Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.     

Triadimenol Increases Nitrate Levels and Nitrate Reductase Activity in Canola Leaves

Nitrate reductase activity in the first true leaves of canola(Brassica napus L.) seedlings grown in one-quarter strengthHoagland's solution from seeds pretreated with triadimenol (0.3or 30 g (a.i.) kg^–1 of seed) was higher than controlsduring the growth period of 15 to 25 d after planting. Triadimenolalso increased chlorophyll levels, the increase being more pronouncedat its lower concentration. The treatment also increased theweight and nitrate content of the leaves. When seedlings weregrown in nutrient solution containing 1 to 20 mM nitrate, theincrease in nitrate reductase activity by triadimenol was higherat lower rather than at higher nitrate concentrations. The nitratelevels and Kjeldahl nitrogen in the triadimenol-treated leaveswas higher than the controls at concentrations of added nitrateabove 2 mM. Addition of nitrate to plants grown in ammonium,increased nitrate reductase activity more in plants grown fromtriadimenol-treated seeds than controls. However, addition of10µM triadimenol for 24 h to ammonium-grown plants hadlittle effect on enzyme activity, both in the absence as wellas the presence of nitrate. This study demonstrates that triadimenolincreases nitrate reductase activity and nitrate accumulationin the leaves and at least part of the increased enzyme activityis independent of nitrate accumulation. Key words: Triazoles, nitrate content, nitrate reductase activity     

Functional Activity of Exoglycans from Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Resistant Mutant M-71 during the Formation of Legume–Rhizobia Symbiosis against a High-Nitrogen Background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71, capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO₃ ^–), was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of the same or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5- to 2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix⁺ phenotype of strain 250a, capable of forming symbiosis with pea plants only against a low-nitrogen background. In study of the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72–75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25–28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

The development of nitrate reductase in Chlorella and its repression by ammonium

Summary Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.     

Effect of nitrate,ammonia and nitrogen starvation on the regulation of nitrate reductase in Cyanidium caldarium

Summary Cells of Cyanidium caldarium grown with ammonia or ammonium nitrate as nitrogen source do not contain appreciable nitrate reductase activity. The alga develops the capacity to synthesize the enzyme when it is transferred from the ammonium medium to a nitrogen-free medium. Nitrate is not needed as an inducer and no enhancement in the rate of enzyme synthesis is observed when it is present. By contrast, whereas the synthesis of the enzyme in nitrogen-free medium proceeds at an increasing rate, in the nitrate medium it attains a stationary level after a short time.Nitrate grown cells possess variable amount of inactive nitrate reductase (from 9 to 60%) whereas in nitrogen-free medium the enzyme occurs principally in a fully active form. Addition of ammonia inactivates reversibly the preexisting enzyme. The inactive enzyme is measurable in the crude extract after activation by heating.It is suggested that in Cyanidium the inactivating effect of ammonia, which is the end product of nitrate reduction, in association with the repression of enzyme controls the level of nitrate reductase activity.     

Aluminium effect on nitrate assimilation in cucumber (Cucumis sativus L.) roots

In vivo effect of aluminium on nitrate uptake and reduction by cucumber seedlings was investigated. The high-performance liquid chromatography was used to analyse the rate of nitrate uptake. Low (0.5 mM) concentration of AlCl₃ in the nutrient solution stimulated nitrate uptake during the first 3 h. On the other hand, 6 h exposure of the cucumber seedlings to 1 or 5 mM of AlCl₃ resulted in inhibition of nitrate uptake and at 5 mM concentration of AlCl₃ the efflux of nitrate was observed. Furthermore, the amount of nitrate accumulated in cucumber roots after aluminium treatment was decreased. The noteworthy fact was observed, that at all concentrations of aluminium tested on increase of the nitrate reductase activity. This stimulation was concentration depended, but independent of the source of the enzyme. The activity of both the cytosolic and the plasma membrane bound nitrate reductase activity was enhanced in vivo. On the other hand, AlCl₃ applied in vitro only slighty decreased nitrate reductase activity.     

