Exploring the co-operativity of secretory structures for defense and pollination in flowering plants.

MAIN CONCLUSION: In flowers multiple secretory systems cooperate to deliver specialized metabolites to support specific roles in defence and pollination. The collective roles of cell types, enzymes, and transporters are discussed. The interplay between reproductive strategies and defense mechanisms in flowering plants has long been recognized, with trade-offs between investment in defense and reproduction predicted. Glandular trichomes and secretory cavities or ducts, which are epidermal and internal structures, play a pivotal role in the secretion, accumulation, and transport of specialized secondary metabolites, and contribute significantly to defense and pollination. Recent investigations have revealed an intricate connection between these two structures, whereby specialized volatile and non-volatile metabolites are exchanged, collectively shaping their respective ecological functions. However, a comprehensive understanding of this profound integration remains largely elusive. In this review, we explore the secretory systems and associated secondary metabolism primarily in Asteraceous species to propose potential shared mechanisms facilitating the directional translocation of these metabolites to diverse destinations. We summarize recent advances in our understanding of the cooperativity between epidermal and internal secretory structures in the biosynthesis, secretion, accumulation, and emission of terpenes, providing specific well-documented examples from pyrethrum (Tanacetum cinerariifolium). Pyrethrum is renowned for its natural pyrethrin insecticides, which accumulate in the flower head, and more recently, for emitting an aphid alarm pheromone. These examples highlight the diverse specializations of secondary metabolism in pyrethrum and raise intriguing questions regarding the regulation of production and translocation of these compounds within and between its various epidermal and internal secretory systems, spanning multiple tissues, to serve distinct ecological purposes. By discussing the cooperative nature of secretory structures in flowering plants, this review sheds light on the intricate mechanisms underlying the ecological roles of terpenes in defense and pollination.

Effects of γ-aminobutyric acid supplementation on glucose control in adults with prediabetes: A double-blind, randomized, placebo-controlled trial.

BACKGROUND: Gamma-aminobutyric acid (GABA) is mainly known as an endogenously produced neurotransmitter. However, GABA intake from dietary sources like tomatoes and fermented foods can be considerable. Studies in rodent models have shown beneficial effects of oral GABA supplementation on glucose homeostasis and cardiovascular health. Still, it is currently unknown whether oral GABA supplementation produces cardiometabolic benefits in humans. OBJECTIVES: This study aimed to investigate whether oral GABA supplementation can improve glucose homeostasis in individuals at risk of developing type 2 diabetes. METHODS: In a randomized, placebo-controlled, double-blind, parallel-arm trial, 52 individuals with prediabetes (classified by impaired glucose tolerance and/or impaired fasting glucose), aged 50 to 70 y with a body mass index ≥25 kg/m2 received either 500 mg GABA 3 times daily or a placebo for 95 days. The primary outcome was the effect of the intervention on glucose response after an OGTT. As exploratory secondary outcomes, markers of glycemic control (glycated hemoglobin, insulin, glucagon, mean amplitude of glycemic excursions, and standard deviation as measured with flash glucose monitoring), cardiovascular health (blood pressure, 24-h blood pressure, circulating triglycerides, cholesterol), and self-reported sleep quality were measured before and after the intervention. RESULTS: Compared with placebo, GABA supplementation for 95 days did not change the postprandial glucose response (0.21 mmol/L; 95% confidence interval: -0.252, 0.674; P = 0.364). After correction for the false discovery rate, all other outcomes (including fasting plasma GABA concentration) showed no significant effects from GABA intervention at a group level. CONCLUSIONS: GABA supplementation does not change the postprandial glucose response in individuals at risk of developing type 2 diabetes. However, based on findings in secondary outcome measures, further research is warranted in other study populations. Research could focus on the effects of GABA in individuals with advanced diabetes or other cardiometabolic disorders. This trial was registered at www. CLINICALTRIALS: gov as NCT04303468.

TcMYC2 regulates Pyrethrin biosynthesis in Tanacetum cinerariifolium.

