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Plants have evolved and adapted under dynamic environmental conditions, particularly to fluctuating light, but plant research has often focused on constant growth conditions. To quantitatively asses the adaptation to fluctuating light, a panel of 384 natural Arabidopsis thaliana accessions was analyzed in two parallel independent experiments under fluctuating and constant light conditions in an automated high-throughput phenotyping system upgraded with supplemental LEDs. While the integrated daily photosynthetically active radiation was the same under both light regimes, plants in fluctuating light conditions accumulated significantly less biomass and had lower leaf area during their measured vegetative growth than plants in constant light. A total of 282 image-derived architectural and/or color-related traits at six common time points, and 77 photosynthesis-related traits from one common time point were used to assess their associations with genome-wide natural variation for both light regimes. Out of the 3000 significant marker-trait associations (MTAs) detected, only 183 (6.1%) were common for fluctuating and constant light conditions. The prevalence of light regime-specific QTL indicates a complex adaptation. Genes in linkage disequilibrium with fluctuating light-specific MTAs with an adjusted repeatability value >0.5 were filtered for gene ontology terms containing "photo" or "light", yielding 15 selected candidates. The candidate genes are involved in photoprotection, PSII maintenance and repair, maintenance of linear electron flow, photorespiration, phytochrome signaling, and cell wall expansion, providing a promising starting point for further investigations into the response of Arabidopsis thaliana to fluctuating light conditions., (© 2024 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Genome-wide association studies (GWAS) identified thousands of genetic loci associated with complex plant traits, including many traits of agronomical importance. However, functional interpretation of GWAS results remains challenging because of large candidate regions due to linkage disequilibrium. High-throughput omics technologies, such as genomics, transcriptomics, proteomics and metabolomics open new avenues for integrative systems biological analyses and help to nominate systems information supported (prime) candidate genes. In the present study, we capitalise on a diverse canola population with 477 spring-type lines which was previously analysed by high-throughput phenotyping of growth-related traits and by RNA sequencing and metabolite profiling for multi-omics-based hybrid performance prediction. We deepened the phenotypic data analysis, now providing 123 time-resolved image-based traits, to gain insight into the complex relations during early vegetative growth and reanalysed the transcriptome data based on the latest Darmor-bzh v10 genome assembly. Genome-wide association testing revealed 61 298 robust quantitative trait loci (QTL) including 187 metabolite QTL, 56814 expression QTL and 4297 phenotypic QTL, many clustered in pronounced hotspots. Combining information about QTL colocalisation across omics layers and correlations between omics features allowed us to discover prime candidate genes for metabolic and vegetative growth variation. Prioritised candidate genes for early biomass accumulation include A06p05760.1_BnaDAR (PIAL1), A10p16280.1_BnaDAR, C07p48260.1_BnaDAR (PRL1) and C07p48510.1_BnaDAR (CLPR4). Moreover, we observed unequal effects of the Brassica A and C subgenomes on early biomass production., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

F1 hybrids derived from a cross between two inbred parental lines often display widespread changes in DNA methylation and gene expression patterns relative to their parents. An emerging challenge is to understand how parental epigenomic differences contribute to these events. Here, we generated a large mapping panel of F1 epigenetic hybrids, whose parents are isogenic but variable in their DNA methylation patterns. Using a combination of multi-omic profiling and epigenetic mapping strategies we show that differentially methylated regions in parental pericentromeres act as major reorganizers of hybrid methylomes and transcriptomes, even in the absence of genetic variation. These parental differentially methylated regions are associated with hybrid methylation remodeling events at thousands of target regions throughout the genome, both locally (in cis) and distally (in trans). Many of these distally-induced methylation changes lead to nonadditive expression of nearby genes and associate with phenotypic heterosis. Our study highlights the pleiotropic potential of parental pericentromeres in the functional remodeling of hybrid genomes and phenotypes., (© 2024 Kakoulidou et al.)

Plant growth is a complex process affected by a multitude of genetic and environmental factors and their interactions. To identify genetic factors influencing plant performance under different environmental conditions, vegetative growth was assessed in Arabidopsis thaliana cultivated under constant or fluctuating light intensities, using high-throughput phenotyping and genome-wide association studies. Daily automated non-invasive phenotyping of a collection of 382 Arabidopsis accessions provided growth data during developmental progression under different light regimes at high temporal resolution. Quantitative trait loci (QTL) for projected leaf area, relative growth rate, and PSII operating efficiency detected under the two light regimes were predominantly condition-specific and displayed distinct temporal activity patterns, with active phases ranging from 2 d to 9 d. Eighteen protein-coding genes and one miRNA gene were identified as potential candidate genes at 10 QTL regions consistently found under both light regimes. Expression patterns of three candidate genes affecting projected leaf area were analysed in time-series experiments in accessions with contrasting vegetative leaf growth. These observations highlight the importance of considering both environmental and temporal patterns of QTL/allele actions and emphasize the need for detailed time-resolved analyses under diverse well-defined environmental conditions to effectively unravel the complex and stage-specific contributions of genes affecting plant growth processes., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Bhutan has demonstrated a trajectory of advances in healthcare, while still remaining true to its culture and traditional forms of medicine. Most recently, Bhutan gained international attention when it implemented a strategic Covid-19 vaccination programme that protected a greater percentage of its population than observed in Western industrialised nations. This accomplishment supports the idea that there are lessons from Bhutan to be shared with the rest of the world. In this work, we delineate our observations of the Bhutanese healthcare system, based on field observations in several Bhutanese cities, and results from surveys of Bhutanese physicians. We identify a number of unique practices that influence patient compliance, health education, and access to care in the Bhutanese system, that may be of particular interest and applicability to other healthcare systems. These include housing multiple health services at one location, fully funded medical visits, using non-physician teachers for health education and use of Gross National Happiness (GNH) measures in care., (© 2023 John Wiley & Sons Ltd.)

