Pedro María Aparicio-Tejo

Drought induces oxidative stress in pea plants

Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (leaf water potential of ≈-1.3 MPa) showed major reductions in photosynthesis (78‰), transpiration (83‰), and glycolate oxidase (EC 1.1.3.1) activity (44‰), and minor reductions (≈18‰) in the contents of chlorophyll a, carotenoids, and soluble protein. Water stress also led to pronounced decreases (72–85‰) in the activities of catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), but resulted in the increase (32–42‰) of non-specific peroxidase (EC 1.11.1.7) and superoxide dismutase (EC 1.15.1.1). Ascorbate peroxidase (EC 1.11.1.11) and monodehydroascorbate reductase (EC 1.6.5.4) activities decreased only by 15‰ and the two enzymes acted in a cyclic manner to remove H2O2, which did not accumulate in stressed leaves. Drought had no effect on the levels of ascorbate and oxidized glutathione in leaves, but caused a 25‰ decrease in the content of reduced glutathione and a 67‰ increase in that of vitamin E. In leaves, average concentrations of catalytic Fe, i.e. Fe capable of catalyzing free-radical generation by redox cycling, were estimated as 0.7 to 7 μM (well-watered plants, depending on age) and 16 μM (water-stressed plants); those of catalytic Cu were ≈4.5 μM and 18 μM, respectively. Oxidation of lipids and proteins from leaves was enhanced two- to threefold under stress conditions and both processes were highly correlated. Fenton systems composed of the purported concentrations of ascorbate, H2O2, and catalytic metal ions in leaves produced hydroxyl radicals, peroxidized membrane lipids, and oxidized leaf proteins. It is proposed that augmented levels and decompartmentation of catalytic metals occurring during water stress are responsible for the oxidative damage observed in vivo.

Ammonium oxidation kinetics in the presence of nitrification inhibitors DCD and DMPP at various temperatures

Use of N-based fertilisers in combination with nitrification inhibitors lengthens N presence in the ammonium form in soil (N-NH4+), with beneficial effects for agriculture and related ecosystems. The efficiency of these inhibitors depends on several factors, the most important being soil temperature. This paper studies the effects of soil temperature on the kinetics of N-NH4+ loss in the presence of the DCD and DMPP nitrification inhibitors. For a 105-day period, 3 chambers, each with 12 containers holding 500 g of dry soil, were incubated at 10, 20, and 30°C. Ammonium sulfate was applied to 4 containers in each chamber; in another 4 containers Basammon Stabil, a N fertiliser with DCD, was used; and Entec 26, a fertiliser with DMPP, was used in the remaining 4 containers. Soil ammonium content was periodically determined for each container. Both DCD and DMPP lengthened ammonium presence in soil in a similar manner. However, their effectiveness was drastically decreased at increased soil temperatures. Thus, when using these inhibitors, soil temperature should be taken into account, especially in warm climate areas.

Pea responses to saline stress is affected by the source of nitrogen nutrition (ammonium or nitrate)

The effect of the source of nitrogen nutrition (ammonium or nitrate), onthe response of pea plants to a moderate saline stress (30 mMNaCl)was studied. Growth declined under saline stress but nitrate-fed plants wereless sensitive to salinity than ammonium-fed plants. This different sensitivitywas due mainly to a better maintenance of root growth in nitrate-fed plants.Organic nitrogen content decreased significantly in roots of ammonium-fedplants. Water relations changed slightly under saline stress leading to adecrease in stomatal conductance, which was correlated to a decline in carbonassimilation rates regardless of nitrogen source. Salinity affects the uptakeofseveral nutrients in a different way, depending on the nitrogen source. Thus,chloride was accumulated mainly in nitrate-fed plants, displacing nitrate,whereas sodium was accumulated mainly in ammonium-fed plants, especially inroots, displacing other cations such as ammonium and potassium. It is concludedthat the nitrogen source (ammonium or nitrate) is a major factor affecting pearesponses to saline stress, plants being more sensitive when ammonium is thesource used. The different sensitivity is discussed in terms of a competitionfor energy between nitrogen assimilation and sodium exclusion processes.

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 mM 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.

