ALBERT

Notes: The influence of salinity on the activity of nitrate reductase (NR, EC 1.6.6.1) and the level of the molybdenum cofactor (MoCo) as affected by the source and concentration of nitrogen was studied in annual ryegrass (Lolium multiflorum cv. Westerwoldicum). Plants grown in sand were irrigated with nutrient solution with an electrical conductivity of 2 or 11.2 dS m−1, containing nitrogen (0.5 or 4.5 mM) in the form of NH4NO3 or NaNO3 Salinity-treated (11.2 dS m−1) plants produced less biomass and more organic nitrogen while accumulating more NO−3 than control plants. Increased nitrogen concentration in the irrigation solutions enhanced biomass and organic nitrogen production as well as NO−3 accumulation irrespective of the electrical conductivity. Salinity inhibited shoot growth and increased shoot NR activity of plants receiving 4.5 mM NH4NO3 or NaNO3. Similar effects were observed in roots of plants grown in 4.5 mM NaNO3. Nitrate added to a complementation medium containing ryegrass MoCo and the NR apoprotein of Neurospora crassa mutant nit-1 stimulated the activity of the reconstituted NR (NADPH-nitrate reductase, EC 1.6.6.3). Increased salinity and nitrogen in the nutrient solutions caused an increase of MoCo content in roots and shoots. Similar results were observed for NR activity in the shoots. The increase of MoCo in response to salinity was more pronounced than that of NR, especially in the roots. We conclude that the pool size of MoCo in ryegrass is not constant, but varies in response to nutritional and environmental factors.

Notes: Our previous work indicated that salinity caused a shift in the predominant site of nitrate reduction and assimilation from the shoot to the root in tomato plants. In the present work we tested whether an enhanced supply of dissolved inorganic carbon (DIC, CO2+ HCO3) to the root solution could increase anaplerotic provision of carbon compounds for the increased nitrogen assimilation in the root of salinity-stressed Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were grown in hydroponic culture with 0 or 100mM NaCl and aeration of the root solution with either ambient or CO2-enriched air (5000 μmol mol−1). The salinity-treated plants accumulated more dry weight and higher total N when the roots were supplied with CO2-enriched aeration than when aerated with ambient air. Plants grown with salinity and enriched DIC also had higher rates of NO−3 uptake and translocated more NO−3 and reduced N in the xylem sap than did equivalent plants grown with ambient DIC. Incorporation of DIC was measured by supplying a 1 -h pulse of H14CO−3 to the roots followed by extraction with 80% ethanol. Enriched DIC increased root incorporation of DIC 10-fold in both salinized and non-salinized plants. In salinity-stressed plants, the products of dissolved inorganic 14C were preferentially diverted into amino acid synthesis to a greater extent than in non-salinized plants in which label was accumulated in organic acids. It was concluded that enriched DIC can increase the supply of N and anaplerotic carbon for amino acid synthesis in roots of salinized plants. Thus enriched DIC could relieve the limitation of carbon supply for ammonium assimilation and thus ameliorate the influence of salinity on NO−3 uptake and assimilation as well as on plant growth.

Notes: Positive influences of high concentrations of dissolved inorganic carbon (DIC) in the growth medium of salinity-stressed plants are associated with carbon assimilation through phosphoenolpyruvate carboxylase (PEPc) activity in roots; and also in salinity-stressed tomato plants, enriched CO2 in the rhizosphere increases NO−3uptake. In the present study, wild-type and nitrate reductase-deficient plants of barley (Hordeum vulgare L. cv. Steptoe) were used to determine whether the influence of enriched CO2 on NO−3 uptake and metabolism is dependent on the activity of nitrate reductase (NR) in the plant. Plants grown in NH4+and aerated with ambient air, were transferred to either NO3− or NH4+ solutions and aerated with air containing between 0 and 6 500 μmol mol−1 CO2. Nitrogen uptake and tissue concentrations of NO3− and NH4+ were measured as well as activities of NR and PEPc.The uptake of NO−3 by the wild-type was increased by increasing CO2. This was associated with increased in vitro NR activity, but increased uptake of NO3− was found also in the NR-deficient genotype when exposed to high CO2 concentrations; so that the influence of CO2 on NO3− uptake was independent of the reduction of NO3− and assimilation into amino acids. The increase in uptake of NO3− in wild-type plants with enriched CO2 was the same at pH 7 as at pH 5, indicating that the relative abundance of HCO3− or CO2 in the medium did not influence NO3− uptake. Uptake of NH4+ was decreased by enriched CO2 in a pH (5 or 7) independent fashion. Thus NO3− and NH+4 uptakes are influenced by the CO2 component of DIC independently of anaplerotic carbon provision for amino acid synthesis, and CO2 may directly affect the uptake of NO3− and NH4+ in ways unrelated to the NR activity in the tissue.

