Short‐term ammonium supply induces cellular defence to prevent oxidative stress in Arabidopsis leaves

Plants can assimilate nitrogen from soil pools of both ammonium and nitrate, and the relative levels of these two nitrogen sources are highly variable in soil. Long‐term ammonium nutrition is known to cause damage to Arabidopsis that has been linked to mitochondrial oxidative stress. Using hydroponic cultures, we analysed the consequences of rapid shifts between nitrate and ammonium nutrition. This did not induce growth retardation, showing that Arabidopsis can compensate for the changes in redox metabolism associated with the variations in nitrogen redox status. During the first 3 h of ammonium treatment, we observed distinct transient shifts in reactive oxygen species (ROS), low‐mass antioxidants, ROS‐scavenging enzymes, and mitochondrial alternative electron transport pathways, indicating rapid but temporally separated changes in chloroplastic, mitochondrial and cytosolic ROS metabolism. The fast induction of antioxidant defences significantly lowered intracellular H₂O₂ levels, and thus protected Arabidopsis leaves from oxidative stress. On the other hand elevated extracellular ROS production in response to ammonium supply may be involved in signalling. The response pattern displays an intricate plasticity of Arabidopsis redox metabolism to minimise stress in responses to nutrient changes.     

Long‐term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity

Ammonium nutrition has been suggested to be associated with alterations in the oxidation‐reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)⁺ ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over‐reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)⁺ ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.     

Nitrogen nutrition and antioxidant metabolism in ammonium-tolerant and -sensitive plants

Ammonium nutrition is of interest as an alternative to that of using nitrate. However, the former has been reported as stressful to many plant species especially to some important crops, as most abiotic stresses may trigger oxidative imbalances in plants. In this work, we investigate the response of oxidative metabolism of two plant species, spinach ( Spinacia oleracea L. cv. Gigante de invierno) and pea ( Pisum sativum L. cv. Rondo), which have distinct tolerance to ammonium. Plants were grown in the presence of 1.5 and 3.0 m M N as ammonium and compared with equivalent nitrate nutrition. The antioxidant enzymes and metabolites as well as oxidative damage to proteins were determined. Protein and amino acid contents in both types of plants were also analysed. Ammonium nutrition in sensitive spinach or in the tolerant pea plants does not alter the redox status of ascorbate and glutathione or the phenolic contents, while no clear effect is seen in the antioxidant enzymes. The results showed that the stress originated from applying ammonium as the only N source is not an oxidative stress, independent of the ammonium tolerance of the plant species studied. Moreover, ammonium stress diminishes oxidative damage to proteins in the spinach plants. The data of the protein oxidation together with those from N metabolism highlight the relation between the stress induced by ammonium and an increased protein turnover.     

Influence of mitochondrial genome rearrangement on cucumber leaf carbon and nitrogen metabolism

The MSC16 cucumber (Cucumis sativus L.) mitochondrial mutant was used to study the effect of mitochondrial dysfunction and disturbed subcellular redox state on leaf day/night carbon and nitrogen metabolism. We have shown that the mitochondrial dysfunction in MSC16 plants had no effect on photosynthetic CO₂ assimilation, but the concentration of soluble carbohydrates and starch was higher in leaves of MSC16 plants. Impaired mitochondrial respiratory chain activity was associated with the perturbation of mitochondrial TCA cycle manifested, e.g., by lowered decarboxylation rate. Mitochondrial dysfunction in MSC16 plants had different influence on leaf cell metabolism under dark or light conditions. In the dark, when the main mitochondrial function is the energy production, the altered activity of TCA cycle in mutated plants was connected with the accumulation of pyruvate and TCA cycle intermediates (citrate and 2-OG). In the light, when TCA activity is needed for synthesis of carbon skeletons required as the acceptors for NH₄ ⁺ assimilation, the concentration of pyruvate and TCA intermediates was tightly coupled with nitrate metabolism. Enhanced incorporation of ammonium group into amino acids structures in mutated plants has resulted in decreased concentration of organic acids and accumulation of Glu.     