Pyrethrins constitute a class of terpene derivatives with high insecticidal activity and are mainly synthesized in the capitula of the horticulturally important plant, Tanacetum cinerariifolium. Treatment of T. cinerariifolium with methyl jasmonate (MeJA) in the field induces pyrethrin biosynthesis, but the mechanism linking MeJA with pyrethrin biosynthesis remains unclear. In this study, we explored the transcription factors involved in regulating MeJA-induced pyrethrin biosynthesis. A single spray application of MeJA to T. cinerariifolium leaves rapidly upregulated the expression of most known pyrethrin biosynthesis genes and subsequently increased the total pyrethrin content in the leaf. A continuous 2-week MeJA treatment resulted in enhanced pyrethrin content and increased trichome density. TcMYC2, a key gene in jasmonate signaling, was screened at the transcriptome after MeJA treatment. TcMYC2 positively regulated expression of the pyrethrin biosynthesis genes TcCHS, TcAOC, and TcGLIP by directly binding to E-box/G-box motifs in the promoters. The stable overexpression of TcMYC2 in T. cinerariifolium hairy roots significantly increased the expression of TcAOC and TcGLIP. Further transient overexpression and viral-induced gene-silencing experiments demonstrated that TcMYC2 positively promoted pyrethrin biosynthesis. Collectively, the results reveal a novel molecular mechanism for MeJA-induced pyrethrin biosynthesis in T. cinerariifolium involving TcMYC2.

The influence of a tomato food matrix on the bioavailability and plasma kinetics of oral gamma-aminobutyric acid (GABA) and its precursor glutamate in healthy men.

Gamma-aminobutyric acid (GABA) and its precursor glutamate play signaling roles in a range of tissues. Both function as neurotransmitters in the central nervous system, but they also modulate pancreatic and immune functioning, for example. Besides endogenous production, both compounds are found in food products, reaching relatively high levels in tomatoes. Recent studies in rodents suggest beneficial effects of oral GABA on glucose homeostasis and blood pressure. However, the bioavailability from food remains unknown. We studied the bioavailability of GABA and glutamate from tomatoes relative to a solution in water. After a fasting blood sample was taken, eleven healthy men randomly received 1 liter of 4 different drinks in a cross-over design with a one-week interval. The drinks were a solution of 888 mg L-1 GABA, a solution of 3673 mg L-1 glutamate, pureed fresh tomatoes and plain water as the control. Following intake, 18 blood samples were taken at intervals for 24 hours. Plasma GABA and glutamate concentrations were determined by ultra-pressure liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Fasting plasma GABA and glutamate concentrations were found to be 16.71 (SD 2.18) ng mL-1 and 4626 (SD 1666) ng mL-1, respectively. Fasting GABA levels were constant (5.8 CV%) between individuals, while fasting glutamate levels varied considerably (23.5 CV%). GABA from pureed tomatoes showed similar bioavailability to that of a solution in water. For glutamate, the absorption from pureed tomatoes occurred more slowly as seen from a longer tmax (0.98 ± 0.14 h vs. 0.41 ± 0.04 h, P = 0.003) and lower Cmax (7815 ± 627 ng mL-1vs. 16 420 ± 2778 ng mL-1, P = 0.006). These data suggest that GABA is bioavailable from tomatoes, and that food products containing GABA could potentially induce health effects similar to those claimed for GABA supplements. The results merit further studies on the bioavailability of GABA from other food products and the health effects of GABA-rich diets. The clinical trial registry number is NCT04086108 (https://clinicaltrials.gov/ct2/show/NCT04303468).

Tissue specificity of (E)-ß-farnesene and germacrene D accumulation in pyrethrum flowers.

Plant defensive mimicry based on the aphid alarm pheromone (E)-ß-farnesene (EßF) was previously shown to operate in Tanacetum cinerariifolium (Asteraceae) flowers. Germacrene D (GD), is another dominant volatile of T. cinerariifolium flowers and may modulate both defense and pollination. Here, we find that the increase in GD/EßF ratio at later developmental stages is correlated with the tissue distribution in the flower head: the total content of EßF and GD is similar, but GD accumulates comparatively more in the upper disk florets. Naphthol and N, N-dimethyl-p-phenylenediamine dihydrochloride (NADI)-stained purple ducts containing EßF and GD, were observed in the five petal lips of the corolla and two-lobed stigma of disk florets. By contrast in the peduncle, EßF accounts for nearly 80% of total terpenes, compared to 5% for GD. EßF is accumulated inside inner cortex cells and parenchyma cells of the pith in young peduncle. This is followed by the formation of terpene-filled axial secretory cavities parallel to the vascular bundles. In conclusion, the observed developmental and diurnal emissions of different EßF/GD ratios appear to be regulated by their tissue distribution.

Development and validation of a UPLC-MS/MS method for the simultaneous determination of gamma-aminobutyric acid and glutamic acid in human plasma.

