Eliana Bianucci

Contribution of phytochelatins to cadmium tolerance in peanut plants

Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 μM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H₂O₂) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.

Antioxidant enzyme activities and gene expression patterns in peanut nodules during a drought and rehydration cycle

Drought stress is one of the most important environmental factors that affect plant growth and limit biomass production. Most studies focus on drought stress development but the reversibility of the effects receives less attention. Therefore, the present work aims to explore the biological nitrogen fixation (BNF) of the symbiotic association between peanut (Arachis hypogaea L.) and Bradyrhizobium sp. during a drought-recovery cycle with a focus on the response of enzyme activity and gene expression of the antioxidant system. Peanuts exposed to drought stress had impaired BNF, as indicated by lower nitrogenase activity, and decreased leghaemoglobin content; the latter was reversed to control values upon rehydration. Previous results demonstrated that reactive oxygen species (O2·- and H2O2) were accumulated as a consequence of drought stress, suggesting that nodules experience oxidative stress. In addition, marker transcripts responsive to drought, abscisic acid and H2O2 were upregulated. Increased transcript levels of glutathione reductase were associated with an increased enzyme activity but superoxide dismutase and glutathione S-transferase activities were unchanged, despite upregulated gene transcription. In contrast, increased activity of ascorbate peroxidase (APX) was unrelated with changes in cytosolic APX transcript levels suggesting isogene specificity. In conclusion, the work exemplarily demonstrates the efficient and dynamic regulation of antioxidant enzymes and marker compounds during drought cycling, which is likely to be a prerequisite for functional optimisation of nodule metabolism.

Involvement of glutathione and enzymatic defense system against cadmium toxicity in Bradyrhizobium sp. strains (peanut symbionts)

In this study, the effects of cadmium (Cd) on cell morphology and antioxidant enzyme activities as well as the distribution of the metal in different cell compartments in Bradyrhizobium sp. strains were investigated. These strains were previously classified as sensitive (Bradyrhizobium sp. SEMIA 6144) and tolerant (Bradyrhizobium sp. NLH25) to Cd. Transmission electron micrographs showed large electron-translucent inclusions in the sensitive strain and electron-dense bodies in the tolerant strain, when exposed to Cd. Analysis of Cd distribution revealed that it was mainly bounded to cell wall in both strains. Antioxidant enzyme activities were significantly different in each strain. Only the tolerant strain was able to maintain a glutathione/oxidized glutathione (GSH/GSSG) ratio by an increase of GSH reductase (GR) and GSH peroxidase (GPX) enzyme activities. GSH S-transferase (GST) and catalase (CAT) activities were drastically inhibited in both strains while superoxide dismutase (SOD) showed a significant decrease only in the sensitive strain. In conclusion, our findings suggest that GSH content and its related enzymes are involved in the Bradyrhizobium sp. tolerance to Cd contributing to the cellular redox balance.

Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium

In this work, we evaluated the effects of cadmium (Cd) on the antioxidant defense system responses and the role of nitrate reductase (NR) in the redox balance maintenance in Bradyrhizobium japonicum strains. For that, B. japonicum USDA110 and its NR defective mutant strain (GRPA1) were used. Results showed that the addition of 10μM Cd did not modify the aerobic growth of the wild type strain while the mutant strain was strongly affected. Anaerobic growth revealed that only the parental strain was able to grow under this condition. Cd reduced drastically the NR activity in B. japonicum USDA110 and increased lipid peroxide content in both strains. Cd decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in B. japonicum USDA110 although, a significant increased was observed in the mutant GRPA1. GSH-related enzymes were induced by Cd, being more evident the increase in the mutant strain. This different behavior observed between strains suggests that NR enzyme plays an important role in the redox balance maintenance in B. japonicum USDA 110 exposed to Cd.

Growth of Bradyrhizobium sp. SEMIA 6144 in Response to Methylglyoxal: Role of Glutathione

We previously showed the important role of glutathione (GSH) in the protection mechanism against different stresses, such as acid pH, saline, and oxidative stress, using a GSH-deficient mutant of Bradyrhizobium sp. (peanut microsymbiont). In this work, we studied the role of GSH in the protection mechanism against methylglyoxal (MG) toxicity. MG is a naturally occurring toxic electrophilic compound, and it has been shown that GSH is involved in the detoxification of MG in Escherichia coli. One recognized component of this detoxification process is the formation of a GSH adduct, which in turn transports potassium (K+) out of bacterial cells. Our results showed that growth of wild-type strain Bradyrhizobium sp. SEMIA 6144 was not affected at a MG concentration of 0.5 mM in the yeast extract–mannitol culture medium. However, a reduction of growth, at concentrations of 1.5 and 2.5 mM MG and reaching complete growth inhibition at 3.0 mM MG, was observed. In wild-type strain, intracellular GSH content decreased, and intracellular K+ content was unchanged, whereas GSH-deficient mutant SEMIA 6144-S7Z was unable to grow at 1.5 mM MG. The addition of external GSH to the incubation medium did not restore the growth rate either in wild-type or mutant strains. Our findings showed that GSH has not proven to be protective against the cell-growth inhibiting activity of MG. Therefore, the response of Bradyrhizobium sp. growth to MG is different from that reported in E. coli and other Gram-negative bacteria.