Key Message: Complementing or replacing genetic markers with transcriptomic data and use of reproducing kernel Hilbert space regression based on Gaussian kernels increases hybrid prediction accuracies for complex agronomic traits in canola. In plant breeding, hybrids gained particular importance due to heterosis, the superior performance of offspring compared to their inbred parents. Since the development of new top performing hybrids requires labour-intensive and costly breeding programmes, including testing of large numbers of experimental hybrids, the prediction of hybrid performance is of utmost interest to plant breeders. In this study, we tested the effectiveness of hybrid prediction models in spring-type oilseed rape (Brassica napus L./canola) employing different omics profiles, individually and in combination. To this end, a population of 950 F 1 hybrids was evaluated for seed yield and six other agronomically relevant traits in commercial field trials at several locations throughout Europe. A subset of these hybrids was also evaluated in a climatized glasshouse regarding early biomass production. For each of the 477 parental rapeseed lines, 13,201 single nucleotide polymorphisms (SNPs), 154 primary metabolites, and 19,479 transcripts were determined and used as predictive variables. Both, SNP markers and transcripts, effectively predict hybrid performance using (genomic) best linear unbiased prediction models (gBLUP). Compared to models using pure genetic markers, models incorporating transcriptome data resulted in significantly higher prediction accuracies for five out of seven agronomic traits, indicating that transcripts carry important information beyond genomic data. Notably, reproducing kernel Hilbert space regression based on Gaussian kernels significantly exceeded the predictive abilities of gBLUP models for six of the seven agronomic traits, demonstrating its potential for implementation in future canola breeding programmes.

We assessed early vegetative growth in a population of 382 accessions of Arabidopsis thaliana using automated non-invasive high-throughput phenotyping. All accessions were imaged daily from 7 d to 18 d after sowing in three independent experiments and genotyped using the Affymetrix 250k SNP array. Projected leaf area (PLA) was derived from image analysis and used to calculate relative growth rates (RGRs). In addition, initial seed size was determined. The generated datasets were used jointly for a genome-wide association study that identified 238 marker-trait associations (MTAs) individually explaining up to 8% of the total phenotypic variation. Co-localization of MTAs occurred at 33 genomic positions. At 21 of these positions, sequential co-localization of MTAs for 2-9 consecutive days was observed. The detected MTAs for PLA and RGR could be grouped according to their temporal expression patterns, emphasizing that temporal variation of MTA action can be observed even during the vegetative growth phase, a period of continuous formation and enlargement of seemingly similar rosette leaves. This indicates that causal genes may be differentially expressed in successive periods. Analyses of the temporal dynamics of biological processes are needed to gain important insight into the molecular mechanisms of growth-controlling processes in plants., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background and Aims: The stomatal conductance (gs) of most plant species decreases in response to elevated atmospheric CO2 concentration. This response could have a significant impact on plant water use in a future climate. However, the regulation of the CO2-induced stomatal closure response is not fully understood. Moreover, the potential genetic links between short-term (within minutes to hours) and long-term (within weeks to months) responses of gs to increased atmospheric CO2 have not been explored., Methods: We used Arabidopsis thaliana recombinant inbred lines originating from accessions Col-0 (strong CO2 response) and C24 (weak CO2 response) to study short- and long-term controls of gs. Quantitative trait locus (QTL) mapping was used to identify loci controlling short- and long-term gs responses to elevated CO2, as well as other stomata-related traits., Key Results: Short- and long-term stomatal responses to elevated CO2 were significantly correlated. Both short- and long-term responses were associated with a QTL at the end of chromosome 2. The location of this QTL was confirmed using near-isogenic lines and it was fine-mapped to a 410-kb region. The QTL did not correspond to any known gene involved in stomatal closure and had no effect on the responsiveness to abscisic acid. Additionally, we identified numerous other loci associated with stomatal regulation., Conclusions: We identified and confirmed the effect of a strong QTL corresponding to a yet unknown regulator of stomatal closure in response to elevated CO2 concentration. The correlation between short- and long-term stomatal CO2 responses and the genetic link between these traits highlight the importance of understanding guard cell CO2 signalling to predict and manipulate plant water use in a world with increasing atmospheric CO2 concentration. This study demonstrates the power of using natural variation to unravel the genetic regulation of complex traits., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company.)

A major challenge of plant biology is to unravel the genetic basis of complex traits. We took advantage of recent technical advances in high-throughput phenotyping in conjunction with genome-wide association studies to elucidate genotype-phenotype relationships at high temporal resolution. A diverse Brassica napus population from a commercial breeding programme was analysed by automated non-invasive phenotyping. Time-resolved data for early growth-related traits, including estimated biovolume, projected leaf area, early plant height and colour uniformity, were established and complemented by fresh and dry weight biomass. Genome-wide SNP array data provided the framework for genome-wide association analyses. Using time point data and relative growth rates, multiple robust main effect marker-trait associations for biomass and related traits were detected. Candidate genes involved in meristem development, cell wall modification and transcriptional regulation were detected. Our results demonstrate that early plant growth is a highly complex trait governed by several medium and many small effect loci, most of which act only during short phases. These observations highlight the importance of taking the temporal patterns of QTL/allele actions into account and emphasize the need for detailed time-resolved analyses to effectively unravel the complex and stage-specific contributions of genes affecting growth processes that operate at different developmental phases., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Main Conclusion: The plasticity of plant growth response to differing nitrate availability renders the identification of biomarkers difficult, but allows access to genetic factors as tools to modulate root systems to a wide range of soil conditions. Nitrogen availability is a major determinant of crop yield. While the application of fertiliser substantially increases the yield on poor soils, it also causes nitrate pollution of water resources and high costs for farmers. Increasing nitrogen use efficiency in crop plants is a necessary step to implement low-input agricultural systems. We exploited the genetic diversity present in the worldwide Arabidopsis thaliana population to study adaptive growth patterns and changes in gene expression associated with chronic low nitrate stress, to identify biomarkers associated with good plant performance under low nitrate availability. Arabidopsis accessions were grown on agar plates with limited and sufficient supply of nitrate to measure root system architecture as well as shoot and root fresh weight. Differential gene expression was determined using Affymetrix ATH1 arrays. We show that the response to differing nitrate availability is highly variable in Arabidopsis accessions. Analyses of vegetative shoot growth and root system architecture identified accession-specific reaction modes to cope with limited nitrate availability. Transcription and epigenetic factors were identified as important players in the adaption to limited nitrogen in a global gene expression analysis. Five nitrate-responsive genes emerged as possible biomarkers for NUE in Arabidopsis. The plasticity of plant growth in response to differing nitrate availability in the substrate renders the identification of morphological and molecular features as biomarkers difficult, but at the same time allows access to a multitude of genetic factors which can be used as tools to modulate and adjust root systems to a wide range of soil conditions.

