Apoplast acidification is not a necessary determinant for the resistance of maize in the first phase of salt stress

In previous studies, a relation between plant growth during the first phase of salt stress and cell‐wall acidification was shown for differently resistant maize genotypes. In the present study, plants of the salt‐sensitive maize (Zea mays L.) cv. Pioneer 3906 and the salt‐resistant genotype SR 12, grown under 100 mM NaCl, showed a similar decrease in plasmalemma H⁺‐ATPase activity, while SR 12 showed less growth reduction than Pioneer 3906. From this it is concluded that maintenance of apoplast acidification is not necessary for better plant growth during the first phase of salt stress.     

Does H+ pumping by plasmalemma ATPase limit leaf growth of maize (Zea mays) during the first phase of salt stress?

In the first phase of salt stress, growth of plants is impaired mainly by osmotic stress. To elucidate the effect of NaCl salinity on elongation growth of maize leaves in the first phase of salt stress, we investigated the effect of NaCl on gene expression and activity of the plasmalemma H⁺ ATPase of elongating leaves of maize (Zea mays L.). Treatment of maize plants with 125 mM NaCl for 3 d decreased leaf growth relative to control plants (1 mM NaCl). Whereas H⁺ ATPase hydrolytic activity was unaffected, the ability of the H⁺ ATPase to establish a pH gradient was strongly reduced. Total mRNA of plasmalemma H⁺ ATPase was slightly increased. However, mRNA of the ATPase isoform MHA1 was significantly reduced and ATPase isoform MHA4 was strongly increased at the mRNA level. Synthesis of total H⁺ ATPase protein was unchanged as revealed by western blot. The results indicate that reduced pumping of H⁺ ATPase in leaf plasmalemma under salt stress may be caused by a switch to gene expression of the specific isoform MHA4, which shows inferior H⁺‐pumping efficiency in comparison to isoforms expressed under control conditions. We propose that reduced H⁺ pumping of plasmalemma H⁺ ATPase is involved in the reduction of leaf growth of maize during the first phase of salt stress.     

Consequences of sodium exclusion for the osmotic potential in the rhizosphere – Comparison of two maize cultivars differing in Na+ uptake

According to the biphasic model of growth response to salinity, growth is first reduced by a decrease in the soil osmotic potential (Ψ_o), i.e., growth reduction is an effect of salt outside rather than inside the plant, and genotypes differing in salt resistance respond identically in this first phase. However, if genotypes differ in Na⁺ uptake as it has been described for the two maize cultivars Pioneer 3906 and Across 8023, this should result in differences in Na⁺ concentrations in the rhizosphere soil solution and thus in the concentration of salt outside the plant. It was the aim of the present investigation to test this hypothesis and to investigate the effect of such potential differences in soil Ψ_o caused by Na⁺ exclusion on plant water relations. Sodium exclusion at the root surface of intact plants growing in soil was investigated by sampling soil solution from the rhizosphere of two maize cultivars (Across 8023, Pioneer 3906). Plants were grown in a model system, consisting of a root compartment separated from the bulk soil compartment by a nylon net (30 μm mesh size), which enabled independent measurements of the change of soil solution composition and soil water content with increasing distance from the root surface (nylon net). Across 8023 accumulated higher amounts of sodium in the shoot compared to the excluder (Pioneer 3906). The lower Na⁺ uptake in the excluder was partly compensated by higher K⁺ uptake. Pioneer 3906 not only excluded sodium from the shoot but also restricted sodium uptake more efficiently from roots relative to Across 8023. This was reflected by higher Na⁺ concentrations in the rhizosphere soil solution of the excluder 34 days after planting (DAP). The difference in Na⁺ concentration in rhizosphere soil solution between cultivars was neither due to differences in transpiration and thus in mass flow, nor due to differences in actual soil water content. As the lower Na⁺ uptake of the excluder (Pioneer 3906) was only partly compensated by increased uptake of K⁺, soil Ψ_o in the rhizosphere of the excluder was more negative compared to Across 8023. However, no significant negative effect of decreased soil Ψ_o on plant water relations (transpiration rate, leaf Ψ_o, leaf water potential, leaf area) could be detected. This may be explained by the fact that significant differences in soil Ψ_o between the two cultivars occurred only towards the end of the experiment (27 DAP, 34 DAP).     