Effect of nitrates and sucrose on the induction of nitrate reductase in etiolated seedling leaves from selected barley genotypes in darkness

The parental genotypes, cv. Aramir and R567 line, as well as the selected DH lines C23, C47/1, C41 and C55, growing in darkness differed significantly in the level of NR activity in crude leaf extracts independently of nitrate concentration in the medium. The highest activity of the enzyme was found in the line C23. When plants grew on the medium with 0.5 mM KNO₃, NR activity in that genotype was almost 10-fold higher than in the parents and lines C41, C55 and also 3.5-fold higher than in the line C47/1. An increase of nitrate concentration in the medium to 10 mM caused a significant increase of NR activity in all the genotypes under study. In the line C23 this enzyme activity was only 20% lower than that found previously in the green leaves of that genotype in light. NR from the leaves of C23 and C41 lines was thermally unstable under in vitro conditions. This enzyme in the leaf extracts from the line C23 was characterized by a considerably lower unstability. The lines DH C23 and C41 growing in the dark on the medium with 0.5 mM KNO₃ did not differ in nitrate accumulation in leaves, whereas a larger nitrate content was found in the leaves of the line C41 when it grew on the medium with 10 mM KNO₃. Independently of nitrate concentration in the medium, leaves of the line C23 were found to have a higher sucrose content than those of the line C41. Excised, etiolated leaves of barley treated with 0.5 and 10 mM KNO₃ in dark under conditions favorable to transpiration had a low NR activity. Leaf treatment with a solution containing 10 mM KNO₃ + 0.2 M sucrose caused, on the average, a 13-fold increase of NR activity in comparison to leaves treated only with 10 mM KNO₃ and about a 6-fold increase of this enzyme in comparison to leaves treated with 0.5 mM KNO₃ + 0.2 M sucrose.     

Evidence for the nitrate-dependent spatial regulation of the nitrate reductase gene in chicory roots

Young chicory plants (Cichorium intybus L. var. Witloof) show a tenfold higher nitrate reductase NR activity in roots compared to leaves. Northern analysis revealed, besides the nitrate inducibility of the nitrate reductase gene (nia), a higher level of expression in the roots. By modifying the external nitrate concentration the NR activity in the leaves remained negligible whereas a maximal activity was observed in the roots when grown in the presence of 5 mM nitrate. Surprisingly, variation of the external nitrate concentration induced changes in the spatial regulation of nia within the root. In-situ hybridization mainly localized nia mRNA in the cortical cells of roots grown at low nitrate concentrations (0.2 mM). At high nitrate concentrations (5 mM), nia mRNA was more abundant in the vascular tissues. The root apex revealed a strong signal under both conditions. The isolation and characterization of the NR structural gene from chicory is also presented. Southern blot analysis revealed the presence of a single nia gene per haploid genome of chicory.     

Fatty acid uptake in Candida tropicalis: induction of a saturable process.

The rates of oleate uptake by Candida tropicalis cells grown on a high oleate concentration (3.5 mM oleate in the presence of 0.50% Brij 58) were higher than those observed in cells grown on glucose; however, oleate uptake was not saturable with substrate concentration. Cells grown at a low oleate concentration (1.0 mM oleate and 2.5% Brij 58) grew to a lower density and at a slightly slower rate; these cells were found to take up oleate at a rate 43-fold higher than cells grown on high oleate concentration. Furthermore, oleate uptake by the cells grown in low oleate medium was a saturable process with Kt and Vmax values of 56 microM and 15 nmol/(min.mg cell protein), respectively. The growth of C. tropicalis under low fatty acid concentration thus clearly results in the induction of a saturable process for its uptake. The total level of acyl-CoA synthetase activity in cells grown on the low oleate concentrations was only twofold higher than in high oleate or glucose grown cells; the level of this enzyme thus does not account for the saturable process and suggests that either the enzyme is regulated in vivo or else a hitherto unidentified enzyme is induced by growth in low concentrations of oleate.     

The Effect of the Plant Growth Stimulant Bactozole on Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Tolerant Mutant M-71 under Different Nitrogen Supply Conditions

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8–2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium are due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.     

Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

Role of Protein Synthesis in Recovering Nitrate Reductase Activity in Wheat Leaves Exposed to Heat Stress

Heating intact leaves of 14–15-day-old seedlings of wheat (Triticum aestivumL.), cv. Albidum 29, for 10 min at 44–45°C brought about a decrease in nitrate reductase activity by 50–90% of the initial level. The complete recovery of the enzyme activity occurred one to two days after the plants were returned to normal temperature conditions. Darkening plants or adding cycloheximide to the nutrient medium did not interfere with the recovery of nitrate reductase activity. The plants grown in darkness or on a nitrate-free medium were devoid of nitrate reductase activity. The transfer of these plants to the light or the addition of nitrate resulted in the induction of enzyme activity. In the untreated plants, nitrate reductase activity attained the control level in 48 h; in the heated plants, this process was considerably retarded. After heating, the activity of the preexisting enzyme recovered at a higher rate than the ability for enzyme induction. This means that the reactivation of nitrate reductase occurred even when the induction of the enzyme was almost entirely suppressed. We conclude that after the short-term effect of high temperatures, the functional activity of nitrate reductase may recover without the de novosynthesis of the enzyme protein.     

Activation of aromatic acids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomonas strain

The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K _m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K _m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K _m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.     

Factors affecting in vivo nitrate reductase activity in triticale

Factors influencing in vivo nitrate reductase activity in triticale (×Triticosecale Wittmack) primary leaves were investigated. Nitrate reductase activity was found to be a function of reaction time or tissue weight. In the range of 1–10 mm, the optimum slice width for nitrate reductase activity in triticale was found to be 1–2 mm. The optimum exogenous nitrate concentration is 300 mM. Substantial nitrite production was obtained even when exogenous nitrate was omitted from the assay. Of the five low molecular weight organic solvents tested, n-propanol is the most effective in enhancing enzyme activity. The optimum n-propanol concentration is 1% (v/v). The concentration of phosphate buffer (pH 6) does not affect nitrate reductase activity. Enzyme activity drops significantly below or above pH 6. In our system, nitrite production is enhanced by incubating under nitrogen, instead of air. The highest level of in vivo activity of nitrate reductase was found to be 10–15 cm from tip, which is close to the basal meristem of triticale primary leaves. Younger but physiologically mature leaves have higher nitrate reductase activity than old leaves.     

The Influence of Temperature on Nitrate Reductase Activity of Agrostis palustris and Cynodon dactylon

The influence of temperature was studied in relation to nitrate reductase activity of creeping bentgrass (Agrostis palustris Huds. cv. ‘Toronto’) a cool season grass and bermudagrass (Cynodon dactylon L. cv. ‘Tifgreen’) a warm season grass. Maximum nitrate reductase activity of both species occurred at 20°C. The nitrate reductase level in bentgrass leaves was reduced when grown at 35°C while bermudagrass leaves were relatively unaffected. The activity per se of the bentgrass enzyme preparation was inhibited rather than synthesis of the enzyme.     

Nitrogenase activity and nitrogen assimilation in Anabaena flos-aquae growing in continuous culture

Summary Anabaena flos-aquae is grown in chemostats under phosphate and urea-limited conditions. Nitrogenase activity in phosphate-limited cells has a maximum activity at a dilution rate of 0.025 h^-1 and is repressed 24-fold by 15 mM KNO₃. Cultures growing on 1.5 mM nitrate obtain 1/2–2/3 of cell nitrogen from N₂. Cells form inducible nitrite assimilating enzymes when grown on nitrate. Algae growing under A or He on limiting urea or phosphate-limited with nitrate have active nitrogenase. The ratio of nitrogenase activity to heterocyst numbers varied 90-fold depending on source of nitrogen, 15 mM KNO₃ gave the smallest ratio. The regulatory mechanisms controlling the activity of nitrogenase in blue-green algae is discussed.     

Isolation,Purification, and Characterization of Nitrate Reductase from a Salt-Tolerant <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> Yeast Strain Grown in the Presence of Tungsten

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.     