Natural selection driven by water availability has resulted in considerable variation for traits associated with drought tolerance and leaf-level water-use efficiency (WUE). In Arabidopsis, little is known about the variation of whole-plant water use (PWU) and whole-plant WUE (transpiration efficiency). To investigate the genetic basis of PWU, we developed a novel proxy trait by combining flowering time and rosette water use to estimate lifetime PWU. We validated its usefulness for large-scale screening of mapping populations in a subset of ecotypes. This parameter subsequently facilitated the screening of water use and drought tolerance traits in a recombinant inbred line population derived from two Arabidopsis accessions with distinct water-use strategies, namely, C24 (low PWU) and Col-0 (high PWU). Subsequent quantitative trait loci mapping and validation through near-isogenic lines identified two causal quantitative trait loci, which showed that a combination of weak and nonfunctional alleles of the FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) genes substantially reduced plant water use due to their control of flowering time. Crucially, we observed that reducing flowering time and consequently water use did not penalize reproductive performance, as such water productivity (seed produced per unit of water transpired) improved. Natural polymorphisms of FRI and FLC have previously been elucidated as key determinants of natural variation in intrinsic WUE (δ 13 C). However, in the genetic backgrounds tested here, drought tolerance traits, stomatal conductance, δ 13 C. and rosette water use were independent of allelic variation at FRI and FLC, suggesting that flowering is critical in determining lifetime PWU but not always leaf-level traits., (© 2019 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.)

Developmental plasticity of root system architecture is crucial for plant performance in nutrient-poor soils. Roots of plants grown under mild nitrogen (N) deficiency show a foraging response characterized by increased root length but mechanisms underlying this developmental plasticity are still elusive. By employing natural variation in Arabidopsis accessions, we show that the brassinosteroid (BR) signaling kinase BSK3 modulates root elongation under mild N deficiency. In particular, a proline to leucine substitution in the predicted kinase domain of BSK3 enhances BR sensitivity and signaling to increase the extent of root elongation. We further show that low N specifically upregulates transcript levels of the BR co-receptor BAK1 to activate BR signaling and stimulate root elongation. Altogether, our results uncover a role of BR signaling in root elongation under low N. The BSK3 alleles identified here provide targets for improving root growth of crops growing under limited N conditions.

Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Chasmataspidids are a rare group of chelicerate arthropods known from 12 species assigned to ten genera, with a geologic range extending from the Ordovician to the Devonian. The Late Ordovician (Richmondian) fauna of the Big Hill Lagerstätte includes a new species of chasmataspidid represented by 55 specimens. This taxon is only the second chasmataspidid described from the Ordovician and preserves morphological details unknown from any of the previously described species., Results: The new chasmataspidid species is described as Hoplitaspis hiawathai gen. et sp. nov.. Comparison with all other known chasmataspidids indicates that Hoplitaspis occupies an intermediate morphological position between the Ordovician Chasmataspis and the Silurian-Devonian diploaspidids. While the modification of appendage VI into a broad swimming paddle allies Hoplitaspis to the Diploaspididae, the paddle lacks the anterior 'podomere 7a' found in other diploaspidids and shows evidence of having been derived from a Chasmataspis-like chelate appendage. Other details, such as the large body size and degree of expression of the first tergite, show clear affinities with Chasmataspis, providing strong support for chasmataspidid monophyly., Conclusions: The large body size and well-developed appendage armature of Hoplitaspis reveals that chasmataspidids occupied a greater breadth of ecological roles than previously thought, with the abundance of available specimens indicating that Hoplitaspis was an important component of the local community. The miniaturization and ecological limiting of diploaspidids potentially coincides with the major radiation of eurypterids and may suggest some degree of competition between the two groups. The geographic distribution of chasmataspidid species suggests the group may have originated in Laurentia and migrated to the paleocontinents of Baltica and Siberia as tectonic processes drew the paleocontinents into close proximity.

Key Message: Polymorphic probes identified via a sequence-based approach are suitable to infer the genotypes of recombinant inbred lines from hybridisation intensities of GeneChip ® transcript profiling experiments. The sequences of the probes of the ATH1 GeneChip ® exactly match transcript sequences of the Arabidopsis thaliana reference genome Col-0, whereas nucleotide differences and/or insertions/deletions may be observed for transcripts of other A. thaliana accessions. Individual probes of the GeneChip ® that show sequence polymorphisms between different A. thaliana accessions may serve as single-feature polymorphism (SFP) markers, provided that the sequence changes cause differences in hybridisation intensity for the accessions of interest. A sequence-based approach identified features on the high-density oligonucleotide array that showed sequence polymorphisms between A. thaliana accessions Col-0 and C24. Hybridisation intensities of polymorphic probes were extracted from genome-wide transcript profiles of Col-0/C24 and C24/Col-0 recombinant inbred lines and assessed after standardisation via sliding window analyses to identify SFP markers. The genotypes of the recombinant inbred lines were determined with the SFP markers and the resulting data were integrated with information, which had been established previously with single nucleotide polymorphism and insertion/deletion markers, to enrich the linkage map of the Col-0/C24 and C24/Col-0 recombinant inbred populations. Congruence between the molecular marker map and the sequence maps of the A. thaliana Col-0 chromosomes proved the reliability of the genotype information which was deduced from the transcript profiles of the Col-0/C24 and C24/Col-0 recombinant inbred lines.

Progressive myoclonus epilepsies (PMEs) are disorders characterized by myoclonic and generalized seizures with progressive neurological deterioration. While several genetic causes for PMEs have been identified, the underlying causes remain unknown for a substantial portion of cases. Here we describe several affected individuals from a large, consanguineous family presenting with a novel PME in which symptoms begin in adolescence and result in death by early adulthood. Whole exome analyses revealed that affected individuals have a homozygous variant in GPR37L1 (c.1047G>T [Lys349Asn]), an orphan G protein-coupled receptor (GPCR) expressed predominantly in the brain. In vitro studies demonstrated that the K349N substitution in Gpr37L1 did not grossly alter receptor expression, surface trafficking or constitutive signaling in transfected cells. However, in vivo studies revealed that a complete loss of Gpr37L1 function in mice results in increased seizure susceptibility. Mice lacking the related receptor Gpr37 also exhibited an increase in seizure susceptibility, while genetic deletion of both receptors resulted in an even more dramatic increase in vulnerability to seizures. These findings provide evidence linking GPR37L1 and GPR37 to seizure etiology and demonstrate an association between a GPR37L1 variant and a novel progressive myoclonus epilepsy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (© 2017 American Society of Plant Biologists. All rights reserved.)