Nitrogen nutritional status of young maize plants (Zea mays) is not limited by NaCl stress

Maize plants (Zea mays L. cv. Pioneer 3906) were grown in hydroponics with four different NaCl treatments (control, 50, 100, 150 mM NaCl). Nitrogen (N) was supplied as 2 mM Ca(NO₃)₂ in the fully concentrated nutrient solution. Plants of half of the pots were treated with additional 1 mM NH₄NO₃ 2 d after start of the NaCl application. After 23 d, the maize plants were harvested and contents and concentrations of nitrate, reduced N as well as chloride were determined in shoots and roots. With increasing NaCl stress net nitrate uptake and net root‐to‐shoot translocation of total N decreased significantly. Under salt stress, decreased nitrate concentrations in shoots probably caused substrate limitation of nitrate reductase. However, the concentrations of reduced N in shoots were not affected by salt stress and no N deficiency was observed. Additional N application to the 100 and 150 mM NaCl treatments did not improve plant growth. A Cl^?/NO antagonism was only weakly pronounced, probably because of the Cl^? exclusion ability of maize. Thus, although net uptake and net translocation of total N were markedly decreased by NaCl application, the smaller maize plants nevertheless took up enough N to meet their demand pointing to other growth‐limiting factors than N nutrition.     

Lupin and pea differ in root cell wall buffering capacity and fractionation of apoplastic calcium

Lupin (Lupinus angustifolius L.) and pea (Pisum sativum L.) differ substantially in their root growth at pH≥6. The mechanisms underlying such a variation are not fully understood. The H⁺ buffering capacity of isolated cell wall and calcium binding property of intact roots of these two species were compared under various experimental conditions. The shape of the H⁺/OH^‐ titration curves of cell wall for lupin and pea showed no major discrepancy except with differed magnitudes. There appeared to be two H⁺‐titratable groups in root cell wall of both species—below pH 6 and above 8. The wall H⁺ buffering capacity of pea roots was lower at pH 4–5, but was greater at pH above 5.5 than that of lupin roots. The fractionation of apoplastic calcium demonstrated that the proportion of easily exchangeable Ca²⁺ was greater while that of tightly bound Ca²⁺ was smaller in pea roots than in lupin roots. In addition, Ca²⁺ in cell wall was more easily exchanged by H⁺ in pea than in lupin roots. The results suggest that the different sensitivity in root growth at pH≥6 of lupin and pea is related to the difference in H⁺ buffering and Ca²⁺ exchange capacities in the root apoplast of these species, and that the greater sensitivity of lupin roots to pH≥6 is partly due to a higher threshold of H⁺ concentration required for cell wall loosening.     

High monosilicic acid supply rapidly increases Na accumulation in maize roots by decreasing external Ca2+ activity

Both calcium (Ca²⁺) and silicon (Si) improve plant performance under salt (NaCl) stress. Although these two mineral elements share numerous similarities, the information on how their extracellular interactions in the root apoplast affect uptake of sodium (Na⁺) is still lacking. Here, we investigated the effect of high Si supply in the bioavailable form of monosilicic acid (H₄SiO₄) on the activity of Ca²⁺ in the external root solution, and subsequent root uptake and compartmentation of Na in maize (Zea mays L.). In the short‐term experiments (6 h), 14‐d‐old maize plants were exposed to various concentrations of Ca²⁺ at three different pH‐values (6.5, 7.5, and 8.5) and two Si concentrations, i.e., low (1 mM) and high (4 mM) supply of H₄SiO₄. The activity of Ca²⁺ and Na⁺ in the external solution as well as the root concentrations of total and cell sap and BaCl₂‐exchangeble apoplastic fractions of both elements were analyzed. The pH of the nutrient solution affected neither the ion activities nor the root accumulation of both Ca²⁺ and Na⁺. At higher pH values (7.5 and 8.5) the interactions of Ca²⁺ and Si at high Si supply led to a decrease of Ca²⁺ activity and, hence, an increase of Na⁺ : Ca²⁺ activity ratio in the external root solution. Concomitantly, despite the elevated exchangeable apoplastic fraction of both Ca²⁺ and Na⁺, the total and cell sap concentrations were remarkably decreased for Ca²⁺ and increased for Na⁺ by the addition of 4 mM H₄SiO₄. This work demonstrates that at high Si supply extracellular Ca‐Si interactions leading to lowered activity of Ca²⁺ might rapidly compromise the ameliorative effect of Ca²⁺ on Na⁺ accumulation in roots. Practically, Si over‐fertilization of saline and, in particular, sodic soils may further promote the accumulation of Na⁺ in root tissues hours after Si application and, hence, increase a potential risk of Na⁺ toxicity.     

