Exploring Thrips Preference and Resistance in Flowers, Leaves, and Whole Plants of Ten Capsicum Accessions.

Capsicum species grown for pepper production suffer severely from thrips damage, urging the identification of natural resistance. Resistance levels are commonly assessed on leaves. However, Capsicum plants are flower-bearing during most of the production season, and thrips also feed on pollen and flower tissues. In order to obtain a comprehensive estimate of elements contributing to thrips resistance, flower tissues should be considered as well. Therefore, we assessed resistance to Frankliniella occidentalis in flowers, leaves, and whole plants of ten Capsicum accessions. Using choice assays, we found that thrips prefer flowers of certain accessions over others. The preference of adult thrips for flowers was positively correlated to trehalose and fructose concentration in anthers as well as to pollen quantity. Resistance measured on leaf discs and thrips population development on whole plants was significantly and positively correlated. Leaf-based resistance thus translates to reduced thrips population development. Results of the flower assays were not significantly correlated with resistance in leaves or on whole plants. This suggests that both leaves and flowers represent a different part of the resistance spectrum and should both be considered for understanding whole plant resistance and the identification of resistant Capsicum varieties.

Quercetin as a Precursor for the Synthesis of Novel Nanoscale Cu (II) Complex as a Catalyst for Alcohol Oxidation with High Antibacterial Activity.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the dietary flavonoids, distributed in medicinal plants, vegetables, and fruits. Quercetin has the ability to bind with several metal ions to increase its biological activities. In the last two decades, quercetin has attracted considerable attention due to the biological and pharmaceutical activities such as antioxidant, antibacterial, and anticancer. In the present study, quercetin and ethanolamine were used for the synthesis Schiff base complex, which was characterized by IR, 1H NMR, and 13C NMR spectroscopy. The Schiff base has been employed as a ligand for the synthesis of novel nanoscale Cu (II) complex. The product was characterized by FT-IR spectroscopy, FESEM, and XRD. Significantly, the product showed remarkable catalytic activity towards the oxidation of primary and secondary alcohols. The antibacterial activity of the final product was assessed against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) bacteria using an inhibition zone test. The synthesized nanoscale Cu (II) complex exhibited a strong antibacterial activity against both Gram-positive and Gram-negative bacteria.

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies.

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-ß-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation).

High Concentrations of Very Long Chain Leaf Wax Alkanes of Thrips Susceptible Pepper Accessions (Capsicum spp).

The cuticular wax layer can be important for plant resistance to insects. Thrips (Frankliniella occidentalis) damage was assessed on 11 pepper accessions of Capsicum annuum and C. chinense in leaf disc and whole plant assays. Thrips damage differed among the accessions. We analyzed the composition of leaf cuticular waxes of these accessions by GC-MS. The leaf wax composition was different between the two Capsicum species. In C. annuum, 1-octacosanol (C28 alcohol) was the most abundant component, whereas in C. chinense 1-triacotanol (C30 alcohol) was the prominent. Thrips susceptible accessions had significantly higher concentrations of C25-C29 n-alkanes and iso-alkanes compared to relatively resistant pepper accessions. The triterpenoids α- and ß-amyrin tended to be more abundant in resistant accessions. Our study suggests a role for very long chain wax alkanes in thrips susceptibility of pepper.

Slug Feeding Triggers Dynamic Metabolomic and Transcriptomic Responses Leading to Induced Resistance in Solanum dulcamara.

Induced plant responses to insect herbivores are well studied, but we know very little about responses to gastropod feeding. We aim to identify the temporal dynamics of signaling- and defense-related plant responses after slug feeding in relation to induced resistance. We exposed Solanum dulcamara plants to feeding by the gray field slug (GFS; Deroceras reticulatum) for different periods and tested disks of local and systemic leaves in preference assays. Induced responses were analyzed using metabolomics and transcriptomics. GFS feeding induced local and systemic responses. Slug feeding for 72 h more strongly affected the plant metabolome than 24 h feeding. It increased the levels of a glycoalkaloid (solasonine), phenolamides, anthocyanins, and trypsin protease inhibitors as well as polyphenol oxidase activity. Phytohormone and transcriptome analyses revealed that jasmonic acid, abscisic acid and salicylic acid signaling were activated. GFS feeding upregulated more genes than that it downregulated. The response directly after feeding was more than five times higher than after an additional 24 h without feeding. Our research showed that GFS, like most chewing insects, triggers anti-herbivore defenses by activating defense signaling pathways, resulting in increased resistance to further slug feeding. Slug herbivory may therefore impact other herbivores in the community.

Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia.

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism.

Metabolomics of Thrips Resistance in Pepper (Capsicum spp.) Reveals Monomer and Dimer Acyclic Diterpene Glycosides as Potential Chemical Defenses.

The development of pesticide resistance in insects and recent bans on pesticides call for the identification of natural sources of resistance in crops. Here, we used natural variation in pepper (Capsicum spp.) resistance combined with an untargeted metabolomics approach to detect secondary metabolites related to thrips (Frankliniella occidentalis) resistance. Using leaf disc choice assays, we tested 11 Capsicum accessions of C. annuum and C. chinense in both vegetative and flowering stages for thrips resistance. Metabolites in the leaves of these 11 accessions were analyzed using LC-MS based untargeted metabolomics. The choice assays showed significant differences among the accessions in thrips feeding damage. The level of resistance depended on plant developmental stage. Metabolomics analyses showed differences in metabolomes among the Capsicum species and plant developmental stages. Moreover, metabolomic profiles of resistant and susceptible accessions differed. Monomer and dimer acyclic diterpene glycosides (capsianosides) were pinpointed as metabolites that were related to thrips resistance. Sucrose and malonylated flavone glycosides were related to susceptibility. To our knowledge, this is the first time that dimer capsianosides of pepper have been linked to insect resistance. Our results show the potential of untargeted metabolomics as a tool for discovering metabolites that are important in plant - insect interactions.

Thrips Resistance Screening Is Coming of Age: Leaf Position and Ontogeny Are Important Determinants of Leaf-Based Resistance in Pepper.

Capsicum is a genus containing important crop species, many of which severely suffer from thrips infestation. Thrips feeding damages leaves and fruits, and often results in virus infections. Only a few insecticides are still effective against thrips, underlining the importance of finding natural resistance in crops. Capsicum is a perennial plant which is usually cultivated for several months, during which time the fruits are harvested. From the young vegetative stage to the mature fruit bearing stage, the plants are at risk to thrips infestation. Constitutive resistance to thrips over the entire ontogenetic development is therefore a key trait for a more sustainable and successful cultivation of the hot and sweet pepper. In addition to ontogeny, leaf position can affect the level of thrips resistance. Pest resistance levels are known to differ between young and old leaves. To our knowledge, no studies have explicitly considered ontogeny and leaf position when screening for constitutive resistance to thrips in Capsicum. In this study we analyze whether ontogeny and leaf position affect leaf-based resistance to Frankliniella occidentalis and Thrips tabaci, in 40 Capsicum accessions, comprising five different species. Our results show that resistance to both thrips species in Capsicum varies with ontogenetic stage. This variation in resistance among ontogenetic stages was not consistent among the accessions. However, accessions with constitutive resistance in both the flowering and fruit ripening stage could be identified. In addition, we found that thrips resistance is overall similar at different leaf positions within the ontogenetic stage. This implies that resistance mechanisms, such as defense compounds, are constitutively present at sufficient levels on all leaf positions. Finally, we found that resistance to F. occidentalis and resistance to T. tabaci were not correlated. This indicates that leaf-based resistance in Capsicum is thrips species-specific. Because of the variation in resistance over ontogeny, identifying Capsicum accessions with resistance over their entire lifespan is challenging. For resistance screening, accounting for leaf position may be less of a concern. To identify the defense mechanisms responsible for thrips resistance, it is important to further analyze and compare resistant and susceptible accessions.

Gastropods and Insects Prefer Different Solanum dulcamara Chemotypes.

Solanum dulcamara (Bittersweet nightshade) shows significant intraspecific variation in glycoalkaloid (GA) composition and concentration. We previously showed that constitutive differences in overall GA levels are correlated with feeding preference of the grey field slug (GFS; Deroceras reticulatum). One particularly preferred accession, ZD11, contained low GA levels, but high levels of previously unknown structurally related uronic acid conjugated compounds (UACs). Here we test whether different slug species as well as insect herbivores show similar feeding preferences among six S. dulcamara accessions with different GA chemotypes. In addition, we investigate whether slug feeding can lead to induced changes in the chemical composition and affect later arriving herbivores. A leaf disc assay using greenhouse-grown plants showed that three slug species similarly preferred accessions with low GA levels. Untargeted metabolomic analyses showed that previous slug feeding consistently increased the levels of N-caffeoyl-putrescine and a structurally related metabolite, but not the levels of GAs and UACs. Slug-induced responses only affected slug preference in one accession. A common garden experiment using the same six accessions revealed that ZD11 received the highest natural gastropod feeding damage, but suffered the lowest damage by specialist flea beetles. The latter preferred to feed on accessions with high GA levels. Our study indicates that different selection pressures imposed by generalist gastropods and specialist insects may explain part of the observed chemical diversity in S. dulcamara.

