Species- and organ-specific responses of agri-environmental plants to residual agricultural pollutants.

Soil pollution by anthropogenic chemicals is a major concern for sustainability of crop production and of ecosystem functions mediated by natural plant biodiversity. The complex effects on plants are however difficult to apprehend. Plant communities of field margins, vegetative filter strips or rotational fallows are confronted with agricultural pollutants through residual soil contamination and/or through drift, run-off and leaching events that result from chemical applications. Exposure to xenobiotics and heavy metals causes biochemical, physiological and developmental effects. However, the range of doses, modalities of exposure, metabolization of contaminants into derived xenobiotics, and combinations of contaminants result in variable and multi-level effects. Understanding these complex plant-pollutant interactions cannot directly rely on toxicological or agronomical approaches that focus on the effects of field-rate pesticide applications. It must take into account exposure at root level, sublethal concentrations of bioactive compounds and functional biodiversity of the plant species that are affected. The present study deals with agri-environmental plant species of field margins, vegetative filter strips or rotational fallows in European agricultural landscapes. Root and shoot physiological and growth responses were compared under controlled conditions that were optimally adjusted for each plant species. Contrasted responses of growth inhibition, no adverse effect or growth enhancement depended on species, organ and nature of contaminant. However, all of the agricultural contaminants under study (pesticides, pesticide metabolites, heavy metals, polycyclic aromatic hydrocarbons) had significant effects under conditions of sublethal exposure on at least some of the plant species. The fungicide tebuconazole and polycyclic aromatic hydrocarbon fluoranthene, which gave highest levels of responses, induced both activation or inhibition effects, in different plant species or in different organs of the same plant species. These complex effects are discussed in terms of dynamics of agri-environmental plants and of ecological consequences of differential root-shoot growth under conditions of soil contamination.

Involvement of polyamines in sucrose-induced tolerance to atrazine-mediated chemical stress in Arabidopsis thaliana.

Treatment of Arabidopsis thaliana seedlings with the PSII-inhibiting herbicide atrazine results in xenobiotic and oxidative stress, developmental arrest, induction of senescence and cell death processes. In contrast, exogenous sucrose supply confers a high level of atrazine stress tolerance, in relation with genome-wide modifications of transcript levels and regulation of genes involved in detoxification, defense and repair. However, the regulation mechanisms related to exogenous sucrose, involved in this sucrose-induced tolerance, are largely unknown. Characterization of these mechanisms was carried out through a combination of transcriptomic, metabolic, functional and mutant analysis under different conditions of atrazine exposure. Exogenous sucrose was found to differentially regulate genes involved in polyamine synthesis. ARGININE DECARBOXYLASE ADC1 and ADC2 paralogues, which encode the rate-limiting enzyme (EC 4.1.1.19) of the first step of polyamine biosynthesis, were strongly upregulated by sucrose treatment in the presence of atrazine. Such regulation occurred concomitantly with significant changes of major polyamines (putrescine, spermidine, spermine). Physiological characterization of a mutant affected in ADC activity and exogenous treatments with sucrose, putrescine, spermidine and spermine further showed that modification of polyamine synthesis and of polyamine levels could play adaptive roles in response to atrazine stress, and that putrescine and spermine had antagonistic effects, especially in the presence of sucrose. This interplay between sucrose, putrescine and spermine is discussed in relation with survival and anti-death mechanisms in the context of chemical stress exposure.

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O2 -driven experimental evolution.

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O2 -evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD+ /NADH, leading to an optimized O2 reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.

Growth of the obligate anaerobe Desulfovibrio vulgaris Hildenborough under continuous low oxygen concentration sparging: impact of the membrane-bound oxygen reductases.

Although obligate anaerobe, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) exhibits high aerotolerance that involves several enzymatic systems, including two membrane-bound oxygen reductases, a bd-quinol oxidase and a cc(b/o)o3 cytochrome oxidase. Effect of constant low oxygen concentration on growth and morphology of the wild-type, single (Δbd, Δcox) and double deletion (Δcoxbd) mutant strains of the genes encoding these oxygen reductases was studied. When both wild-type and deletion mutant strains were cultured in lactate/sulfate medium under constant 0.02% O2 sparging, they were able to grow but the final biomasses and the growth yield were lower than that obtained under anaerobic conditions. At the end of the growth, lactate was not completely consumed and when conditions were then switched to anaerobic, growth resumed. Time-lapse microscopy revealed that a large majority of the cells were then able to divide (over 97%) but the time to recover a complete division event was longer for single deletion mutant Δbd than for the three other strains. Determination of the molar growth yields on lactate suggested that a part of the energy gained from lactate oxidation was derived toward cells protection/repairing against oxidative conditions rather than biosynthesis, and that this part was higher in the single deletion mutant Δbd and, to a lesser extent, Δcox strains. Our data show that when DvH encounters oxidative conditions, it is able to stop growing and to rapidly resume growing when conditions are switched to anaerobic, suggesting that it enters active dormancy sate under oxidative conditions. We propose that the pyruvate-ferredoxin oxidoreductase (PFOR) plays a central role in this phenomenon by reversibly switching from an oxidative-sensitive fully active state to an oxidative-insensitive inactive state. The oxygen reductases, and especially the bd-quinol oxidase, would have a crucial function by maintaining reducing conditions that permit PFOR to stay in its active state.