Invertase activity limits grain yield of maize under salt stress

Salt stress reduces grain yield of maize (Zea mays L.) due to poor kernel setting but not due to decreased grain filling. In the present study, it was tested whether acid invertase activity is decreased in developing kernels of maize under salt stress, and if assimilate supply is limited. The relatively salt‐sensitive maize hybrid Pioneer 3906 was compared with the more salt‐resistant hybrid SR 12. Salt stress caused a significant decrease in grain yield which was due to a 50% decrease in kernel number. No source limitation was observed, as the sucrose concentrations in kernels were significantly increased under salt stress for both genotypes. In contrast, glucose and fructose concentrations in kernels were significantly decreased. Salt stress caused a significant inhibition of soluble acid invertase activity to 19% in hydroponics 5 d after pollination (5 DAP) and to 50% in the soil culture experiment (2 DAP). The decrease in enzyme activity was the same for both genotypes. In the soil experiment, the highest soluble acid invertase activity was found 2 DAP with a steep decline until 8 DAP in Pioneer 3906. It is concluded that a decrease in acid invertase activity is a key factor associated with limited kernel setting under salt stress but additional factors may be responsible for genotypic differences.     

H+‐atpase and H+‐transport activities in tonoplast vesicles from barley roots under salt stress and influence of calcium and abscisic acid 1

Seedlings of two barley cultivars differing in NaCl sensitivity were treated with low (100 mM) or high (400 mM) concentration of NaCl for 6 days. Tonoplast vesicles were prepared from roots, and H⁺‐ATPase and H⁺‐transport activities associated with tonoplast were assayed. Both H⁺‐ATPase and H⁺‐transport activities in the two cultivars were increased at 100 mM NaCl. These activities also increased in the salt‐tolerant cultivar at 400 mM NaCl, but in salt‐sensitive cultivar were decreased. In vivo treatment with 10 mM Ca²⁺ stimulated H⁺‐ATPase and H⁺‐transport activities at two levels of NaCl, however, treatment with 10⁵M (±) abscisic acid (ABA) inhibited these activities. From these results we propose that the increase of the vacuolar H⁺ pumps in barley roots reflects an adaptation to salt stress. The stimulation of HVATPase and H⁺‐transport activities by calcium (Ca) depends mainly on its effect in maintaining stability of membrane under salt stress.     

Is the salt tolerance of maize related to sodium exclusion? I. Preliminary screening of seven cultivars

Maize (Zea mays L.) plants in the early stage of development were treated with 80 mM sodium chloride (NaCl) with or without supplemental calcium (Ca²⁺) (8.75 mM) for a seven day period. The effects of salinity on dry matter production and shoot and root concentrations of sodium (Na⁺), Ca²⁺, and potassium (K⁺) were measured for seven Pioneer maize cultivars. Salinity significantly reduced total dry weight, leaf area, and shoot and root dry weight below control levels. For all seven cultivars, Na⁺concentrations were reduced and leaf area was significantly increased by supplementing salinized nutrient solutions with 8.75 mM calcium chloride (CaCl₂). The two cultivars with the lowest shoot and root Na⁺ concentrations under NaCl‐salinity showed the greatest increases in total, shoot and root dry weights with the addition of supplemental Ca. Shoot fresh weight/dry weight ratios for all cultivars were decreased significantly by both salinity treatments, but supplemental Ca²⁺ increased the ratio relative to salinity treatments without supplemental Ca. Root fresh weight/dry weight ratios were decreased only by salinity treatments with supplemental Ca. With NaCl‐salinity, cultivars which had lower shoot and root Na⁺ concentrations were found to be more salt sensitive and had significantly lower amounts of dry matter production than those cultivars which had higher shoot and root Na⁺ concentrations. It was concluded that Na⁺ exclusion from the shoot was not correlated with and was an unreliable indicator of salt tolerance for maize.     

Changes of H+ pumps of tonoplast vesicle from wheat roots in vivo and in vitro under aluminum treatment and effect of calcium

Wheat seedlings were treated with 0.1 mM Al for 7 days. Tonoplast vesicles were isolated from apical segments of roots and activities of H⁺‐ATPase and H⁺‐PPase measured. Compared with the control, 0.1 mM Al increased H⁺‐ATPase activity and decreased H⁺‐PPase activity. High external supply of calcium (Ca) (5 mM) diminished the extent of the stimulation of H⁺‐ATPase activity and alleviated the reduction of H⁺‐PPase activity under Al treatment. However, 0.1 mM Al treatment in vitro resulted in the inhibition of H⁺‐ATPase activity with the decrease of Vmax and Km for ATP. In vitro treatment with 0.1 mM Al also decreased the H⁺‐PPase activity, but increased the Km for PPi.     