Gamma-aminobutyric acid (GABA) and its precursor glutamic acid are important neurotransmitters. Both are also present in peripheral tissues and the circulation, where abnormal plasma concentrations have been linked to specific mental disorders. In addition to endogenous synthesis, GABA and glutamic acid can be obtained from dietary sources. An increasing number of studies suggest beneficial cardio-metabolic effects of GABA intake, and therefore GABA is being marketed as a food supplement. The need for further research into their health effects merits accurate and sensitive methods to analyze GABA and glutamic acid in plasma. To this end, an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantification of GABA and glutamic acid in human plasma. Samples were prepared by a protein precipitation step and subsequent solid phase extraction using acetonitrile. Chromatographic separation was achieved on an Acquity UPLC HSS reversed phase C18 column using gradient elution. Analytes were detected using electrospray ionization and selective reaction monitoring. Standard curve concentrations for GABA ranged from 3.4 to 2500 ng/mL and for glutamic acid from 30.9 ng/mL to 22,500 ng/mL. Within- and between-day accuracy and precision were <10% in quality control samples at low, medium and high concentrations for both GABA and glutamic acid. GABA and glutamic acid were found to be stable in plasma after freeze-thaw cycles and up to 12 months of storage. The validated method was applied to human plasma from 17 volunteers. The observed concentrations ranged between 11.5 and 20.0 ng/ml and 2269 and 7625 ng/ml for respectively GABA and glutamic acid. The reported method is well suited for the measurement of plasma GABA and glutamic acid in pre-clinical or clinical studies.

The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay.

The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein-coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments.

Defense of pyrethrum flowers: repelling herbivores and recruiting carnivores by producing aphid alarm pheromone.

(E)-ß-Farnesene (EßF) is the predominant constituent of the alarm pheromone of most aphid pest species. Moreover, natural enemies of aphids use EßF to locate their aphid prey. Some plant species emit EßF, potentially as a defense against aphids, but field demonstrations are lacking. Here, we present field and laboratory studies of flower defense showing that ladybird beetles are predominantly attracted to young stage-2 pyrethrum flowers that emitted the highest and purest levels of EßF. By contrast, aphids were repelled by EßF emitted by S2 pyrethrum flowers. Although peach aphids can adapt to pyrethrum plants in the laboratory, aphids were not recorded in the field. Pyrethrum's (E)-ß-farnesene synthase (EbFS) gene is strongly expressed in inner cortex tissue surrounding the vascular system of the aphid-preferred flower receptacle and peduncle, leading to elongated cells filled with EßF. Aphids that probe these tissues during settlement encounter and ingest plant EßF, as evidenced by the release in honeydew. These EßF concentrations in honeydew induce aphid alarm responses, suggesting an extra layer of this defense. Collectively, our data elucidate a defensive mimicry in pyrethrum flowers: the developmentally regulated and tissue-specific EßF accumulation and emission both prevents attack by aphids and recruits aphid predators as bodyguards.

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.

Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.

In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10-carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

An Integrated System for the Automated Recording and Analysis of Insect Behavior in T-maze Arrays.

Host-plant resistance to insects like thrips and aphids is a complex trait that is difficult to phenotype quickly and reliably. Here, we introduce novel hardware and software to facilitate insect choice assays and automate the acquisition and analysis of movement tracks. The hardware consists of an array of individual T-mazes allowing simultaneous release of up to 90 insect individuals from their individual cage below each T-maze with choice of two leaf disks under a video camera. Insect movement tracks are acquired with computer vision software (EthoVision) and analyzed with EthoAnalysis, a novel software package that allows for automated reporting of highly detailed behavior parameters and statistical analysis. To validate the benefits of the system we contrasted two Arabidopsis accessions that were previously analyzed for differential resistance to western flower thrips. Results of two trials with 40 T-mazes are reported and we show how we arrived at optimized settings for the different filters and statistics. The statistics are reported in terms of frequency, duration, distance and speed of behavior events, both as sum totals and event averages, and both for the total trial period and in time bins of 1 h. Also included are higher level analyses with subcategories like short-medium-long events and slow-medium-fast events. The time bins showed how some behavior elements are more descriptive of differences between the genotypes during the first hours, whereas others are constant or become more relevant at the end of an 8 h recording. The three overarching behavior categories, i.e., choice, movement, and halting, were automatically corrected for the percentage of time thrips were detected and 24 out of 38 statistics of behavior parameters differed by a factor 2-6 between the accessions. The analysis resulted in much larger contrasts in behavior traits than reported previously. Compared to leaf damage assays on whole plants or detached leaves that take a week or more to complete, results were obtained in 8 h, with more detail, fewer individuals and higher significance. The potential value of the new integrated system, named EntoLab, for discovery of genetic traits in plants and insects by high throughput screening of large populations is discussed.

Automated high-throughput individual tracking system for insect behavior: Applications on memory retention in parasitic wasps.