Background: Automated plant phenotyping has been established as a powerful new tool in studying plant growth, development and response to various types of biotic or abiotic stressors. Respective facilities mainly apply non-invasive imaging based methods, which enable the continuous quantification of the dynamics of plant growth and physiology during developmental progression. However, especially for plants of larger size, integrative, automated and high throughput measurements of complex physiological parameters such as photosystem II efficiency determined through kinetic chlorophyll fluorescence analysis remain a challenge., Results: We present the technical installations and the establishment of experimental procedures that allow the integrated high throughput imaging of all commonly determined PSII parameters for small and large plants using kinetic chlorophyll fluorescence imaging systems (FluorCam, PSI) integrated into automated phenotyping facilities (Scanalyzer, LemnaTec). Besides determination of the maximum PSII efficiency, we focused on implementation of high throughput amenable protocols recording PSII operating efficiency (Φ PSII ). Using the presented setup, this parameter is shown to be reproducibly measured in differently sized plants despite the corresponding variation in distance between plants and light source that caused small differences in incident light intensity. Values of Φ PSII obtained with the automated chlorophyll fluorescence imaging setup correlated very well with conventionally determined data using a spot-measuring chlorophyll fluorometer. The established high throughput operating protocols enable the screening of up to 1080 small and 184 large plants per hour, respectively. The application of the implemented high throughput protocols is demonstrated in screening experiments performed with large Arabidopsis and maize populations assessing natural variation in PSII efficiency., Conclusions: The incorporation of imaging systems suitable for kinetic chlorophyll fluorescence analysis leads to a substantial extension of the feature spectrum to be assessed in the presented high throughput automated plant phenotyping platforms, thus enabling the simultaneous assessment of plant architectural and biomass-related traits and their relations to physiological features such as PSII operating efficiency. The implemented high throughput protocols are applicable to a broad spectrum of model and crop plants of different sizes (up to 1.80 m height) and architectures. The deeper understanding of the relation of plant architecture, biomass formation and photosynthetic efficiency has a great potential with respect to crop and yield improvement strategies.

Primary root growth in Arabidopsis and a number of other species has previously been shown to be remarkably sensitive to the presence of external glutamate, with glutamate signalling eliciting major changes in root architecture. Using two recombinant inbred lines from reciprocal crosses between Arabidopsis accessions C24 and Col-0, we have identified one large-effect quantitative trait locus (QTL), GluS1, and two minor QTLs, GluS2 and GluS3, which together accounted for 41% of the phenotypic variance in glutamate sensitivity. The presence of the GluS1 locus on chromosome 3 was confirmed using a set of C24/Col-0 isogenic lines. GluS1 was mapped to an interval between genes At3g44830-At3g46880. When QTL mapping was repeated under a range of environmental conditions, including temperature, shading and nitrate supply, a strong genotype-by-environment interaction in the controls for the glutamate response was identified. Major differences in the loci controlling this trait were found under different environmental conditions. Here we present evidence for the existence of loci on chromosomes 1 and 5 epistatically controlling the response of the GluS1 locus to variations in ambient temperature, between 20°C and 26°C. In addition, a locus on the long arm of chromosome 1 was found to play a major role in controlling the ability of external nitrate signals to antagonize the glutamate effect. We conclude that there are multiple loci controlling natural variation in glutamate sensitivity in Arabidopsis roots and that epistatic interactions play an important role in modulating glutamate sensitivity in response to changes in environmental conditions., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

To gain insight into genetic factors controlling seed metabolic composition and its relationship to major seed properties, an Arabidopsis recombinant inbred line (RIL) population, derived from accessions Col-0 and C24, was studied using an MS-based metabolic profiling approach. Relative intensities of 311 polar primary metabolites were used to identify associated genomic loci and to elucidate their interactions by quantitative trait locus (QTL) mapping. A total of 786 metabolic QTLs (mQTLs) were unequally distributed across the genome, forming several hotspots. For the branched-chain amino acid leucine, mQTLs and candidate genes were elucidated in detail. Correlation studies displayed links between metabolite levels, seed protein content, and seed weight. Principal component analysis revealed a clustering of samples, with PC1 mapping to a region on the short arm of chromosome IV. The overlap of this region with mQTL hotspots indicates the presence of a potential master regulatory locus of seed metabolism. As a result of database queries, a series of candidate regulatory genes, including bZIP10, were identified within this region. Depending on the search conditions, metabolic pathway-derived candidate genes for 40-61% of tested mQTLs could be determined, providing an extensive basis for further identification and characterization of hitherto unknown genes causal for natural variation of Arabidopsis seed metabolism., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Fumarate and malate are known intermediates of the TCA cycle, a mitochondrial metabolic pathway generating NADH for respiration. Arabidopsis thaliana and other Brassicaceae contain an additional cytosolic fumarase (FUM2) that functions in carbon assimilation and nitrogen use. Here, we report the identification of a hitherto unknown FUM2 promoter insertion/deletion (InDel) polymorphism found between the Col-0 and C24 accessions, which also divides a large number of Arabidopsis accessions carrying either the Col-0 or the C24 allele. The polymorphism consists of two stretches of 2.1 and 3.8 kb, which are both absent from the promotor region of Col-0 FUM2. By analysing mutants as well as mapping and natural populations with contrasting FUM2 alleles, the promotor insertion was linked to reduced FUM2 mRNA expression, reduced fumarase activity and reduced fumarate/malate ratio in leaves. In a large population of 174 natural accessions, the polymorphism was also found to be associated with the fumarate/malate ratio, malate and fumarate levels, and with dry weight at 15 days after sowing (DAS). The association with biomass production was confirmed in an even larger (251) accession population for dry weight at 22 DAS. The dominant Col-0 allele that results in increased fumarate/malate ratios and enhanced biomass production is predominantly found in central/eastern European accessions, whereas the C24 type allele is prevalent on the Iberian Peninsula, west of the Rhine and in the British Isles. Our findings support the role of FUM2 in diurnal carbon storage, and point to a growth advantage of accessions carrying the FUM2 Col-0 allele., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Cocaine blocks plasma membrane monoamine transporters and increases extracellular levels of dopamine (DA), norepinephrine (NE) and serotonin (5-HT). The addictive properties of cocaine are mediated primarily by DA, while NE and 5-HT play modulatory roles. Chronic inhibition of dopamine β-hydroxylase (DBH), which converts DA to NE, increases the aversive effects of cocaine and reduces cocaine use in humans, and produces behavioral hypersensitivity to cocaine and D2 agonism in rodents, but the underlying mechanism is unknown. We found a decrease in β-arrestin2 (βArr2) in the nucleus accumbens (NAc) following chronic genetic or pharmacological DBH inhibition, and overexpression of βArr2 in the NAc normalized cocaine-induced locomotion in DBH knockout (Dbh -/-) mice. The D2/3 agonist quinpirole decreased excitability in NAc medium spiny neurons (MSNs) from control, but not Dbh -/- animals, where instead there was a trend for an excitatory effect. The Gαi inhibitor NF023 abolished the quinpirole-induced decrease in excitability in control MSNs, but had no effect in Dbh -/- MSNs, whereas the Gαs inhibitor NF449 restored the ability of quinpirole to decrease excitability in Dbh -/- MSNs, but had no effect in control MSNs. These results suggest that chronic loss of noradrenergic tone alters behavioral responses to cocaine via decreases in βArr2 and cellular responses to D2/D3 activation, potentially via changes in D2-like receptor G-protein coupling in NAc MSNs., (© 2014 Society for the Study of Addiction.)

