Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression

Background: Anthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana. Results: Increased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production. Conclusions: Cadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants

Cadmium-induced effects on the ethylene pathway and the link with oxidative stress in Arabidopsis thaliana

Elevated concentrations of toxic metals such as cadmium (Cd) in soils, primarily caused by anthropogenic sources (e.g. mining), are a severe problem worldwide. Plants grown on contaminated soils accumulate Cd, which consequently enters the food chain, eliciting a major threat to the public health. In plants, Cd disturbs several developmental (e.g. growth) and physiological (e.g. photosynthesis) processes. Despite its non redox-active character, Cd is also capable of inducing the production of reactive oxygen species (ROS) at the cellular level, resulting in an oxidative challenge. Excessive ROS react with virtually all biomolecules, causing cellular damage. However, controlled levels of ROS, maintained by the antioxidative defence system, act as signal transduction molecules contributing to plant acclimation to abiotic stress such as exposure to Cd. Increasing evidence suggests an existing relation between cellular redox signalling and phytohormones, key regulators of plant growth and development, in order to control defence responses. Furthermore, the phytohormone ethylene, often considered as the ‘stress hormone’, is known to mediate hormone and redox signalling processes during abiotic stress. The stress-induced oxidative burst as well as the biosynthesis of glutathione (GSH), an important antioxidant during Cd stress, were already shown to be mediated by ethylene. Therefore, the aim of the current work was to unravel the involvement of ethylene biosynthesis and signalling during the Cd-induced oxidative challenge induced by sublethal Cd concentrations (5 and 10 µM Cd) in Arabidopsis thaliana.   Verhoogde gehaltes aan toxische metalen zoals cadmium (Cd) in bodems zijn voornamelijk veroorzaakt door menselijk activiteiten zoals mijnbouw en vormen een wereldwijd probleem. Planten die op deze verontreinigde bodems groeien, stapelen Cd op en via consumptie van dit gecontamineerd plantaardig voedsel komt Cd vervolgens in de voedselketen terecht. Hierdoor vormt het een ernstige bedreiging voor de volksgezondheid. Planten blootgesteld aan Cd ondervinden verstoringen in verschillende fysiologische processen (vb. fotosynthese) met een verminderde groei tot gevolg. Hoewel Cd geen redox-actieve eigenschappen bezit, veroorzaakt het toch een toename van reactieve zuurstofvormen (ROS) op cellulair niveau. Een overmaat aan ROS kan enerzijds oxidatieve schade toebrengen aan verschillende cellulaire componenten, maar anderzijds zijn het belangrijke signaalmoleculen die bijdragen tot de bescherming van de plant tegen abiotische stress zoals Cd. Activatie van het antioxidatief verdedigingssysteem zorgt voor het behoud van de hoeveelheid ROS binnen fysiologische grenzen. Uit recent onderzoek blijkt dat er een relatie bestaat tussen redox-signalisatie en planthormonen in de verdediging van de plant tegen stress. Daarnaast zijn deze planthormonen ook belangrijk tijdens de groei en ontwikkeling van de plant. Ethyleen wordt vaak beschouwd als stresshormoon en is zowel betrokken bij de productie van ROS als bij de biosynthese van glutathion (GSH), een belangrijk antioxidant tijdens Cd stress. Het doel van deze studie is bijgevolg de rol van ethyleenproductie en -signalisatie te ontrafelen in Arabidopsis thaliana planten die oxidatieve stress ervaren als gevolg van blootstelling aan sublethale Cd concentraties (5 en 10 µM)

Cadmium-induced effects on the ethylene pathway and the link with oxidative stress in Arabidopsis thaliana

Elevated concentrations of toxic metals such as cadmium (Cd) in soils, primarily caused by anthropogenic sources (e.g. mining), are a severe problem worldwide. Plants grown on contaminated soils accumulate Cd, which consequently enters the food chain, eliciting a major threat to the public health. In plants, Cd disturbs several developmental (e.g. growth) and physiological (e.g. photosynthesis) processes. Despite its non redox-active character, Cd is also capable of inducing the production of reactive oxygen species (ROS) at the cellular level, resulting in an oxidative challenge. Excessive ROS react with virtually all biomolecules, causing cellular damage. However, controlled levels of ROS, maintained by the antioxidative defence system, act as signal transduction molecules contributing to plant acclimation to abiotic stress such as exposure to Cd. Increasing evidence suggests an existing relation between cellular redox signalling and phytohormones, key regulators of plant growth and development, in order to control defence responses. Furthermore, the phytohormone ethylene, often considered as the ‘stress hormone’, is known to mediate hormone and redox signalling processes during abiotic stress. The stress-induced oxidative burst as well as the biosynthesis of glutathione (GSH), an important antioxidant during Cd stress, were already shown to be mediated by ethylene. Therefore, the aim of the current work was to unravel the involvement of ethylene biosynthesis and signalling during the Cd-induced oxidative challenge induced by sublethal Cd concentrations (5 and 10 µM Cd) in Arabidopsis thaliana.  

