Vacuolar-Iron-Transporter1-Like Proteins Mediate Iron Homeostasis in Arabidopsis

Iron deficiency is a nutritional problem in plants and reduces crop productivity, quality and yield. With the goal of improving the iron (Fe) storage properties of plants, we have investigated the function of three Arabidopsis proteins with homology to Vacuolar Iron Transporter1 (AtVIT1). Heterologous expression of Vacuolar Iron Transporter-Like1 (AtVTL1; At1g21140), AtVTL2 (At1g76800) or AtVTL5 (At3g25190) in the yeast vacuolar Fe transport mutant, Δccc1, restored growth in the presence of 4 mM Fe. Isolated vacuoles from yeast expressing either of the VTL genes in the Δccc1 background had a three- to four-fold increase in Fe concentration compared to vacuoles isolated from the untransformed mutant. Transiently expressed GFP-tagged AtVTL1 was localized exclusively and AtVTL2 was localized primarily to the vacuolar membrane of onion epidermis cells. Seedling root growth of the Arabidopsis nramp3/nramp4 and vit1-1 mutants was decreased compared to the wild type when seedlings were grown under Fe deficiency. When expressed under the 35S promoter in the nramp3/nramp4 or vit1-1 backgrounds, AtVTL1, AtVTL2 or AtVTL5 restored root growth in both mutants. The seed Fe concentration in the nramp3/nramp4 mutant overexpressing AtVTL1, AtVTL2 or AtVTL5 was between 50 and 60% higher than in non-transformed double mutants or wild-type plants. We conclude that the VTL proteins catalyze Fe transport into vacuoles and thus contribute to the regulation of Fe homeostasis in planta.     

Nitric Oxide Interferes with Islet Cell Zinc Homeostasis

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function.     

