Werner M. Kaiser

On the origins of nitric oxide

Nitric oxide (NO) is widely recognized for its role as signaling compound. However, the metabolic mechanisms that determine changes in the level of NO in plants are only poorly understood, despite this knowledge being crucial to understanding the signal function of NO. To date, at least seven possible pathways of NO biosynthesis have been described for plants, although the molecular and enzymatic components are resolved for only one of these. Currently, this represents the most significant bottleneck for NO research. In this review, we provide an overview of the multiplicity of NO production and scavenging pathways in plants. Furthermore, we discuss which areas should be focused on in future studies to investigate the origin of fluctuations in the level of NO in plants.

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell–specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity–dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

Nitric oxide participates in cold‐responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.

Correlation between changes in photosynthetic activity and changes in total protoplast volume in leaf tissue from hygro-, meso- and xerophytes under osmotic stress

Rates of photosynthesis of leaf slices from various hygro-, meso- and xerophytes were measured in the absence of stomatal control in various stages of osmotic dehydration. The external osmotic potential π° for a 50% inhibition of photosynthesis varied between 20 bar in some hygrophytes up to 50 bar in xerophytes. The response of photosynthetic enzymes to increased salt concentrations in the reaction medium was similar in leaf extracts from hygro-, meso- and xerophytes. The total protoplast volume in vacuum-infiltrated leaf discs from various plants was measured as the difference between 3H2O-labeled space and [14C]sorbitol-labeled space. In all plants, the protoplast volume could be reduced to about 55% of the maximum volume of tissue in equilibrium with water, without decreasing photosynthesis. Reduction of the maximal protoplast volume below 55% decreased photosynthesis in all tissues to the same decreased photosynthesis in all tissues to the same degree. At 20% maximal volume, photosynthesis of all plants was completely inhibited. The differential decrease of protoplast volumes of various leaf tissues in response to changes in π° was mainly due to the different osmotic potential of the cell sap (πcs). The relative contribution of sugars to the overall osmolarity of the cell sap was up to nineteen times higher in xerophytes than in hygrophytes. Short-term recovery of photosynthesis after hypertonic stress was good in xerophytes, incomplete in mesophytes and absent in hygrophytes. There was also a large discrepancy between the partial recovery of protoplast volumes and the complete absence of a recovery of photosynthesis in hygrophytes.

Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana

Wild type (WT), and nitrate reductase (NR)‐ and nitrite‐reductase (NiR)‐deficient cells of Chlorella sorokiniana were used to characterize nitric oxide (NO) emission. The NO emission from nitrate‐grown WT cells was very low in air, increased slightly after addition of nitrite (200 μM), but strongly under anoxia. Importantly, even completely NR‐free mutants, as well as cells grown on tungstate, emitted NO when fed with nitrite under anoxia. Therefore, this NO production from nitrite was independent of NR and other molybdenum cofactor enzymes. Cyanide and inhibitors of mitochondrial complex III, myxothiazol or antimycin A, but not salicylhydroxamic acid (inhibitor of alternative oxidase) inhibited NO production by NR‐free cells. In contrast, NiR‐deficient cells growing on nitrate accumulated nitrite and emitted NO at very high equal rates in air and anoxia. This NO emission was 50% inhibited by salicylhydroxamic acid, indicating that in these cells the alternative oxidase pathway had been induced and reduced nitrite to NO.

Photosynthesis under osmotic stress : Inhibition of photosynthesis of intact chloroplasts, protoplasts, and leaf slices at high osmotic potentials.

Abstract1. Photosynthesis of leaf slices, mesophyll protoplasts, and intact chloroplasts of spinach was inhibited in hypertonic sorbitol solutions. Sorbitol could be replaced by other nonpenetrating osmotica such as sucrose or glycinebetaine. As a penetrating solute, ethyleneglycol was also inhibitory, but osmolarities required for inhibition of photosynthesis were considerably higher than in the case of non-penetrating osmotica.-2. With leaf slices and protoplasts, 50% inhibition by sorbitol was usually observed at osmotic potentials between 25 and 40 bar. With isolated intact chloroplasts, the osmotic potentials producing 50% inhibition varied considerably. Depending on the growth conditions of the plant material, 50% inhibition occurred between 14 and 40 bar. The integrity of the chloroplast envelope as measured by the accessibility of the thylakoid system for ferricyanide was not affected by osmotic stress.-3. Quantum requirements for CO2 assimilation and reduction of 3-phosphoglycerate or nitrite by intact chloroplasts increased under osmotic stress. The increase was larger for CO2 reduction than for reduction of 3-phosphoglycerate or nitrite.-4. In intact chloroplasts, electron transport to methylviologen was not much affected by osmotic stress. Basal electron transport was not stimulated, suggesting absence of uncoupling.-5. The increase in ATP/ADP ratios on illumination of intact chloroplasts was slower at an osmotic potential of 36 bar than at 11 bar.-6. The results indicate that inhibition of photosynthesis is not caused by the sensitivity of a single photosynthetic reaction to increased osmotic potentials. Rather, several reactions are sensitive to water stress. Osmotic stress acts on the photosynthetic apparatus mainly at the level of dark reactions and ATP synthesis, and much less on primary photoreactions or electron transport, between water and the primary oxidant of photosystem I.-7. The different sensitivity of chloroplasts to penetrating and non-penetrating solutes and the observed variability of chloroplast sensitivity to stress suggests that the reduction in water potential is not directly responsible for damage to the photosynthetic apparatus during osmotic stress. Rather, the composition of the chloroplasts appears to be a decisive factor which determines sensitivity or resistance to osmotic stress.

