Per- and Polyfluoroalkyl Substances (PFAS) Accumulation, Reproductive Impairment, and Associations with Nestling Body Condition in Great (Parus major)- and Blue Tits (Cyanistes caeruleus) Living near a Hotspot in Belgium.

Due to the limited number of field studies investigating associations between environmentally relevant per- and polyfluoroalkyl substances (PFAS) mixtures and reproductive impairment, there is uncertainty as to whether birds are affected by PFAS pollution, whether species differ in sensitivity to PFAS, and whether the observed reproductive impairment is caused by PFAS or rather due to other potential confounding variables. Therefore, we investigated PFAS concentrations in eggs and blood plasma of great tit (Parus major) and blue tit (Cyanistes caeruleus) nestlings near a PFAS hotspot in Belgium, reproductive impairment, and associations between the accumulated levels and nestling body condition. In total, 29 eggs and 22 blood plasma samples of great tit clutches, and 10 egg and 10 blood plasma samples of blue tit clutches, were collected. Despite more types of PFAS being detected in eggs compared to plasma, only minor differences in profiles were observed between species. On the other hand, tissue-specific differences were more pronounced and likely reflect a combination of maternal transfer and dietary exposure post-hatching. Despite the high concentrations detected in both species, limited reproductive impairment was observed. Our results support previous findings that great tits and blue tits may not be very susceptible to PFAS pollution and provide evidence that other factors, including ecological stoichiometry, may be more important in explaining inter-species variation in PFAS accumulation and reproductive impairment.

Arinole, a novel auxin-stimulating benzoxazole, affects root growth and promotes adventitious root formation.

The triple response phenotype is characteristic for seedlings treated with the phytohormone ethylene or its direct precursor 1-aminocyclopropane-carboxylic acid, and is often employed to find novel chemical tools to probe ethylene responses. We identified a benzoxazole-urea derivative (B2) partially mimicking ethylene effects in a triple response bioassay. A phenotypic analysis demonstrated that B2 and its closest analogue arinole (ARI) induced phenotypic responses reminiscent of seedlings with elevated levels of auxin, including impaired hook development and inhibition of seedling growth. Specifically, ARI reduced longitudinal cell elongation in roots, while promoting cell division. In contrast to other natural or synthetic auxins, ARI mostly acts as an inducer of adventitious root development, with only limited effects on lateral root development. Quantification of free auxins and auxin biosynthetic precursors as well as auxin-related gene expression demonstrated that ARI boosts global auxin levels. In addition, analyses of auxin reporter lines and mutants, together with pharmacological assays with auxin-related inhibitors, confirmed that ARI effects are facilitated by TRYPTOPHAN AMINOTRANSFERASE1 (TAA1)-mediated auxin synthesis. ARI treatment in an array of species, including Arabidopsis, pea, tomato, poplar, and lavender, resulted in adventitious root formation, which is a desirable trait in both agriculture and horticulture.

A UPLC-MS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

New Understanding of Meta-Topolin Riboside Metabolism in Micropropagated Woody Plants.

Topolin cytokinins have emerged as valuable tools in micropropagation. This study investigates the metabolism of meta-topolin riboside (mTR) in three distinct tree species: Handroanthus guayacan and Tabebuia rosea (Bignoniaceae), and Tectona grandis (Lamiaceae). Employing labeled N15 mTR, we unraveled the complex mechanisms underlying cytokinin homeostasis, identifying N9-glucosylation as the principal deactivation pathway. Our findings demonstrate a capacity in T. rosea and H. guayacan to reposition the hydroxyl group on the cytokinin molecule, a previously unexplored metabolic pathway. Notably, this study reveals remarkable interfamilial and interspecies differences in mTR metabolism, challenging established perspectives on the role of callus tissue in cytokinin storage. These insights not only illuminate the metabolic intricacies of mTR, a cytokinin with interesting applications in plant tissue culture, but also enhances our understanding of cytokinin dynamics in plant systems, thereby enriching the scientific discourse on plant physiology and cytokinin biology.