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

The signaling role of hydrogen gas (H₂) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H₂ in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H₂, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K⁺ channel (GORK). ABA elicited a rapid and sustained H₂ release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H₂ production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impaired. In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H₂-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved.Stomata are responsible for leaves of terrestrial plants taking in carbon dioxide for photosynthesis and likewise regulate how much water plants evaporate through the stomatal pores (Chaerle et al., 2005). When experiencing water-deficient conditions, surviving plants balance photosynthesis with controlling water loss through the stomatal pores, which relies on turgor changes by pairs of highly differentiated epidermal cells surrounding the stomatal pore, called the guard cells (Haworth et al., 2011; Loutfy et al., 2012).Besides the characterization of the significant roles of abscisic acid (ABA) in regulating stomatal movement, the key factors in guard cell signal transduction have been intensively investigated by performing forward and reverse genetics approaches. For example, both reactive oxygen species (ROS) and nitric oxide (NO) have been identified as vital intermediates in guard cell ABA signaling (Bright et al., 2006; Yan et al., 2007; Suzuki et al., 2011; Hao et al., 2012). The key ROS-producing enzymes in Arabidopsis (Arabidopsis thaliana) guard cells are the respiratory burst oxidase homologs (Rboh) D and F (Kwak et al., 2003; Bright et al., 2006; Mazars et al., 2010; Marino et al., 2012). Current available data suggest that there are at least two distinct pathways responsible for NO synthesis involved in ABA signaling in guard cells: the nitrite reductase (NR)- and l-Arg-dependent pathways (Desikan et al., 2002; Besson-Bard et al., 2008). Genetic evidence further demonstrated that removal of the major known sources of either ROS or NO significantly impairs ABA-induced stomatal closure. ABA fails to induce ROS production in the atrbohD/F double mutant (Kwak et al., 2003; Wang et al., 2012) and NO synthesis in the NR-deficient mutant nitrate reductase1/2 (nia1/2; Bright et al., 2006; Neill et al., 2008), both of which lead to impaired stomatal closure in Arabidopsis. Most importantly, ROS and NO, which function both synergistically and independently, have been established as ubiquitous signal transduction components to control a diverse range of physiological pathways in higher plants (Bright et al., 2006; Tossi et al., 2012).The guard cell outward-rectifying K⁺ channel (GORK) encodes the exclusive voltage-gated outwardly rectifying K⁺ channel protein, which was located in the guard cell membrane (Ache et al., 2000; Dreyer and Blatt, 2009). Expression profiles revealed that this gene is up-regulated upon the onset of drought, salinity, and cold stress and ABA exposure (Becker et al., 2003; Tran et al., 2013). Reverse genetic evidence further showed that GORK plays an important role in the control of stomatal movements and allows the plant to reduce transpirational water loss significantly (Hosy et al., 2003) and participates in the regulation of salinity tolerance by preventing salt-induced K⁺ loss (Jayakannan et al., 2013). Due to the high complexity of guard cell signaling cascades, whether and how ABA-triggered GORK up-regulation is attributed to the generation of cellular secondary messengers, such as ROS and NO, is less clear.Hydrogen gas (H₂) was recently revealed as a signaling modulator with multiple biological functions in clinical trails (Ohsawa et al., 2007; Itoh et al., 2009; Ito et al., 2012). It was previously found that a hydrogenase system could generate H₂ in bacteria and green algae (Meyer, 2007; Esquível et al., 2011). Although some earlier studies discovered the evolution of H₂ in several higher plant species (Renwick et al., 1964; Torres et al., 1984), it was also proposed that the eukaryotic hydrogenase-like protein does not metabolize H₂ (Cavazza et al., 2008; Mondy et al., 2014). Since the explosion limit of H₂ gas is about 4% to 72.4% (v/v, in the air), the direct application of H₂ gas in experiments is flammable and dangerous. Regardless of these problems to be resolved, the methodology, such as using exogenous hydrogen-rich water (HRW) or hydrogen-rich saline, which is safe, economical, and easily available, provides a valuable approach to investigate the physiological function of H₂ in animal research and clinical trials. For example, hydrogen dissolved in Dulbecco’s modified Eagle’s medium was found to react with cytotoxic ROS and thus protect against oxidative damage in PC12 cells and rats (Ohsawa et al., 2007). The neuroprotective effect of H₂-loaded eye drops on retinal ischemia-reperfusion injury was also reported (Oharazawa et al., 2010). In plants, corresponding results by using HRW combined with gas chromatography (GC) revealed that H₂ could act as a novel beneficial gaseous molecule in plant responses against salinity (Xie et al., 2012; Xu et al., 2013), cadmium stress (Cui et al., 2013), and paraquat toxicity (Jin et al., 2013). More recently, the observation that HRW could delay the postharvest ripening and senescence of kiwifruit (Actinidia deliciosa) was reported (Hu et al., 2014).Considering the fact that the signaling cascades for salt, osmotic, and drought stresses share a common cascade in an ABA-dependent pathway, it would be noteworthy to identify whether and how H₂ regulates the bioactivity of ABA-induced downstream components and, thereafter, biological responses, including stomatal closure and drought tolerance. To resolve these scientific questions, rbohD, rbohF, nia1/2, nitric oxide associated1 (noa1; Van Ree et al., 2011), nia1/2/noa1, and gork mutants were utilized to investigate the relationship among H₂, ROS, NO, and GORK in the guard cell signal transduction network. By the combination of pharmacological and biochemical analyses with this genetics-based approach, we provide comprehensive evidence to show that H₂ might be a newly identified bioeffective modulator involved in ABA signaling responsible for drought tolerance, that HRW-promoted stomatal closure was mainly attributed to the modulation of ROS-dependent NO generation, and that GORK might be the downstream target protein of H₂ signaling.     

