Detection of Ascorbate Peroxidase (APX) Activity in Plant Tissues: Using Non-denaturing PAGE and Spectrophotometric Assay.

At the cellular level, the generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), due to different abiotic or biotic stress, causes oxidative stress that induces an imbalance in the metabolism. Among the different H2O2-scavenging enzymatic antioxidants, ascorbate peroxidase (APX) is a heme-peroxidase that plays an important role in the ascorbate-glutathione pathway using ascorbate to reduce H2O2 to water. Using non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with a spectrophotometric assay for APX activity, the protocol allows identifying diverse APX isozymes present in different organs and plant species.

Can nutrients act as signals under abiotic stress?

Plant cells are in constant communication to coordinate development processes and environmental reactions. Under stressful conditions, such communication allows the plant cells to adjust their activities and development. This is due to intercellular signaling events which involve several components. In plant development, cell-to-cell signaling is ensured by mobile signals hormones, hydrogen peroxide (H2O2), nitric oxide (NO), or hydrogen sulfide (H2S), as well as several transcription factors and small RNAs. Mineral nutrients, including macro and microelements, are determinant factors for plant growth and development and are, currently, recognized as potential signal molecules. This review aims to highlight the role of nutrients, particularly calcium, potassium, magnesium, nitrogen, phosphorus, and iron as signaling components with special attention to the mechanism of response against stress conditions.

Potassium (K+) Starvation-Induced Oxidative Stress Triggers a General Boost of Antioxidant and NADPH-Generating Systems in the Halophyte Cakile maritima.

Potassium (K+) is an essential macro-element for plant growth and development given its implication in major processes such as photosynthesis, osmoregulation, protein synthesis, and enzyme function. Using 30-day-old Cakile maritima plants as halophyte model grown under K+ deprivation for 15 days, it was analyzed at the biochemical level to determine the metabolism of reactive oxygen species (ROS), key photorespiratory enzymes, and the main NADPH-generating systems. K+ starvation-induced oxidative stress was noticed by high malondialdehyde (MDA) content associated with an increase of superoxide radical (O2•-) in leaves from K+-deficient plants. K+ shortage led to an overall increase in the activity of hydroxypyruvate reductase (HPR) and glycolate oxidase (GOX), as well as of antioxidant enzymes catalase (CAT), those of the ascorbate-glutathione cycle, peroxidase (POX), and superoxide dismutase (SOD), and the main enzymes involved in the NADPH generation in both leaves and roots. Especially remarkable was the induction of up to seven CuZn-SOD isozymes in leaves due to K+ deficiency. As a whole, data show that the K+ starvation has associated oxidative stress that boosts a biochemical response leading to a general increase of the antioxidant and NADPH-generating systems that allow the survival of the halophyte Cakile maritima.

Mechanical wounding promotes local and long distance response in the halophyte Cakile maritima through the involvement of the ROS and RNS metabolism.

Mechanical wounding in plants, which are capable of generating defense responses possibly associated with nitro-oxidative stress, can be caused by (a)biotic factors such as rain, wind, herbivores and insects. Sea rocket (Cakile maritima L.), a halophyte plant belonging to the mustard family Brassicaceae, is commonly found on sandy coasts throughout Europe. Using 7-day-old Cakile maritima L. seedlings, mechanical wounding was induced in hypocotyls by pinching with a striped-tip forceps; after 3 h, several biochemical parameters were analyzed in both the damaged and unwounded organs (green cotyledons and roots). We thus determined NO production, H2O2 content, lipid oxidation as well as protein nitration patterns; we also identified several antioxidant enzymes including catalase, superoxide dismutase (SOD) isozymes, peroxidases, ascorbate-glutathione cycle enzymes and NADP-dehydrogenases. All these parameters were differentially modulated in the damaged (hypocotyls) and unwounded organs, which clearly indicated an induction of CuZnSOD V in the three organs, an increase in protein nitration in green cotyledons and an induction of NADP-isocitrate dehydrogenase activity in roots. On the whole, our results indicate that the wounding of hypocotyls, which showed an active ROS metabolism and oxidative stress, causes long-distance signals that also trigger responses in unwounded tissues with a more active RNS metabolism. These data therefore confirm the existence of local and long-distance responses which counteract negative effects and provide appropriate responses, enabling the wounded seedlings to survive.