Growth Hormones in Broad Bean (Vicia faba L.) and Radish (Raphanus raphanistrum subsp. sativus L.) Are Associated with Accumulated Concentrations of Perfluoroalkyl Substances.

In this study, we grew radish (Raphanus raphanistrum subsp. sativus L.) and broad beans (Vicia faba L.) in a greenhouse on soils spiked with a mixture of 15 per- and polyfluoroalkyl substances (PFASs) and investigated the association between accumulated ∑PFAS concentrations, growth, and hormone levels. Short-chained PFASs dominated aboveground tissues, whereas long-chained PFASs were most abundant in the plant roots. Our results showed that the presence or absence of exodermal Casparian strips, as well as the hydrophobicity and anion exchange capacities of PFASs, could explain the translocation of PFASs within plants. Significant associations found between accumulated PFAS concentrations and levels of gibberellins (GA1 and GA15), methionine, and indole-3-acetic acid (IAA) imply potential effects of PFASs on plant development and growth. This study provides the first evidence of associations between PFAS accumulation in plants and growth hormone levels, possibly leading to growth reduction of the apical dome and effects on the cell cycle in pericycle cells and methionine metabolism in plants.

The Effect of Topophysis on the In Vitro Development of Handroanthus guayacan and on Its Metabolism of Meta-Topolin Riboside.

An important factor affecting the uniformity of in vitro cultures is the topophysical position of the original explant. We investigated this phenomenon in Handroanthus guayacan, a tropical woody tree species. Shoots from a stock culture were separated into upper, middle and basal sections and transferred to a modified MS medium containing meta-topolin-riboside and indole-butyric acid. After 8 weeks, the middle section produced the most shoots, the longest shoots and the highest number of nodes per plant. Shoots derived from the upper section were elongated, but had the shortest internodes, while those from the basal section formed the largest callus. None of the three types of explants rooted during the proliferation phase. The topophysically dependent spatial distribution of endogenous cytokinins and auxins was determined. The topophysical effect observed could not be explained solely by analyzing the endogenous isoprenoid and auxin. However, the metabolism and distribution of the aromatic cytokinin could provide an explanation. The concentration of the meta hydroxy-substituted topolins was highest in shoots derived from the middle section. Aromatic N- and O-glucosides were much more concentrated in the leaves than in the stems. In conclusion, it is recommended to consider the explant's topophysis when developing a multiplication protocol to avoid heterogeneity in an in vitro culture.

Assessment of poly- and perfluoroalkyl substances (PFAS) in commercially available drinking straws using targeted and suspect screening approaches.

Many food contact materials (FCMs) and reusable plastics in the food industry contain poly- and perfluoroalkyl substances (PFAS), a group of synthetic pollutants that are known to be potentially harmful for wildlife, humans, and the environment. PFAS may migrate from FCMs to food consumed by humans. As a replacement for plastics, often paper and other plant-based materials are used in commercial settings. This also applies to drinking straws, where plant-based and other presumably eco-friendly straws are increasingly used to reduce plastic pollution. In order to make these materials water-repellent, PFAS are added during manufacturing but can also already be present early in the supply chain due to the use of contaminated raw materials. In the present study, we examined the PFAS concentrations in 39 different brands of straws, made from five materials (i.e. paper, bamboo, glass, stainless steel, and plastic) commercially available on the Belgian market. We combined both targeted and suspect-screening approaches to evaluate a wide range of PFAS. PFAS were found to be present in almost all types of straws, except for those made of stainless steel. PFAS were more frequently detected in plant-based materials, such as paper and bamboo. We did not observe many differences between the types of materials, or the continents of origin. The presence of PFAS in plant-based straws shows that they are not necessarily biodegradable and that the use of such straws potentially contributes to human and environmental exposure of PFAS.

Accumulation and effects of perfluoroalkyl substances in Arabidopsis thaliana in a temperature-dependent manner: an in vitro study.