Notes: Three-week-old seedlings of carob (Ceratonia siliqua L. cv. Mulata) were grown for 9 weeks under different root temperatures (20, 30 and 40°C) at pH values of 5, 7 and 9 with nitrate or ammonium as nitrogen source. Nitrogen uptake rates were determined by depletion from the medium and decreased with distance from the apex. The decline of nitrogen uptake rates along the roots depended on the form of inorganic nitrogen in the medium as well as on pH and temperature, such that the NO−3 and NH+4 ions were taken up essentially by the root tips (0–2 cm) through processes requiring energy. The uncharged NH3 species entered passively, through the mature parts of the root (2–10 cm). Root zone temperature and pH affect the NH+4/NH3 equilibrium in the nutrient solution and, consequently, the uptake areas of the root for these ions. Furthermore. while root tip uptake of nitrogen is energy dependent, uptake through mature root areas is essentially passive and seems to depend on a well developed apparent free space.

Notes: We investigated the influence of an increased inorganic carbon supply in the root medium on NO−3 uptake and assimilation in seedlings of Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were pre-grown for 4 to 7 days with 0 or 100 mM NaCl in hydroponic culture using 0.2 mM NO−3 (group A) or 0.2 mM NH+4 (group B) as nitrogen source. The nutrient solution for group A plants was aerated with air or with air containing 4 800 μumol mol−1 CO2. Nitrate uptake rate and root and leaf malate contents in these plants were determined. The plants of group B were subdivided into two sets. Plants of one set were transferred either to N-free solution containing 0 or 5 mM NaHCO3, or to a medium containing 2 mM NO−3 and 5 mM NaHCO3. Both sets of group B plants were grown for 12 h in darkness prior to 2 h of illumination, and were assayed for malate content and NO−3 uptake rate (only for plants grown in N-free solution). The second set of group B plants was labeled with 14C by a 1-h pulse of H14CO−3 which was added to a 5 mM NaHCO3 solution containing 0 or 100 mM NaCl and 0 or 2 mM NO−3, and 14C-assimilates were extracted and fractionated.The roots of group B plants growing in carbonated medium accumulated twice as much malate as did control plants. This malate was accumulated only when NO−3 was absent from the root medium. Both a high level of root malate and aeration with CO2-enriched air stimulated NO−3 uptake. Analysis of 14C-assimilates indicated that with no NO−3 in the medium, the 14C was present mainly in organic acids, whereas with NO−3, a large proportion of 14C was incorporated into amino acids. Transport of root-incorporated 14C to the shoot was enhanced by NO−3, while the amino acid fraction was the major 14C-assimilates in the shoot. It is concluded that inorganic carbon fixed through phosphoenolpyruvate carboxylase (EC 4.1.1.31) in roots of tomato plants may have two fates: (a) as a carbon skeleton for amino acid synthesis; and (b) to accumulate, mainly as malate, in the roots, in the absence of a demand for the carbon skeleton. Inorganic carbon fixation in the root provides carbon skeletons for the assimilation of the NH+4 resulting from NO3 reduction, and the subsequent removal of amino acids through the xylem. This ‘removal’ of NO−3 from the cytoplasm of the root cells may in turn increase NO−3 uptake.

Notes: The effects of NO−3 and NH+4 nutrition on the rates of dark incorporation of inorganic carbon by roots of hydroponically grown Zea mays L. cv. 712 and on the metabolic products of this incorporation, were determined in plants supplied with NaH14CO3 in the nutrient solution. The shoots and roots of the plants supplied with NaH14CO3 in the root medium for 30 min were extracted with 80%; (v/v) ethanol and fractionated into soluble and insoluble fractions. The soluble fraction was further separated into the neutral, organic acid, amino acid and non-polar fractions. The amino acid fraction was then analyzed to determine quantities and the 14C content of its individual components.The rates of dark incorporation of inorganic carbon calculated from H14CO−3 fixation and attributable to the activity of phosphoenolpyuvate carboxylase (EC 4.1.1.31), were 5-fold higher in ammonium-fed plants than in nitrate-fed plants after a 30-min pulse of 14C. This activity forms a small, but significant component of the carbon budget of the root. The proportion of 14C located in the shoots was also significantly higher in ammonium-fed plants than in nitrate-fed plants, indicating more rapid translocation of the products of dark fixation to the shoots in plants receiving NH+/sp4 nutrition.Ammonium-fed plants favoured incorporation of 14C into amino acids, while nitrate-fed plants allocated relatively more 14C into organic acids. The amino acid composition was also dependent on the type of nitrogen supplied, and asparagine was found to accumulate in ammonium-fed plants. The 14C labelling of the amino acids was consistent with the diversion of 14C-oxaloacetate derived from carboxlyation of phosphoenolpyruvate into the formation of both asparatate and glutamate. The results support the conclusion that inorganic carbon fixation in the roots of maize plants provides an important anaplerotic source of carbon for NH+4 assimilation.