Responses of Batis maritima plants challenged with up to two‐fold seawater NaCl salinity

Batis maritima is a promising halophyte for sand‐dune stabilization and saline‐soil reclamation. This species has also applications in herbal medicine and as an oilseed crop. Here, we address the plant response to salinity reaching up to two‐fold seawater concentration (0–1000 mM NaCl), with a particular emphasis on growth, water status, mineral nutrition, proline content, and photosystem II integrity. Plant biomass production was maximal at 200 mM NaCl, and the plants survived even when challenged with 1000 mM NaCl. Plant water status was not impaired by the high accumulation of sodium in shoots, suggesting that Na⁺ compartmentalization efficiently took place in vacuoles. Concentrations of Mg²⁺ and K⁺ in shoots were markedly lower in salt‐treated plants, while that of Ca²⁺ was less affected. Soluble‐sugar and chlorophyll concentrations were hardly affected by salinity, whereas proline concentration increased significantly in shoots of salt‐treated plants. Maximum quantum efficiency (F_v/F_m), quantum yield of PSII (Φ_PSII), and electron‐transport rate (ETR) were maximal at 200–300 mM NaCl. Both nonphotochemical quenching (NPQ) and photochemical quenching (qP) were salt‐independent. Interestingly, transferring the plants previously challenged with supraoptimal salinities (400–1000 mM NaCl) to the optimal salinity (200 mM NaCl) substantially restored their growth activity. Altogether, our results indicate that B. maritima is an obligate halophyte, requiring high salt concentrations for optimal growth, and surviving long‐term extreme salinity. Such a performance could be ascribed to the plant capability to use sodium for osmotic adjustment, selective absorption of K⁺ over Na⁺ in concomitance with the stability of PSII functioning, and the absence of photosynthetic pigment degradation.     

Effect of sodium chloride stress on the plasma membrane ATPase of barley roots: Probable cause for decrease in ATPase activity

The regulation of plasma membrane ATPase activity by salt stress was investigated in barley roots. The plasma membrane fractions were prepared from the roots treated with or without 200 mM sodium chloride (NaCl) for one day. After salt treatment, ATPase activity reduced by 20 to 30% as compared with that of control roots. No significant changes in the content of total phospholipid and sterol were detected in the plasma membrane fraction by salt stress. After extraction of most of the phospholipids in the plasma membrane vesicles with a solution containing 1% (W/V) octylglucoside and 1% (W/V) Triton X‐100, the ATPase activity in salt‐stressed roots was lower than that of control roots. After reconstitution of detergent‐extracted protein into liposome, the reduction of ATPase activity by salt stress did not recover. Based on immunoblott analysis, the relative amount of H⁺‐ATPase in plasma membrane fraction prepared, from NaCl‐stressed roots was smaller than that of control roots. These results indicate that the reduction of H⁺‐ATPase activity by salt stress was caused by the decrease in the amount of H⁺‐ATPase rather than the modification of ATPase.     

Effect of salinity on metalloenzymes of oxygen metabolism in two leguminous plants

Abstract

Superoxide dismutase (SOD) pattern, catalase, Cyt c oxidase and fumarase activity were studied in leaves of Phaseolus vulgaris and Vigna unguiculata plants growth in two sodium chloride (NaCl) concentrations (35 mM and 100 mM). In bean plants growth with NaCl, leaf chloride (Cl^?) contents were higher than in control plants, and the same was found for sodium (Na⁺) and potassium (K⁺) contents, although to a lesser degree. In cowpea leaves, Na⁺ and Cl^? had a similar increase due to salt‐growth conditions. Under salinity, all changes in the antioxidant (SOD and catalase) enzymes levels were smaller in bean than in cowpea plants. In Phaseolus at 15 days growth, Cu, Zn‐SOD I showed an increase by the effect of salt treatment, but this induction did not occur at 30 days growth, and both Mn‐SOD and Cu, Zn‐SOD II did not show variations due to salt‐stress. In Vigna, Mn‐SOD was decreased by salinity but this was compensated by an increase in Cu, Zn‐SOD I activity in plants at 30 days growth, whereas in young leaves under saline conditions, both isozymes were also decreased. Likewise, there was a rise in cytochrome c oxidase and fumarase activity in leaves of NaCl‐treated plants compared to the control. The activity changes observed are discused in term of their possible relevance to plant sensitivity to saline conditions.     