Jasmonate: A decision maker between cell death and acclimation in the response of plants to singlet oxygen.

Under stress conditions that bring about excessive absorption of light energy in the chloroplasts, the formation of singlet oxygen ( (1)O2) can be strongly enhanced, triggering programmed cell death. However, the (1)O2 signaling pathway can also lead to acclimation to photooxidative stress, when (1)O2 is produced in relatively low amounts. This acclimatory response is associated with a strong downregulation of the jasmonate biosynthesis pathway and the maintenance of low jasmonate levels, even under high light stress conditions that normally induce jasmonate synthesis. These findings suggest a central role for this phytohormone in the orientation of the (1)O2 signaling pathway toward cell death or acclimation. This conclusion is confirmed here in an Arabidopsis double mutant obtained by crossing the (1)O2-overproducing mutant ch1 and the jasmonate-deficient mutant dde2. This double mutant was found to be constitutively resistant to (1)O2 stress and to display a strongly stimulated growth rate compared with the single ch1 mutant. However, the involvement of other phytohormones, such as ethylene, cannot be excluded.

Light-induced acclimation of the Arabidopsis chlorina1 mutant to singlet oxygen.

Singlet oxygen (¹O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, ¹O2-induced transcriptomic changes result in acclimation to ¹O2. Here, using a chlorophyll b-less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of ¹O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to ¹O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing ¹O2 formation, indicating acclimation to ¹O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in ¹O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

Physiology and toxicology of hormone-disrupting chemicals in higher plants.

Higher plants are exposed to natural environmental organic chemicals, associated with plant-environment interactions, and xenobiotic environmental organic chemicals, associated with anthropogenic activities. The effects of these chemicals result not only from interaction with metabolic targets, but also from interaction with the complex regulatory networks of hormone signaling. Purpose-designed plant hormone analogues thus show extensive signaling effects on gene regulation and are as such important for understanding plant hormone mechanisms and for manipulating plant growth and development. Some natural environmental chemicals also act on plants through interference with the perception and transduction of endogenous hormone signals. In a number of cases, bioactive xenobiotics, including herbicides that have been designed to affect specific metabolic targets, show extensive gene regulation effects, which are more in accordance with signaling effects than with consequences of metabolic effects. Some of these effects could be due to structural analogies with plant hormones or to interference with hormone metabolism, thus resulting in situations of hormone disruption similar to animal cell endocrine disruption by xenobiotics. These hormone-disrupting effects can be superimposed on parallel metabolic effects, thus indicating that toxicological characterisation of xenobiotics must take into consideration the whole range of signaling and metabolic effects. Hormone-disruptive signaling effects probably predominate when xenobiotic concentrations are low, as occurs in situations of residual low-level pollutions. These hormone-disruptive effects in plants may thus be of importance for understanding cryptic effects of low-dosage xenobiotics, as well as the interactive effects of mixtures of xenobiotic pollutants.

Nonenzymic carotenoid oxidation and photooxidative stress signalling in plants.

Carotenoids play a crucial protective role in photosynthetic organisms as quenchers of singlet oxygen ((1)O(2)). This function occurs either via a physical mechanism involving thermal energy dissipation or via a chemical mechanism involving direct oxidation of the carotenoid molecule. The latter mechanism can produce a variety of aldehydic or ketonic cleavage products containing a reactive carbonyl group. One such molecule, the volatile ß-carotene derivative ß-cyclocitral, triggers changes in the expression of (1)O(2)-responsive genes and leads to an enhancement of photooxidative stress tolerance. Thus, besides their well-known functions in light harvesting and photoprotection, carotenoids can also play a role through their nonenzymic oxidation in the sensing and signalling of reactive oxygen species and photooxidative stress in photosynthetic organisms. Enzymic carotenoid oxidation does not seem to play a significant role in this phenomenon. Elucidation of the carotenoid-mediated (1)O(2) signalling pathway could provide new targets for improving photooxidative stress tolerance of plants.

Xenobiotic sensing and signalling in higher plants.

