that in reality there is a continuous spectrum of fruit ripening behaviour.

Deoxyxylulose 5-Phosphate Synthase Controls Flux through the Methylerythritol 4-Phosphate Pathway in Arabidopsis.

The 2-C-methylerythritol 4-phosphate (MEP) pathway supplies precursors for plastidial isoprenoid biosynthesis including carotenoids, redox cofactor side chains, and biogenic volatile organic compounds. We examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), using metabolic control analysis. Multiple Arabidopsis (Arabidopsis thaliana) lines presenting a range of DXS activities were dynamically labeled with 13CO2 in an illuminated, climate-controlled, gas exchange cuvette. Carbon was rapidly assimilated into MEP pathway intermediates, but not into the mevalonate pathway. A flux control coefficient of 0.82 was calculated for DXS by correlating absolute flux to enzyme activity under photosynthetic steady-state conditions, indicating that DXS is the major controlling enzyme of the MEP pathway. DXS manipulation also revealed a second pool of a downstream metabolite, 2-C-methylerythritol-2,4-cyclodiphosphate (MEcDP), metabolically isolated from the MEP pathway. DXS overexpression led to a 3- to 4-fold increase in MEcDP pool size but to a 2-fold drop in maximal labeling. The existence of this pool was supported by residual MEcDP levels detected in dark-adapted transgenic plants. Both pools of MEcDP are closely modulated by DXS activity, as shown by the fact that the concentration control coefficient of DXS was twice as high for MEcDP (0.74) as for 1-deoxyxylulose 5-phosphate (0.35) or dimethylallyl diphosphate (0.34). Despite the high flux control coefficient for DXS, its overexpression led to only modest increases in isoprenoid end products and in the photosynthetic rate. Diversion of flux via MEcDP may partly explain these findings and suggests new opportunities to engineer the MEP pathway.

The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with ß-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for ß-carotene production in orange-fleshed melon varieties.

Plastid ancestors lacked a complete Entner-Doudoroff pathway, limiting plants to glycolysis and the pentose phosphate pathway.

The Entner-Doudoroff (ED) pathway provides an alternative to glycolysis. It converts 6-phosphogluconate (6-PG) to glyceraldehyde-3-phosphate and pyruvate in two steps consisting of a dehydratase (EDD) and an aldolase (EDA). Here, we investigate its distribution and significance in higher plants and determine the ED pathway is restricted to prokaryotes due to the absence of EDD genes in eukaryotes. EDDs share a common origin with dihydroxy-acid dehydratases (DHADs) of the branched chain amino acid pathway (BCAA). Each dehydratase features strict substrate specificity. E. coli EDD dehydrates 6-PG to 2-keto-3-deoxy-6-phosphogluconate, while DHAD only dehydrates substrates from the BCAA pathway. Structural modeling identifies two divergent domains which account for their non-overlapping substrate affinities. Coupled enzyme assays confirm only EDD participates in the ED pathway. Plastid ancestors lacked EDD but transferred metabolically promiscuous EDA, which explains the absence of the ED pathway from the Viridiplantae and sporadic persistence of EDA genes across the plant kingdom.

Arabidopsis TGA256 Transcription Factors Suppress Salicylic-Acid-Induced Sucrose Starvation.

Salicylic acid (SA) is produced by plants in response to pathogen infection. SA binds the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES (NPR) family of receptors to regulate both positive (NPR1) and negative (NPR3/4) plant immune responses by interacting with the clade II TGACG (TGA) motif-binding transcription factors (TGA2, TGA5, and TGA6). Here, we report that the principal metabolome-level response to SA treatment in Arabidopsis is a reduction in sucrose and other free sugars. We observed nearly identical effects in the tga256 triple mutant, which lacks all clade II TGA transcription factors. The tga256 mutant presents reduced leaf blade development and elongated hypocotyls, roots, and petioles consistent with sucrose starvation. No changes were detected in auxin levels, and mutant seedling growth could be restored to that of wild-type by sucrose supplementation. Although the retrograde signal 2-C-methyl-D-erythritol-2,4-cyclodiphosphate is known to stimulate SA biosynthesis and defense signaling, we detected no negative feedback by SA on this or any other intermediate of the 2-C-methyl-D-erythritol-4-phosphate pathway. Trehalose, a proxy for the sucrose regulator trehalose-6-phosphate (T6P), was highly reduced in tga256, suggesting that defense-related reductions in sugar availability may be controlled by changes in T6P levels. We conclude that the negative regulatory roles of TGA2/5/6 include maintaining sucrose levels in healthy plants. Disruption of TGA2/5/6-NPR3/4 inhibitory complexes by mutation or SA triggers sucrose reductions in Arabidopsis leaves, consistent with the 'pathogen starvation' hypothesis. These findings highlight sucrose availability as a mechanism by which TGA2/5/6 balance defense and development.

