Andrea Galatro

Plant Survival in a Changing Environment: The Role of Nitric Oxide in Plant Responses to Abiotic Stress

Nitric oxide in plants may originate endogenously or come from surrounding atmosphere and soil. Interestingly, this gaseous free radical is far from having a constant level and varies greatly among tissues depending on a given plant’s ontogeny and environmental fluctuations. Proper plant growth, vegetative development, and reproduction require the integration of plant hormonal activity with the antioxidant network, as well as the maintenance of concentration of reactive oxygen and nitrogen species within a narrow range. Plants are frequently faced with abiotic stress conditions such as low nutrient availability, salinity, drought, high ultraviolet (UV) radiation and extreme temperatures, which can influence developmental processes and lead to growth restriction making adaptive responses the plant’s priority. The ability of plants to respond and survive under environmental-stress conditions involves sensing and signaling events where nitric oxide becomes a critical component mediating hormonal actions, interacting with reactive oxygen species, and modulating gene expression and protein activity. This review focuses on the current knowledge of the role of nitric oxide in adaptive plant responses to some specific abiotic stress conditions, particularly low mineral nutrient supply, drought, salinity and high UV-B radiation.

Effect of nitric oxide and plant antioxidants on microsomal content of lipid radicals

The antioxidant ability of nitric oxide (NO) generated by a chemical donor and of commercially available antioxidant preparations was assayed. SNAP (S-Nitroso-N-acetylpenicilamine) was used as the NO donor, and Ginkgo biloba, wheat and alfalfa preparations were tested. Lipid peroxidation was assayed by EPR employing a reaction system consisting of rat liver microsomes, ADP, FeCl3, NADPH and POBN in phosphate buffer, pH=7.4. In vitro NO exposure decreased microsomal lipid peroxidation in a dose-dependent manner. The dose responsible for inhibiting the microsomal content of lipid radical adducts by 50% (LD50) for SNAP was 550 microM (NO generation rate 0.1 microM/min). The addition of 50 microM hemoglobin to the incubation media prevented NO effect on lipid peroxidation. The addition of an amount of the antioxidant preparations equivalent to the LD50 doses inhibited lipid peroxidation by 21, 15, and 33% for wheat, alfalfa, ginkgo biloba preparations respectively in the presence of 550 microM SNAP. We detected a decrease in the content of lipid radical adducts after simultaneous supplementation, although it was less than 50%, even when LD50 doses of the products were added. This suggests that NO and the natural antioxidants inhibit lipid peroxidation by a mechanism that has both common and non-shared features.

Antioxidant capacity of a 3-deoxyanthocyanidin from soybean

Soybean cotyledons directly exposed to UV-C (190-280 nm) contained a colored pigment in those areas of the epidermis directly exposed to UV-C. Ethanolic extracts from UV-C irradiated cotyledons showed a significant peak at 532 nm at pH=10, but not seen at pH=6, successive changes in pH were accompanied by reversible changes in the spectra. The identity of the pigment isolated from soybean cotyledons was established as apigeninidin by comparing the features of standard of a apigeninidin (from sorghum) previously characterized by FAB-MS, UV, HPLC, 1H NMR, and IR spectroscopy. To characterize antioxidant activity of this compound, its ability to scavenge radical species in vitro was tested. In the concentration range tested (up to 200 microg ml (-1)), apigeninidin did not show any scavenger activity towards hydroxyl radical, quinones or NO. However, ascorbyl radical and lipid radicals were effectively quenched in a dose-dependent manner. Overall, UV-C radiation triggers molecular signals that lead in soybean cotyledons to the synthesis and accumulation of an antioxidant pigment, apigeninidin, that shows scavenger activity against ascorbyl and lipid radicals in in vitro studies.