The replacement of long-chained per- and polyfluoroalkyl substances (PFAS) with their short-chained homologues may have an impact on the accumulation in plants. The extent to which PFAS are absorbed by plants may differ among species and may depend on environmental factors, including temperature. The effect of an increased temperature on root uptake and translocation of PFAS in plants has been poorly studied. In addition, very few studies have examined toxicity of environmentally realistic PFAS concentrations to plants. Here, we investigated the bioaccumulation and tissue-distribution of fifteen PFAS in Arabidopsis thaliana L. grown in vitro at two different temperatures. Additionally, we examined the combined effects of temperature and PFAS accumulation on plant growth. Short-chained PFAS mainly accumulated in the leaves. The perfluorocarboxylic acid (PFCA) concentrations in roots and leaves, and the relative contribution of PFCAs to the ΣPFAS concentrations increased with carbon chain length regardless of temperature, with the exception of perfluorobutanoic acid (PFBA). An increased uptake of PFAS in leaves and roots at higher temperatures was observed for PFAS containing either eight or nine carbon atoms and could hence potentially result in higher risks for human intake. Leaf:root ratios of PFCAs followed a U-shaped pattern with carbon chain length, which is attributed to both hydrophobicity and anion exchange. Overall, no combined effects of realistic PFAS concentrations and temperature on the growth of A. thaliana were observed. PFAS exposure positively affected early root growth rates and root hair lengths, indicating a potential effect on factors involved in root hair morphogenesis. However, this effect on root growth rate became negligible later on in the exposure, and solely a temperature effect was observed after 6 days. Temperature also affected the leaf surface area. The underlying mechanisms on how PFAS stimulates root hair growth require further examination.

PFAS accumulation in several terrestrial plant and invertebrate species reveals species-specific differences.

Despite the known persistence and bioaccumulation potential of perfluoroalkyl substances (PFAS), much uncertainty exists regarding their bioavailability in the terrestrial environment. Therefore, this study investigated the influence of soil characteristics and PFAS concentrations on the adsorption of PFAS to soil and their influence on the PFAS bioavailability to terrestrial plants and invertebrates. PFAS concentrations and profile were compared among different invertebrate and plant species and differences between leaves and fruits/nuts of the plant species were assessed. Soil concentrations were primarily affected by organic carbon content. The PFAS accumulation in biota was, except for PFOA concentrations in nettles, unrelated to the soil concentrations, as well as to the soil characteristics. The PFAS profiles in soil and invertebrates were mainly dominated by PFOA and PFOS, whereas short-chained PFAS were more abundant in plant tissues. Our results show that different invertebrate taxa accumulate different PFAS, likely due to dietary differences. Both long-chained and, to lesser extent, short-chained PFAS were observed in herbivorous invertebrate taxa, whereas the carnivorous invertebrates only accumulated long-chained PFAS. Correlations were observed between PFOA concentrations in herbivorous invertebrates and in the leaves of some plant species, whereas such relationships were absent for the carnivorous spiders. It is essential to continuously monitor PFAS exposure in terrestrial organisms, taking into account differences in bioaccumulation, and subsequent potential toxicity, among taxa, in order to protect the terrestrial ecosystem.

Mild and severe salt stress responses are age-dependently regulated by abscisic acid in tomato.

Salt stress hampers plant growth and development through both osmotic and ionic imbalances. One of the key players in modulating physiological responses towards salinity is the plant hormone abscisic acid (ABA). How plants cope with salinity largely depends on the magnitude of the soil salt content (stress severity), but also on age-related developmental processes (ontogeny). Here we studied how ABA directs salt stress responses in tomato plants for both mild and severe salt stress in leaves of different ages. We used the ABA-deficient mutant notabilis, which contains a null-mutation in the gene of a rate-limiting ABA biosynthesis enzyme 9-cis-epoxycarotenoid dioxygenase (NCED1), leading to impaired stomatal closure. We showed that both old and young leaves of notabilis plants keep a steady-state transpiration and photosynthesis rate during salt stress, probably due to their dysfunctional stomatal closure. At the whole plant level, transpiration declined similar to the wild-type, impacting final growth. Notabilis leaves were able to produce osmolytes and accumulate ions in a similar way as wild-type plants, but accumulated more proline, indicating that osmotic responses were not impaired by the NCED1 mutation. Besides NCED1, also NCED2 and NCED6 are strongly upregulated under salt stress, which could explain why the notabilis mutant did not show a lower ABA content upon salt stress, except in young leaves. This might be indicative of a salt-mediated feedback mechanism on NCED2/6 in notabilis and might explain why notabilis plants seem to perform better under salt stress compared to wild-type plants with respect to biomass and water content accumulation.

