An Update on Nitric Oxide Production and Role Under Phosphorus Scarcity in Plants.

Phosphate (P) is characterized by its low availability and restricted mobility in soils, and also by a high redistribution capacity inside plants. In order to maintain P homeostasis in nutrient restricted conditions, plants have developed mechanisms which enable P acquisition from the soil solution, and an efficient reutilization of P already present in plant cells. Nitric oxide (NO) is a bioactive molecule with a plethora of functions in plants. Its endogenous synthesis depends on internal and environmental factors, and is closely tied with nitrogen (N) metabolism. Furthermore, there is evidence demonstrating that N supply affects P homeostasis and that P deficiency impacts on N assimilation. This review will provide an overview on how NO levels in planta are affected by P deficiency, the interrelationship with N metabolism, and a summary of the current understanding about the influence of this reactive N species over the processes triggered by P starvation, which could modify P use efficiency.

Nitric oxide and plant mineral nutrition: current knowledge.

Plants under conditions of essential mineral deficiency trigger signaling mechanisms that involve common components. Among these components, nitric oxide (NO) has been identified as a key participant in responses to changes in nutrient availability. Usually, nutrient imbalances affect the levels of NO in specific plant tissues, via modification of its rate of synthesis or degradation. Changes in the level of NO affect plant morphology and/or trigger responses associated with nutrient homeostasis, mediated by its interaction with reactive oxygen species, phytohormones, and through post-translational modification of proteins. NO-related events constitute an exciting field of research to understand how plants adapt and respond to conditions of nutrient shortage. This review summarizes the current knowledge on NO as a component of the multiple processes related to plant performance under conditions of deficiency in mineral nutrients, focusing on macronutrients such as nitrogen, phosphate, potassium, and magnesium, as well as micronutrients such as iron and zinc.

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.

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.

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.

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.

Chloroplast functionality has a positive effect on nitric oxide level in soybean cotyledons.

The subcellular localization of NO generation in soybean cotyledons, and the relationship between NO synthesis and in vivo chloroplast performance were studied. Employing the NO probe 4-aminomethyl-2',7'-difluorofluorescein diacetate (DAF-FM DA) and fluorescence microscopy, a strongly punctuated fluorescence was detected in mesophyll cells. The co-localization of DAF-FM and chlorophyll fluorescence, in confocal laser microscopy images, indicated the presence of NO in the chloroplasts. NO visualization was dependent on light, seedling age, and chloroplast function throughout cotyledons lifespan. The addition of herbicides with action in chloroplasts (DCMU and paraquat) dramatically reduced the quantum yield of photosystem II (φ(PSII)), and lead to images with absence of punctuated green fluorescence. Moreover, electron paramagnetic resonance signals corresponding to NO-spin trap adduct observed in cotyledon homogenates decreased significantly by the treatment with herbicides, as compared to controls. Neither chloroplast function nor NO content were significantly different in cotyledons from plants growing in the presence of ammonium or nitrate as the nitrogen source. These findings suggest that chloroplasts are organelles that contribute to NO synthesis in vivo, and that their proper functionality is essential for maintaining NO levels in soybean cotyledons.

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.

Labile iron pool and ferritin content in developing rat brain gamma-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.

Mitochondrial ferritin in animals and plants.

Ferritins play a role in preventing Fe toxicity because of their ability to sequester several thousand Fe atoms in their central cavity in a soluble, non-toxic bioavailable form. The identification of ferritin in mitochondria, an organelle with a constant generation of O2(-) as a by-product of the electron transfer, and the presence of a mitochondrial nitric oxide synthase activity opened up brand new metabolic interactions to be analyzed. In spite of cytosolic ferritins in mammals being ubiquitous, mitochondrial ferritin (mtF) expression is restricted to the testis, neuronal cells, islets of Langerhans, and as recently described to mice normal retinas. None was detected in major storage organs such as liver and spleen. MtF has about 80% identity to cytosolic H-chain and 55% to L-chain in its coding region. There has been reported some differences in the Fe binding and oxidation properties between mtF and cytosolic H-ferritin suggesting that mtF functions differently as an Fe storage protein within the mitochondria and perhaps has other function(s) in Fe homeostasis as well. Recently it was also presented evidence for the presence of ferritins in plant mitochondria. The understanding of the role of mitochondrial ferritin in Fe oxidative metabolism may be useful in approaching clinical situations such as the treatment of Friedreich's ataxia, X-linked sideroblastic anemia, and in other neurodegenerative disorders.