BACKGROUND: Insects are important models to study learning and memory formation in both an ecological and neuroscience context due to their small size, behavioral flexibility and ecological diversity. Measuring memory retention is often done through simple time-consuming set-ups, producing only a single parameter for conditioned behavior. We wished to obtain higher sample sizes with fewer individuals to measure olfactory memory retention more efficiently. NEW METHOD: The high-throughput individual T-maze uses commercially available tracking software, Ethovision XT®, in combination with a Perspex stack of plates as small as 18 × 18 cm, which accommodates 36 olfactory T-mazes, where each individual wasp could choose between two artificial odors. Various behavioral parameters, relevant to memory retention, were acquired in this set-up; first choice, residence time, giving up time and zone entries. From these parameters a performance index was calculated as a measure of memory retention. Groups of 36 wasps were simultaneously tested within minutes, resulting in efficient acquisition of sufficiently high sample sizes. RESULTS: This system was tested with two very different parasitic wasp species, the larval parasitoid Cotesia glomerata and the pupal parasitoid Nasonia vitripennis, and has proven to be highly suitable for testing memory retention in both these species. COMPARISON WITH EXISTING METHODS: Unlike other bioassays, this system allows for both high-throughput and recording of detailed individual behavior. CONCLUSIONS: The high-throughput individual T-maze provides us with a standardized high-throughput, labor-efficient and cost-effective method to test various kinds of behavior, offering excellent opportunities for comparative studies of various aspects of insect behavior.

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.

Modification of chrysanthemum odour and taste with chrysanthemol synthase induces strong dual resistance against cotton aphids.

Aphids are pests of chrysanthemum that employ plant volatiles to select host plants and ingest cell contents to probe host quality before engaging in prolonged feeding and reproduction. Changes in volatile and nonvolatile metabolite profiles can disrupt aphid-plant interactions and provide new methods of pest control. Chrysanthemol synthase (CHS) from Tanacetum cinerariifolium represents the first committed step in the biosynthesis of pyrethrin ester insecticides, but no biological role for the chrysanthemol product alone has yet been documented. In this study, the TcCHS gene was over-expressed in Chrysanthemum morifolium and resulted in both the emission of volatile chrysanthemol (ca. 47 pmol/h/gFW) and accumulation of a chrysanthemol glycoside derivative, identified by NMR as chrysanthemyl-6-O-malonyl-ß-D-glucopyranoside (ca. 1.1 mM), with no detrimental phenotypic effects. Dual-choice assays separately assaying these compounds in pure form and as part of the headspace and extract demonstrated independent bioactivity of both components against the cotton aphid (Aphis gossypii). Performance assays showed that the TcCHS plants significantly reduced aphid reproduction, consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram (EPG) studies. In open-field trials, aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait. The results suggest that expression of the TcCHS gene induces a dual defence system, with both repellence by chrysanthemol odour and deterrence by its nonvolatile glycoside, introducing a promising new option for engineering aphid control into plants.

SIEVE ELEMENT-LINING CHAPERONE1 Restricts Aphid Feeding on Arabidopsis during Heat Stress.

The role of phloem proteins in plant resistance to aphids is still largely elusive. By genome-wide association mapping of aphid behavior on 350 natural Arabidopsis thaliana accessions, we identified the small heat shock-like SIEVE ELEMENT-LINING CHAPERONE1 (SLI1). Detailed behavioral studies on near-isogenic and knockout lines showed that SLI1 impairs phloem feeding. Depending on the haplotype, aphids displayed a different duration of salivation in the phloem. On sli1 mutants, aphids prolonged their feeding sessions and ingested phloem at a higher rate than on wild-type plants. The largest phenotypic effects were observed at 26°C, when SLI1 expression is upregulated. At this moderately high temperature, sli1 mutants suffered from retarded elongation of the inflorescence and impaired silique development. Fluorescent reporter fusions showed that SLI1 is confined to the margins of sieve elements where it lines the parietal layer and colocalizes in spherical bodies around mitochondria. This localization pattern is reminiscent of the clamp-like structures observed in previous ultrastructural studies of the phloem and shows that the parietal phloem layer plays an important role in plant resistance to aphids and heat stress.

Genetic architecture of plant stress resistance: multi-trait genome-wide association mapping.