The sensitivity to ammonium nutrition is related to nitrogen accumulation

Abstract Ammonium and NO3− can be utilized as nitrogen sources by most plant species although the plant response to continuous ammonium nutrition is species dependent. The effect of the nitrogen source (nitrate and ammonium) on growth, photosynthetic parameters and nitrogen content in spinach (Spinacea oleracea L.), sunflower (Helianthus annuus L.) and pea (Pisum sativum L.) was studied. Results showed spinach to be highly sensitive, sunflower moderately sensitive and pea tolerant to ammonium nutrition. Ammonium accumulation in shoots was closely correlated to growth reduction. Moreover, ammonium accumulation was correlated to an increase of organic nitrogen content. The data suggest that the site of ammonium assimilation is a key factor controlling tolerance to ammonium nutrition in the different plant species, with plants being more tolerant when ammonium is assimilated in roots.

Short-term ammonium supply stimulates glutamate dehydrogenase activity and alternative pathway respiration in roots of pea plants

Summary The changes of C and N metabolism after switching Pisum sativum L. plants from nitrate to ammonium were studied. Pea plants were grown for three weeks in nutrient solution containing 0.5 mmol/L nitrate, and then randomly divided into five sets for five different nitrogen treatments: control (0.5 mmol/L nitrate) and four ammonium concentrations (0.5, 1, 2.5 and 5 mmol/L). After 72 hours, activities of enzymes related to C and N metabolism were measured. Ammonium content in roots increased with ammonium concentration showing saturation from a concentration of 2.5 mmol/L. Increasing external ammonium concentration also increased free amino acid content (mainly glutamine and asparagine), whereas starch content decreased and neither organic acid or soluble carbohydrates changed. Glutamine synthetase (GS; EC 6.3.1.2) activity decreased and root glutamate dehydrogenase (GDH; EC 1.4.1.2) activity increased with ammonium regardless of the concentration used. Root respiration rate increased with ammonium, due mainly to an increase of the alternative pathway. These results could be consistent with the assumption of a possible role for GDH in ammonium detoxification. Our results show a close relationship between GDH activity and respiration rate through alternate pathways in order to ensure the supply of C skeletons for ammonium assimilation, whereas surplus NADH is oxidised directly via the non-phosphorylating route.

Effects of low and high levels of magnesium on the response of sunflower plants grown with ammonium and nitrate

The effect of the nitrogen source (ammonium and nitrate) and its interaction with magnesium on various physiological processes was studied in sunflower plants (Helianthus annuusL.). Plants were grown in hydroponic culture with nitrate (5 mM) or ammonium (5 mM) and four concentrations of magnesium (0.1, 0.8, 5 and 10 mM). After 2 weeks, growth, gas exchange and fluorescence parameters, soluble carbohydrates, free amino acids, soluble protein and mineral elements were determined. Ammonium nutrition resulted in a reduction of dry matter accumulation, as well as in a decrease in the CO2 assimilation. Moreover, ammonium-fed plants showed a greater content of free amino acids, soluble protein, Rubisco and anions, and a lower cation content, mostly Mg2+. The presence of high levels of Mg2+ in the nutrient solution containing NH4+ resulted in a stimulation of growth and CO2 assimilation to the levels observed in nitrate-fed plants. The lower photosynthetic rate of ammonium-fed plants grown with low level of magnesium does not seem to be due to a lower photosynthetic pigment content, or a deficiency in Photosystem II activity, or to lower Rubisco content. Hence, Rubisco activity or other enzymes involved in CO2 fixation could have been affected in ammonium-fed plants.

Physiological consequences of continuous, sublethal imazethapyr supply to pea plants