Organisms adopt a wide range of strategies to adapt to change. Gene silencing describes the ability of organisms to modulate the expression of susceptible genes at certain times at the transcriptional or the translational level. In all known eukaryotic organisms 21-nt long short interfering RNAs (siRNAs) are the effector molecules of post-transcriptional gene silencing (PTGS), while 24-nt long siRNAs are involved in PTGS in plants. Mutant studies in Caenorhabditis elegans lead to the identification of the enzyme ERI (Enhancer of RNAinterference) with enhanced PTGS. Although the genes involved in growth vigor and growth rate are still unknown, it becomes clearer that the population of small RNAs plays a role in the very early phase of plant development. To pinpoint the link between growth and siRNAs, the expression of Arabidopsis uni-gene Enhancer of RNAi (ERI) homolog from C. elegans was modulated. Increased degradation of small RNAs was achieved by ectopic AtERI overexpression in planta. Based on global small RNA analysis, AtERI overexpression affects mainly the population of 21 mers, excluding miRNAs. To identify target genes, AtERI gain-of-function mutants were analyzed, and differentially abundant small RNAs were identified. Plants with an elevated level of AtERI were bigger in all three light intensities analyzed, indicating an inhibitory function of particular small RNAs in plant growth, with differences in relative growth rates depending on developmental stage and light intensity. Understanding the role of these siRNAs could open new avenues for enhancing plant growth.

Detailed and standardized protocols for plant cultivation in environmentally controlled conditions are an essential prerequisite to conduct reproducible experiments with precisely defined treatments. Setting up appropriate and well defined experimental procedures is thus crucial for the generation of solid evidence and indispensable for successful plant research. Non-invasive and high throughput (HT) phenotyping technologies offer the opportunity to monitor and quantify performance dynamics of several hundreds of plants at a time. Compared to small scale plant cultivations, HT systems have much higher demands, from a conceptual and a logistic point of view, on experimental design, as well as the actual plant cultivation conditions, and the image analysis and statistical methods for data evaluation. Furthermore, cultivation conditions need to be designed that elicit plant performance characteristics corresponding to those under natural conditions. This manuscript describes critical steps in the optimization of procedures for HT plant phenotyping systems. Starting with the model plant Arabidopsis, HT-compatible methods were tested, and optimized with regard to growth substrate, soil coverage, watering regime, experimental design (considering environmental inhomogeneities) in automated plant cultivation and imaging systems. As revealed by metabolite profiling, plant movement did not affect the plants' physiological status. Based on these results, procedures for maize HT cultivation and monitoring were established. Variation of maize vegetative growth in the HT phenotyping system did match well with that observed in the field. The presented results outline important issues to be considered in the design of HT phenotyping experiments for model and crop plants. It thereby provides guidelines for the setup of HT experimental procedures, which are required for the generation of reliable and reproducible data of phenotypic variation for a broad range of applications.

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.

Prosaposin (also known as SGP-1) is an intriguing multifunctional protein that plays roles both intracellularly, as a regulator of lysosomal enzyme function, and extracellularly, as a secreted factor with neuroprotective and glioprotective effects. Following secretion, prosaposin can undergo endocytosis via an interaction with the low-density lipoprotein-related receptor 1 (LRP1). The ability of secreted prosaposin to promote protective effects in the nervous system is known to involve activation of G proteins, and the orphan G protein-coupled receptors GPR37 and GPR37L1 have recently been shown to mediate signaling induced by both prosaposin and a fragment of prosaposin known as prosaptide. In this review, we describe recent advances in our understanding of prosaposin, its receptors and their importance in the nervous system., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Seedling establishment is inhibited on media containing high levels (∼ 6%) of glucose or fructose. Genetic loci that overcome the inhibition of seedling growth on high sugar have been identified using natural variation analysis and mutant selection, providing insight into sugar signaling pathways. In this study, a quantitative trait locus (QTL) analysis was performed for seedling sensitivity to high sugar in a Col/C24 F2 population of Arabidopsis thaliana. A glucose and fructose-sensing QTL, GSQ11, was mapped through selective genotyping and confirmed in near-isogenic lines in both Col and C24 backgrounds. Allelism tests and transgenic complementation showed that GSQ11 lies within the ANAC060 gene. The Col ANAC060 allele confers sugar insensitivity and was dominant over the sugar-sensitive C24 allele. Genomic and mRNA analyses showed that a single-nucleotide polymorphism (SNP) in Col ANAC060 affects the splicing patterns of ANAC060 such that 20 additional nucleotides are present in the mRNA. The insertion created a stop codon, resulting in a truncated ANAC60 protein lacking the transmembrane domain (TMD) that is present in the C24 ANAC060 protein. The absence of the TMD results in the nuclear localization of ANAC060. The short version of the ANAC060 protein is found in ∼ 12% of natural Arabidopsis accessions. Glucose induces GSQ11/ANAC060 expression in a process that requires abscisic acid (ABA) signaling. Chromatin immunoprecipitation-qPCR and transient expression analysis showed that ABI4 directly binds to the GSQ11/ANAC060 promoter to activate transcription. Interestingly, Col ANAC060 reduced ABA sensitivity and Glc-induced ABA accumulation, and ABI4 expression was also reduced in Col ANAC060 lines. Thus, the sugar-ABA signaling cascade induces ANAC060 expression, but the truncated Col ANAC060 protein attenuates ABA induction and ABA signaling. This negative feedback from nuclear ANAC060 on ABA signaling results in sugar insensitivity.

Botulinum neurotoxins (BoNTs) are the most poisonous substances known and cause the severe disease botulism. BoNTs have also been remarkably effective as therapeutics in treating many neuronal and neuromuscular disorders. One of the hallmarks of BoNTs, particularly serotype A, is its long persistence of 2-6 months in patients at concentrations as low as fM or pM. The mechanisms for this persistence are currently unclear. In this study we determined the persistence of the BoNT/A subtypes 1 through 5 in primary rat spinal neurons. Remarkably, the duration of intracellular enzymatic activity of BoNT/A1, /A2, /A4 and /A5 was shown to be at least 10 months. Conversely, the effects of BoNT/A3 were observed for up to ∼5 months. An intermittent dosing with BoNT/E showed intracellular activity of the shorter acting BoNT/E for 2-3 weeks, followed by reoccurrence and persistence of BoNT/A-induced SNAP-25 cleavage products.