Redox Regulation of the NPR1-TGA1 System of Arabidopsis thaliana by Nitric Oxide

The role of reactive oxygen and nitrogen species in local and systemic defense reactions is well documented. NPR1 and TGA1 are key redox-controlled regulators of systemic acquired resistance in plants. NPR1 monomers interact with the reduced form of TGA1, which targets the activation sequence-1 (as-1) element of the promoter region of defense proteins. Here, we report the effect of the physiological nitric oxide donor S-nitrosoglutathione on the NPR1/TGA1 regulation system in Arabidopsis thaliana. Using the biotin switch method, we demonstrate that both NPR1 and TGA1 are S-nitrosylated after treatment with S-nitrosoglutathione. Mass spectrometry analyses revealed that the Cys residues 260 and 266 of TGA1 are S-nitrosylated and S-glutathionylated even at GSNO concentrations in the low micromolar range. Furthermore, we showed that S-nitrosoglutathione protects TGA1 from oxygen-mediated modifications and enhances the DNA binding activity of TGA1 to the as-1 element in the presence of NPR1. In addition, we observed that the translocation of NPR1 into the nucleus is promoted by nitric oxide. Taken together, our results suggest that nitric oxide is a redox regulator of the NPR1/TGA1 system and that they underline the importance of nitric oxide in the plant defense response.     

Iron, Nitric Oxide, and Myeloperoxidase in Asthmatic Patients

Plasma nitric oxide (NO), myeloperoxidase (MPO), and iron (Fe) levels were determined in bronchial asthma. The relations among these parameters in different stages of asthma were interpreted. Their association with airway inflammation observed in patients with bronchial asthma as well as the roles and the contributions to the pathological processes were evaluated. A total of 62 individuals, 32 asthmatics and 30 controls, were included into the scope of this study. Plasma nitric oxide metabolites (NOx) and MPO and Fe levels were determined by the Griess reaction, ELISA, and the automated TPTZ (2,4,6-tri[2-pyridyl]-5-triazine) method, respectively. In the asthmatic individuals, plasma NOx, MPO, and Fe concentrations were 133 +/- 13 microM, 95 +/- 20 ng/ml, and 159 +/- 20 microg/dl, respectively; in the control group these values were 82 +/- 11 microM, 62 +/- 11 ng/ml, and 96 +/- 9 microg/dl. Increased values were detected for plasma MPO (p > 0.05), NOx (p < 0.01), and Fe (p < 0.01) concentrations in asthmatic individuals. Considering the facts that NO modulates the catalytic activity of MPO and induces the expression of heme oxygenase as important contributors to the mechanisms causing free Fe release, it is concluded that elevated NOx, MPO, and Fe levels observed in the asthmatic group act in a concerted manner and appear to be involved in the pathogenesis of asthma.     

Production of Nitric Oxide and Nitrous Oxide During Denitrification by Corynebacterium nephridii

Resting cells of Corynebacterium nephridii reduce nitrate, nitrite, and nitric oxide to nitrous oxide under anaerobic conditions. Nitrous oxide production from nitrite was optimal from pH 7.0 to 7.4. The stoichiometry of nitrous oxide production from nitrite was 99% of the theoretical-two moles of nitrite was used for each mole of nitrous oxide detected. Hydroxylamine increases gas evolution from nitrite but inhibits the reduction of nitric oxide to nitrous oxide. Hydroxylamine is converted to nitrogenous gas(es) by resting cells only in the presence of nitrite. Under certain conditions nitric oxide, as well as nitrous oxide, was detected.     