Perfluoroalkylated compounds in the eggs and feathers of resident and migratory seabirds from the Antarctic Peninsula.

In this study, we investigated factors that influence the differences in exposure of perfluoroalkyl acids (PFAAs) from eight species of Antarctic seabirds, including Pygoscelis penguins, Stercorarius maccormicki, and Macronectes giganteus. We analyzed the relationship between foraging ecology (based on δ13C, δ15N, and δ34S values) and PFAAs accumulated in eggs and breast feathers. Ten out of 15 targeted PFAAs were detected in eggs compared to eight in feathers. Mean ∑PFAA concentrations in feathers ranged from 0.47 in P. antarcticus to 17.4 ng/g dry weight (dw) in S. maccormicki. In eggs, ∑PFAA concentrations ranged from 3.51 in P. adeliae to 117 ng/g dw in S. maccormicki. The highest concentrations of most PFAAs were found in trans-equatorial migrators such as S. maccormicki, probably due their high trophic position and higher concentrations of PFAAs in the Northern Hemisphere compared to the Southern Hemisphere. Based on stable isotopes correlations, our results suggest that the trophic position (δ15N) and the foraging area (δ13C and δ34S) influence PFAAs concentrations in Antarctic seabirds. Our results point to the possibility that long-distance migratory birds may have as bio-vectors in the transport of pollutants, including PFCAs, in Antarctic environments, although this must be further confirmed in future studies using a mass balanced approach, such as extractable organofluorine (EOF).

Modeling reveals posttranscriptional regulation of GA metabolism enzymes in response to drought and cold.

The hormone gibberellin (GA) controls plant growth and regulates growth responses to environmental stress. In monocotyledonous leaves, GA controls growth by regulating division-zone size. We used a systems approach to investigate the establishment of the GA distribution in the maize leaf growth zone to understand how drought and cold alter leaf growth. By developing and parameterizing a multiscale computational model that includes cell movement, growth-induced dilution, and metabolic activities, we revealed that the GA distribution is predominantly determined by variations in GA metabolism. Considering wild-type and UBI::GA20-OX-1 leaves, the model predicted the peak in GA concentration, which has been shown to determine division-zone size. Drought and cold modified enzyme transcript levels, although the model revealed that this did not explain the observed GA distributions. Instead, the model predicted that GA distributions are also mediated by posttranscriptional modifications increasing the activity of GA 20-oxidase in drought and of GA 2-oxidase in cold, which we confirmed by enzyme activity measurements. This work provides a mechanistic understanding of the role of GA metabolism in plant growth regulation.

Downsizing in plants-UV light induces pronounced morphological changes in the absence of stress.

Ultraviolet (UV) light induces a stocky phenotype in many plant species. In this study, we investigate this effect with regard to specific UV wavebands (UV-A or UV-B) and the cause for this dwarfing. UV-A- or UV-B-enrichment of growth light both resulted in a smaller cucumber (Cucumis sativus L.) phenotype, exhibiting decreased stem and petiole lengths and leaf area (LA). Effects were larger in plants grown in UV-B- than in UV-A-enriched light. In plants grown in UV-A-enriched light, decreases in stem and petiole lengths were similar independent of tissue age. In the presence of UV-B radiation, stems and petioles were progressively shorter the younger the tissue. Also, plants grown under UV-A-enriched light significantly reallocated photosynthates from shoot to root and also had thicker leaves with decreased specific LA. Our data therefore imply different morphological plant regulatory mechanisms under UV-A and UV-B radiation. There was no evidence of stress in the UV-exposed plants, neither in photosynthetic parameters, total chlorophyll content, or in accumulation of damaged DNA (cyclobutane pyrimidine dimers). The abscisic acid content of the plants also was consistent with non-stress conditions. Parameters such as total leaf antioxidant activity, leaf adaxial epidermal flavonol content and foliar total UV-absorbing pigment levels revealed successful UV acclimation of the plants. Thus, the UV-induced dwarfing, which displayed different phenotypes depending on UV wavelengths, occurred in healthy cucumber plants, implying a regulatory adjustment as part of the UV acclimation processes involving UV-A and/or UV-B photoreceptors.

