Exogenous Nitrate Research Articles

The Effect of Exogenous Nitrate on LCO Signalling, Cytokinin Accumulation, and Nodule Initiation in Medicago truncatula.

Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant Mtein2/sickle, as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.

Dynamic thermal imaging confirms local but not fast systemic ABA responses.

Abscisic acid (ABA) signals regulating stomatal aperture and water loss are usually studied in detached leaves or isolated epidermal peels and at infrequent timepoints. Measuring stomatal ABA responses in attached leaves across a time course enables the study of stomatal behaviour in the physiological context of the plant. Infrared thermal imaging is often used to characterize steady-state stomatal conductance via comparisons of leaf surface temperature but is rarely used to capture stomatal responses over time or across different leaf surfaces. We used dynamic thermal imaging as a robust, but sensitive, tool to observe stomatal ABA responses in a whole plant context. We detected stomatal responses to low levels of ABA in both monocots and dicots and identified differences between the responses of different leaves. Using whole plant thermal imaging, stomata did not always behave as described previously for detached samples: in Arabidopsis, we found no evidence for fast systemic ABA-induced stomatal closure, and in barley, we observed no requirement for exogenous nitrate during ABA-induced stomatal closure. Thus, we recommend dynamic thermal imaging as a useful approach to complement detached sample assays for the study of local and systemic stomatal responses and molecular mechanisms underlying stomatal responses to ABA in the whole plant context.

The role of denitrification genes in anaerobic growth and virulence of Flavobacterium columnare.

Comparative genomics analyses indicated that the Flavobacterium columnare genome has unique denitrification genes relative to Flavobacterium psychrophilum and Flavobacterium johnsoniae, including nasA (nitrate reductase), nirS (nitrite reductase), norB (nitric oxide reductase) and nosZ (nitrous oxide reductase). The current study determines the roles of nasA, nirS, norB and nosZ in anaerobic growth, nitrate reduction, biofilm formation and virulence. Four in-frame deletion mutants invirulent F. columnare strain 94-081 were constructed by allelic exchange using pCP29 plasmid. Compared with parent strain 94-081, FcΔnasA,FcΔnirS and FcΔnosZ mutants did not grow as well anaerobically, whereas the growth of FcΔnorB strain was similar to the parent strain (FcWT). Exogenous nitrate was not significantly consumed under anaerobic conditions in FcΔnasA, FcΔnirS and FcΔnosZ compared to parent strain 94-081. Under anaerobic conditions, Fc∆nasA, Fc∆norB and Fc∆nosZ formed significantly less biofilm than the wild type strain at 24 and 96h, but FcΔnirS was not significantly affected. The nitrite reductase mutant FcΔnirS was highly attenuated incatfish, whereas FcΔnasA, FcΔnorB and FcΔnosZ had similar virulence to FcWT. These results show, for the first time, that denitrification genes enable F. columnare to grow anaerobically using nitrate as an electron acceptor, and nitrite reductase contributes to F. columnare virulence. These findings indicate potential for F. columnare to grow in nitrate-rich anaerobic zones in catfish production ponds, and they suggest that a Fc∆nirS strain could be useful as a safe live vaccine if it protects catfish against columnaris disease.

Ameliorative effects of exogenous ascorbic acid and potassium nitrate on antioxidant defence system and mineral nutrients uptake in tomatoes under salt stress

Ameliorative effects of exogenous ascorbic acid and potassium nitrate on antioxidant defence system and mineral nutrients uptake in tomatoes under salt stress

Does the Oral Microbiome Play a Role in Hypertensive Pregnancies?