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status

Abstract. The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg2+ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NRact) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NRmax). Both values together permit an estimation of the NR-phosphorylation state. Changes in NRact and NRmax were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal, NRmax decreased slowly without a large change in the phosphorylation state. This decrease of NRmax was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NRmax without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NRmax and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation.

Is nitrate reductase a major player in the plant NO (nitric oxide) game

Nitric oxide (NO) is a diffusible, very reactive gas that is involved in the regulation of many processes in plants. Several enzymatic sources of NO production have been identified in recent years. Nitrate reductase (NR) is one of them and it has been shown that this well-known plant protein, apart from its role in nitrate reduction and assimilation, can also catalyse the reduction of nitrite to NO. This reaction can produce large amounts of NO, or at least more than is needed for signalling, as some escape of NO to the outside medium can be detected after NR activation. A role for NO and NR in stomata functioning in response to abscisic acid has also been proposed. The question that remains is whether this NR-derived NO is a signalling molecule or the mere product of an enzymatic side reaction like the products generated by the oxygenase activity of RuBisCO.

Interaction of nitrogen and phosphorus nutrition in determining growth

In this paper we discuss the differences and similarities in the growth response of tomato plants to N and P limitation, and to their interaction. Two detailed growth experiments, with varied N or P supply, were conducted in order to unravel the effects of N and P limitation on growth of young tomato plants (Lycopersicon esculentum Mill.). Relative growth rate (RGR) initially increased sharply with increasing plant P concentration but leveled off at higher plant P concentrations. In contrast, RGR increased gradually with increasing plant N concentration before it leveled off at higher plant N concentrations. The relationship of RGR with organic leaf N and P showed the same shape as with total N and P concentrations, respectively. The difference in response is most likely due to the different roles of N and P in the machinery of the plants energy metabolism (e.g., photosynthesis, respiration). Plant N concentration decreased with increasing P limitation. We show that this decrease cannot be explained by a shift in dry-mass partitioning. Our results suggest that the decrease in N concentration with increasing P limitation may be mediated by a decrease in leaf cytokinin levels and is less likely due to decreased energy availability at low P conditions. Dry-mass partitioning to the roots was closely linearly related to the leaf reduced-N concentration. However, treatments that were severely P limited deviated from this relationship.

Inhibition of aconitase by nitric oxide leads to induction of the alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids

Nitric oxide (NO) is a free radical molecule involved in signalling and in hypoxic metabolism. This work used the nitrate reductase double mutant of Arabidopsis thaliana (nia) and studied metabolic profiles, aconitase activity, and alternative oxidase (AOX) capacity and expression under normoxia and hypoxia (1% oxygen) in wild-type and nia plants. The roots of nia plants accumulated very little NO as compared to wild-type plants which exhibited ∼20-fold increase in NO emission under low oxygen conditions. These data suggest that nitrate reductase is involved in NO production either directly or by supplying nitrite to other sites of NO production (e.g. mitochondria). Various studies revealed that NO can induce AOX in mitochondria, but the mechanism has not been established yet. This study demonstrates that the NO produced in roots of wild-type plants inhibits aconitase which in turn leads to a marked increase in citrate levels. The accumulating citrate enhances AOX capacity, expression, and protein abundance. In contrast to wild-type plants, the nia double mutant failed to show AOX induction. The overall induction of AOX in wild-type roots correlated with accumulation of glycine, serine, leucine, lysine, and other amino acids. The findings show that NO inhibits aconitase under hypoxia which results in accumulation of citrate, the latter in turn inducing AOX and causing a shift of metabolism towards amino acid biosynthesis.