Perfluoroalkylated acids (PFAAs) accumulate in field-exposed snails (Cepaea sp.) and affect their oxidative status.

Perfluoroalkyl acids (PFAAs) are a group of synthetic persistent chemicals with distinctive properties, such as a high thermal and chemical stability, that make them suitable for a wide range of applications. They have been produced since the 1950s, resulting in a global contamination of the environment and wildlife. They are resistant to biodegradation and have the tendency to bio-accumulate in organisms and bio-magnify in the food chain. However, little is known about the bioaccumulation of PFAAs in terrestrial invertebrates, including how they affect the physiology and particularly oxidative status. Therefore, we studied the bioaccumulation of PFAAs in snails that were exposed for 3 and 6 weeks along a distance gradient radiating from a well-known fluorochemical hotspot (3M). In addition, we examined the potential effects of PFAAs on the oxidative status of these snails. Finally, we tested for relationships between the concentrations of PFAAs in snails with those in soil and nettles they were feeding on and the influence of soil physicochemical properties on these relationships. Our results showed higher concentrations of PFOA and/or PFOS in almost every matrix at the 3M site, but no concentration gradient along the distance gradient. The PFOS concentrations in snails were related to those in the nettles and soil, and were affected by multiple soil properties. For PFOA, we observed no relationships between soil and biota concentrations. Short-chained PFAAs were dominant in nettles, whereas in soil and snails long-chained PFAAs were dominant. We found a significant positive correlation between peroxidase, catalase and peroxiredoxins and PFAA concentrations, suggesting that snails, in terms of oxidative stress (OS) response, are possibly susceptible to PFAAs pollution. CAPSULE: We observed a positive correlation between the levels of PFAAs and the antioxidants peroxidase, catalase and peroxiredoxins in snails, exposed on nettles grown at contaminated sites.

A rapid method for the detection and quantification of legacy and emerging per- and polyfluoroalkyl substances (PFAS) in bird feathers using UPLC-MS/MS.

The bioaccumulation and toxicity of per- and polyfluoroalkyl substances (PFAS) have raised scientific and public concern in recent decades, leading to regulatory measures for some PFAS (e.g. perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA)). In addition, the discovery of new PFAS alternatives in the environment has led to growing concern about the presence of numerous other PFAS that are used unrestricted. Feathers have been successfully applied as non-destructive indicators for various contaminants, mostly metals and persistent organic pollutants (POPs), whereas their suitability as an indicator for PFAS is still discussed. Previous studies on PFAS in feathers have focused primarily on perfluoroalkyl sulfonic acids (PFSAs) and perfluoroalkyl carboxylic acids (PFCAs); analytical methods for other groups of PFAS or PFAS alternatives in feathers are still lacking. Hence, this study aimed to develop a rapid, sensitive and reliable analytical method for determining a broad range of PFAS (N = 32) in feathers, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An extraction duration of 24 h was found to be sufficient to extract the majority of PFAS from the feathers. The extraction recovery of the internal standards ranged on average from 68% (PFBA) to 97% (PFOS). The spike recovery was within an acceptable range of at least 70% for most of the target analytes and the precision was often > 80%. A further extract clean-up using weak anion exchange (WAX) solid phase extraction (SPE), was proven unnecessary, as it resulted in a similar or lower spike recovery, and, as a consequence, a lower precision and higher quantification limit. The analytical method allows detection of low PFAS concentrations in a low quantity of matrix (i.e. small feathers). The developed LC-MS/MS method was validated and shown to be a fast, sensitive and reliable method for determining a broad range of legacy and emerging PFAS in feathers.