Chronic hypertension during gestation is associated with an increased risk of adverse pregnancy outcomes including pre-eclampsia, fetal growth restriction and preterm birth. Research into new chemotherapeutic regimes for the treatment of hypertension in pregnancy is limited due to concerns about fetal toxicity and teratogenicity, and new therapeutic avenues are being sought in alternative physiological pathways. Historically, generation of the vasodilator nitric oxide was believed to be solely from L-arginine by means of nitric oxide synthase enzymes. Recently, a novel pathway for the reduction of dietary inorganic nitrate to nitrite by the bacteria in the oral cavity and subsequently to vasodilatory nitric oxide within the body has been uncovered. Dietary nitrate is abundant in green leafy vegetables, including beetroot and spinach, and reduction of exogenous nitrate to nitrite by oral bacteria can increase nitric oxide in the vasculature, lessening hypertension. Supplements rich in nitrate may be an attractive choice for treatment due to fewer side effects than drugs that are currently used to treat hypertensive pregnancy disorders. Additionally, manipulation of the composition of the oral microbiota using pro- and prebiotics in tandem with additional dietary interventions to promote cardiovascular health during gestation may offer a safe and effective means of treating hypertensive pregnancy disorders including gestational hypertension and pre-eclampsia. The use of dietary inorganic nitrate as a supplement during pregnancy requires further exploration and large scale studies before it may be considered as part of a treatment regime. The aim of this article is to review the current evidence that oral microbiota plays a role in hypertensive pregnancies and whether it could be manipulated to improve patient outcomes.

Dietary Nitrate Counteracts The Elevated Blood Pressure Response To Nitric Oxide Synthase Inhibition In Humans

Decreased production of nitric oxide (NO) via the endogenous NO-synthase (NOS) pathway is a hallmark of impaired endothelial function and is associated with elevated blood pressure. Potentiation of the alternative nitrate-nitrite-NO pathway via the diet has been suggested as a possible therapeutic strategy to counteract NOS dysfunction, but proof-of-concept that exogenous nitrate treatment compensates in situations of NOS inhibition in humans is lacking. PURPOSE: To determine whether dietary supplementation with nitrate-rich beetroot juice attenuates the detrimental effects of acute NOS inhibition achieved by intravenous infusion of Nω-monomethyl-L-arginine (L-NMMA) on vascular function. METHODS: Seven male volunteers (age 23 ± 3 years, body mass 77.9 ± 6.8 kg) completed four conditions in a double-blind, randomised cross-over design: 1) 5-d dietary placebo supplementation with acute saline infusion (PL-CON), 2) 5-d placebo supplementation with acute L-NMMA infusion (3 mg/kgBM over 5 min followed by 55 μg/kgBM/min; PL-LNMMA), 3) 5-d dietary nitrate supplementation (12 mmol/d) with acute saline infusion (BR-CON), and 4) 5-d nitrate supplementation with acute L-NMMA infusion (BR-LNMMA). Heart rate (HR), brachial mean arterial pressure (MAP), and femoral artery blood flow (FBF) were measured every 5 min during 20 min of infusion and compared between conditions using repeated measures ANOVA. RESULTS: L-NMMA infusion resulted in decreases from baseline in HR (PL-LNMMA -5 ± 3, BR-LNMMA -6 ± 5 bpm) and FBF (PL-LNMMA -114 ± 85, BR-LNMMA -95 ± 71 mL/min) that were greater than after saline infusion (HR: PL-CON -2 ± 5, BR-CON -1 ± 4 bpm, P<0.05; FBF: 20 ± 40 and 18 ± 36 mL/min, P<0.05), with no effects of dietary supplementation. MAP was elevated by L-NMMA (PL-LNMMA 9 ± 2, BR-LNMMA 4 ± 2 mmHg) compared to saline infusion (PL-CON -2 ± 2, BR-CON 0 ± 2 mmHg, P<0.05), and this response was attenuated by ~56% following BR ingestion (P<0.05). CONCLUSIONS: Acute NOS blockade through systemic infusion of L-NMMA resulted in reduced HR and FBF, and elevated MAP, in healthy humans, resembling cardiovascular impairments observed in conditions of chronic NOS-dysfunction. BR attenuated the elevation in MAP, highlighting the potential for exogenous nitrate to improve vascular control in situations where NOS function is impaired.