DELLA蛋白参与拟南芥幼苗对一氧化氮逆境的抵抗

DELLA蛋白是赤霉素信号途径中的一类对植物生长起抑制作用的重要蛋白质,在拟南芥（Arabidopsis thaliana）基因组中已经鉴定出5个DELLA蛋白基因。目前研究发现,DELLA蛋白在抗逆中也起了重要的作用。近年来,一氧化氮（nitric oxide,NO）的研究工作取得重要进展,低浓度的NO能够促进植物的生长,但在高浓度下它对植物生长起抑制作用甚至导致细胞死亡。通过外施一氧化氮供体硝普钠（sodium nitro prusside,SNP）,研究高浓度NO对拟南芥幼苗生长的影响,发现植物体内H2O2积累,幼苗死亡。通过研究DELLA蛋白基因表达的变化及其相关突变体的表型,证明DELLA蛋白在抵抗NO逆境中起了重要作用。研究结果揭示了DELLA蛋白与NO逆境的关系,为今后科学指导农业生产提供了理论依据。     

一氧化氮在拟南芥气孔发育中的功能研究

为了研究一氧化氮(nitric oxide,NO)在气孔发育中的功能,本实验利用NO的释放剂硝普钠(SNP)处理拟南芥野生型,结果显示,SNP处理后的子叶气孔指数(SI)和%(GMC+M)都较未处理组明显升高。继续检测拟南芥内源一氧化氮升高的突变体nox1和内源一氧化氮降低的突变体noa1的气孔参数,结果显示,nox1的气孔指数(SI)和%(GMC+M)相对野生型明显提高,noa1的气孔指数(SI)和%(GMC+M)低于野生型。荧光定量PCR结果进一步显示,NO抑制了气孔发育相关基因MUTE,SCRM,SCRM2的表达。综上结果表明,NO通过调控气孔发育相关基因的表达影响气孔发育。     

Nitric Oxide Alleviates Photochemical Damage Induced by Cadmium Stress in Pea Seedlings

Cadmium (Cd), a life threatening hazardous heavy metal is abundant in nature. Cd amounts are greater in leaves than other plant parts, and it shows considerable effects on photosynthesis. Nitric oxide (NO), a free radical present in living organisms, is now known as an important signaling molecule playing various physiological processes in plants. In this study, the possible ameliorative effect of NO on photosynthesis was examined on pea seedlings grown under Cd stress. Results showed that chlorophyll, net photosynthetic rate, transpiration rate, stomatal conductance, photochemical efficiency of Photosystem II and Photosystem I decreased, and Fo and non-photochemical parameters for PSII and PSI significantly increased due to Cd stress. This suggests that Cd affects the photochemistry efficiency at both the PSII and PSI levels. Nitric oxide supplementation through SNP ameliorated Cd stress by enhancing all the above mentioned parameters but causing a reduction in the Fo, and non-photochemical parameters of PSII and PSI in pea plants. These data indicate that the exogenous application of NO was useful in mitigating Cd-induced damage to photosynthesis in pea seedling.     

Nitric Oxide Regulates Dark-Induced Leaf Senescence Through EIN2 in Arabidopsis

The nitric oxide (NO)-deficient mutant nos1/noa1 exhibited an early leaf senescence phenotype. ETHY-LENE INSENSITIVE 2 (EIN2) was previously reported to function as a positive regulator of ethylene-induced senescence. The aim of this study was to address the question of how NO interacts with ethylene to regulate leaf senescence by characterizing the double mutant ein2-1 nos1/noa1 (Arabidopsis thaliana). Double mutant analysis revealed that the nos1/noa1-mediated, dark-induced early senescence phenotype was suppressed by mutations in EIN2, suggesting that EIN2 is involved in nitric oxide signaling in the regulation of leaf senescence. The results showed that chlorophyll degradation in the double mutant leaves was significantly delayed. In addition, nos1/noa1-mediated impairment in photochemical efficiency and integrity of thylakoid membranes was reverted by EIN2 mutations. The rapid upregulation of the known senescence marker genes in the nos1/noa1 mutant was severely inhibited in the double mutant during leaf senescence. Interestingly, the response of dark-grown nos1/noa1 mutant seedlings to ethylene was similar to that of wild type seedlings. Taken together, our findings suggest that EIN2 is involved in the regulation of early leaf senescence caused by NO deficiency, but NO deficiency caused by NOS1/NOA1 mutations does not affect ethylene signaling.