De rol van plant-geassocieerde bacteriën voor de verbetering van fytoremediatie van gemengde (cadmium-tolueen) verontreiniging door wilg

Contamination of toxic metals and organic xenobiotics threatens the environment and human health. Phytoremediation is an eco-friendly remediation method, using plants and their associated microorganisms to deal with polluted soil and groundwater. Although phytoremediation is a promising method, it still faces some obstacles. These can be tackled by exploiting characteristics of plant-associated bacteria. This project aims to improve the phytoremediation potential of willow for a mixed (Cd ' toluene) contamination. In a first part, willow-associated bacteria were isolated and characterized genotypically/phenotypically. A selection of Cd-resistant bacteria, possessing the appropriate characteristics (siderophores, IAA, organic acids), were conjugated with a bacterium possessing the TOM plasmid coding for toluene degradation. Two resulting transconjugants were inoculated separately as well as together into willows, exposed to low or high concentrations of Cd and toluene. In a second par

De rol van plant-geassocieerde bacteriën voor de verbetering van fytoremediatie van gemengde (cadmium-tolueen) verontreiniging door wilg

Contamination of toxic metals and organic xenobiotics threatens the environment and human health. Phytoremediation is an eco-friendly remediation method, using plants and their associated microorganisms to deal with polluted soil and groundwater. Although phytoremediation is a promising method, it still faces some obstacles. These can be tackled by exploiting characteristics of plant-associated bacteria. This project aims to improve the phytoremediation potential of willow for a mixed (Cd ' toluene) contamination. In a first part, willow-associated bacteria were isolated and characterized genotypically/phenotypically. A selection of Cd-resistant bacteria, possessing the appropriate characteristics (siderophores, IAA, organic acids), were conjugated with a bacterium possessing the TOM plasmid coding for toluene degradation. Two resulting transconjugants were inoculated separately as well as together into willows, exposed to low or high concentrations of Cd and toluene. In a second par

Can bacteria associated with willow explain differences in Cd-accumulation capacity between different cultivars?

Phyoremediation has been proposed as an economically interesting alternative for remediation of metal contaminated soils. It can be applied on extended and diffusely contaminated areas and targets the "bioavailable" fraction of metals, which is the most relevant fraction from an environmental risk assessment perspective. The most important drawback is the long time period required for remediation process. Main limiting factors for an efficient phytoextraction are plant availability of metals, metal uptake and phytotoxicity. These constraints can be conquered by (1) choosing plant cultivars with both high metal uptake capacity and high biomass production; by (2) increasing metal availability and uptake using plant-associated bacteria capable of producing siderophores, organic acids and/or auxins; or by (3) reducing metal phytotoxicity exploiting endophytes equipped with a metal sequestration system. In this work two different willow clones growing on a Cd-contaminated site were chosen.; one with a rather high and one with a rather low Cd-accumulation capacity. All cultivable bacteria associated with both willow clones were isolated and identified using 16SrDNA ARDRA analysis followed by 16SrDNA sequencing. Further all isolated bacteria were tested for their capacity to produce siderophores, organic acids and indol acetic acid for their Cd-resistance. Both willow-associated bacterial populations were compared and if possible, differences in phenotypic characteristics were related to the different Cd-accumulation capacities of their hosts. From this bacterial collection, bacteria with the potential to improve Cd-phytoremediation will be inoculated in willow cuttings and their Cd-extraction capacity and Cd-phytotoxicity will be evaluated in a greenhouse experiment

Ethylene signalling is mediating the early cadmium-induced oxidative challenge in Arabidopsis thaliana

Cadmium (Cd) induces the generation of reactive oxygen species (ROS) and stimulates ethylene biosynthesis. The phytohormone ethylene is a regulator of many developmental and physiological plant processes as well as stress responses. Previous research indicated various links between ethylene signalling and oxidative stress. Our results support a correlation between the Cd-induced oxidative challenge and ethylene signalling in Arabidopsis thaliana leaves. The effects of 24 or 72 h exposure to 5 M Cd on plant growth and several oxidative stress-related parameters were compared between wild-type (WT) and ethylene insensitive mutants (etr1-1, ein2-1, ein3-1). Cadmium-induced responses observed in WT plants were mainly affected in etr1-1 and ein2-1 mutants, of which the growth was less inhibited by Cd exposure as compared to WT and ein3-1 mutants. Both etr1-1 and ein2-1 showed a delayed response in the glutathione (GSH) metabolism, including GSH levels and transcript levels of GSH synthesising and recycling enzymes. Furthermore, the expression of different oxidative stress marker genes was significantly lower in Cd-exposed ein2-1 mutants, evidencing that ethylene signalling is involved in early responses to Cd stress. A model for the cross-talk between ethylene signalling and oxidative stress is proposed.This work was supported by the research fund of Hasselt University (BOF08G01) through a PhD grant for Kerim Schellingen and a postdoctoral grant (FWO) for Els Keunen. Additional funding was obtained from Research Foundation Flanders (FWO) (G080709N; G.0D34.14N), the Methusalem project (08M03VGRJ) and tUL-Impuls financing. DVDS acknowledges funding from Ghent University (Bijzonder Onderzoeksfonds, BOF12/BAS/021). DVDS acknowledges funding from Ghent University. The authors wish to thank Carine Put and Ann Wijgaerts for their skilful technical assistance.Arabidopsis thaliana; cadmium; ethylene signalling; glutathione; oxidative stres