NRT1.1-Mediated Nitrate Suppression of Root Coiling Relies on PIN2- and AUX1-Mediated Auxin Transport.

Asymmetric root growth (ARG) on tilted plates, or root coiling on horizontally placed plates, is proposed to be a combination of gravitropism, mechanical sensing, and “circumnutation,” a word designated by Charles Darwin to describe the helical movement that all plant organs make around the growth direction. ARG is developmentally controlled in which microtubule-regulating proteins and the phytohormone auxin participates. Nutrient deficiency influences ARG. However, it is unclear which nutrients play key roles in regulating ARG, what endogenous components are involved in responding to nutrient deficiency for ARG, and how nutrient deficiency is translated into endogenous responses. We report here that nitrate deficiency resulted in a strong ARG in Arabidopsis. Nitrate deficiency caused root coiling on horizontal plates, which is inhibited by an auxin transport inhibitor, and by mutations in PIN-FORMED2 (PIN2) and AUXIN RESISTANT 1 (AUX1). We further show that suppression of ARG by nitrate is mediated by the nitrate transporter/sensor NRT1.1. In addition, PIN2- and AUX1-mediated auxin transports are epistatic to NRT1.1 in nitrate deficiency-induced ARG. This study reveals a signaling pathway in root growth by responding to exogenous nitrate and relaying it into altered auxin transport.

Genome-wide responses to shoot nitrate satiety are attenuated by external ammonium in Arabidopsis thaliana

ABSTRACT The supply of exogenous nitrate to nitrate-depleted plants transiently changes the expression of nitrate-responsive genes within minutes. In addition to this so-called primary nitrate response, nitrate is suspected to play a long-term regulatory role, acting as a nitrogen (N) status signal for the entire plant. In our recent study using Arabidopsis thaliana, the manipulation of the internal nitrate levels independently of internal organic N without external N indicated that nitrate accumulation in the shoot alone causes genome-wide responses systemically, including the induction of nitrate assimilation genes and repression of N starvation-inducible genes. These responses mimic high nitrate growth situations for plants, suggesting that shoot nitrate acts as a N satiety signal. On the other hand, it is widely known that external supply of ammonium as another major N source also triggers N-rich responses, which led us to hypothesize that the existence of external ammonium may interfere with the genome-wide responses regarding shoot nitrate satiety. To clarify this, we compared transcriptional responses to shoot nitrate satiety with and without external ammonium both in Arabidopsis shoots and roots. Further, in order to determine the major role of nitrate satiety signaling, we extracted those genes regulated by shoot nitrate level regardless of the presence or absence of external ammonium and annotated them by two independent enrichment analyses. Here, we show that genome-wide transcriptional responses to shoot nitrate satiety are significantly lowered in the presence of external ammonium especially in the shoot. A major component for the ammonium-dependent attenuation of the nitrate satiety responses comprised a set of genes encoding the machinery for translation, glucosinolate biosynthesis, and photosynthesis. On the other hand, the nitrate satiety responses commonly observed irrespective of the presence/absence of ammonium included the components for translation and nitrate assimilation, whose transcriptional responses could be mediated partly via NIN-LIKE PROTEIN (NLP), NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1), and cytokinin signaling on the basis of our transcriptional profiling and cytokinin determination.

Nitrate uptake and metabolism in human skeletal muscle cell cultures

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

Biphasic effect of nitrate on anaerobic ammonium oxidation (anammox) and related kinetic modeling

