Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro.

NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.     

The occurrence and control of nitric oxide generation by the plant mitochondrial electron transport chain

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Q_i‐site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Q_o‐site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate‐grown wild‐type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium‐grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate‐grown plants. Hence the ETC, likely the Q‐cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non‐energy‐conserving electron sink upstream of Complex III.     

Nitrate reductase activity in leaves and roots ofAlnus glutinosa (L) Gaertner

Summary Thein vivo nitrate reductase activity (NRA) was determined inAlnus glutinosa plants grown nonsymbiotically on ammonium, nitrate, a combination of both, or symbiotically with atmospheric nitrogen as the only nitrogen source. Root NRA was absent when ammonium or atmospheric nitrogen was the nitrogen source. With nitrate in the culture solution the roots showed a high NRA. However, the leaf NRA behaved quite differently: with negligible activities on all nitrogen sources except atmospheric nitrogen. The foliar NRA measured, however, is likely not due to the activity of the plant but of microbial origin. Methods commonly used to facilitate produced nitrite to leak out of the tissue, such as addition of propanol and cutting the plant material, did not increase the nitrite release from the leaves. A turbidity developed when testing the samples for nitrite which was positively correlated with the NRA. Populations of microorganisms in the phyllosphere did not differ between the nutritional treatments. Bacteria, able to grow on a low-nitrogen medium, were present on the leaves. Nitrifiers could not be detected. The bacteria on the leaves appear to produce nitrite when incubated with leaf material. Grassland Species Research Group, Publication no. 106     

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ

At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.     

Nitrate reductase from <Emphasis Type="Italic">Triticum aestivum</Emphasis> leaves: Regulation of activity and possible role in production of nitric oxide

Nitrate reductase (NR) and peroxidase (POX) are important enzymes involved in the metabolism of reactive oxygen (ROS) and nitrogen species in leaves of wheat (Triticum aestivum L.) seedlings. It has been confirmed that NR activity in wheat leaves depends on the light conditions and the presence of nitrates during the cultivation of the seedlings, and it is regulated by the molybdenum cofactor and phosphorylation. In the present study, confocal microscopy and EPR spectroscopy studies showed that the addition of nitrite, a product of NR, increased the level of nitric oxide (NO). This increase was prevented by the addition of sodium azide, an inhibitor of NR. The results suggest that in wheat leaves one of the key functions of NR is the formation of the signaling NO molecule. Cultivation of green plants under conditions of prolonged (4 days) darkness, a strong stress factor for photosynthesizing cells, decreased the activity of NR. Moreover, darkness induced significant elevation of the POX activity that was prevented by the addition of nitrate to the growth medium. It is proposed that the changes in light conditions result in the competition between nitrate- and ROS-metabolizing activities of POX in leaves, and a possible interaction between NR and POX controls the levels of NO and ROS in the leaf tissue.     

Apoplastic synthesis of nitric oxide by plant tissues

Nitric oxide (NO) is an important signaling molecule in animals and plants. In mammals, NO is produced from Arg by the enzyme NO synthase. In plants, NO synthesis from Arg using an NO synthase-type enzyme and from nitrite using nitrate reductase has been demonstrated previously. The data presented in this report strongly support the hypothesis that plant tissues also synthesize NO via the nonenzymatic reduction of apoplastic nitrite. As measured by mass spectrometry or an NO-reactive fluorescent probe, Hordeum vulgare (barley) aleurone layers produce NO rapidly when nitrite is added to the medium in which they are incubated. NO production requires an acid apoplast and is accompanied by a loss of nitrite from the medium. Phenolic compounds in the medium can increase the rate of NO production. The possible significance of apoplastic NO production for germinating grain and for plant roots is discussed.     

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport

Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.     

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.Abbreviations GS glutamine synthetase - NR nitrate reductase - NiR nitrite reductase     

Decrease of Nitrate Reductase Activity in Spinach Leaves during a Light-Dark Transition

In leaves of spinach plants (Spinacia oleracea L.) performing CO₂ and NO₃⁻ assimilation, at the time of sudden darkening, which eliminates photosystem I-dependent nitrite reduction, only a minor temporary increase of the leaf nitrite content is observed. Because nitrate reduction does not depend on redox equivalents generated by photosystem I activity, a continuation of nitrate reduction after darkening would result in a large accumulation of nitrite in the leaves within a very short time, which is not observed. Measurements of the extractable nitrate reductase activity from spinach leaves assayed under standard conditions showed that in these leaves the nitrate reductase activity decreased during darkening to 15% of the control value with a half-time of only 2 minutes. Apparently, in these leaves nitrate reductase is very rapidly inactivated at sudden darkness avoiding an accumulation of the toxic nitrite in the cells.     

Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.     

