José M. Maldonado

Properties of the in vivo Nitrate Reductase Assay in Maize, Soybean, and Spinach Leaves

Summary The effect of different parameters on the in vivo nitrate reductase activity in leaves of maize, soybean, and spinach has been examined. Leaf sections infiltrated under vacuum with an assay medium containing CO 2 tension and NO - 3 and incubated under air, excreted CO 2 tension and NO - 2 in the dark but not in the light. Nevertheless, addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, a potent inhibitor of the photosynthetic electron transport, promoted an immediate excretion of CO 2 tension and NO - 2 in the light. Under those conditions, subsequent addition of ascorbate-reduced dichlorophenolindophenol or N,N,N′,N′-tetramethylphenylenediamine, which can supply electrons to photosystem I, abolished the excretion of CO 2 tension and NO - 2 by the leaf sections. These results support the view that, in green tissues, the reducing equivalents photogenerated in the chloroplasts are more directly used for NO - 2 reduction than for NO - 2 reduction. Incubation of leaf discs under O 2 -atmosphere severely impaired NO - 2 accumulation. Under strict anaerobic conditions, the rate of NO - 2 production was only slightly enhanced as compared with that observed in the assay under air. Exogenous NO - 3 stimulated the activity, more markedly in soybean than in the other two species. Concentrations of exogenous NO - 3 between 50 and 100 mM were required for maximal activities in all the three species. The external pH optimum for in vivo NO - 3 reduction was 7.5 for maize and spinach and 7.0 for soybean. Inclusion of 1% (v/v) n -propanol in the infiltration-incubation medium enhanced the activity about 15%. However, 7% (v/v) n -propanol caused a drastic decrease in the rate of NO - 2 accumulation which could be greatly overcome by inclusion in the medium of NADH or reduced flavin mononucleotide.

Reversible inactivation of maize leaf nitrate reductase

Abstract Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.

PHOTOREGULATION OF NITRATE UTILIZATION IN GREEN ALGAE AND HIGHER PLANTS

Abstract— Nitrate reductase from eukaryotes can be reversibly inactivated, blue light being an effective activating agent both in vitro and in vivo. Hydroxylamine proved to be a powerful inactivating agent of Ankistrodesmus braunii nitrate reductase. Irradiation with blue light of NH2OH‐inactivated nitrate reductase, specially in the presence of μM amounts of FAD, promoted the recovery of the enzyme activity. Similarly, photoexcited methylene blue reactivated spinach nitrate reductase. On the other hand, in vitro nitrate reductase is highly susceptible to photodynamic inactivation caused by singlet O2. Aerobic incubation of the active spinach enzyme with either FMN or methylene blue under either blue or red light respectively led to its irreversible inactivation. Irradiation of frozen and thawed spinach leaf discs also promoted, in situ, an irreversible inactivation of nitrate reductase, provided that 62 was present in the incubation mixture. Thus, either in vitro or in situ, light can cause two quite different responses of nitrate reductase, its blue light‐dependent photoactivation in a flavin sensitized reaction and its photodynamic inactivation in a singlet O2‐dependent process.

Nitrate uptake and reduction by durum wheat (Triticum turgidum) and Tritordeum (Hordeum chilense x Triticum turgidum)

Summary Tritordeum is a fertile amphiploid between a wild South American barley ( Hordeum chilense Roem. et Schultz.) and durum wheat ( Triticum turgidum L. conv. durum ). An outstanding characteristic of tritordeum is its high protein content in the grain, which is substantially higher than that of its wheat parent. This study was conducted to gain information on the properties of the nitrate uptake system of two tritordeum lines (HT8 and HT31) and their wheat parent, and to find differences among genotypes in nitrate uptake and/ or the activity of the nitrate-reducing enzymes as possible factors related to the protein content. Exposure of intact tritordeum plants to environmental nitrate induced nitrate uptake by roots, and nitrate reductase (EC 1.6.6.1) and nitrite reductase (EC 1.7.7.1) activities in roots and leaves. Ammonium did not induce the nitrate uptake system. Moreover, pretreatment of the plants with nitrate plus ammonium resulted in a decreased induction as compared with nitrate alone. The apparent K s , values for net nitrate uptake in T. turgidum , tritordeum HT8 and tritordeum HT31 were 128, 112 and 90 μM, respectively, and the corresponding V max values were 6.5, 9 and 12 μmol h -1 g -1 fresh weight, indicating that tritordeum lines, and specially HT31, absorb nitrate more efficiently than the wheat parent. Nitrate reductase and nitrite reductase activities in roots after nitrate induction ranged from 0.8 to 2.2 and from 158 to 266 μmol h -1 g -1 fresh weight, respectively. Therefore, nitrate to nitrite reduction appears to be the rate-limiting step of nitrate assimilation. Tritordeum HT8, whose grain protein concentration is substantially higher than that of tritordeum HT31, exhibited higher nitrate reductase activity both in roots and leaves but lower efficiency for nitrate absorption than tritordeum HT31. Hence, protein accumulation appears to be more related to nitrate reduction than to nitrate uptake rate.

Relation between photosynthesis and dark respiration in cereal leaves

Summary From the light response curves of net photosynthesis at 30 °C, the values of dark respiration, apparent quantum yield and light compensation point were calculated for leaves of Triticum turgidum, Hordeum chilense (H1, H35 and H71 lines), and tritordeum HT31, an amphiploid from H. chilense and T. turgidum . Tritordeum HT31 has an unusually low light compensation point and dark respiration rate; at high irradiance, its rate of O 2 evolution was within the range of a typical sun plant. In the five genotypes, both the light compensation point and the apparent quantum yield were positively correlated with the dark respiration rate. T. turgidum and H. chilense H71 had a larger chlorophyll content than tritordeum HT31. Leaf chlorophyll content per unit fresh mass was correlated to dark respiration rate, apparent quantum yield and light compensation point. These results indicate that in leaves from cereals grown under identical environmental conditions, there is a close association between chlorophyll content, dark respiration rate and apparent quantum yield.