Cinnamaldehyde mitigates placental vascular dysfunction of gestational diabetes and protects from the associated fetal hypoxia by modulating placental angiogenesis, metabolic activity and oxidative stress.

Gestational diabetes mellitus (GDM) is a major pregnancy-related disorder with an increasing prevalence worldwide. GDM is associated with altered placental vascular functions and has severe consequences for fetal growth. There is no commonly accepted medication for GDM due to safety considerations. Actions of the currently limited therapeutic options focus exclusively on lowering the blood glucose level without paying attention to the altered placental vascular reactivity and remodelling. We used the fat-sucrose diet/streptozotocin (FSD/STZ) rat model of GDM to explore the efficacy of cinnamaldehyde (Ci; 20 mg/kg/day), a promising antidiabetic agent for GDM, and glyburide/metformin-HCl (Gly/Met; 0.6 + 100 mg/kg/day), as a reference drug for treatment of GDM, on the placenta structure and function at term pregnancy after their oral intake one week before mating onward. Through genome-wide transcriptome, biochemical, metabolome, metal analysis and histopathology we obtained an integrated understanding of their effects. GDM resulted in maternal and fetal hyperglycemia, fetal hyperinsulinemia and placental dysfunction with subsequent fetal anemia, hepatic iron deficiency and high serum erythropoietin level, reflecting fetal hypoxia. Differentially-regulated genes were overrepresented for pathways of angiogenesis, metabolic transporters and oxidative stress. Despite Ci and Gly/Met effectively alleviated the maternal and fetal glycemia, only Ci offered substantial protection from GDM-associated placental vasculopathy and prevented the fetal hypoxia. This was explained by Ci's impact on the molecular regulation of placental angiogenesis, metabolic activity and redox signaling. In conclusion, Ci provides a dual impact for the treatment of GDM at both maternal and fetal levels through its antidiabetic effect and the direct placental vasoprotective action. Lack of Gly/Met effectiveness to restore it's impaired functionality demonstrates the vital role of the placenta in developing efficient medications for GDM.

Ineffective humoral anti-tick IgY-response in birds: reaction against pathogen constituents?

Background: Variation in parasite burdens among hosts is typically related to differences in adaptive immunity. Comprehension of underlying mechanisms is hence necessary to gain better insights into endemic transmission cycles. Here we investigate whether wild songbirds that have never been exposed to ticks develop adaptive humoral immunity against endemic Ixodes ricinus ticks. Methods: Blue tits were exposed three times in succession to wild Ixodes ricinus ticks. For each infestation, serum samples were obtained. An enzyme-linked immunosorbent assay was developed, using tick salivary antigens, in order to quantify the bird's IgY response against ticks. In addition, at every sampling occasion the birds' body weight (corrected for body size) and haematocrit level was determined. Results: Individual IgY levels against the ticks' salivary proteins increased over three consecutive tick infestations, and large among-individual variation was observed. The responses were specifically directed against I. ricinus; cross-reactivity against the congeneric tree-hole tick Ixodes arboricola was negligibly low. IgY responses did not impinge on tick feeding success (engorgement weight and attachment success). Yet, those birds with the highest immune responses were more capable to reduce the acute harm (blood depletions) by compensating erythrocyte loss. Furthermore, at the end of the experiment, these birds had gained more body weight than birds with lower IgY levels. Conclusions: Latter observations can be considered as an effect of host quality and/or tolerance mechanisms. Birds anticipate the (future) costs of the activation of the immune system by ticks and/or ongoing tick-borne pathogen infections. Furthermore, although unsuccessful against tick feeding, the IgY responses may indirectly protect birds against tick-borne disease by acting against salivary protein secretions on which pathogens rely for transmission.

Glutathione Is Required for the Early Alert Response and Subsequent Acclimation in Cadmium-Exposed Arabidopsis thaliana Plants.