Anthropogenic changes and chemical pollution confront plant communities with various xenobiotic compounds or combinations of xenobiotics, involving chemical structures that are at least partially novel for plant species. Plant responses to chemical challenges and stimuli are usually characterized by the approaches of toxicology, ecotoxicology, and stress physiology. Development of transcriptomics and proteomics analysis has demonstrated the importance of modifications to gene expression in plant responses to xenobiotics. It has emerged that xenobiotic effects could involve not only biochemical and physiological disruption, but also the disruption of signalling pathways. Moreover, mutations affecting sensing and signalling pathways result in modifications of responses to xenobiotics, thus confirming interference or crosstalk between xenobiotic effects and signalling pathways. Some of these changes at gene expression, regulation and signalling levels suggest various mechanisms of xenobiotic sensing in higher plants, in accordance with xenobiotic-sensing mechanisms that have been characterized in other phyla (yeast, invertebrates, vertebrates). In higher plants, such sensing systems are difficult to identify, even though different lines of evidence, involving mutant studies, transcription factor analysis, or comparative studies, point to their existence. It remains difficult to distinguish between the hypothesis of direct xenobiotic sensing and indirect sensing of xenobiotic-related modifications. However, future characterization of xenobiotic sensing and signalling in higher plants is likely to be a key element for determining the tolerance and remediation capacities of plant species. This characterization will also be of interest for understanding evolutionary dynamics of stress adaptation and mechanisms of adaptation to novel stressors.

Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants.

(1)O(2) (singlet oxygen) is a reactive O(2) species produced from triplet excited chlorophylls in the chloroplasts, especially when plants are exposed to excess light energy. Similarly to other active O(2) species, (1)O(2) has a dual effect: It is toxic, causing oxidation of biomolecules, and it can act as a signal molecule that leads to cell death or to acclimation. Carotenoids are considered to be the main (1)O(2) quenchers in chloroplasts, and we show here that light stress induces the oxidation of the carotenoid ß-carotene in Arabidopsis plants, leading to the accumulation of different volatile derivatives. One such compound, ß-cyclocitral, was found to induce changes in the expression of a large set of genes that have been identified as (1)O(2) responsive genes. In contrast, ß-cyclocitral had little effect on the expression of H(2)O(2) gene markers. ß-Cyclocitral-induced reprogramming of gene expression was associated with an increased tolerance to photooxidative stress. The results indicate that ß-cyclocitral is a stress signal produced in high light that is able to induce defense mechanisms and represents a likely messenger involved in the (1)O(2) signaling pathway in plants.

Chemical quenching of singlet oxygen by carotenoids in plants.

Carotenoids are considered to be the first line of defense of plants against singlet oxygen ((1)O(2)) toxicity because of their capacity to quench (1)O(2) as well as triplet chlorophylls through a physical mechanism involving transfer of excitation energy followed by thermal deactivation. Here, we show that leaf carotenoids are also able to quench (1)O(2) by a chemical mechanism involving their oxidation. In vitro oxidation of ß-carotene, lutein, and zeaxanthin by (1)O(2) generated various aldehydes and endoperoxides. A search for those molecules in Arabidopsis (Arabidopsis thaliana) leaves revealed the presence of (1)O(2)-specific endoperoxides in low-light-grown plants, indicating chronic oxidation of carotenoids by (1)O(2). ß-Carotene endoperoxide, but not xanthophyll endoperoxide, rapidly accumulated during high-light stress, and this accumulation was correlated with the extent of photosystem (PS) II photoinhibition and the expression of various (1)O(2) marker genes. The selective accumulation of ß-carotene endoperoxide points at the PSII reaction centers, rather than the PSII chlorophyll antennae, as a major site of (1)O(2) accumulation in plants under high-light stress. ß-Carotene endoperoxide was found to have a relatively fast turnover, decaying in the dark with a half time of about 6 h. This carotenoid metabolite provides an early index of (1)O(2) production in leaves, the occurrence of which precedes the accumulation of fatty acid oxidation products.

Carbon dynamics, development and stress responses in Arabidopsis: involvement of the APL4 subunit of ADP-glucose pyrophosphorylase (starch synthesis).

An Arabidopsis thaliana T-DNA insertional mutant was identified and characterized for enhanced tolerance to the singlet-oxygen-generating herbicide atrazine in comparison to wild-type. This enhanced atrazine tolerance mutant was shown to be affected in the promoter structure and in the regulation of expression of the APL4 isoform of ADP-glucose pyrophosphorylase, a key enzyme of the starch biosynthesis pathway, thus resulting in decrease of APL4 mRNA levels. The impact of this regulatory mutation was confirmed by the analysis of an independent T-DNA insertional mutant also affected in the promoter of the APL4 gene. The resulting tissue-specific modifications of carbon partitioning in plantlets and the effects on plantlet growth and stress tolerance point out to specific and non-redundant roles of APL4 in root carbon dynamics, shoot-root relationships and sink regulations of photosynthesis. Given the effects of exogenous sugar treatments and of endogenous sugar levels on atrazine tolerance in wild-type Arabidopsis plantlets, atrazine tolerance of this apl4 mutant is discussed in terms of perception of carbon status and of investment of sugar allocation in xenobiotic and oxidative stress responses.