Expression of Organic Anion Transporter 1 or 3 in Human Kidney Proximal Tubule Cells Reduces Cisplatin Sensitivity.

Cisplatin is a cytostatic drug used for treatment of solid organ tumors. The main adverse effect is organic cation transporter 2 (OCT2)-mediated nephrotoxicity, observed in 30% of patients. The contribution of other renal drug transporters is elusive. Here, cisplatin-induced toxicity was evaluated in human-derived conditionally immortalized proximal tubule epithelial cells (ciPTEC) expressing renal drug transporters, including OCT2 and organic anion transporters 1 (OAT1) or 3 (OAT3). Parent ciPTEC demonstrated OCT2-dependent cisplatin toxicity (TC50 34 ± 1 µM after 24-hour exposure), as determined by cell viability. Overexpression of OAT1 and OAT3 resulted in reduced sensitivity to cisplatin (TC50 45 ± 6 and 64 ± 11 µM after 24-hour exposure, respectively). This effect was independent of OAT-mediated transport, as the OAT substrates probenecid and diclofenac did not influence cytotoxicity. Decreased cisplatin sensitivity in OAT-expressing cells was associated directly with a trend toward reduced intracellular cisplatin accumulation, explained by reduced OCT2 gene expression and activity. This was evaluated by Vmax of the OCT2-model substrate ASP+ (23.5 ± 0.1, 13.1 ± 0.3, and 21.6 ± 0.6 minutes-1 in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3, respectively). Although gene expression of cisplatin efflux transporter multidrug and toxin extrusion 1 (MATE1) was 16.2 ± 0.3-fold upregulated in ciPTEC-OAT1 and 6.1 ± 0.7-fold in ciPTEC-OAT3, toxicity was unaffected by the MATE substrate pyrimethamine, suggesting that MATE1 does not play a role in the current experimental set-up. In conclusion, OAT expression results in reduced cisplatin sensitivity in renal proximal tubule cells, explained by reduced OCT2-mediated uptake capacity. In vitro drug-induced toxicity studies should consider models that express both OCT and OAT drug transporters.

Glycoalkaloid composition explains variation in slug resistance in Solanum dulcamara.

In natural environments, plants have to deal with a wide range of different herbivores whose communities vary in time and space. It is believed that the chemical diversity within plant species has mainly arisen from selection pressures exerted by herbivores. So far, the effects of chemical diversity on plant resistance have mostly been assessed for arthropod herbivores. However, also gastropods, such as slugs, can cause extensive damage to plants. Here we investigate to what extent individual Solanum dulcamara plants differ in their resistance to slug herbivory and whether this variation can be explained by differences in secondary metabolites. We performed a series of preference assays using the grey field slug (Deroceras reticulatum) and S. dulcamara accessions from eight geographically distinct populations from the Netherlands. Significant and consistent variation in slug preference was found for individual accessions within and among populations. Metabolomic analyses showed that variation in steroidal glycoalkaloids (GAs) correlated with slug preference; accessions with high GA levels were consistently less damaged by slugs. One, strongly preferred, accession with particularly low GA levels contained high levels of structurally related steroidal compounds. These were conjugated with uronic acid instead of the glycoside moieties common for Solanum GAs. Our results illustrate how intraspecific variation in steroidal glycoside profiles affects resistance to slug feeding. This suggests that also slugs should be considered as important drivers in the co-evolution between plants and herbivores.

Quantification of Thrips Damage Using Ilastik and ImageJ Fiji.

Quantification of insect damage is an essential measurement for identifying resistance in plants. In screening for host plant resistance against thrips, the total damaged leaf area is used as a criterion to determine resistance levels. Here we present an objective novel method for analyzing thrips damage on leaf disc using the freely available software programs Ilastik and ImageJ. The protocol was developed in order to screen over 40 Capsicum lines for resistance against Frankliniella occidentalis (Western Flower Thrips) and Thrips tabaci (Onion thrips).

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.

HAWAIIAN SKIRT controls size and floral organ number by modulating CUC1 and CUC2 expression.