Labile iron pool and ferritin content in developing rat brain γ-irradiated in utero

This study was aimed to assess the content of total Fe, Ferritin (Ft) and labile Fe pool (LIP) in developing rat brain exposed in utero to 1 Gy of gamma-irradiation. A significant increase (2.3-fold) in the total Fe content of the fetal rat brain irradiated in utero was observed from 1 to 4h post-irradiation, as compared to the content in non-irradiated brain. Ft was analyzed by immunoblotting. The Ft protein was composed by 20 kDa subunits. According to the analysis of the band density in the Western blot, the Ft content decreased by 77+/-15% 2h after gamma-irradiation, as compared to the values in non-irradiated samples. The effect of gamma-irradiation on the LIP was studied by both electron paramagnetic resonance (EPR) and by a fluorescence technique employing calcein (CA). A reduction on the LIP was detected at 2h post-irradiation, independently of the methodology employed for the assay. Since NO content increased in the same time frame of LIP decreasing, a protective role for NO is suggested in fetal rat brain exposed to gamma-irradiation. The data presented in this work are the first experimental evidence suggesting that, as part of the network of the cellular response to limit irradiation-dependent injury, a complex interaction between Fe and NO could be triggered.

Soybean Ferritin: Isolation, Characterization, and Free Radical Generation

The main aim of this work was to assess the multi-task role of ferritin (Ft) in the oxidative metabolism of soybean (Glycine max). Soybean seeds incubated for 24 h yielded 41 ± 5 μg Ft/g fresh weight. The rate of in vitro incorporation of iron (Fe) into Ft was tested by supplementing the reaction medium with physiological Fe chelators. The control rate, observed in the presence of 100 μM Fe, was not significantly different from the values observed in the presence of 100 μM Fe-his. However, it was significantly higher in the presence of 100 μM Fe-citrate (approximately 4.5-fold) or of 100 μM Fe-ATP (approximately 14-fold). Moreover, a substantial decrease in the Trp-dependent fluorescence of the Ft protein was determined during Fe uptake from Fe-citrate, as compared with the control. On the other hand, Ft addition to homogenates from soybean embryonic axes reduced endogenously generated ascorbyl radical, according to its capacity for Fe uptake. The data presented here suggest that Ft could be involved in the generation of free radicals, such as hydroxyl radical, by Fe-catalyzed reactions. Moreover, the scavenging of these radicals by Ft itself could then lead to protein damage. However, Ft could also prevent cellular damage by the uptake of catalytically active Fe.

Measurement of Nitric Oxide (NO) Generation Rate by Chloroplasts Employing Electron Spin Resonance (ESR)

Chloroplasts are among the more active organelles involved in free energy transduction in plants (photophosphorylation). Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was measured as an endogenous product assessed by electron paramagnetic resonance (ESR) spin-trapping technique. ESR spectroscopy is a methodology employed to detect species with unpaired electrons (paramagnetic). This technology has been successfully applied to different plant tissues and subcellular compartments to asses both, NO content and generation. The spin trap MGD-Fe(2+) is extensively employed to efficiently detect NO. Here, we describe a simple methodology to asses NO generation rate by isolated chloroplasts in the presence of either L-Arginine or nitrite (NO2 (-)) as substrates, since these compounds are required for enzymatic activities considered as the possible sources of NO generation in plants.

The stay-green phenotype of TaNAM-RNAi wheat plants is associated with maintenance of chloroplast structure and high enzymatic antioxidant activity

TaNAM transcription factors play an important role in controlling senescence, which in turn, influences the delivery of nitrogen, iron and other elements to the grain of wheat (Triticum aestivum) plants, thus contributing to grain nutritional value. While lack or diminished expression of TaNAMs determines a stay-green phenotype, the precise effect of these factors on chloroplast structure has not been studied. In this work we focused on the events undergone by chloroplasts in two wheat lines having either control or diminished TaNAM expression due to RNA interference (RNAi). It was found that in RNAi plants maintenance of chlorophyll levels and maximal photochemical efficiency of photosystem II were associated with lack of chloroplast dismantling. Flow cytometer studies and electron microscope analysis showed that RNAi plants conserved organelle ultrastructure and complexity. It was also found that senescence in control plants was accompanied by a low leaf enzymatic antioxidant activity. Lack of chloroplast dismantling in RNAi plants was associated with maintenance of protein and iron concentration in the flag leaf, the opposite being observed in control plants. These data provide a structural basis for the observation that down regulation of TaNAMs confers a functional stay-green phenotype and indicate that the low export of iron and nitrogen from the flag leaf of these plants is concomitant, within the developmental window studied, with lack of chloroplast degradation and high enzymatic antioxidant activity.