Alleviation of Cr(VI)-induced oxidative stress in maize (Zea mays L.) seedlings by NO and H2S donors through differential organ-dependent regulation of ROS and NADPH-recycling metabolisms.

Chromium (Cr) contamination in soil is a growing concern in relation to sustainable agricultural production and food safety. Nitric oxide (NO) and, more recently, hydrogen sulfide (H2S) are considered to be new signalling molecules with biotechnological applications in the agronomical sector. Using 9-day-old maize (Zea mays) seedlings exposed to 200µM Cr(VI), the potential mitigating effects of exogenous NO and H2S on chromium-induced stress in maize seedlings were investigated in roots, cotyledons and coleoptiles. Analysis of Cr content, lipid peroxidation, antioxidant enzymes (catalase and superoxide dismutase isozymes), peroxisomal H2O2-producing glycolate oxidase and the main NADPH-regenerating system revealed that chromium causes oxidative stress, leading to a general increase in these activities in coleptiles and roots, with the latter organ being the most affected. However, cotyledons behaved in an opposite manner. Moreover, exogenous applications of NO and H2S to Cr-stressed maize seedlings triggered a significant response, involving the virtual restoration of the values for all these activities to those observed in unstressed seedlings, although their specific impact on ROS and NADPH-recycling metabolisms depends on the seedling organ involved. Taken together, the data indicate that gas transmitters, NO and H2S, which act as a defence against the negative effects of hexavalent chromium contamination, are alternative compounds with potential biotechnological applications.

Glyphosate-induced oxidative stress in Arabidopsis thaliana affecting peroxisomal metabolism and triggers activity in the oxidative phase of the pentose phosphate pathway (OxPPP) involved in NADPH generation.

Glyphosate is a broad-spectrum systemic herbicide used worldwide. In susceptible plants, glyphosate affects the shikimate pathway and reduces aromatic amino acid synthesis. Using Arabidopsis seedlings grown in the presence of 20µM glyphosate, we analyzed H2O2, ascorbate, glutathione (GSH) and protein oxidation content as well as antioxidant catalase, superoxide dismutase (SOD) and ascorbate-glutathione cycle enzyme activity. We also examined the principal NADPH-generating system components, including glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-malic enzyme (NADP-ME) and NADP-isocitrate dehydrogenase (NADP-ICDH). Glyphosate caused a drastic reduction in growth parameters and an increase in protein oxidation. The herbicide also resulted in an overall increase in GSH content, antioxidant enzyme activity (catalase and all enzymatic components of the ascorbate-glutathione cycle) in addition to the two oxidative phase enzymes, G6PDH and 6PGDH, in the pentose phosphate pathway involved in NADPH generation. In this study, we provide new evidence on the participation of G6PDH and 6PGDH in the response to oxidative stress induced by glyphosate in Arabidopsis, in which peroxisomal enzymes, such as catalase and glycolate oxidase, are positively affected. We suggest that the NADPH provided by the oxidative phase of the pentose phosphate pathway (OxPPP) should serve to maintain glutathione reductase (GR) activity, thus preserving and regenerating the intracellular GSH pool under glyphosate-induced stress. It is particularly remarkable that the 6PGDH activity was unaffected by pro-oxidant and nitrating molecules such as H202, nitric oxide or peroxynitrite.

Modulation of superoxide dismutase (SOD) isozymes by organ development and high long-term salinity in the halophyte Cakile maritima.

Superoxide dismutase (SOD) activity catalyzes the disproportionation of superoxide radicals into hydrogen peroxide and oxygen. This enzyme is considered to be a first line of defense for controlling the production of reactive oxygen species (ROS). In this study, the number and type of SOD isozymes were identified in the principal organs (roots, stems, leaves, flowers, and seeds) of Cakile maritima. We also analyzed the way in which the activity of these SOD isozymes is modulated during development and under high long-term salinity (400 mM NaCl) stress conditions. The data indicate that this plant contains a total of ten SOD isozymes: two Mn-SODs, one Fe-SOD, and seven CuZn-SODs, with the Fe-SOD being the most prominent isozyme in the different organs analyzed. Moreover, the modulation of SOD isozymes, particularly CuZn-SODs, was only detected during development and under severe salinity stress conditions. These data suggest that, in C. maritima, the occurrence of these CuZn-SODs in roots and leaves plays an adaptive role since this CuZn-SOD isozyme might replace the diminished Fe-SOD activity under salinity stress to overcome this adverse environmental condition.