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT (HWS) affects organ growth and the timing of floral organ abscission. The loss-of-function hws-1 mutant exhibits fused sepals and increased organ size. To understand the molecular mechanisms of HWS during plant development, we mutagenized hws-1 seeds with ethylmethylsulphonate (EMS) and screened for mutations suppressing hws-1 associated phenotypes. We isolated the shs1/hws-1 (suppressor of hws-1) mutant in which hws-1 sepal fusion phenotype was suppressed. The shs1/hws-1 mutant carries a G→A nucleotide substitution in the MIR164 binding site of CUP-SHAPED COTYLEDON 1 (CUC1) mRNA. CUC1 and CUP-SHAPED COTYLEDON 2 (CUC2) transcript levels were altered in shs1, renamed cuc1-1D, and in hws-1 mutant. Genetic interaction analyses using single, double and triple mutants of cuc1-1D, cuc2-1D (a CUC2 mutant similar to cuc1-1D), and hws-1, demonstrate that HWS, CUC1 and CUC2 act together to control floral organ number. Loss of function of HWS is associated with larger petal size due to alterations in cell proliferation and mitotic growth, a role shared with the CUC1 gene.

Molecular, genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana.

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17-Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F-box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col-FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South-Eastern Europe approximately 5000 years ago and the FRI-Col allele to North-West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The 'American haplotype' was identified from several European localities, potentially due to return migration.

A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity.

Drug-induced nephrotoxicity still hampers drug development, because current translation from in vitro or animal studies to human lacks high predictivity. Often, renal adverse effects are recognized only during clinical stages of drug development. The current study aimed to establish a robust and a more complete human cell model suitable for screening of drug-related interactions and nephrotoxicity. In addition to endogenously expressed renal organic cation transporters and efflux transporters, conditionally immortalized proximal tubule epithelial cells (ciPTEC) were completed by transduction of cells with the organic anion transporter (OAT) 1 or OAT3. Fluorescence-activated cell sorting upon exposure to the OAT substrate fluorescein successfully enriched transduced cells. A panel of organic anions was screened for drug-interactions in ciPTEC-OAT1 and ciPTEC-OAT3. The cytotoxic response to the drug-interactions with antivirals was further examined by cell viability assays. Upon subcloning, concentration-dependent fluorescein uptake was found with a higher affinity for ciPTEC-OAT1 (Km = 0.8 ± 0.1 µM) than ciPTEC-OAT3 (Km = 3.7 ± 0.5 µM). Co-exposure to known OAT1 and/or OAT3 substrates (viz. para-aminohippurate, estrone sulfate, probenecid, furosemide, diclofenac, and cimetidine) in cultures spanning 29 passage numbers revealed relevant inhibitory potencies, confirming the robustness of our model for drug-drug interactions studies. Functional OAT1 was directly responsible for cytotoxicity of adefovir, cidofovir, and tenofovir, while a drug interaction with zidovudine was not associated with decreased cell viability. Our data demonstrate that human-derived ciPTEC-OAT1 and ciPTEC-OAT3 are promising platforms for highly predictive drug screening during early phases of drug development.

Multi-Level Interactions Between Heat Shock Factors, Heat Shock Proteins, and the Redox System Regulate Acclimation to Heat.

High temperature has become a global concern because it seriously affects the growth and reproduction of plants. Exposure of plant cells to high temperatures result in cellular damage and can even lead to cell death. Part of the damage can be ascribed to the action of reactive oxygen species (ROS), which accumulate during abiotic stresses such as heat stress. ROS are toxic and can modify other biomacromolecules including membrane lipids, DNA, and proteins. In order to protect the cells, ROS scavenging is essential. In contrast with their inherent harms, ROS also function as signaling molecules, inducing stress tolerance mechanisms. This review examines the evidence for crosstalk between the classical heat stress response, which consists of heat shock factors (HSFs) and heat shock proteins (HSPs), with the ROS network at multiple levels in the heat response process. Heat stimulates HSF activity directly, but also indirectly via ROS. HSFs in turn stimulate the expression of HSP chaperones and also affect ROS scavenger gene expression. In the short term, HSFs repress expression of superoxide dismutase scavenger genes via induction of miRNA398, while they also activate scavenger gene expression and stabilize scavenger protein activity via HSP induction. We propose that these contrasting effects allow for the boosting of the heat stress response at the very onset of the stress, while preventing subsequent oxidative damage. The described model on HSFs, HSPs, ROS, and ROS scavenger interactions seems applicable to responses to stresses other than heat and may explain the phenomenon of crossacclimation.

Modulating crossover positioning by introducing large structural changes in chromosomes.