Effect of Chloramphenicol on Denitrification in Flexibacter canadensis and "Pseudomonas denitrificans"

It was recently reported that chloramphenicol inhibits existing denitrification enzyme activity in sediments and carbon-starved cultures of "Pseudomonas denitrificans." Therefore, we studied the effect of chloramphenicol on denitrification by Flexibacter canadensis and "P. denitrificans." Production of N(inf2)O from nitrate by F. canadensis cells decreased as the concentration of chloramphenicol was increased, and 10.0 mM chloramphenicol completely inhibited N(inf2)O production. "P. denitrificans" was less sensitive to chloramphenicol, and production of N(inf2)O from nitrate was inhibited by only about 50% even in the presence of 10.0 mM chloramphenicol. These results suggested that inhibition of denitrification enzyme activity depended on the concentration of chloramphenicol. Increasing the concentration of chloramphenicol decreased the rate of production of nitrite from nitrate by F. canadensis cells, and the concentration of chloramphenicol which resulted in 50% inhibition of production of nitrite from nitrate was 2.5 mM. In contrast, the rates of production of nitrite from nitrate by intact cells and cell extracts of "P. denitrificans" were inhibited by only 58 and 54%, respectively, at a chloramphenicol concentration of 10.0 mM. Chloramphenicol caused accumulation of NO from nitrite but not from nitrate and inhibited NO consumption in F. canadensis; however, it had neither effect in "P. denitrificans." Chloramphenicol did not affect N(inf2)O consumption by these organisms. We concluded that chloramphenicol inhibits denitrification at the level of nitrate reduction and, in F. canadensis, also at the level of NO reduction.     

Effect of nitrogen concentration on the activity of manganese peroxidase ofPhanerochœte chrysosporium

Manganese peroxidase as an extracellular enzyme is produced by the white rot fungusPhanerochœte chrysosporium under nutrient nitrogen or carbon limitation. The effect of nitrogen concentration on the activity of manganese peroxidase was studied using ammonium nitrate andl-asparagine as nitrogen sources. The highest activity of the enzyme was observed in cultures grown in a medium containing 75 mg/L ammonium nitrate and 0.15 g/Ll-asparagine. Manganese peroxidase was not detectable in cultures grown in the presence 0.5 g/L ammonium nitrate and 1 g/Ll-asparagine.     

The effect of nitrogen status on the regulation of plant-mediated proteolysis in ingested forage; an assessment using non-nodulating white clover

Production of nitrogenous waste by livestock agriculture is a significant environmental concern in terms of pollution of land and water. In the rumens of cattle and sheep, the excessive proteolysis which contributes to inefficiency of nutrient use involves both the rumen microbial population and the intrinsic plant proteases that can mediate protein degradation in ingested fresh forage on exposure to the environmental stresses of the rumen. Here, white clover (Trifolium repens) plants that do not form root nodules, and so are dependent on nitrate supplied to the roots, have been used to determine how nitrogen status of the plant affects the rate of plant‐mediated proteolysis in forage under conditions that simulate ingestion by grazing ruminants. Plants were grown from seed and supplied with nutrient solution containing 2.5, 5.0, 7.5 or 10 mM nitrate. Protein, free amino acid and protease activity were determined in leaves which had been placed in an in vitro system designed to simulate conditions experienced in the rumen (anaerobic phosphate buffer maintained at 39°C in the dark). Foliar protein content increased with increasing nitrate supply, while in vitro incubation of leaves resulted in time‐dependent decreases in protein concentration and increases in amino acid concentration. Regardless of nitrate supply, 50% of the protein was degraded in 6 h and 80% after 24 h. As the extent of protein decrease was determined by initial protein content, more protein degradation occurred in those plants grown with the highest nitrate supply: after 6 h, 130.7 mg g^?1 dry matter (DM) was degraded in leaves grown at 10 mM nitrate but only 52.3 mg g^?1 DM in leaves grown at 2.5 mM nitrate. Hence, although the percentage of proteolysis is independent of foliar protein concentration, the latter is critical to the quantity of protein degraded. Heat‐stable serine and cysteine proteases were active throughout the term of the in vitro incubation. Although proteolysis in ingested forage can continue for many hours, mediated by heat‐stable proteases, maximum amino acid accumulation accounted for less than 40% of initial protein. Therefore, it is proposed that continued and extensive proteolysis occurs following leaf excision and exposure to rumen conditions because amino acid accumulation is insufficient to initiate those feedback systems which sense cytoplasmic amino acid concentration and prevent excessive proteolysis during normal source–sink relations.