Plasma membrane-generated ROS and their possible contribution to leaf cell growth of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) MSC16 mitochondrial mutant

Reactive oxygen species (ROS) generally regarded as harmful products of oxygenic metabolism causing oxidative stress and cell damage are also important for control and regulation of biological processes. ROS can be generated by various enzymatic activities and removed by an array of ROS-scavenging molecules in the cell. In plants, the generation of ROS initiated by the plasma membrane NADPH oxidase can be used for controlled polymer breakdown leading to cell wall loosening during extension growth. The mosaic (MSC16) mitochondrial mutant of cucumber (Cucumis sativus L.) has marked phenotypic changes, including a slower growth rate which partially may result from disturbed leaf carbon and energy metabolism and ROS/antioxidants equilibrium. Cytochemical localization of H₂O₂ in leaf cells showed lower total level of H₂O₂ particularly in the apoplast of MSC16 leaf cells as compared to WT. The activity of plasma membrane NADPH oxidase (EC 1.6.3.1) was about 30% lower in plasmalemma vesicles isolated from MSC16 leaf tissue as compared to WT. The total foliar ascorbate pool (reduced and oxidized) was about 35% higher in MSC16 compared to WT leaves due to an increased content of the oxidized form. About 3% of the whole-leaf ascorbate was localized in the apoplast but in MSC16 it was considerably more reduced. We conclude that the lower apoplastic ROS content caused by decreased activity of plasma membrane NADPH oxidase and lower amounts of H₂O₂ in the apoplast may also contribute to altered growth of the MSC16 cucumber mutant.     

Photosynthesis and carbohydrate metabolism in tobacco leaves during an incompatible interaction with Phytophthora nicotianae

The metabolic and cellular changes in source leaves of Nicotiana tabacum L. cv SNN during an incompatible interaction with Phytophthora nicotianae van Breda de Haan were investigated and compared with defence reactions. Hypersensitive cell death was preceded by a rapid and highly localized shift to non-assimilatoric metabolism. During the first 6 h post infection (hpi), reactive oxygen species (ROS) accumulated. Callose was deposited at the interface of adjacent mesophyll cells (≥1 hpi), the export of sucrose collapsed and its content in the apoplast increased. Stomata closed and photosynthetic flux was reallocated from CO₂ assimilation in favour of photorespiration. This was accompanied by an increase in respiration, glucose-6-phosphate dehydrogenase (G6PDH) activity, apoplastic invertase and hexose content. Later (>6 hpi) the photosynthetic electron transport chain was interrupted and photosynthesis completely collapsed. This was accompanied by a further increase in apoplastic invertase and carbohydrates, respiration and oxidative pentose phosphate pathway (OPPP) and followed by further burst in ROS release. Hypersensitive cell death did not appear until photosynthesis completely declined. Photosynthesis was visualized by chlorophyll-a fluorescence imaging on a macro- and microscopic scale. Decline in photosynthesis and defence reactions were highly localized processes, which occur in single mesophyll cells. We propose that in photoautotrophic leaves, photosynthesis and assimilatory metabolism must be switched off to initiate respiration and other processes required for defence. An early blockage of intercellular sugar transportation, due to callose deposition, in conjunction with enhanced apoplastic invertase activity could facilitate this metabolic shift.     