Natural variation reveals relationships between pre-stress carbohydrate nutritional status and subsequent responses to xenobiotic and oxidative stress in Arabidopsis thaliana.

BACKGROUND: Soluble sugars are involved in responses to stress, and act as signalling molecules that activate specific or hormone cross-talk transduction pathways. Thus, exogenous sucrose treatment efficiently induces tolerance to the herbicide atrazine in Arabidopsis thaliana plantlets, at least partially through large-scale modifications of expression of stress-related genes. METHODS: Availability of sugars in planta for stress responses is likely to depend on complex dynamics of soluble sugar accumulation, sucrose-starch partition and organ allocation. The question of potential relationships between endogenous sugar levels and stress responses to atrazine treatment was investigated through analysis of natural genetic accessions of A. thaliana. Parallel quantitative and statistical analysis of biochemical parameters and of stress-sensitive physiological traits was carried out on a set of 11 accessions. KEY RESULTS: Important natural variation was found between accessions of A. thaliana in pre-stress shoot endogenous sugar levels and responses of plantlets to subsequent atrazine stress. Moreover, consistent trends and statistically significant correlations were detected between specific endogenous sugar parameters, such as the pre-stress end of day sucrose level in shoots, and physiological markers of atrazine tolerance. CONCLUSIONS: These significant relationships between endogenous carbohydrate metabolism and stress response therefore point to an important integration of carbon nutritional status and induction of stress tolerance in plants. The specific correlation between pre-stress sucrose level and greater atrazine tolerance may reflect adaptive mechanisms that link sucrose accumulation, photosynthesis-related stress and sucrose induction of stress defences.

Differential patterns of reactive oxygen species and antioxidative mechanisms during atrazine injury and sucrose-induced tolerance in Arabidopsis thaliana plantlets.

BACKGROUND: Besides being essential for plant structure and metabolism, soluble carbohydrates play important roles in stress responses. Sucrose has been shown to confer to Arabidopsis seedlings a high level of tolerance to the herbicide atrazine, which causes reactive oxygen species (ROS) production and oxidative stress. The effects of atrazine and of exogenous sucrose on ROS patterns and ROS-scavenging systems were studied. Simultaneous analysis of ROS contents, expression of ROS-related genes and activities of ROS-scavenging enzymes gave an integrative view of physiological state and detoxifying potential under conditions of sensitivity or tolerance. RESULTS: Toxicity of atrazine could be related to inefficient activation of singlet oxygen (1O2) quenching pathways leading to 1O2 accumulation. Atrazine treatment also increased hydrogen peroxide (H2O2) content, while reducing gene expressions and enzymatic activities related to two major H2O2-detoxification pathways. Conversely, sucrose-protected plantlets in the presence of atrazine exhibited efficient 1O2 quenching, low 1O2 accumulation and active H2O2-detoxifying systems. CONCLUSION: In conclusion, sucrose protection was in part due to activation of specific ROS scavenging systems with consequent reduction of oxidative damages. Importance of ROS combination and potential interferences of sucrose, xenobiotic and ROS signalling pathways are discussed.

Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana: novel insights into atrazine toxicity and sucrose-induced tolerance.

BACKGROUND: Soluble sugars, which play a central role in plant structure and metabolism, are also involved in the responses to a number of stresses, and act as metabolite signalling molecules that activate specific or hormone-crosstalk transduction pathways. The different roles of exogenous sucrose in the tolerance of Arabidopsis thaliana plantlets to the herbicide atrazine and oxidative stress were studied by a transcriptomic approach using CATMA arrays. RESULTS: Parallel situations of xenobiotic stress and sucrose-induced tolerance in the presence of atrazine, of sucrose, and of sucrose plus atrazine were compared. These approaches revealed that atrazine affected gene expression and therefore seedling physiology at a much larger scale than previously described, with potential impairment of protein translation and of reactive-oxygen-species (ROS) defence mechanisms. Correlatively, sucrose-induced protection against atrazine injury was associated with important modifications of gene expression related to ROS defence mechanisms and repair mechanisms. These protection-related changes of gene expression did not result only from the effects of sucrose itself, but from combined effects of sucrose and atrazine, thus strongly suggesting important interactions of sucrose and xenobiotic signalling or of sucrose and ROS signalling. CONCLUSION: These interactions resulted in characteristic differential expression of gene families such as ascorbate peroxidases, glutathione-S-transferases and cytochrome P450s, and in the early induction of an original set of transcription factors. These genes used as molecular markers will eventually be of great importance in the context of xenobiotic tolerance and phytoremediation.