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca(2+). However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca(2+)-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca(2+)-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca(2+) enhanced mGluR1α-mediated intracellular Ca(2+) responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca(2+) diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca(2+), respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca(2+) binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca(2+). These studies reveal that binding of extracellular Ca(2+) to the predicted Ca(2+)-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.

GPR37 (also known as Pael-R) and GPR37L1 are orphan G protein-coupled receptors that are almost exclusively expressed in the nervous system. We screened these receptors for potential activation by various orphan neuropeptides, and these screens yielded a single positive hit: prosaptide, which promoted the endocytosis of GPR37 and GPR37L1, bound to both receptors and activated signaling in a GPR37- and GPR37L1-dependent manner. Prosaptide stimulation of cells transfected with GPR37 or GPR37L1 induced the phosphorylation of ERK in a pertussis toxin-sensitive manner, stimulated (35)S-GTPγS binding, and promoted the inhibition of forskolin-stimulated cAMP production. Because prosaptide is the active fragment of the secreted neuroprotective and glioprotective factor prosaposin (also known as sulfated glycoprotein-1), we purified full-length prosaposin and found that it also stimulated GPR37 and GPR37L1 signaling. Moreover, both prosaptide and prosaposin were found to protect primary astrocytes against oxidative stress, with these protective effects being attenuated by siRNA-mediated knockdown of endogenous astrocytic GPR37 or GPR37L1. These data reveal that GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin.

Most natural Arabidopsis thaliana accessions are susceptible to one or more isolates of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). However, Arabidopsis C24 has proved resistant to all Hpa isolates tested so far. Here we describe the complex genetic basis of broad-spectrum resistance in C24. The genetics of C24 resistance to three Hpa isolates was analyzed by segregation analysis and quantitative trait locus (QTL) mapping on recombinant inbred and introgression lines. Resistance of C24 to downy mildew was found to be a multigenic trait with complex inheritance. Many identified resistance loci were isolate-specific and located on different chromosomes. Among the C24 resistance QTLs, we found dominant, codominant and recessive loci. Interestingly, none of the identified loci significantly contributed to resistance against all three tested Hpa isolates. Our study demonstrates that broad-spectrum resistance of Arabidopsis C24 to Hpa is based on different combinations of multiple isolate-specific loci. The identified quantitative resistance loci are particularly promising as they provide an important basis for the cloning of susceptibility- and immunity-related genes., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

In the Arabidopsis multiparent recombinant inbred line mapping population, a limited number of plants were detected that lacked axillary buds in most of the axils of the cauline (stem) leaves, but formed such buds in almost all rosette axils. Genetic analysis showed that polymorphisms in at least three loci together constitute this phenotype, which only occurs in late-flowering plants. Early flowering is epistatic to two of these loci, called REDUCED SHOOT BRANCHING1 (RSB1) and RSB2, which themselves do not affect flowering time. Map-based cloning and confirmation by transformation with genes from the region where RSB1 was identified by fine-mapping showed that a specific allele of AGAMOUS-Like6 from accession C24 conferred reduced branching in the cauline leaves. Site-directed mutagenesis in the Columbia allele revealed the causal amino acid substitution, which behaved as dominant negative, as was concluded from a loss-of-function mutation that showed the same phenotype in the late-flowering genetic background. This causal allele occurs at a frequency of 15% in the resequenced Arabidopsis thaliana accessions and correlated with reduced stem branching only in late-flowering accessions. The data show the importance of natural variation and epistatic interactions in revealing gene function.

Like protein-coding genes, loci that produce microRNAs (miRNAs) are generally considered to be under purifying selection, consistent with miRNA polymorphisms being able to cause disease. Nevertheless, it has been hypothesized that variation in miRNA genes may contribute to phenotypic diversity. Here we demonstrate that a naturally occurring polymorphism in the MIR164A gene affects leaf shape and shoot architecture in Arabidopsis thaliana, with the effects being modified by additional loci in the genome. A single base pair substitution in the miRNA complementary sequence alters the predicted stability of the miRNA:miRNA(∗) duplex. It thereby greatly reduces miRNA accumulation, probably because it interferes with precursor processing. We demonstrate that this is not a rare exception and that natural strains of Arabidopsis thaliana harbor dozens of similar polymorphisms that affect processing of a wide range of miRNA precursors. Our results suggest that natural variation in miRNA biogenesis resulting from cis mutations is a common contributor to phenotypic variation in plants., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To contribute to a further insight into heterosis we applied an integrative analysis to a systems biological network approach and a quantitative genetics analysis towards biomass heterosis in early Arabidopsis thaliana development. The study was performed on the parental accessions C24 and Col-0 and the reciprocal crosses. In an over-representation analysis it was tested if the overlap between the resulting gene lists of the two approaches is significantly larger than expected by chance. Top ranked genes in the results list of the systems biological analysis were significantly over-represented in the heterotic QTL candidate regions for either hybrid as well as regarding mid-parent and best-parent heterosis. This suggests that not only a few but rather several genes that influence biomass heterosis are located within each heterotic QTL region. Furthermore, the overlapping resulting genes of the two integrated approaches were particularly enriched in biomass related pathways. A chromosome-wise over-representation analysis gave rise to the hypothesis that chromosomes number 2 and 4 probably carry a majority of the genes involved in biomass heterosis in the early development of Arabidopsis thaliana.