Assessing the Benefits and Costs of the Hydrogen Cyanide Antiherbivore Defense in Trifolium repens.

Understanding the evolution of plant defenses against herbivores requires identifying the benefits and costs of defense. Here, we tested the hypothesis that the benefits and costs of hydrogen cyanide (HCN) defense against herbivory on white clover (Trifolium repens) are temperature dependent. We first tested how temperature affected HCN production in vitro, and then examined how temperature influenced the efficacy of HCN defense of T. repens against a generalist slug (Deroceras reticulatum) herbivore using no-choice and choice feeding trial assays. To understand how temperature affected the costs of defense, plants were exposed to freezing, and HCN production, photosynthetic activity, and ATP concentration were quantified. HCN production increased linearly from 5 °C to 50 °C, and cyanogenic plants experienced reduced herbivory compared to acyanogenic plants only at warmer temperatures when fed upon by young slugs. Freezing temperatures induced cyanogenesis in T. repens and decreased chlorophyll fluorescence. Cyanogenic plants experienced lower ATP levels than acyanogenic plants due to freezing. Our study provides evidence that the benefits of HCN defense against herbivores are temperature dependent, and freezing may inhibit ATP production in cyanogenic plants, but the physiological performance of all plants recovered quickly following short-term freezing. These results contribute to understanding how varying environments alter the benefits and costs of defense in a model system for the study of plant chemical defenses against herbivores.

Improving the Documentation of Penicillin Allergy Labels Among Pediatric Inpatients.

BACKGROUND AND OBJECTIVES: Penicillin allergy is the most common medication allergy, and the penicillin allergy label is commonly over-applied without adequate reaction history inquiry or documentation. Because penicillin allergy labels are often applied in childhood and carried into adulthood, we sought to increase the completeness of reaction history documentation from 20% to 70% for pediatric hospital medicine patients and from 20% to 50% for all other pediatric inpatients within 12 months. As a secondary outcome, we also aimed to increase the proportion of delabeling unnecessary penicillin labels to 20% for all pediatric inpatients. METHODS: To address our aims, our quality improvement initiative included education for pediatric faculty and staff, development and implementation of a clinical pathway for allergy risk stratification, and electronic health record optimizations. Statistical process control charts were used to track the impact of the interventions facilitated by an automated dashboard. RESULTS: Within 12 months of interventions, the completeness of allergy labels improved from 20% to 64% among patients admitted to the pediatric hospital medicine service and improved from 20% to 45% for all other pediatric inpatients. The frequency of penicillin allergy delabeling remained unchanged; however, 98 patients were risk stratified and 34 received outpatient allergy referrals for further testing. The number of adverse drug reactions to penicillin, a balancing measure, did not change during the study period. CONCLUSIONS: We increased the completeness of penicillin allergy documentation using a standardized workflow facilitated by a multidisciplinary clinical pathway. With ongoing efforts, more penicillin delabeling in low-risk patients is anticipated.

A single gene encodes isopentenyl diphosphate isomerase isoforms targeted to plastids, mitochondria and peroxisomes in Catharanthus roseus.

Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.

Soapbark Triterpenes: Quillaja brasiliensis Cell Culture Sapogenin and Free Sterol Analysis by GCMS.

Triterpene saponins of the genus Quillaja (Quillajaceae) are known for their immunoadjuvant, hypocholesterolemic, and anti-inflammatory activity. Plant cell cultures are useful for the study of saponin metabolism and industrial production of these bioactive compounds. While structurally related phytosterols are primary metabolites essential to growth and development, saponins are responsive to pathogen and abiotic stress, fulfilling roles in plant specialized metabolism. For cell culture production of saponins, phytosterols may be considered a competing pathway which relies on a common pool of cytosolic isoprenoid precursors.Understanding the metabolic allocation of resources between these two related pathways is key to maximizing saponin production in in vitro production systems. Sterols and saponins naturally occur in multiple conjugated forms, which complicate separation and quantification. The acid hydrolysis of conjugated sterols and saponins to their free forms is a useful technique to simplify their analysis by gas chromatography. Here we provide the workflow for the quantification of free sterols and sapogenins in cell cultures of Quillaja brasiliensis .