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite

Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation. Received: 8 May 2000 / Accepted: 24 August 2000     

Integrative response of plant mitochondrial electron transport chain to nitrogen source

Nitrogen (N) availability is widely known as a determinant of plant growth and respiration rate. However, less attention has been paid to the effect of the type of N source (nitrate, nitrite or ammonium) on the respiratory system. This review summarizes the latest findings on this topic, with an emphasis on the effect of ammonium and nitric oxide (NO) on the respiratory system, and the physiological role of alternative oxidase (AOX). First, concentrated ammonium has been found to increase plant respiration rate (ammonium-dependent respiratory increase, ARI). We will introduce two hypotheses to explain ARI, futile ammonium cycling and excess reducing equivalents, and verify the validity of each hypothesis. We suggest that these two hypotheses are not necessarily mutually exclusive. Second, gene expression of AOX is suppressed when N is predominately available as nitrate instead of ammonium. We will discuss possible signaling pathways leading to this expression pattern. Third, while AOX expression is induced by NO, AOX activity itself is insensitive to NO. In contrast, activity of cytochrome c oxidase (COX) is sensitive to NO. We outline the NO production pathway, focusing on nitrite-dependent NO production, and discuss the physiological significance of the fact that AOX activity is insensitive to NO. Finally, this review aims to build an integrated scheme of the respiratory response to the type of N source, considering leaves in high light conditions or hypoxic roots.     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

Effect of Elevated Temperature on Nitrite and Nitrate Reduction in Leaves and Intact Chloroplasts

Barley (Hordeum vulgare L.) leaves and intact spinach (Spinacia oleracea L.) chloroplasts were exposed to short-term heating, and the aftereffects of heat treatment on in vitro andin vivo activities of nitrate reductase and noncyclic electron transport associated with nitrite reduction were studied. Heating of leaves at temperatures above 40°C led to a monotonic decrease in nitrate reductase in vitro activity. On the contrary, the in vivo enzyme activity, assayed in intact leaf tissues after 5-min heat treatment, increased 1.5 times upon elevating the pretreatment temperature from 37 to 40°C and gradually decreased at higher temperatures. Noncyclic electron transport related to CO₂ fixation in intact chloroplasts decreased gradually after heat exposures above 39°C, unlike the electron transport to nitrite as a terminal acceptor, which was stimulated by heating of intact chloroplast suspensions in the temperature range from 33 to 40°C. The heating at higher temperatures inhibited nitrite photoreduction. It is concluded that the heating of phototrophic cells at sublethal temperatures stimulates the mobilization of inorganic nitrogen and thereby facilitates the repair of thermally induced injuries of proteinaceous cell structures. The stimulation of nitrate reductase activity in vivo at the temperature range 37–40°C provides an evidence for the increase in the availability of reductants in the cytosolic compartment of the leaf cell.     

Apoplastic pH and Fe(3+) reduction in intact sunflower leaves

It has been hypothesized that under NO(3)(-) nutrition a high apoplastic pH in leaves depresses Fe(3+) reductase activity and thus the subsequent Fe(2+) transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO(3)(-) nutrition significantly increased apoplastic pH at distinct interveinal sites (pH >/= 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO(3)(-)/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH(4)(+) or NH(4)NO(3) nutrition. Complementary to pH measurements, the formation of Fe(2+)-ferrozine from Fe(3+)-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe(3+) reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5. 0. In analogy to the monitoring of Fe(3+) reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe(3+) reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena)

Both the in vivo (+ nitrate) nitrate reductase (NR) activity (leaf disks incubated in the presence of KNO₃) and the in vivo (? nitrate) NR activity (leaf disks incubated without KNO₃) in leaves of eggplant (Solanum melongena L. cv. Bonica) were affected by rapidly growing fruits. Plants with a fruit load showed more pronounced diurnal variation in (+ nitrate) NR activity and higher (? nitrate) NR activity than plants without fruit. The higher (? nitrate) NR activity was accompanied by higher nitrate and lower sucrose and starch contents of leaves. The more pronounced diurnal changes in (+ nitrate) NR activity were paralleled by more pronounced diurnal variation in carbohydrate content of leaves. Fruit removal led to a decrease in both (? nitrate) NR activity and nitrate concentration in leaves, while the carbohydrate content increased. Plants supplied with ammonium instead of nitrate showed only slightly lower (+ nitrate) but no (? nitrate) NR activity. As for plants treated with nitrate, diurnal changes in (+ nitrate) NR activity were most pronounced in leaves of plants with fruit and this again was paralleled by a more pronounced diurnal variation in the carbohydrate concentration in the leaves. Increasing the oxygen level of the atmosphere to 50% led to a dramatic decrease in the (+ nitrate) NR activity and to an increase in both (? nitrate) NR activity and nitrate concentration, which was accompanied by decreasing carbohydrate contents of the leaves. Low light intensities and extended dark periods caused similar changes in NR activity and nitrate and carbohydrate concentrations in leaves. Increasing the nitrate concentration in the nutrient solution led to a rise in (+ nitrate) and (? nitrate) NR activity, but only the (? nitrate) NR activity paralleled the nitrate concentration in the leaves. This increase in the nitrate concentration was accompanied by a decrease in the carbohydrate content of the leaves. It is concluded that the level of and the diurnal changes in both (+ nitrate) and (? nitrate) NR activity and the concentration of nitrate in the leaves are dependent upon their carbohydrate status.     