Pollution by cadmium (Cd) is a worldwide problem, posing risks to human health and impacting crop yield and quality. Cadmium-induced phytotoxicity arises from an imbalance between antioxidants and pro-oxidants in favour of the latter. The Cd-induced depletion of the major antioxidant glutathione (GSH) strongly contributes to this imbalance. Rather than being merely an adverse effect of Cd exposure, the rapid depletion of root GSH levels was proposed to serve as an alert response. This alarm phase is crucial for an optimal stress response, which defines acclimation later on. To obtain a better understanding on the importance of GSH in the course of these responses and how these are defined by the rapid GSH depletion, analyses were performed in the GSH-deficient cadmium-sensitive 2-1 (cad2-1) mutant. Cadmium-induced root and leaf responses related to oxidative challenge, hydrogen peroxide (H2O2), GSH, ethylene, and 1-aminocyclopropane-1-carboxylic acid (ACC) were compared between wild-type (WT) and mutant Arabidopsis thaliana plants. Although the cad2-1 mutant has significantly lower GSH levels, root GSH depletion still occurred, suggesting that the chelating capacity of GSH is prioritised over its antioxidative function. We demonstrated that responses related to GSH metabolism and ACC production were accelerated in mutant roots and that stress persisted due to suboptimal acclimation. In general, the redox imbalance in cad2-1 mutant plants and the lack of proper transient ethylene signalling contributed to this suboptimal acclimation, resulting in a more pronounced Cd effect.

Toxin variation among salamander populations: discussing potential causes and future directions.

Amphibians produce defensive chemicals which provide protection against both predators and infections. Within species, populations can differ considerably in the composition and amount of these chemical defenses. Studying intraspecific variation in toxins and linking it to environmental variables may help us to identify the selective drivers of toxin evolution, such as predation pressure and infection risk. Recently, there has been a renewed interest in the unique toxins produced by salamanders from the genus Salamandra: the samandarines. Despite this attention, intraspecific variation has largely been ignored within Salamandra-species. The aim of this study was to investigate whether geographic variation in profiles of samandarines exists, by sampling 4 populations of Salamandra atra over its range in the Dinaric Alps. In addition, we preliminary explored whether potential variation could be explained by predation (counting the number of snake species) and infection risk (cultivation and genomic analyses of collected soil samples). Salamanders from the 4 populations differed in toxin composition and in the size of their poison glands, although not in overall toxin quantity. Nor predation nor infection risk could explain this variation, as populations barely differed in these variables. Sampling over a much broader geographic range, using better estimators for predation and infection risk, will contribute to an improved understanding of how environment may shape variation in chemical defenses. Nevertheless, as the 4 populations of S. atra did differ in their toxin profiles, we propose that this species provides an interesting opportunity for further ecological and evolutionary studies on amphibian toxins.

Identifying the Pressure Points of Acute Cadmium Stress Prior to Acclimation in Arabidopsis thaliana.

The toxic metal cadmium (Cd) is a major soil pollutant. Knowledge on the acute Cd-induced stress response is required to better understand the triggers and sequence of events that precede plant acclimation. Therefore, we aimed to identify the pressure points of Cd stress using a short-term exposure set-up ranging from 0 h to 24 h. Acute responses related to glutathione (GSH), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and the oxidative challenge were studied at metabolite and/or transcript level in roots and leaves of Arabidopsis thaliana either exposed or not to 5 µM Cd. Cadmium rapidly induced root GSH depletion, which might serve as an alert response and modulator of H2O2 signalling. Concomitantly, a stimulation of root ACC levels was observed. Leaf responses were delayed and did not involve GSH depletion. After 24 h, a defined oxidative challenge became apparent, which was most pronounced in the leaves and concerted with a strong induction of leaf ACC synthesis. We suggest that root GSH depletion is required for a proper alert response rather than being a merely adverse effect. Furthermore, we propose that roots serve as command centre via a.o. root-derived ACC/ethylene to engage the leaves in a proper stress response.