Effect of ammonium or nitrate nutrition on net photosynthesis,growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry,raspberry and strawberry

Blueberry, raspberry and strawberry may have evolved strategies for survival due to the different soil conditions available in their natural environment. Since this might be reflected in their response to rhizosphere pH and N form supplied, investigations were carried out in order to compare effects of nitrate and ammonium nutrition (the latter at two different pH regimes) on growth, CO2 gas exchange, and on the activity of key enzymes of the nitrogen metabolism of these plant species. Highbush blueberry (Vaccinium corymbosum L. cv. 13–16–A), raspberry (Rubus idaeus L. cv. Zeva II) and strawberry (Fragaria × ananassa Duch. cv. Senga Sengana) were grown in 10 L black polyethylene pots in quartz sand with and without 1% CaCO3 (w: v), respectively. Nutrient solutions supplied contained nitrate (6 mM) or ammonium (6 mM) as the sole nitrogen source. Compared with strawberries fed with nitrate nitrogen, supply of ammonium nitrogen caused a decrease in net photosynthesis and dry matter production when plants were grown in quartz sand without added CaCO3. In contrast, net photosynthesis and dry matter production increased in blueberries fed with ammonium nitrogen, while dry matter production of raspberries was not affected by the N form supplied. In quartz sand with CaCO3, ammonium nutrition caused less deleterious effects on strawberries, and net photosynthesis in raspberries increased as compared to plants grown in quartz sand without CaCO3 addition. Activity of nitrate reductase (NR) was low in blueberries and could only be detected in the roots of plants supplied with nitrate nitrogen. In contrast, NR activity was high in leaves, but low in roots of raspberry and strawberry plants. Ammonium nutrition caused a decrease in NR level in leaves. Activity of glutamine synthetase (GS) was high in leaves but lower in roots of blueberry, raspberry and strawberry plants. The GS level was not significantly affected by the nitrogen source supplied. The effects of nitrate or ammonium nitrogen on net photosynthesis, growth, and activity of enzymes in blueberry, raspberry and strawberry cultivars appear to reflect their different adaptability to soil pH and N form due to the conditions of their natural environment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of different N fertilizers on the activity of <Emphasis Type="Italic">Glomus mosseae</Emphasis> and on grapevine nutrition and berry composition

Grapevine N fertilization may affect and be affected by arbuscular mycorrhizal (AM) fungal colonization and change berry composition. We studied the effects of different N fertilizers on AM fungal grapevine root colonization and sporulation, and on grapevine growth, nutrition, and berry composition, by conducting a 3.5-year pot study supplying grapevine plants with either urea, calcium nitrate, ammonium sulfate, or ammonium nitrate. We measured the percentage of AM fungal root colonization, AM fungal sporulation, grapevine shoot dry weight and number of leaves, nutrient composition (macro- and micronutrients), and grapevine berry soluble solids (total sugars or °Brix) and total acidity. Urea suppressed AM fungal root colonization and sporulation. Mycorrhizal grapevine plants had higher shoot dry weight and number of leaves than non-mycorrhizal and with a higher growth response with calcium nitrate as the N source. For the macronutrients P and K, and for the micronutrient B, leaf concentration was higher in mycorrhizal plants. Non-mycorrhizal plants had higher concentration of microelements Zn, Mn, Fe, and Cu than mycorrhizal. There were no differences in soluble solids (°Brix) in grapevine berries among mycorrhizal and non-mycorrhizal plants. However, non-mycorrhizal grapevine berries had higher acid content with ammonium nitrate, although they did not have better N nutrition and vegetative growth.     

Replacement of nitrate by ammonium as the nitrogen source increases the salt sensitivity of pea plants. II. Inter- and intracellular solute compartmentation in leaflets