Nitrate is a byproduct of the anaerobic ammonium oxidation (anammox) process and is related to its electron transfer. However, little is known about the influence of nitrate on the anammox process. In this work, the biphasic effect of exogenous nitrate on the anammox process was investigated in an upflow biofilter (UBF) reactor with ammonium as the sole electron donor. The responses of anammox to increased nitrate were analyzed by one-way ANOVA test and found to be significantly different under a constant and decreased nitrite condition (p < 0.01). With a single increase in nitrate and constant ammonium and nitrite in the influent, the total nitrogen removal rate (TNRR) of anammox was uninhibited, but stoichiometry deviated and nitrate production always showed a linear decrease. In contrast, anammox exhibited a range of activity with constant ammonium and simultaneously increased nitrate and decreased nitrite in the influent, including a continuous reduction of TNRR, a nonpersistent ammonium overconsumption and a pronounced nonlinear response of nitrate production. Correlation analysis shows that the lack of ammonium overconsumption was accompanied by the disappearance of nitrate underproduction. Kinetic models of product formation were effectively used to explore the nitrate production behavior of anammox subjected to increased nitrate, and the metabolite of nitrate was divided into a growth negative coupling type and growth (partial) coupling type under a constant and decreased nitrite condition, respectively. These findings collectively suggest that nitrate has a biphasic effect on the anammox process and is correlated with the availability of nitrite.

Shoot nitrate underlies a perception of nitrogen satiety to trigger local and systemic signaling cascades in Arabidopsis thaliana

ABSTRACT Nitrate is an important ion for plant growth and development. It serves not only as a building block for amino acid synthesis but also as a signaling molecule. Changes in the exogenous nitrate concentrations affect the expression of nitrate-responsive genes within minutes. Following these rapid transcriptional events, nitrate and its downstream organic nitrogen (N) compounds accumulate in the plant body, inducing secondary responses to the internal N level. Nevertheless, the respective roles of nitrate and organic N in triggering plant responses to internal N remain to be clarified. Several studies have implied that internal nitrate levels regulate root N uptake independent of the levels of N assimilation products. However, little is known about the specific effects of internal nitrate levels on plant growth and gene expression. To manipulate the internal nitrate levels independently of internal organic N, we grew wild-type Arabidopsis and a nitrate reductase (NR)-null mutant under a series of modified N conditions. Using their shoots and roots, we performed analyses of plant growth and RNA sequencing. The results showed that elevated shoot nitrate accumulation in the NR-null mutant was accompanied by increased expression of nitrate assimilatory genes in the shoots, decreased gene expression of high-affinity nitrate and ammonium uptake transporters in the roots, and decreased lateral root growth. Furthermore, the genes normally induced by N deficiency were significantly downregulated both in the shoots and roots of the NR-null mutant, compared with the wild-type. Our transcriptional profiling suggests that the transcription factors NLP7 and NIGT mediate a wide range of these transcriptional responses. Taken together, we conclude that shoot nitrate acts as a N satiety signal to trigger local and systemic signaling cascades in A. thaliana. The present study illustrates an adaptive strategy of plants to survive in N-limited environments, depending on the residual nitrate storage.

PO-089 Effects of 6-days nitrate supplementation on [Ca2+] and CRT in skeletal muscle of exhausted rats