Notes: Peanuts (Arachis hypogaea L. cv. Shulamit) grown with NO3− and saline water in hydroponics responded positively to addition of nitrogen (N) in their vegetative growth, but not in desert dune sand. In order to clarify these conflicting results, peanut plants were grown in a greenhouse pot experiment with fine calcareous sand. The nutrient solution contained 0 or 50 mM NaCl and 2 or 6 mM N in the form of Ca(NO3)2, NH4NO3 or (NH4)2SO4. Three replicates were harvested after 48 days (beginning of reproductive stage) and three after 109 days (pod filling). In addition, gynophores were treated with 0, 50, 100, 150 or 200 mM NaCl outside the growth pot to check their sensitivity to salt. Shoot dry weight became greater with increasing NH4+/NO3− ratio. Increasing the N concentration from 2 to 6 mM did not change shoot dry weight of the NH4NO3 or NH4+-fed plants, but caused a reduction in shoot dry weight of NO3−-fed plants. Shoot dry weight was not affected by increasing the NaCl concentration to 50 mM. Salt caused an increase in the number of gynophores per plant and a reduction of the mean pod weight. A NaCl concentration of 100 mM and above reduced gynophore vitality. It is concluded that the salt sensitivity of peanut plants resides mainly in the sensitivity of the reproductive organs.

Notes: A fast-growing normal and a slow-growing gibberellin-deficient mutant of Lycopersicon esculentum (L.) Mill. cv. Moneymaker were used to test the hypothesis that slow-growing plants reduce NO3− in the root to a greater extent than do fast-growing plants. Plants that reduce NO3− in the root may grow more slowly due to the higher energetic and carbon costs associated with root-based NO3− reduction compared to photosynthetically driven shoot NO3− reduction. The plants were grown hydroponically with a complete nutrient solution containing 10 mM NO3− and the biomass production, gas exchange characteristics, root respiratory O2 consumption, nitrate reductase activity and translocation of N in the xylem were measured. The gibberellin-deficient mutants accumulated more total N unit−1 dry weight than did the faster-growing normal plants. There were no significant differences between the genotypes in the rates of photosynthesis expressed on a leaf dry weight basis. The plants differed in the proportion of photosynthetic carbon available to growth due to a greater proportion of daily photo-synthate production being consumed by respiration in the slow-growing genotype. This difference in allocation of carbon was associated with differences in the specific leaf area and specific root length. In addition, a greater leaf weight ratio in the fast-growing than in the slow-growing plants indicates a greater investment of carbon into biomass supporting photosynthetic production in the former. We did not find differences in the activity or distribution of nitrate reductase or in the N composition of the xylem sap between the genotypes. We thus conclude that the growth rate was determined by the efficiency of carbon partitioning and that the site of NO3− reduction and assimilation was not related to the growth rate of these plants.

Notes: Most of the nitrate reductase activity (80%;) in carob (Ceratonia siliqua L. cv. Mulata) is localised in the roots. The nitrate concentration in the leaves is relatively low compared to that in the roots, suggesting that nitrate influx into the leaf may be a major factor limiting the levels of nitrate reductase in the shoot. Transport of nitrate from root to shoot appears limited by the entrance of nitrate into the xylem. In order to study this problem, we determined the nitrate concentrations and nitrate reductase activities along the roots of nitrate-grown plants, as well as the composition of the xylem sap and the nitrate levels in the leaves. Some of the the bypocotyl, in order to bypass the loading of nitrate into the xylem of the roots. The results show that the loading of nitrate into the xylem is a limiting step.The cation and anion concentrations of nitrate- and ammonium-fed plants were similar, showing almost no production of organic anions. In both nitrate- and ammonium-fed plants, the transport of nitrogen from root to shoot was in the form of organic nitrogen compounds. The nitrate reductase activity in the roots was more than sufficient to explain all the efflux of OH− into the root medium of nitrate-fed plants. In carob plants the K-shuttle may thus be operative to a limited extent only, corresponding to between 11 and 27%; of the nitrate taken up. Potassium seems to be the cation accompanying stored nitrate in the roots of carob seedlings, since they accumulate nearly stoichiometric amounts of K+ and NO−3.