Pea plants (Pisum sativum L. cv. ‘Kleine Rheinländerin’) grown on ammonium or nitrate as the sole nitrogen source were treated with 50 mol m⁻³ NaCl. Four days after salt addition, ammonium-grown plants developed the first visible damage symptoms (wilting of leaflets, starting from the margins). Salt-treated, nitrate-grown plants were not affected during the experimental period. In order to obtain a better understanding of this differential salt sensitivity, we investigated the inter- and intracellular ion compartmentation of leaflets under both nutritional conditions by analysing ion concentrations in the apoplastic space, in chloroplasts and in protoplasts. When the leaves of nitrate- and ammonium-grown plants had attained similar sodium and chloride contents (after different times of exposure to salinity), the latter had a considerably lower chloroplastic chloride (and also sulphate) concentration. The results suggest that the intracellular compartmentation capacity of ammonium-grown plants is considerably lower than that of nitrate-grown plants. Ion toxicity appeared to initiate breakdown of metabolism in parts of the mesophyll tissue of ammonium-grown plants, causing an abrupt release of solutes into the apoplast, which coincided with the appearance of visible damage. Although the ammonium concentrations in leaves increased dramatically in the later phases of damage development, they were too low to cause the collapse of electrochemical gradients at the time at which damage became visible. Thus, the reason for a lower compartmentation capacity under ammonium nutrition remains as yet unclear.     

Effect of phosphoros nutrition in peat on tomato plant growth and fruit development

Summary The effect of P nutrition on the growth of tomato plants in peat was examined. Initially, plants received an adequate supply of P and then received either nil, 0.78 or 2.34 kg superphosphate per m³ in combination with either 50 g N/ml (N₁) or 300 g N/ml (N₂) as ammonium nitrate in a liquid feed. Vegetative growth was restricted in the lower P treatmentsi.e. inhibited shoot growth, reduced duration of leaf expansion phase, thinner stems and reduced vegetative dry wt. Plants receiving N₂ showed a greater restriction in growth compared with N₁ plants when the P supply was limiting. P deficiency disrupted protein metabolism in the leaves, in that soluble leaf protein was reduced and trichloroacetic acid-soluble N accumulated. Flower development was accelerated by low P applications but the final numbers of flowers and the fruit-setting efficiency were reduced. Lowering the N supply reduced the fruit yield by 36 per cent while an intermediate P level reduced yields by about 15 per cent. Maximum fruit yields and good vegetative growth occurred when plants contained 0.4 per cent P or above in the mature leaves, and this value was achieved by adding the highest level (2.34 kg/m³) of superphosphate to the peat.     

Biochemical and quantitative proteomics investigations in Arabidopsis ggt1 mutant leaves reveal a role for the gamma‐glutamyl cycle in plant's adaptation to environment

The existence of a gamma‐glutamyl cycle consisting of intracellular GSH synthesis, extrusion to the apoplastic space and recovery by gamma‐glutamyl transferase (GGT)‐assisted degradation into its constituent amino acids, has been demonstrated in plants. To address the significance of this cycle in plant cells, we performed integrated biochemical, immunocytochemical, and quantitative proteomics analyses in the Arabidopsis thaliana ggt1 knockout mutant (lacking apoplastic GGT1 isoform) and its corresponding wild‐type (WT). The ggt1 knockout leaves exhibited an increased ascorbate and GSH content, increased apoplastic GSH content, and enhanced protein carbonylations in the low‐molecular weight range compared to WT. The combined iTRAQ and LC‐MS/MS‐based quantitative proteomics approach identified 70 proteins (out of 1013 identified proteins) whose abundance was significantly different in leaves of ggt1 mutant compared to WT, with a fold change ≥1.5. Mining of the proteome data for GSH‐associated genes showed that disruption of gamma‐glutamyl cycle in ggt1 knockout‐leaves was associated with the induction of genes encoding four GSTs in the phi class (GSTF2, GSTF6, GSTF9, and GSTF10), a GSH peroxidase (GPX1), and glyoxylase II. Proteins with a lower abundance compared to the WT are involved in chloroplast functions, carbohydrate/maltose metabolism, and vegetative storage protein synthesis. Present findings suggest that GGT1 plays a role in redox signaling. The disruption of the gamma‐glutamyl cycle in the ggt1 mutant results in pleiotropic effects related to biotic and abiotic stress response, antioxidant metabolism, senescence, carbohydrate metabolism, and photosynthesis, with strong implications for plant adaptation to the environment.     