Objective There are many active ingredients in sports nutrition, and nitrate is gradually being valued by sports nutrition and product developers. Supplementation of nitrate is a practical method to increase circulating plasma nitrite, thereby increasing NO bioavailability. However, the existing research has rarely reported the dose effect between nitrate supplementation and changes in exercise efficiency and capacity. The mechanism of action of nitrate is not fully understood yet. The aim of this study was to analyze the effects of different doses of nitrate on the exercise capacity of rats, as well as the detection of [Ca2+] and calreticulin (CRT) expression in the gastrocnemius, soleus and extensor digitorum longus, trying to figure out the effects of different doses of nitrate supplementation on calcium homeostasis in different types of muscle fibers.&#x0D; Methods 40 SD rats (8-week-old) weighing 270-290 grams were randomly divided into control group (C group, 8), exercise control group (EC group, 8), exercise with low-dose supplementation group (ELN group, 8), exercise with medium-dose supplementation group (EMN group, 8) and exercise with high-dose supplementation group (EHN group, 8). Sodium nitrate was used as exogenous nitrate for oral gavage. The intragastric concentration was 0.3 mmol/day/kg body weight in the ELN group, 0.7 mmol/day/kg body weight in the EMN group, and 1.0 mmol/day/kg body weight in the EHN group. The others were orally administered with normal saline. All exercise groups (EC, ELN, EMN and EHN) underwent 3 days adaptive low-intensity treadmill training with slope 0°, speed 16 meter/min, and the time is 5 min, 10 min and 15 min incrementally. 24 hours after the end of the last gavage, a one-time exhaustion treadmill experiment was started. The running platform slope was -16°, the speed was 16 meter/min. Exhaustive experiment participants were not aware of the grouping of rats. Immediately after the end of exhaustive exercise, the rats were weighed and anesthetized with sodium pentobarbital solution. Blood is collected for testing [NO2-]. The gastrocnemius, soleus and extensor digitorum longus were collected for testing NOS activity, tissue [Ca2+] and CRT expression.&#x0D; Results (1) Compared with the EC group, the exhaustion time of the other exercise groups was prolonged. The exhaustion time of the EMN group was very significantly prolonged from that of the EC group (P&lt;0.01). The exhaustion time of the EMN group was significantly prolonged from that of the EC group (P&lt; 0.05). At the same time, the difference between the EMN group and the ELN group was statistically significant (P&lt;0.05). (2) Serum [NO2-] in the supplemented nitrate groups (ELN, EMN and EHN) was higher than that in the EC group, and the difference was statistically significant (P&lt;0.01). The serum [NO2-] in different groups (ELN, EMN and EHN) raised with the increase of nitrate concentration, and the difference between each adjacent concentration group was statistically significant (P&lt;0.05). (3) In the gastrocnemius, soleus and extensor digitorum longus respectively, there were no significant differences in TNOS, iNOS and cNOS activities between the groups (P&gt;0.05). (4) In the gastrocnemius, compared with the EC group, the [Ca2+] (P&lt;0.05) and CRT expression (P&lt;0.05) in the EMN group were significantly decreased. There was no difference between the ELN vs EC group (P&gt;0.05) and the EHN vs EC group (P&gt;0.05). (5) In the soleus, the [Ca2+] (P&lt;0.05) and CRT expression (P&lt;0.05) in the EMN group were significantly decreased. There was no difference between the ELN vs EC group (P&gt;0.05) and the EHN vs EC group (P&gt;0.05). (6) In the extensor digitorum longus, compared with the EC group, the [Ca2+] (P&lt;0.05) and CRT expression (P&lt;0.05) in the EHN group were significantly decreased. There was no difference between ELN and EC group (P&gt;0.05). The expression of CRT in EMN group was significantly decreased (P&lt;0.05), but there is no difference of [Ca2+] between EMN and EC group (P&lt;0.05).&#x0D; Conclusions (1) 6-day sodium nitrate supplementation is a reliable method to increase serum [NO2-] concentration. (2) 6-day sodium nitrate supplementation can prolong the duration of one-time exhaustive exercise in rats, and the dose of 0.7mmol/kg/d is the best. (3) Sodium nitrate supplementation can affect the [Ca2+] and the expression of CRT in skeletal muscle after one-time exhaustive exercise. Different concentrations of sodium nitrate have different effects on different types of muscle fibers.

Reduced blood pressure responsiveness to skeletal muscle metaboreflex activation in older adults following inorganic nitrate supplementation

