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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−1 vs. 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 eff

This thesis describes the development of a G Protein-Coupled Receptor (GPCR) screening technology that combines a receptor cell array (~300 spots) with microfluidics. This technology was developed for the purpose of sensing the taste of, or active components in complex samples. GPCR activation was monitored using a genetically encoded calcium indicator (GECI) which was based on a change in Förster Resonance Energy Transfer (FRET) between two fluorescent proteins linked by a calcium binding domain which, upon binding of calcium, induces a conformational change between the fluorophores. The receptor cell arrays were created by reverse transfection of printed plasmid DNA. The arrays were assembled in a flowcell, connected to a microfluidic system, and mounted on a stereo fluorescence microscope. This setup allowed for controlled and importantly, repeated sample exposure while monitoring the changes in intracellular calcium in real-time. GPCRs play an important role in many physiological or disease-related processes. These membrane proteins have evolved to sense a wide range of molecules that can be of either exogenous or endogenous origin. Their sensing mechanisms are complex and potentially involve many cellular signalling events depending the cell type. The introductory chapter of this thesis presents a brief overview of the GPCR types and their signalling pathways with a focus on taste signalling. This chapter also places the microfluidic receptomics technology within the framework of existing receptor screening technologies. The second chapter explores the general principles, setup and characterization of the microfluidic biosensor to measure GPCR activation via imaging of [Ca2+] changes in recombinant human HEK293 cells. These cells expressed a combination of the Neurokinin 1-receptor and Cameleon YC3.6 protein as calcium indicator. Here, a stable cell line was employed for robust expression with little variation Next to GPCRs, the system was also used for the det

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.

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 andstatistical 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, whereasothers 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 t

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.

In a mission to Rwanda in December 2017, important stakeholders from government, research institutes, universities and private enterprises were interviewed to elaborate on Public Private Partnerships (PPP), or triple helix collaborations for further development of plant extracts in the agricultural sector of Rwanda. Findings of the missions were shared in a debriefing with the ambassador and the agricultural council of the Kingdom of the Netherlands in Rwanda. During the mission, potential leads were shared with the University of Rwanda. Through the social enterprise Crosswise Works, most important issues were discussed and brought further in a workshop in January 2018, aimed at a follow up mission to the Netherlands in June 2018, and the start-up of PPP in Rwanda. To increase business opportunities for developing plant extract chains in Rwanda, further research, preferably in PPP would be required. Pyrethrum productivity can be increased, based on increasing pyrethrin content of the flowers (now 2% in Rwanda compared to 4% in other parts of the world), and improvement of the cropping system (direct seeding instead of transplanting). Local products from Pyrethrum might also be an option to explore. Artemisia extracts for the pharmaceutical industry has the potential to diversify the current plant extract portfolio of Rwandan organizations. Government bodies in collaboration with universities and research institutes, together with the private sector should investigate whether and how the development of the value chain can be accelerated, and how a good connection to the international market can be obtained.

The sesquiterpenoid polygodial, belonging to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C-12 hydroxylation, and further oxidations at both C-11 and C-12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol only. Co-expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12-hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro-infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyzes the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C-12 position of drimendiol is therefore likely further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C-11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED50 ) of ~200-400 μg gFW-1 against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed

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.

IntroductionMetabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds.ObjectiveWe aimed to combine LC–MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics.MethodsWe developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Capsicum varieties. Two were selected with contrasting degrees of spiciness for characterization by HPLC coupled to high mass resolution MS. Subsequently, the biosensor was then used to link individual pepper compounds with TRPV1 activity.ResultsAmong the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Capsicum fruits as well as a commercial product. Sensitivity of the biosensor to this commercial product was similar to the sensory threshold of a human sensory panel.ConclusionOur results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC–MS.

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, andWRKY22 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 susceptibilityto 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 genesinvolved in plant growth and cell-wall loosening. In addition, echanostimulation of knockout plants by clip cages up-regulated jasmonic acid (JA)-responsive genes, resulting in substantial negative JA–SA crosstalk. Based on thisand 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 signallingmake WRKY22 a potential target for aphids to manipulate host plant defences.

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 highthroughput, 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.

In a number of studies, the Dutch vegetable breeding industry has been described as a highly innovative sector, but the root causes for its innovativeness have not yet been analysed systematically. In order to understand the factors that affect innovation and business performance, the Sectoral Innovation System (SIS) framework was used to analyse the linkages and interactions among the different actors in the Dutch vegetable breeding industry. Within SIS, five interacting domains are recognized and analysed: the business domain, the research & education domain, the intermediate organizations, the market demand, and the infrastructure & framework conditions, resulting in an integrated picture of the innovation system. It was found that the business domain, the research & education domain and the intermediate organisations do not only show an outstanding individual performance, but more importantly, they closely collaborate via public-private partnerships (PPP), research consortia, etc. It is shown that especially the cluster characteristics of the Dutch vegetable breeding industry, i.e. the geographical proximity and the high level of intra- and interconnectivity within and between domains, induces an intensive knowledge flow, a key indicator for the innovation level of a sector.

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Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. Results We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. Conclusions We developed a marker-free transformation pipeline to select potato plants functionally expressing a s