An Update to the Understanding of Nitric Oxide Metabolism in Plants

Nitric oxide (NO) is an inorganic free radical gaseous molecule which has been shown to play an unprecedented range of roles in biological systems. The potential reactions of NO are numerous and depend on many different factors. The site and source of production, as well as the concentration of NO collectively determine whether NO will elicit direct or indirect effects. In animals, NO is generated by the activity of nitric oxide synthase (NOS). In plants, neither the gene nor protein similar to known NOS has been found. However, different pathways producing NO in plants have been described, and can be classified as either oxidative or reductive steps. These sources of NO seem to cooperate to the growth and development, and to respond to several stress situations like abiotic stress. Chloroplasts are key organelles in plant metabolism and they seem to be involved in NO production, thus, proposed pathways for NO generation in chloroplasts are discussed.

Key acclimation responses to phosphorus deficiency in maize plants are influenced by exogenous nitric oxide

Improving phosphorus (P) acquisition and utilization in crops is of great importance in order to achieve a good plant nutritional state and maximize biomass production while minimizing the addition of fertilizers, and the concomitant risk of eutrophication. This study explores to which extent key processes involved in P-acquisition, and other acclimation mechanisms to low P supply in maize (Zea mays L.) plants, are affected by the addition of a nitric oxide (NO) donor (S-nitrosoglutathione, GSNO). Plants grown in a complete culture solution were exposed to four treatments performed by the combination of two P levels (0 and 0.5 mM), and two GSNO levels (0 and 0.1 mM), and responses to P-deprivation were then studied. Major plant responses related to P-deprivation were affected by the presence of the NO donor. In roots, the activity of acid phosphatases was significantly increased in P-depleted plants simultaneously exposed to GSNO. Acidification of the culture solution also increased in plants that had been grown in the presence of the NO donor. Furthermore, the potential capability displayed by roots of P-deprived plants for P-uptake, was higher in the plants that had been treated with GSNO. These results indicate that exogenous NO addition affects fundamental acclimation responses of maize plants to P scarcity, particularly and positively those that help plants to sustain P-acquisition under low P availability.

Nitric oxide and membrane lipid peroxidation in photosynthetic and non-photosynthetic organisms under several stress conditions

Oxidative damage to lipids was characterized in terms of the nature of the oxidant, the type of lipid, and the severity of the oxidation (Simontacchi et al., 2011). Even though malondialdehyde detection with the thiobarbituric acid reactive substances test (TBARS) is the most currently used assay for the determination of lipid oxidation, it is unspecific since the reaction can be reproduced by other biological compounds (Simontacchi et al., 2011). On the other hand, electron paramagnetic resonance (EPR) spectroscopy showed the capacity of detecting the presence of the lipid radicals (LR•) formed during peroxidation, by yielding unique and stable products with spin traps (Malanga and Puntarulo, 2012). Nitric oxide (NO) is recognized both, as a signaling molecule that regulates many enzyme activities, but as a toxic agent as well. It has been found that NO is able to protect animal and plant cell types from oxidative damage resulting from superoxide (O−2), hydrogen peroxide (H2O2) and alkyl peroxides by acting as a terminator of free radical chain reactions (Wink et al., 1995, 1996; Yalowich et al., 1999; Beligni and Lamattina, 2002; Sharpe et al., 2003). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) interact through the reaction of O−2 with NO, to generate peroxynitrite (ONOO−) at a rate close to diffusion. ONOO− acts as both, a nitrating agent and a powerful oxidant capable of modifying proteins (formation of nitrotyrosine), lipids (lipid oxidation, lipid nitration), and nucleic acids (DNA oxidation and DNA nitration) (Gisone et al., 2004). The purpose of this commentary is to point out that NO complex interactions with other cellular components lead to a wide range of effects depending on the biological system under study and the oxidative stress condition.