The vasoactive molecule nitric oxide (NO) contributes to regulation of blood pressure (BP) at rest and during exercise. Age-related exaggerated increased BP responses during exercise have been proposed to be due in part to a decreased NO bioavailability and possibly an enhanced skeletal muscle metaboreflex. In the present study we sought to determine if age-related differences in BP responses to skeletal muscle metaboreflex activation exist. Additionally, since NO bioavailability can be improved with exogenous nitrate (NO3−) via the nitrate-nitrite-NO pathway, we tested the hypothesis that inorganic NO3− supplementation would reduce BP responses to muscle metaboreflex activation in healthy older adults. 13 older adults (67 ± 1 years) participated in a randomized, double-blind, placebo controlled crossover study consisting of four weeks of NO3− supplementation [beetroot powder; 250 mg (∼4.03 mmol) of NO3− and 20 mg (∼0.29 mmol) of NO2−] and four weeks of placebo (beetroot powder devoid of NO3−/NO2−). Skeletal muscle metaboreflex testing consisted of isometric handgrip exercise (IHG) at 30% of maximal voluntary contraction immediately followed by post exercise forearm ischemia (PEI), which was achieved by inflation of a rapid pressure cuff (240 mmHg) around the upper arm. BP responses were analyzed as the change (Δ) from baseline to the end of IHG and PEI. An additional 10 young adults (25 ± 1 years) were recruited to serve as a reference cohort and address if BP responses to skeletal muscle metaboreflex activation were greater with aging. BP responses to IHG were similar between the young and older adults. However, older adults demonstrated a greater increase in systolic BP during PEI (P < 0.05). Plasma NO3− and NO2−were increased following NO3− supplementation in older adults (P < 0.01). ΔSystolic BP (19 ± 2 vs. 13±3 mmHg, P < 0.05), ΔDiastolic BP (7 ± 1 vs. 5±1 mmHg, P < 0.05) and ΔMean arterial pressure (11 ± 1 vs. 8±2 mmHg, P < 0.05) were reduced during PEI following four weeks of NO3−supplementation, whereas placebo had no effect on ΔSystolic BP (16 ± 2 vs. 17±2 mmHg), ΔDiastolic BP (5 ± 1 vs. 7±1 mmHg), and ΔMean arterial pressure (8 ± 1 vs. 10±1 mmHg) during PEI (all P > 0.05). These data suggest that inorganic NO3− supplementation attenuates skeletal muscle metaboreflex mediated increases in BP during exercise in older adults.

Индуцирование синтеза NO в корнях проростков пшеницы и развития их теплоустойчивости экзогенными L-аргинином и нитратом

Индуцирование синтеза NO в корнях проростков пшеницы и развития их теплоустойчивости экзогенными L-аргинином и нитратом

Nitrate Sensing and Metabolism Inhibit Biofilm Formation in the Opportunistic Pathogen Burkholderia pseudomallei by Reducing the Intracellular Concentration of c-di-GMP.

The opportunistic pathogen Burkholderia pseudomallei is a saprophytic bacterium and the causative agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality. Although melioidosis is most prevalent during the rainy season in endemic areas, domestic gardens and farms can also serve as a reservoir for B. pseudomallei during the dry season, in part due to irrigation and fertilizer use. In the environment, B. pseudomallei forms biofilms and persists in soil near plant root zones. Biofilms are dynamic bacterial communities whose formation is regulated by extracellular cues and corresponding changes in the nearly universal secondary messenger cyclic dimeric GMP. Recent studies suggest B. pseudomallei loads are increased by irrigation and the addition of nitrate-rich fertilizers, whereby such nutrient imbalances may be linked to the transmission epidemiology of this important pathogen. We hypothesized that exogenous nitrate inhibits B. pseudomallei biofilms by reducing the intracellular concentration of c-di-GMP. Bioinformatics analyses revealed B. pseudomallei 1026b has the coding capacity for nitrate sensing, metabolism, and transport distributed on both chromosomes. Using a sequence-defined library of B. pseudomallei 1026b transposon insertion mutants, we characterized the role of denitrification genes in biofilm formation in response to nitrate. Our results indicate that the denitrification pathway is implicated in B. pseudomallei biofilm growth dynamics and biofilm formation is inhibited by exogenous addition of sodium nitrate. Genomics analysis identified transposon insertional mutants in a predicted two-component system (narX/narL), a nitrate reductase (narGH), and a nitrate transporter (narK-1) required to sense nitrate and alter biofilm formation. Additionally, the results presented here show that exogenous nitrate reduces intracellular levels of the bacterial second messenger c-di-GMP. These results implicate the role of nitrate sensing in the regulation of a c-di-GMP phosphodiesterase and the corresponding effects on c-di-GMP levels and biofilm formation in B. pseudomallei 1026b.