Summary Imazethapyr (IM) is a herbicide that inhibits the branched-chain amino acid (BCAA) biosynthesis through the specific inhibition of acetolactate synthase activity. This herbicide acts very slowly and several weeks are required for complete plant death. From the BCAA biosynthesis inhibition to the growth inhibition and plant death, the processes involved are not fully understood. Starvation for BCAAs and/or starvation for carbohydrates in sinks. have been proposed as part of the death mechanisms. In this study, a permanent acetolactate synthase inhibition is used in order to (1) determine whether the growth inhibition effects can be attributed to a reduction in BCAA content and/or to starvation of carbohydrates; and (2) to analyse the physiological changes induced. Sublethal doses of IM were continuously supplied in the nutrient solution of nodulated pea plants. These conditions led to a significant decline in plant growth. The herbicide also caused a decline in nodule initiation, but had little effect on nodule development. However, plants were not nitrogen-limited and net photosynthesis was only slightly affected at the higher herbicide concentration. Total soluble sugars and starch were accumulated in both leaves and roots following herbicide supply. These results were also found in non-nodulated, nitrate-fed plants. In relation with a likely BCAA starvation, a significant increase was observed in the free amino acid pool, with a marked imbalance among different amino acids, although among BCAAs, only valine pool declined as a consequence of IM supply. It is concluded that acetolactate synthase inhibition by continuous, sublethal IM supply does not induce carbohydrate or a specific BCAA starvation in pea plants.

Function of antioxidant enzymes and metabolites during maturation of pea fruits

In plant cells, antioxidants keep reactive oxygen species at low concentrations, avoiding oxidative damage while allowing them to play crucial functions in signal transduction. However, little is known about the role of antioxidants during fruit maturation, especially in legumes. Snap pea (Pisum sativum) plants, which have edible fruits, were grown under nodulating and non-nodulating conditions. Fruits were classified in three maturity stages and antioxidants were determined in the seeds and seedless pods. Maturation or prolonged storage of fruits at 25 °C led to a decline in antioxidant activities and metabolites and in γ-glutamylcysteine synthetase protein. Notable exceptions were superoxide dismutase activity and glutathione peroxidase protein, which increased in one or both of these processes. During maturation, cytosolic peroxiredoxin decreased in seeds but increased in pods, and ascorbate oxidase activity was largely reduced in seeds. In stored fruits, ascorbate oxidase activity was nearly abolished in seeds but doubled in pods. It is concluded that symbiotic nitrogen fixation is as effective as nitrogen fertilization in maintaining the antioxidant capacity of pea fruits and that, contrary to climacteric fruits, a general decrease in antioxidants during maturation does not involve oxidative stress. Results underscore the importance of the antioxidant system in reproductive organs and point to ascorbate–glutathione metabolism and cytosolic peroxiredoxin as key players in pea fruit development.

Depletion of the heaviest stable N isotope is associated with NH4+/NH3 toxicity in NH4+-fed plants

BackgroundIn plants, nitrate (NO3-) nutrition gives rise to a natural N isotopic signature (δ15N), which correlates with the δ15N of the N source. However, little is known about the relationship between the δ15N of the N source and the 14N/15N fractionation in plants under ammonium (NH4+) nutrition. When NH4+ is the major N source, the two forms, NH4+ and NH3, are present in the nutrient solution. There is a 1.025 thermodynamic isotope effect between NH3 (g) and NH4+ (aq) which drives to a different δ15N. Nine plant species with different NH4+-sensitivities were cultured hydroponically with NO3- or NH4+ as the sole N sources, and plant growth and δ15N were determined. Short-term NH4+/NH3 uptake experiments at pH 6.0 and 9.0 (which favours NH3 form) were carried out in order to support and substantiate our hypothesis. N source fractionation throughout the whole plant was interpreted on the basis of the relative transport of NH4+ and NH3.ResultsSeveral NO3--fed plants were consistently enriched in 15N, whereas plants under NH4+ nutrition were depleted of 15N. It was shown that more sensitive plants to NH4+ toxicity were the most depleted in 15N. In parallel, N-deficient pea and spinach plants fed with 15NH4+ showed an increased level of NH3 uptake at alkaline pH that was related to the 15N depletion of the plant. Tolerant to NH4+ pea plants or sensitive spinach plants showed similar trend on 15N depletion while slight differences in the time kinetics were observed during the initial stages. The use of RbNO3 as control discarded that the differences observed arise from pH detrimental effects.ConclusionsThis article proposes that the negative values of δ15N in NH4+-fed plants are originated from NH3 uptake by plants. Moreover, this depletion of the heavier N isotope is proportional to the NH4+/NH3 toxicity in plants species. Therefore, we hypothesise that the low affinity transport system for NH4+ may have two components: one that transports N in the molecular form and is associated with fractionation and another that transports N in the ionic form and is not associated with fractionation.