The biochemical diversity in the plant kingdom is estimated to well exceed 100,000 distinct compounds (Weckwerth, 2003) and 4000 to 20,000 metabolites per species seem likely (Fernie et al., 2004). In recent years extensive progress has been made towards the identification of enzymes and regulatory genes working in a complex network to generate this large arsenal of metabolites. Genetic loci influencing quantitative traits, e.g. metabolites or biomass, may be mapped to associated molecular markers, a method called quantitative trait locus mapping (QTL mapping), which may facilitate the identification of novel genes in biochemical pathways. Arabidopsis thaliana, as a model organism for seed plants, is a suitable target for metabolic QTL (mQTL) studies due to the availability of highly developed molecular and genetic tools, and the extensive knowledge accumulated on the metabolite profile. While intensely studied, in particular since the availability of its complete sequence, the genome of Arabidopsis still comprises a large proportion of genes with only tentative function based on sequence homology. From a total number of 33,518 genes currently listed (TAIR 9, http://www.arabidopsis.org), only about 25% have direct experimental evidence for their molecular function and biological process, while for more than 30% no biological data are available. Modern metabolomics approaches together with continually extended genomic resources will facilitate the task of assigning functions to those genes. In our previous study we reported on the identification of mQTL (Lisec et al., 2008). In this paper, we summarize the current status of mQTL analyses and causal gene identification in Arabidopsis and present evidence that a candidate gene located within the confidence interval of a fumarate mQTL (AT5G50950) encoding a putative fumarase is likely to be the causal gene of this QTL. The total number of genes molecularly identified based on mQTL studies is still limited, but the advent of multi-parallel analysis techniques for measurement of gene expression, as well as protein and metabolite abundances and for rapid gene identification will assist in the important task of assigning enzymes and regulatory genes to the growing network of known metabolic reactions., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Plants that constitutively express otherwise inducible disease resistance traits often suffer a depressed seed yield in the absence of a challenge by pathogens. This has led to the view that inducible disease resistance is indispensable, ensuring that minimal resources are diverted from growth, reproduction and abiotic stress tolerance. The Arabidopsis genotype C24 has enhanced basal resistance, which was shown to be caused by permanent expression of normally inducible salicylic acid (SA)-regulated defences. However, the seed yield of C24 was greatly enhanced in comparison to disease-resistant mutants that display identical expression of SA defences. Under both water-replete and -limited conditions, C24 showed no difference and increased seed yield, respectively, in comparison with pathogen-susceptible genotypes. C24 was the most drought-tolerant genotype and showed elevated water productivity, defined as seed yield per plant per millilitre water consumed, and achieved this by displaying adjustments to both its development and transpiration efficiency (TE). Therefore, constitutive high levels of disease resistance in C24 do not affect drought tolerance, seed yield and seed viability. This study demonstrates that it will be possible to combine traits that elevate basal disease resistance and improve water productivity in crop species, and such traits need not be mutually exclusive., (© 2010 Blackwell Publishing Ltd.)

Background: Excitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI) have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood., Methods: We used Western blot procedures to test whether in vivo TBI in rat altered the protein expression of hippocampal GABA-A receptor subunits alpha1, alpha2, alpha3, alpha5, beta3, and gamma2 at 3 h, 6 h, 24 h, and 7 days post-injury. We then used pre-injury injections of MK-801 to block calcium influx through the NMDA receptor, diltiazem to block L-type voltage-gated calcium influx, or diazepam to enhance chloride conductance, and re-examined the protein expressions of alpha1, alpha2, alpha3, and gamma2, all of which were altered by TBI in the first study and all of which are important constituents in benzodiazepine-sensitive GABA-A receptors., Results: Western blot analysis revealed no injury-induced alterations in protein expression for GABA-A receptor alpha2 or alpha5 subunits at any time point post-injury. Significant time-dependent changes in alpha1, alpha3, beta3, and gamma2 protein expression. The pattern of alterations to GABA-A subunits was nearly identical after diltiazem and diazepam treatment, and MK-801 normalized expression of all subunits 24 hours post-TBI., Conclusions: These studies are the first to demonstrate that GABA-A receptor subunit expression is altered by TBI in vivo, and these alterations may be driven by calcium-mediated cascades in hippocampal neurons. Changes in GABA-A receptors in the hippocampus after TBI may have far-reaching consequences considering their essential importance in maintaining inhibitory balance and their extensive impact on neuronal function.

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype x environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

A recombinant inbred line (RIL) population, derived from two Arabidopsis thaliana accessions, and the corresponding testcrosses with these two original accessions were used for the development and validation of machine learning models to predict the biomass of hybrids. Genetic and metabolic information of the RILs served as predictors. Feature selection reduced the number of variables (genetic and metabolic markers) in the models by more than 80% without impairing the predictive power. Thus, potential biomarkers have been revealed. Metabolites were shown to bear information on inherited macroscopic phenotypes. This proof of concept could be interesting for breeders. The example population exhibits substantial mid-parent biomass heterosis. The results of feature selection could therefore be used to shed light on the origin of heterosis. In this respect, mainly dominance effects were detected.

The main objective of this study was to identify genomic regions involved in biomass heterosis using QTL, generation means, and mode-of-inheritance classification analyses. In a modified North Carolina Design III we backcrossed 429 recombinant inbred line and 140 introgression line populations to the two parental accessions, C24 and Col-0, whose F (1) hybrid exhibited 44% heterosis for biomass. Mid-parent heterosis in the RILs ranged from -31 to 99% for dry weight and from -58 to 143% for leaf area. We detected ten genomic positions involved in biomass heterosis at an early developmental stage, individually explaining between 2.4 and 15.7% of the phenotypic variation. While overdominant gene action was prevalent in heterotic QTL, our results suggest that a combination of dominance, overdominance and epistasis is involved in biomass heterosis in this Arabidopsis cross.

GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is an orphan G-protein-coupled receptor (GPCR) that exhibits poor plasma membrane expression when expressed in most cell types. We sought to find ways to enhance GPR37 trafficking to the cell surface to facilitate studies of GPR37 functional activity in heterologous cells. In truncation studies, we found that removing the GPR37 N-terminus (NT) dramatically enhanced the receptor's plasma membrane insertion. Further studies on sequential NT truncations revealed that removal of the first 210 amino acids increased the level of surface expression nearly as much as removal of the entire NT. In studies examining the effects of coexpression of GPR37 with a variety of other GPCRs, we observed significant increases in the level of GPR37 surface expression when the receptor was coexpressed with adenosine receptor A(2A)R or dopamine receptor D(2)R. Co-immunoprecipitation experiments revealed that full-length GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly associating with D(2)R, resulting in modestly altered D(2)R affinity for both agonists and antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds, we observed a specific interaction between GPR37 and syntenin-1, which resulted in a dramatic increase in the level of GPR37 surface expression in HEK-293 cells. These findings reveal three independent approaches (N-terminal truncation, coexpression with other receptors, and coexpression with syntenin-1) by which GPR37 surface trafficking in heterologous cells can be greatly enhanced to facilitate functional studies with this orphan receptor.