Stimulatory effect of exogenous nitrate on soil denitrifiers and denitrifying activities in submerged paddy soil

Paddy soil is submerged under water for most of the rice growing season, but our understanding of the driving mechanisms of nitrogen cycling, N2O production, and N2O consumption under submerged conditions is limited. In this study, intact paddy soil cores were sampled and an incubation experiment was conducted with nitrate amendments under flooding. N2O concentrations in the soil profile and N2O flux rates were measured by gas chromatography. The community compositions and abundances of narG- and nosZ-containing denitrifiers were analyzed by terminal restriction fragment length polymorphism (T-RFLP) and real-time quantitative polymerase chain reactions (qPCR), respectively. The results showed that N2O emissions and N2O concentrations in the submerged soil profile were significantly affected by the nitrate inputs. Higher NO3-N additions stimulated a sharp increase in N2O flux rate, which suggested an obvious stimulation caused by the increasing nitrate input. This effect was closely related to the significant increases in the population size of narG-containing denitrifiers and obvious alterations in its community composition. Therefore, high nitrate concentrations in submerged paddy soil can stimulate much higher N2O production and emissions, and in this process, narG- rather than nosZ-containing denitrifiers are the important drivers. We also observed that N2O concentrations in the 0–5cm soil layer were clearly lower than those in the 5–10cm layer, but the nitrate contents in these layers were reversed, indicating large N2O losses occur from the 0–5cm layer. The emitted N2O is derived mainly from the uppermost soil layer (0–5cm).

451 - Endogenous vs. Dietary Nitrate and Nitrite Distribution in Rat Skeletal Muscle during Exercise

Nitric oxide (NO), small gaseous molecule generated enzymatically by a family of NOS enzymes, is a potent vasodilator. At low oxygen conditions, NO is generated in a series of nonenzymatic reactions from nitrite and nitrate. In the past decade, these two ions started to be considered as storage and precursors molecules for NO. We recently showed that skeletal muscle is the largest body reservoir of nitrate with levels 2-3 times higher than those found in blood or any other internal organ. We proposed that this endogenous nitrate serves as a precursor for NO in muscle during the exercise-induced hypoxia and following active hyperemia. It is known that dietary nitrate intake increases exercise endurance, even if the exact mechanism is still up to debate. In our study, using the combination of dietary nitrate manipulation and exercise, we try to understand relative roles of skeletal muscle endogenous nitrate and exogenous (dietary) nitrate at resting state and during exercise. We showed that the endogenous nitrate stored in skeletal muscle is extensively used by the whole body at times of low nitrate intake in both, resting and exercising state. Dietary nitrate excess is delivered by bloodstream mainly into liver (and likely into other internal organs), but only in very low extent into muscle tissue. However, when on excess of nitrate, nitrite drastically increased in all studied organs. We hypothesize that it is the nitrite, formed in liver from dietary nitrate excess that is transported into skeletal muscle during exercise where it improves performance. The mechanism by which exercise enhances the transport of nitrite derived from dietary sources into skeletal muscle is still unclear.

Nitrate Reverses Severe Nitrite Inhibition of Anaerobic Ammonium Oxidation (Anammox) Activity in Continuously-Fed Bioreactors