Plants are exposed to combinations of various biotic and abiotic stresses, but stress responses are usually investigated for single stresses only. Here, we investigated the genetic architecture underlying plant responses to 11 single stresses and several of their combinations by phenotyping 350 Arabidopsis thaliana accessions. A set of 214 000 single nucleotide polymorphisms (SNPs) was screened for marker-trait associations in genome-wide association (GWA) analyses using tailored multi-trait mixed models. Stress responses that share phytohormonal signaling pathways also share genetic architecture underlying these responses. After removing the effects of general robustness, for the 30 most significant SNPs, average quantitative trait locus (QTL) effect sizes were larger for dual stresses than for single stresses. Plants appear to deploy broad-spectrum defensive mechanisms influencing multiple traits in response to combined stresses. Association analyses identified QTLs with contrasting and with similar responses to biotic vs abiotic stresses, and below-ground vs above-ground stresses. Our approach allowed for an unprecedented comprehensive genetic analysis of how plants deal with a wide spectrum of stress conditions.

AtWRKY22 promotes susceptibility to aphids and modulates salicylic acid and jasmonic acid signalling.

Aphids induce many transcriptional perturbations in their host plants, but the signalling cascades responsible and the effects on plant resistance are largely unknown. Through a genome-wide association (GWA) mapping study in Arabidopsis thaliana, we identified WRKY22 as a candidate gene associated with feeding behaviour of the green peach aphid, Myzus persicae The transcription factor WRKY22 is known to be involved in pathogen-triggered immunity, and WRKY22 gene expression has been shown to be induced by aphids. Assessment of aphid population development and feeding behaviour on knockout mutants and overexpression lines showed that WRKY22 increases susceptibility to M. persicae via a mesophyll-located mechanism. mRNA sequencing analysis of aphid-infested wrky22 knockout plants revealed the up-regulation of genes involved in salicylic acid (SA) signalling and down-regulation of genes involved in plant growth and cell-wall loosening. In addition, mechanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA-SA crosstalk. Based on this and previous studies, WRKY22 is considered to modulate the interplay between the SA and JA pathways in response to a wide range of biotic and abiotic stimuli. Its induction by aphids and its role in suppressing SA and JA signalling make WRKY22 a potential target for aphids to manipulate host plant defences.

Automated video tracking of thrips behavior to assess host-plant resistance in multiple parallel two-choice setups.

BACKGROUND: Piercing-sucking insects cause severe damage in crops. Breeding for host-plant resistance can significantly reduce the yield losses caused by these insects, but host-plant resistance is a complex trait that is difficult to phenotype quickly and reliably. Current phenotyping methods mainly focus on labor-intensive and time-consuming end-point measurements of plant fitness. Characterizing insect behavior as a proxy for host-plant resistance could be a promising time-saving alternative to end-point measurements. RESULTS: We present a phenotyping platform that allows screening for host-plant resistance against Western flower thrips (WFT, Frankliniella occidentalis (Pergande)) in a parallel two-choice setup using automated video tracking of thrips behavior. The platform was used to establish host-plant preference of WFT with a large plant population of 345 wild Arabidopsis accessions and the method was optimized with two extreme accessions from this population that differed in resistance towards WFT. To this end, the behavior of 88 WFT individuals was simultaneously tracked in 88 parallel two-choice arenas during 8 h. Host-plant preference of WFT was established both by the time thrips spent on either accession and various behavioral parameters related to movement (searching) and non-movement (feeding) events. CONCLUSION: In comparison to 6-day end-point choice assays with whole plants or detached leaves, the automated video-tracking choice assay developed here delivered similar results, but with higher time- and resource efficiency. This method can therefore be a reliable and effective high throughput phenotyping tool to assess host-plant resistance to thrips in large plant populations.

High-throughput phenotyping of plant resistance to aphids by automated video tracking.

BACKGROUND: Piercing-sucking insects are major vectors of plant viruses causing significant yield losses in crops. Functional genomics of plant resistance to these insects would greatly benefit from the availability of high-throughput, quantitative phenotyping methods. RESULTS: We have developed an automated video tracking platform that quantifies aphid feeding behaviour on leaf discs to assess the level of plant resistance. Through the analysis of aphid movement, the start and duration of plant penetrations by aphids were estimated. As a case study, video tracking confirmed the near-complete resistance of lettuce cultivar 'Corbana' against Nasonovia ribisnigri (Mosely), biotype Nr:0, and revealed quantitative resistance in Arabidopsis accession Co-2 against Myzus persicae (Sulzer). The video tracking platform was benchmarked against Electrical Penetration Graph (EPG) recordings and aphid population development assays. The use of leaf discs instead of intact plants reduced the intensity of the resistance effect in video tracking, but sufficiently replicated experiments resulted in similar conclusions as EPG recordings and aphid population assays. One video tracking platform could screen 100 samples in parallel. CONCLUSIONS: Automated video tracking can be used to screen large plant populations for resistance to aphids and other piercing-sucking insects.

Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 µm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 µm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 µg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.