Reassessment of the in vivo Assay for Nitrate Reductase in Leaves

The in vivo assay procedure for nitrate reductase and its dependence on the concentration of nitrate and other ions were examined. It was found that high ion concentrations led to an increased release of nitrite to the reaction media which could be interpreted as a stimulated nitrate reductase activity. This phenomenon is not an osmotic effect, since equivalent concentrations of mannitol did not lead to identical results. The effect of ions on the enhanced nitrite production was attributed to changes in cell membrane permeability rather than to a supply of substrate. This conclusion is based on several findings: (a) in in vitro assays, the rate of nitrite production was not affected by ion concentrations: (b) the stimulation of nitrite production was obtained by various ions and not only by nitrate; (c) pretreatment of alfalfa leaves with nitrate did not increase the NO₂^? release rate to the external solution; and (d) nitrate and nitrite export from leaf discs to the solution was stimulated even in discs which were enzymatically inactive. Calcium ions in the presence of KNO₃ inhibited the enhanced nitrite production, probably due to alteration of membrane stability. The effect of ions on the rate of nitrite production was reversible and the high rate of nitrite production was reduced to the control rate when discs were transferred to a solution of low concentration.     

Nitrate (NO3) and nitrite (NO2) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate (NO₃⁻) and nitrite (NO₂⁻) on the activity of dopaminergic neuron in zebrafish embryos and larvae. Both nitrate and nitrite exposure decreased the expression of tyrosine hydroxylase (TH) in dopaminergic neurons at 48 hpf. Only nitrite decreased the response to tactile stimulation at 72 hpf, whereas both nitrate and nitrite decreased the swimming activity at 6 dpf. When the embryos were exposed to nitrate or nitrite together with an estrogen receptor blocker (ICI 182,780), the decreases in TH expression and motor behavior caused by nitrate or nitrite alone were reversed suggesting the effects of nitrate and nitrite were mediated through estrogen receptor (ER). The result of co-incubation with an oxidoreductase inhibitor, diphenyleneiodonium, indicated the conversion to nitric oxide (NO) is likely to be responsible for the effects of nitrate and nitrite, which was further supported by the increased staining for NO after exposure. The present study demonstrates that nitrate and nitrite are neurotoxicants acting as an endocrine disruptor possibly through conversion to NO to downregulate the activity of dopaminergic neuron in early development of zebrafish.     

Iron availability in plant tissues-iron chlorosis on calcareous soils

The article describes factors and processes which lead to Fe chlorosis (lime chlorosis) in plants grown on calcareous soils. Such soils may contain high HCO₃ ^- concentrations in their soil solution, they are characterized by a high pH, and they rather tend to accumulate nitrate than ammonium because due to the high pH level ammonium nitrogen is rapidly nitrified and/or even may escape in form of volatile NH₃. Hence in these soils plant roots may be exposed to high nitrate and high bicarbonate concentrations. Both anion species are involved in the induction of Fe chlorosis.Physiological processes involved in Fe chlorosis occur in the roots and in the leaves. Even on calcareous soils and even in plants with chlorosis the Fe concentration in the roots is several times higher than the Fe concentration in the leaves. This shows that the Fe availability in the soil is not the critical process leading to chlorosis but rather the Fe uptake from the root apoplast into the cytosol of root cells. This situation applies to dicots as well as to monocots. Iron transport across the plasmamembrane is initiated by Fe^III reduction brought about by a plasmalemma located Fe^III reductase. Its activity is pH dependent and at alkaline pH supposed to be much depressed. Bicarbonate present in the root apoplast will neutralize the protons pumped out of the cytosol and together with nitrate which is taken up by a H⁺/nitrate cotransport high pH levels are provided which hamper or even block the Fe^III reduction.Frequently chlorotic leaves have higher Fe concentrations than green ones which phenomenon shows that chlorosis on calcareous soils is not only related to Fe uptake by roots and Fe translocation from the roots to the upper plant parts but also dependent on the efficiency of Fe in the leaves. It is hypothesized that also in the leaves Fe^III reduction and Fe uptake from the apoplast into the cytosol is affected by nitrate and bicarbonate in an analogous way as this is the case in the roots. This assumption was confirmed by the highly significant negative correlation between the leaf apoplast pH and the degree of iron chlorosis measured as leaf chlorophyll concentration. Depressing leaf apoplast pH by simply spraying chlorotic leaves with an acid led to a regreening of the leaves.