Effect of Freezing and Thawing on in situ Nitrate Reductase Activity in Spinach and Maize Leaves

Summary Spinach leaf discs subjected to freezing and thawing, and subsequently incubated with a buffer containing nitrate and NADH, excreted nitrite into the medium linearly for at least 90 min. Chemically-reduced FMN was also an efficient added electron donor for in situ nitrate reduction. The freeze-thaw nitrate reductase assay did not require anaerobic conditions. Nitrite production occurred equally in the light and in the dark; consequently, DCMU did not inhibit the assay in light. The freeze-thaw treatment completely abolished the photosynthetic oxygen evolution by leaf discs. In maize leaf sections, freezing and thawing caused irreversible inactivation of nitrate reductase. This inactivation was greatly prevented when both freeze-thaw treatment and further incubation were carried out under anaerobic conditions. Alternatively, the freeze-thaw assay in maize leaves could be improved by performing both the freeze-thaw treatment and the incubation in the presence of compounds which protect the enzyme against the inactivating effects of phenolics.

MITOCHONDRIAL DNA POLYMORPHISM AND GYNODIOECY IN A NATURAL POPULATION OF ROSMARINUS OFFICINALIS L.

Mitochondrial genome variability was studied in a gynodioecious population of Rosmarinus officinalis L. Analysis of mitochondrial DNA organization using Southern hybridization with probes for mitochondrial genes coxll, cob. and atp A revealed a high level of mitochondrial polymorphism; 11 mitotypes were detected in a total of 27 individuals studied. One mitotype was particularly common among male fertile individuals. The remaining mitotypes were found in only one or two individuals, generally male sterile. Polymorphism was thus greater in male sterile than in male fertile individuals. The data presented here suggest that male sterility in Rosmarinus is associated with the appearance of mitochondrial genome variants which differ from the standard pattern for male fertile plants.

Properties of the glutamine synthetase isoforms isolated from sunflower leaves

Summary Glutamine synthetase (GS) from sunflower leaves was very unstable in vitro. The decay of GS activity was prevented by the presence of thiols and protease inhibitors in the extraction buffer. Properties of cytosolic (GS1) and chloroplastic (GS2) isoforms were studied after separation by ion-exchange chromatography. Transferase assay pH optima were 7.0 for GS1, and 7.5 for GS2. Affinities of GS2 for L-glutamine and hydroxylamine were somewhat higher than those of GS1. The activity of both isoforms was absolutely dependent on Mn2+, but GS2 required a much lower Mn2+ concentration. GS1 exhibited maximum activity at 47 °C, with an activation energy of 37 kJ mol−1 and a Q10 of 1.63. Maximum activity of GS2 was shown at 43 °C, with an activation energy of 43 kJ mol−1 and a Q10 of 1.76. GS2 was much more heatlabile than GS1. The presence of Mn+2-ADP greatly protected the isoenzyme against thermal denaturation. Both isoforms were inactivated by p-hydroxymercuribenzoate and subsequently reactivated by a short preincubation with dithioerythritol. Nevertheless, GS2 showed a higher sensitivity to thiol reagents.

Red-light effects sensitized by methylene blue on nitrate reductase from spinach (Spinacia oleracea L.) leaves

Abstract Nitrate reductase from spinach (Spinacia oleracea L.) leaves, which had been inactivated in vitro by incubation with NADH and cyanide, was fully reactivated in minutes when irradiated in anaerobic conditions with red light in the presence of methylene blue. Both the rate and the extent of reactivation increased with light intensity (6 to 100 W·m-2) and dye concentration (1 to 10 μM). On the contrary, photoreactivation was completely abolished when NADH or ethylenediaminetetra-acetic acid were present during irradiation. We propose that methylene blue, when photo excited, exhibits a redox potential positive enough to reoxidise the CN--re-duced molybdenum complex settled in the inactive enzyme, thus causing its reactivation. On the other hand, prolonged irradiation of nitrate reductase, under air and in the presence of methylene blue, promoted an oxygen-dependent irreversible inactivation of the two partial activities of the enzyme. This inactivation was markedly enhanced in 77% deuterated water and greatly prevented by azide, which indicates that singlet oxygen is the species primarily involved in the photooxidative inactivation of the enzyme.

Effects of phosphate on in vitro nitrate reductase activity from tobacco leaves

Abstract Extracts from tobacco (Nicotiana tabacum L. cv. Gatersleben 1) leaves harvested 30 min before the beginning of the light period (‘dark extract’) and after 4 h illumination (‘light extract’) were used to study the effects of phosphate (Pi) on in vitro nitrate reductase (NR, E.C. 1.6.6.1) activity. Addition of 25 mM Pi increased (−Mg2+)NR activity from ‘light and dark extracts’, this effect being specific of this anion and not due to an increased ionic strength. However, (+Mg2+)NR activity decreased when the extracts were preincubated with 25 mM Pi. This Pi-inactivation was insensitive to staurosporine, which indicates that inactivation was not the result of NR protein phosphorylation. NR activity (±Mg2+) from ‘dark extracts’ increased when preincubated for 1 h and reached similar activities as those from ‘light extracts’ at the end of the preincubation. This increase of NR activity from ‘dark extracts’ was abolished in the presence of okadaic acid, reflecting a dephosphorylation of NR protein by endogenous phosphatases in a time-dependent process. These data are discussed concluding that Pi affects the in vitro NR activity in tobacco plants.