AMMONIUM ION-INDUCED CHANGES IN FORM AND HYDROXYPROLINE CONTENT OF WALL PROTEIN IN THE LIVERWORT,GYMNOCOLEA INFLATA

Ammonium ion induces phenovariation in Gymnocolea inflata (Huds.) Dum. (Lophoziaceae) similar in nature to that induced in representatives of four other families of leafy liverworts by antagonists of hydroxyproline-protein metabolism. That is, in the presence of ammonium ion, ventral leaves developed and the lateral leaf morphology and the branching pattern of the plants changed. These changes, which reflect altered patterns of morphogenesis, were correlated with statistically significant (P ≥ 0.01) differences in peptidyl-hydroxyproline between normal and NH₄⁺-induced phenovariant plants. The results support the hypothesis that cell wall-associated hydroxyproline-protein plays a morphoregulatory role and that any of a number of factors, genetic, epigenetic or environmental, that could alter the synthesis/distribution of this moiety could cause changes in form.     

Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O₂ conditions. However, an increase in the stoichiometry of photorespiratory CO₂ release was found during O₂-dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD⁺ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.     

Importance of seed Zn content for wheat growth on Zn-deficient soil

Eggplants (Solanum melongena L. cv. Bonica) were grown in a glasshouse during summer under natural light with one unbranched shoot or one shoot with 3 to 4 branches and with or without fruit in quartz sand buffered and not buffered with 0.5% CaCO₃ (w : v), respectively. Nutrient solutions supplied contained nitrate or ammonium as the sole nitrogen source. Compared with nutrient solutions containing nitrate (10 mM), solutions containing ammonium (10 mM) caused a decrease in net photosynthesis of eggplants during early stages of vegetative growth when grown in quartz sand not buffered with CaCO₃. The decrease was not observed before leaves showed interveinal chlorosis. In contrast, net photosynthesis after bloom at first increased more rapidly in eggplants supplied with ammonium than with nitrate nitrogen. However, even in this case, net photosynthesis decreased four weeks later when ammonium nutrition was continued. The decrease was accompanied by epinasty and interveinal chlorosis on the lower leaves and later by severe wilting, leaf drop, stem lesions, and hampered growth of stems, roots, and fruits. These symptoms appeared later on plants not bearing fruits than on plants bearing fruits. If nutrient solutions containing increasing concentrations of ammonium (0.5–30 mM) were supplied after the time of first fruit ripening, shoot growth and set of later flowers and fruits were promoted. In contrast, vegetative growth and reproduction was only slightly affected by increasing the concentration of nitrate in the nutrient solutions. In quartz sand buffered with CaCO₃ ammonium nutrition caused deleterious effects only under low light conditions (shade) and on young plants during rapid fruit growth. If eggplants were supplied with ammonium nitrogen before bloom, vegetative growth was promoted, and set of flowers and fruit occurred earlier than on plants supplied with nitrate. Furthermore, the number of flowers and fruit yield increased. These effects of ammonium nutrition were more pronounced when plants were grown with branched shoots than with unbranched shoots. The results indicate that vegetative and reproductive growth of eggplants may be manipulated without causing injury to the plants by supplying ammonium nitrogen as long as the age of the plants, carbohydrate reserves of the roots, quantity of ammonium nitrogen supplied, and pH of the growth medium are favourable. T W Rufty Section editor     

THE INFLUENCE OF LOW PLANT WATER POTENTIAL ON THE GROWTH AND NITROGEN METABOLISM OF THE NATIVE CALIFORNIA SHRUB LOTUS SCOPARIUS (NUTT. IN T & G) OTTLEY