Salinity‐induced changes in growth,superoxide dismutase activity,and ion content of two olive cultivars

Olive trees (Olea europaea L.) are considered moderately tolerant to salinity, with clear differences found among cultivars. One‐year‐old self‐rooted olive plants of the Croatian cv. Oblica and Italian cv. Leccino were grown for 90 d in nutrient solutions containing 0, 66, or 166 mM NaCl, respectively. The shoot length and the number of nodes and leaves for both cultivars were not affected by salinity up to 66 mM NaCl. However, at 166 mM NaCl, growth of Leccino was reduced earlier and to a higher extent than growth of Oblica. After 10 d of exposure to 66 and 166 mM NaCl, increased activity of superoxide dismutase (SOD) was observed in Leccino, whereas there was almost no response in Oblica. Reduced SOD activity in Leccino at 166 mM NaCl was observed after prolonged stress (90 d), whereas in Oblica SOD was increased at 66 mM compared to control or 166 mM NaCl. Electrolyte and K⁺ leakage were increased and relative water content decreased as NaCl concentration increased with similar intensity of response measured in both cultivars. Oblica exhibited an ability to keep a higher K⁺ : Na⁺ ratio at all salinity levels compared to Leccino, but since no difference was found in leaf K⁺ concentration, this was mainly achieved by less Na⁺ ions reaching the younger leaves. The antioxidative system represents a component of the complex olive salt‐tolerance mechanism, and it seems that the role of SOD in protection from oxidative stress depends on sodium accumulation in leaves.     

Intra‐specific variation for salt tolerance in a potential oil‐seed crop,brown mustard (Brassica juncea (l.) Czern. and Coss.)

Thirty eight accessions of brown mustard (Brassica juncea (L.) Czern. and Coss.) were screened after two weeks growth in solution culture containing 120 mol m‐³ NaCl. Considerable variation for salt tolerance was observed in this set of germplasm, since some accessions showed relatively vigorous growth in saline medium.

In order to determine the consistency of degree of salt tolerance at different growth stages of crop life cycle two salt tolerant accessions, P‐15 and KS‐51 and two salt sensitive 85362 and 85605 were tested at the adult stage in 0(control), 100 and 200 mol m^‐3 NaCl. Both the tolerant accessions produced significantly greater fresh and dry biomass and had considerably higher seed yield than those of the salt sensitive accessions. Analysis of different ions in the leaves showed that salt tolerant accessions contained greater amounts of Na⁺, K⁺ and Ca²⁺ than the salt sensitive accessions, although they did not differ significantly for leaf Cl^‐. Only one salt tolerant accession P‐15 had greater leaf K/Na ratio and K⁺ versus Na⁺ selectivity compared with the tolerant KS‐51 and the two salt sensitive accessions.

From this study it was established that there is a considerable variation for salt tolerance in B.juncea which can be exploited by selection and breeding for improvement of its salt tolerance. Since the degree of salt tolerance in B.juncea does not change at different growth stages of the crop life cycle, selection for salt tolerance at the initial growth stages could provide individuals that would be tolerant at all other growth stages. Accumulation of Na⁺, K⁺ and Ca²⁺ in the leaves are important components of salt tolerance in B.juncea.     

Limits imposed by salt to the growth of the halophyte Sesuvium portulacastrum

The growth of the halophyte Sesuvium portulacastrum, commonly known as sea purslane, is impeded by NaCl only at high (600–1000 mM) concentration. Therefore, the goal of this investigation was to identify the mechanisms which set the limit of the salt resistance of S. portulacastrum. 21‐day‐old cuttings were grown for 45–50 d under split‐root conditions in which one half of the root system was immersed in complete nutrient solution supplemented with 800 mM NaCl, while the other half was immersed in a NaCl‐free medium, containing all nutrients or being deprived of potassium or calcium or nitrogen. Using this approach, we demonstrate that K⁺ and N uptake was impaired in roots exposed to NaCl. Concerning Ca²⁺, there was no indication of uptake inhibition by NaCl. However, restriction of K⁺ uptake by roots was compensated by an increase in the K⁺‐use efficiency, so that growth was not inhibited. Concerning N, our analysis shows that NO and/or NH uptake, but not their assimilation, was limited by salt treatment. Thus, we conclude that at high salinity levels, the growth of S. portulacastrum is limited by the restrictions imposed by NaCl on N uptake, perhaps in addition to inhibiting effects of excessive Na⁺ accumulation in shoot.     