BACKGROUND: Crossing over assures the correct segregation of the homologous chromosomes to both poles of the dividing meiocyte. This exchange of DNA creates new allelic combinations thus increasing the genetic variation present in offspring. Crossovers are not uniformly distributed along chromosomes; rather there are preferred locations where they may take place. The positioning of crossovers is known to be influenced by both exogenous and endogenous factors as well as structural features inherent to the chromosome itself. We have introduced large structural changes into Arabidopsis chromosomes and report their effects on crossover positioning. RESULTS: The introduction of large deletions and putative inversions silenced recombination over the length of the structural change. In the majority of cases analyzed, the total recombination frequency over the chromosomes was unchanged. The loss of crossovers at the sites of structural change was compensated for by increases in recombination frequencies elsewhere on the chromosomes, mostly in single intervals of one to three megabases in size. Interestingly, two independent cases of induced structural changes in the same chromosomal interval were found on both chromosomes 1 and 2. In both cases, compensatory increases in recombination frequencies were of similar strength and took place in the same chromosome region. In contrast, deletions in chromosome arms carrying the nucleolar organizing region did not change recombination frequencies in the remainder of those chromosomes. CONCLUSIONS: When taken together, these observations show that changes in the physical structure of the chromosome can have large effects on the positioning of COs within that chromosome. Moreover, different reactions to induced structural changes are observed between and within chromosomes. However, the similarity in reaction observed when looking at chromosomes carrying similar changes suggests a direct causal relation between induced change and observed reaction.

Flooding and fragment size interact to determine survival and regrowth after fragmentation in two stoloniferous Trifolium species.

Clonal plants, which reproduce by means of stolons and rhizomes, are common in frequently flooded habitats. Resilience to disturbance is an important trait enabling plants to survive in such highly disturbed habitats. Resource storage is thought to enable clonal plants to resume growth after clonal fragmentation caused by disturbance. Here we investigated if submergence prior to disturbance reduces survival and regrowth of clonal fragments and whether or not genotypes originating from highly disturbed riverine habitats are more resistant to mechanical disturbance than genotypes from less disturbed coastal dune slack habitats. We further tested if variation in survival and regrowth was affected by internode size. Clones from contrasting habitats of two closely related Trifolium species were first genotypically characterized by amplification fragment length polymorphism and then subjected to soil flooding and subsequent clonal fragmentation. These species differ with respect to their abundance in riverine and dune slack habitats, with Trifolium repens mainly occurring in riverine grasslands and Trifolium fragiferum in coastal dune slacks. Soil flooding decreased survival and regrowth by up to 80 %. Plants originating from riverine grasslands were less negatively affected by fragmentation than plants from dune slack habitats. Surprisingly, ramets did not always benefit from being attached to a larger internode, as internode size was often negatively correlated with survival after fragmentation. Regrowth, on the other hand, was generally positively correlated with internode size. These unexpected results indicate that there may be contrasting selection pressures on internode size in stoloniferous species growing in severely disturbed habitats.

Humoral signalling compounds in remote ischaemic preconditioning of the kidney, a role for the opioid receptor.

BACKGROUND: Renal ischaemia-reperfusion injury (IRI) is a common clinical problem associated with significant mortality and morbidity. One strategy to reduce this damage is remote ischaemic preconditioning (RIPC), in which brief ischaemia of a limb protects the kidney against a prolonged ischaemic insult. The mechanism of renal RIPC has not yet been elucidated. Here, we address the gap in our understanding of renal RIPC signalling, using a rat model of renal IRI and RIPC by brief hind limb ischaemia. METHODS: Rats were treated with either no RIPC, RIPC+vehicle or RIPC+ an inhibitor or antagonist of one of the following candidate signalling molecules: noradrenalin, cannabinoids, glucocorticoids, inducible nitric oxide synthase, calcitonin gene-related peptide, ganglion-mediated signalling, haem oxygenase and free radicals. Subsequently, the animals underwent 25 min of renal ischaemia and 2 days of reperfusion, after which renal function and damage were assessed. RESULTS: RIPC by three 4 min cycles of hind limb ischaemia effectively reduced renal IRI. Pre-treatment with the opioid receptor antagonist naloxone completely blocked this protective effect, when compared with animals treated with RIPC+vehicle; serum creatinine and urea increased (307.8±43.7 versus 169.5±16.7 µmol/L and 42.2±4.9 versus 27.6±2.2 mmol/L, respectively), as did the renal histological damage (score 4.2±0.7 versus 2.8±0.5) and expression of kidney injury molecule-1 (KIM-1; relative-fold increase in mRNA expression 164±18 versus 304±33). All other antagonists were without effect. CONCLUSIONS: Renal RIPC by brief hind limb ischaemia may be the result of endorphin release from the hind limb. The importance of opioid signalling in renal RIPC provides vital clues for its successful translation to the clinical setting.