Reduced plant water potential, induced by polyethylene glycol in hydroponics, inhibited growth and decreased the number of leaves per branch in the southern California drought-deciduous species Lotus scoparius (Nutt. in T & G) Ottley. Decreasing plant water potential diminished the proportion of large leaves per branch and therefore reduced the leaf area. Nitrate uptake rate decreased with decreasing water potential, although the nitrate ion concentration increased in the roots and the leaves. Ammonium ion concentration increased significantly in the roots at −5 bars and lower osmotic potentials in the root medium. Kjeldahl nitrogen remained the same in all treatments and tissues over the experimental period. It is suggested that the increase in ammonium ion in the roots was due to a decreased rate of ammonium transport caused by low plant water potential. The slight increase in nitrate ion in the roots may correspond to a decrease in nitrate reductase activity. This study indicates that some of the changes in nitrogen metabolism associated with low water potentials in agricultural plants occur also in a plant which experiences frequent droughts in its native habitat.     

Ammonium and nitrate nutrition inPlantago lanceolata L. andPlantago major L. ssp.major

With the aims (1) to test whether the different natural occurrence of twoPlantago species in grasslands is explained by a different preference of the species for nitrate or ammonium; (2) to test whether the different occurrence is explained by differences in the flexibility of the species towards changes in the nitrogen form; (3) to find suitable parameters as a tool to study ammonium and nitrate utilization of these species at the natural sites in grasslands, plants ofPlantago lanceolata andP. major ssp.major were grown with an abundant supply of nitrate, ammonium or nitrate+ammonium as the nitrogen source (0.5 mM). The combination of ammonium and nitrate gave a slightly higher final plant weight than nitrate or ammonium alone. Ammonium lowered the shoot to root ratio inP. major. Uptake of nitrate per g root was faster than that of ammonium, but from the mixed source ammonium and nitrate were taken up at the same rate. In vivo nitrate reductase activity (NRA) was present in both shoot and roots of plants receiving nitrate. When ammonium was applied in addition to nitrate, NRA of the shoot was not affected, but in the root the activity decreased. Thus, a larger proportion of total NRA was present in the shoot than with nitrate alone. In vitro glutamate dehydrogenase activity (GDHA) was enhanced by ammonium, both in the shoot and in the roots.In vitro glutamine synthetase activity (GSA) was highest in roots of plants receiving ammonium. Both GDHA and GSA were higher inP. lanceolata than inP. major. The concentration of ammonium in the roots increased with ammonium, but it did not accumulate in the shoot. The concentration of amino acids in the roots was also enhanced by ammonium. Protein concentration was not affected by the form of nitrogen. Nitrate accumulated in both the shoot and the roots of nitrate grown plants. When nitrate in the solution was replaced by ammonium, the nitrate concentration in the roots decreased rapidly. It also decreased in the shoot, but slowly. It is concluded that the nitrogen metabolism of the twoPlantago species shows a similar response to a change in the form of the nitrogen source, and that differences in natural occurrence of these species are not related to a differential adaptation of nitrogen metabolism towards the nitrogen form. Suitable parameters for establishing the nitrogen source in the field are thein vivo NRA, nitrate concentrations in tissues and xylem exudate, and the fraction of total reduced nitrogen in the roots that is in the soluble form, and to some extent thein vitro GDHA and GSA of the roots. Grassland Species Research Group. Publ. no 118.     

Effects of Exogenously Supplied Ammonium on Root Development of Scots Pine (Pinus sylvestris L.) Seedlings

The effect of potentially toxic concentrations of ammonium on root development of Scots pine seedlings raised on Perlite was investigated during growth periods of 3 or 10 weeks after sowing. It was shown that imbalanced ammonium nutrition led to conspicuous changes of root morphology provided the pH value in the medium was allowed to decrease to 3.9 due to the NH⁺₄-dependent proton excretion into the rhizosphere. Ammonium toxicity could not be observed with seedlings treated either with ammonium nitrate or with ammonium chloride at pH 5.3 ? 6.8. While the supply of NH⁺₄ considerably inhibited root development the biomass production of the shoot was increased. Determination of the endogenous level of ammonium in roots and the leaf whorl exclude a simple causal correlation between ammonium toxicity and accumulated ammonium as has been postulated for herbaceous plants.