Interactive effects of salinity and potassium availability on growth,water status,and ionic composition of Hordeum maritimum

The interactive effects of salinity and potassium (K⁺) availability on biomass production, water status, and ionic composition were investigated in Hordeum maritimum, an annual grass growing natively on saline soils. Plants were grown for 7 weeks on Hewitt nutrient solution supplied with NaCl (0, 100, 150, 200, and 300 mM) combined with low (0.232 mM) or high (5.8 mM) K⁺ levels. Independent of potassium availability, dry matter of both roots and shoots decreased consistently with increasing NaCl levels in the culture medium, in association with a significant reduction of the shoot water content. This salt‐induced growth reduction did not result from a restriction of K⁺ nutrition, since H. maritimum expressed similar growth under both low and high K⁺ supply. NaCl decreased shoot K⁺ concentrations. This effect was more pronounced in plants grown at high K⁺ supply than in plants grown at low K⁺ supply. This result suggests that the absorption systems were strongly selective for K⁺, and that this selectivity was enhanced by salt.     

Effect of ammonium and phosphorus supply on h+ production in gel by two tropical forage grasses

The effect of the supply of ammonium (NH₄ ⁺) and phosphorus (P) in gel on the amounts of hydrogen ion (H⁺) excreted from plant roots was studied with Brachiaria humidicola (a highly acid‐soil tolerant tropical grass) and B. brizantha (less acid‐soil tolerant) grown in soil in a glasshouse. The H⁺ production was measured over 24 h in agar gel containing full nutrient solution with a range of NH/‐N levels (0, 0.25, 0.5, and 5.0 mM NH₄ ⁺‐N). Highly soluble P, K₂HPO₄, or relatively insoluble P, rock P, was supplied at four concentrations (0, 11.5, 34.5, or 115 μM p) in the gel. Increasing NH₄ ⁺ concentration in the gel increased H⁺ production for both grasses, but there was some inhibition of growth for B. brizantha at the highest N concentration. For B. humidicola, but not B. brizantha H⁺ production was greater with 34.5 μM K₂HPO₄ than 11.5 μM K₂HPO₄. At 34.5 μM P for both grasses there was no difference in H⁺ production when P was supplied as rock P or K₂HPO₄. With 11.5 μM P both grasses produced less acid in the gel with the rock P compared with K₂HPO₄. The reduced H⁺ production is probably due to a lower availability of P in the rock P compared with K₂HPO₄. This effect was greater with B. brizantha than B. humidicola, implying that 11.5 μM rock P was not able to supply sufficient P for the growth of B. brizantha. Brachiaria humidicola was able to dissolve more rock P than B. brizantha or alternatively, the growth of B. humidicola was less adversely affected by the low P supply from rock P than B. brizantha. Plant‐induced acidity does not seem to occur as a response to a lack of available P, but rather these grasses only produce acid if there are enough nutrients for growth, i.e., both NH₄ ⁺ and P. If either N or P is limiting, growth is limited as is NH₄ ⁺ uptake, so that H⁺ production is curtailed.     

INFLUENCE OF CHEMICAL FORM AND CONCENTRATION OF NITROGEN ON APOPLASTIC pH OF LEAVES

The objective of this study was to investigate the effects of various forms of nitrogen (NO^? ₃, NH⁺ ₄) supplied to the roots via a nutrient solution on the apoplastic pH in intact leaves determined by fluorescence ratio imaging. In contrast to NH⁺ ₄, higher apoplastic pH values in leaves of Phaseolus vulgaris and Helianthus annuus were measured with NO^? ₃ nutrition. In this context no significant differences were found in leaves of Vicia faba and Zea mays supplied with the various forms of N. Comparative studies on apoplastic pH in leaves of Vicia faba, Zea mays and Helianthus annuus demonstrated that NO^? ₃ reductase activity in roots was responsible for the differences in NO^? ₃ concentration and pH in the leaf apoplast. Light-induced pH changes in the leaf apoplast also occur and may overlap the effects of various forms of N. Increasing concentrations of NO^? ₃ supply to the roots did not significantly affect apoplastic pH in leaves of Helianthus annuus. Depletion of NO^? ₃ in the nutrient solution led to lower apoplastic pH in leaves of Zea mays. Leaf fertilization with NH⁺ ₄ led to a decline in apoplastic pH of leaves whereas NH₃ gas exposure caused a biphasic response in apoplastic pH.