Dynamic responses of photosynthesis and the antioxidant system during a drought and rehydration cycle in peanut plants

Drought stress is one of the most important environmental factors that adversely affect the productivity and quality of crops. Most studies focus on elucidating plant responses to this stress but the reversibility of these effects is less known. The aim of this work was to evaluate whether drought-stressed peanut (Arachis hypogaea L.) plants were capable of recovering their metabolism upon rehydration, with a focus on their antioxidant system. Peanut plants in the flowering phase (30 days after sowing) were exposed to drought stress by withholding irrigation during 14 days and subsequent rehydration during 3 days. Under these conditions, physiological status indicators, reactive oxygen species production and antioxidant system activity were evaluated. Under drought stress, the stomatal conductance, photosynthetic quantum yield and 13C:12C ratio of the peanut plants were negatively affected, and also they accumulated reactive oxygen species. The antioxidant system of peanut plants showed increases in superoxide dismutase-, ascorbate peroxidase- and glutathione reductase-specific activities, as well as the total ascorbate content. All of these responses were reversed upon rehydration at 3 days. The efficient and dynamic regulation of variables related to photosynthesis and the antioxidant system during a drought and rehydration cycle in peanut plants was demonstrated. It is suggested that the activation of the antioxidant system could mediate the signalling of drought stress responses that enable the plant to survive and recover completely within 3 days of rehydration.

Metabolic features involved in drought stress tolerance mechanisms in peanut nodules and their contribution to biological nitrogen fixation

Legumes belong to the most important crops worldwide. They increase soil fertility due their ability to establish symbiotic associations with soil microorganisms, known as rhizobia, capable of fixing nitrogen from the atmosphere. However, they are frequently exposed to abiotic stress conditions in particular drought. Such adverse conditions impair the biological nitrogen fixation (BNF) and depend largely on the legume. Therefore, two peanut cultivars with contrasting tolerance to drought, namely the more tolerant EC-98 and the sensitive Granoleico, were investigated to elucidate the relative contribution of BNF to the tolerance to drought. The tolerant cultivar EC-98 sustained growth and BNF similar to the control condition despite the reduced water potential and photosynthesis, suggesting the functioning of distinct metabolic pathways that contributed to enhance the tolerance. The biochemical and metabolomics approaches revealed that nodules from the tolerant cultivar accumulated trehalose, proline and gamma-aminobutyric acid (GABA), metabolites with known function in protecting against drought stress. The amide metabolism was severely affected in nodules from the sensitive cultivar Granoleico as revealed by the low content of asparagine and glutamine in the drought stressed plants. The sensitive cultivar upon rehydration was unable to re-establish a metabolism similar to well-watered plants. This was evidenced by the low level of metabolites and, transcripts and specific activities of enzymes from the carbon (sucrose synthase) and nitrogen (glutamine synthetase) metabolism which decreased below the values of control plants. Therefore, the increased content of metabolites with protective functions under drought stress likely is crucial for the full restoration upon rehydration. Smaller changes of drought stress-related metabolites in nodule are another trait that contributes to the effective control of BNF in the tolerant peanut cultivar (EC-98).

Importance of Glutathione in the Legume-Rhizobia Symbiosis

Glutathione (GSH) is essential for the proper development of root nodules during the symbiotic association of legume and rhizobia. It is involved in the antioxidant defense, the detoxification of xenobiotics, and the tolerance to abiotic and biotic stresses. The high level of GSH in root nodules and the presence of an active ascorbate-glutathione (AsA-GSH) cycle suggest that GSH participates in the protection of the nitrogen-fixing process against reactive oxygen species (ROS) resulting from the active nodule metabolism. Glutathione-related enzymes also play a critical role in defense against ROS: (a) glutathione peroxidase (GPX) is a H2O2 scavenger that uses GSH as a reductant, (b) glutathione reductase (GR) reduces GSSG using NADPH as a source of reducing power and maintaining the GSH/GSSG ratio in cells, (c) glutathione-S-transferase (GST) catalyzes the nucleophilic conjugation of GSH with several electrophilic substrates, and (d) glutaredoxins (GRXs), small redox proteins from the thioredoxin (TRX) superfamily, use GSH as electron donor. In this chapter, the role of GSH and its related enzymes was analyzed in free-living rhizobia and in the symbiosis with the legumes as well as the responses to different abiotic stresses (acid pH, saline, drought, and heavy metals/metalloids).

Signaling Role of ROS in Modulating Drought Stress Tolerance

Tolerance of plants to drought stress is a consequence of the activation of multicomponent signaling pathways to achieve cellular homeostasis and promote survival. Evidence of regulatory systems that link sensing and signaling of environmental conditions and the intracellular redox status have shed light on reactive oxygen species (ROS) as key components of transduction pathways. Thus, ROS play a dual role in the response of plants to abiotic stresses functioning as toxic by-products of stress metabolism, as well as important signal transduction molecules. The signaling pathway is accomplished by the balance between the intensity, duration, and subcellular localization of ROS generation. In this sense, the subcellular compartmentalization of oxidants and the associated changes in redox state are important aspects to be considered beyond the overall ROS cellular content. Hydrogen peroxide (H2O2), the main signaling molecule studied so far, is the most likely ROS to act as messenger because of its relative stability, and it can cross membranes through aquaporins. Nowadays, signaling mechanisms involve not only toxic molecules but also detoxification systems. Effective ROS signaling may require increased flux through antioxidant components, notably those that are thiol dependent. For signal transduction, ROS can interact with other signaling pathways such as activation of NADPH oxidase dependent on monomeric G protein, lipid-derived signals, induction of MAPK, redox-sensitive transcription factors, regulation of Ca2+, and hormones. In this chapter, aspects of reactive oxygen species as signaling molecules modulating drought stress tolerance are reported.