Nitrite (NO2-) substrate under certain conditions can cause failure of N-removal processes relying on anaerobic ammonium oxidizing (anammox) bacteria. Detoxification of NO2- can potentially be achieved by using exogenous nitrate (NO3-). In this work, continuous experiments in bioreactors with anammox bacteria closely related to "Candidatus Brocadia caroliniensis" were conducted to evaluate the effectiveness of short NO3- additions to reverse NO2- toxicity. The results show that a timely NO3- addition immediately after a NO2- stress event completely reversed the NO2- inhibition. This reversal occurs without NO3- being metabolized as evidence by lack of any 30N2 formation from 15N-NO3-. The maximum recovery rate was observed with 5 mM NO3- added for 3 days; however, slower but significant recovery was also observed with 5 mM NO3- for 1 day or 2 mM NO3- for 3 days. Without NO3- addition, long-term NO2- inhibition of anammox biomass resulted in irreversible damage of the cells. These results suggest that a short duration dose of NO3- to an anammox bioreactor can rapidly restore the activity of NO2--stressed anammox cells. On the basis of the results, a hypothesis about the detoxification mechanism related to narK genes in anammox bacteria is proposed and discussed.

N availability modulates the role of NPF3.1, a gibberellin transporter, in GA-mediated phenotypes in Arabidopsis.

AtNPF3.1 gene expression is promoted by limiting nitrogen nutrition. Atnpf3.1 mutants are affected in hypocotyl elongation and seed germination under conditions of low-nitrate availability. The NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) family encodes nitrate or peptides transporters, some of which are also able to transport hormones. AtNPF3.1 has been described as a nitrate/nitrite/gibberellin transporter. Until now only its gibberellins (GAs) transport capacity have been proven in planta. We further analyzed its substrate specificity towards different GA species using a yeast heterologous system which revealed that (1) NPF3.1 transported not only bioactive GAs but also their precursors and metabolites and (2) the GAs' import activity of NPF3.1 was not affected by the presence of exogenous nitrate. Gene expression analysis along with germination assays and hypocotyl length measurements of loss of function mutants was used to understand the in planta role of NPF3.1. GUS staining revealed that this gene is expressed mainly in the endodermis of roots and hypocotyls, in shoots, stamens, and dry seeds. Germination assays in the presence of paclobutrazol, a GA biosynthesis inhibitor, revealed that the germination rate of npf3.1 mutants was lower compared to wild type when GA was added at the same time. Likewise, hypocotyl length measurements showed that the npf3.1 mutants were less sensitive to exogenous GA addition in the presence of paclobutrazol, compared to wild type. Moreover, this phenotype was observed only when plants were grown on low-nitrate supply. In addition, NPF3.1 gene expression was upregulated by low exogenous nitrate concentrations and the npf3.1 mutants exhibited a not yet described GA-related phenotype under these conditions. All together, these results indicated that NPF3.1 is indeed involved in GAs transport in planta under low-nitrate conditions.

Exogenously applied nitrate improves the photosynthetic performance and nitrogen metabolism in tomato (Solanum lycopersicum L. cv Pusa Rohini) under arsenic (V) toxicity

Tomato (Solanum lycopersicum L.) being a widespread and most commonly consumed vegetable all over the world has an important economic value for its producers and related food industries. It is a serious matter of concern as its production is affected by arsenic present in soil. So, the present study, investigated the toxicity of As(V) on photosynthetic performance along with nitrogen metabolism and its alleviation by exogenous application of nitrate. Plants were grown under natural conditions using soil spiked with 25mg and 20mM, As(V) and nitrate, respectively. Our results revealed that plant growth indices, photosynthetic pigments, and other major photosynthetic parameters like net photosynthetic rate and maximum quantum efficiency (Fv/Fm ) of photosystem II (PSII) were significantly (P≤0.05) reduced under As(V) stress. However, nitrate application significantly (P≤0.05) alleviated As(V) toxicity by improving the aforesaid plant responses and also restored the abnormal shape of guard cells. Nitrogen metabolism was assessed by studying the key nitrogen-metabolic enzymes. Exogenous nitrate revamped nitrogen metabolism through a major impact on activities of NR, NiR, GS and GOGAT enzymes and also enhanced the total nitrogen and NO content while malondialdehyde content, and membrane electrolytic leakage were remarkably reduced. Our study suggested that exogenous nitrate application could be considered as a cost effective approach in ameliorating As(V) toxicity.