Two mapping populations of a cross between the Arabidopsis thaliana accessions Col-0 and C24 were cultivated and analyzed with respect to the levels of 181 metabolites to elucidate the biological phenomenon of heterosis at the metabolic level. The relative mid-parent heterosis in the F(1) hybrids was <20% for most metabolic traits. The first mapping population consisting of 369 recombinant inbred lines (RILs) and their test cross progeny with both parents allowed us to determine the position and effect of 147 quantitative trait loci (QTL) for metabolite absolute mid-parent heterosis (aMPH). Furthermore, we identified 153 and 83 QTL for augmented additive (Z(1)) and dominance effects (Z(2)), respectively. We identified putative candidate genes for these QTL using the aracyc database (http://www.arabidopsis.org/biocyc), and calculated the average degree of dominance, which was within the dominance and over-dominance range for most metabolites. Analyzing a second population of 41 introgression lines (ILs) and their test crosses with the recurrent parent, we identified 634 significant differences in metabolite levels. Nine per cent of these effects were classified as over-dominant, according to the mode of inheritance. A comparison of both approaches suggested epistasis as a major contributor to metabolite heterosis in Arabidopsis. A linear combination of metabolite levels was shown to significantly correlate with biomass heterosis (r = 0.62).

Rationale: Diphenhydramine (DPH) is an over-the-counter medication used in the treatment of allergic symptoms. While DPH abuse is infrequent, recent preclinical evidence suggests that DPH and cocaine combinations may have enhanced reinforcing properties., Objective: The aims were to assess the reinforcing effectiveness of cocaine and DPH alone or in combination under a second-order schedule of reinforcement and to examine the neurochemical basis of this interaction using in vivo microdialysis in awake rhesus monkeys., Materials and Methods: Cocaine (0.03-0.3 mg/kg per injection), DPH (0.3-3.0 mg/kg per injection), or a combination was available under a second-order schedule of intravenous drug reinforcement (n = 3). In microdialysis studies, noncontingent cocaine (0.1-1.0 mg/kg, iv), DPH (1.7 and 3.0 mg/kg, iv), or a combination was administered and changes in extracellular dopamine levels in the caudate nucleus were examined (n = 3-5)., Results: Cocaine and DPH dose-dependently maintained operant responding. Dose combinations of 1.0 or 1.7 mg/kg per injection DPH and 0.03 mg/kg per injection cocaine maintained greater rates of operant responding than 0.03 mg/kg per injection cocaine alone in the second component of the behavioral session. In microdialysis studies, cocaine dose-dependently increased extracellular dopamine levels, but no dose of DPH tested significantly increased dopamine levels above baseline. Moreover, combining DPH with cocaine did not enhance cocaine-induced dopamine increases., Conclusions: The results support previous evidence of enhanced reinforcement with cocaine and DPH combinations and extend this finding to operant behavior maintained under a second-order schedule. However, the reinforcing effects of DPH alone or in combination with cocaine do not appear to be mediated via changes in dopamine overflow.

Libraries of near-isogenic lines (NILs) are a powerful plant genetic resource to map quantitative trait loci (QTL). Nevertheless, QTL mapping with NILs is mostly restricted to genetic main effects. Here we propose a two-step procedure to map additive-by-additive digenic epistasis with NILs. In the first step, a generation means analysis of parents, their F(1) hybrid, and one-segment NILs and their triple testcross (TTC) progenies is used to identify in a one-dimensional scan loci exhibiting QTL-by-background interactions. In a second step, one-segment NILs with significant additive-by-additive background interactions are used to produce particular two-segment NILs to test for digenic epistatic interactions between these segments. We evaluated our approach by analyzing a random subset of a genomewide Arabidopsis thaliana NIL library for growth-related traits. The results of our experimental study illustrated the potential of the presented two-step procedure to map additive-by-additive digenic epistasis with NILs. Furthermore, our findings suggested that additive main effects as well as additive-by-additive digenic epistasis strongly influence the genetic architecture underlying growth-related traits of A. thaliana.

Background: Hybrids represent a cornerstone in the success story of breeding programs. The fundamental principle underlying this success is the phenomenon of hybrid vigour, or heterosis. It describes an advantage of the offspring as compared to the two parental lines with respect to parameters such as growth and resistance against abiotic or biotic stress. Dominance, overdominance or epistasis based models are commonly used explanations., Conclusion/significance: The heterosis level is clearly a function of the combination of the parents used for offspring production. This results in a major challenge for plant breeders, as usually several thousand combinations of parents have to be tested for identifying the best combinations. Thus, any approach to reliably predict heterosis levels based on properties of the parental lines would be highly beneficial for plant breeding., Methodology/principal Findings: Recently, genetic data have been used to predict heterosis. Here we show that a combination of parental genetic and metabolic markers, identified via feature selection and minimum-description-length based regression methods, significantly improves the prediction of biomass heterosis in resulting offspring. These findings will help furthering our understanding of the molecular basis of heterosis, revealing, for instance, the presence of nonlinear genotype-phenotype relationships. In addition, we describe a possible approach for accelerated selection in plant breeding.

In our analysis of 5' and 3' end formation in plant mitochondria, we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis (Arabidopsis thaliana) accessions Columbia (Col), C24, and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2, no transcripts at all were detected in Ler. This and further analyses suggest that the atp6-2 gene arrangement is absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5' termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24, and Ler/C24 F(1) hybrids revealed the differing transcript patterns of ccmC to be inherited maternally, suggesting these to arise from differences in the mitochondrial DNA. Biparental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB, and rpl5, indicating these differences to be caused by nuclear-encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible, as well as the mitochondrial cis-elements required, for 5' end generation of mitochondrial transcripts.

Leaf senescence is a developmentally regulated process that contributes to nutrient redistribution during reproductive growth and finally leads to tissue death. Manipulating leaf senescence through breeding or genetic engineering may help to improve important agronomic traits, such as crop yield and the storage life of harvested organs. Here, we studied natural variations in the regulation of plant senescence among 16 Arabidopsis thaliana accessions. Chlorophyll content and the proportion of yellow leaves were used as indicator parameters to determine leaf and plant senescence respectively. Our study indicated significant genotype effects on the onset and development of senescence. We selected three late- and five early-senescence accessions for further physiological studies. The relationship between leaf and plant senescence was accession-dependent. There was a significant correlation between plant senescence and the total number of leaves, siliques and plant bolting age. We monitored expression of two senescence marker genes, SAG12 and WRKY53, to evaluate progression of senescence. Our data revealed that chlorophyll content does not fully reflect leaf age, because even fully green leaves had already commenced senescence at the molecular level. Integrating senescence parameters, such as the proportion of senescent leaves, at the whole plant level provided a better indication of the molecular status of the plant than single leaf senescence parameters.