Nitrite reduction is inhibited by anaerobiosis in barley aleurone layers (5) and wheat roots (18), whereas the reduction of nitrate to nitrite by nitrate reductase is enhanced (4). Nitrite accumulates under such conditions and the anaerobic production of nitrite has been used to assay nitrate reductase in situ in a variety of plant tissues (4, 11-13, 17, 22, 24, 25). The reaction was found to be linear for more than 1 hr and dependent upon the addition of exogenous nitrate (4,13,24
Abstract. Nitrate reductase activity in barley (Hordeum vulgare L. cv. Himalaya) aleurone layers has been determined in the intact tissue, using two different methods. The first method measures the rate of appearance of H2180 produced during the reduction of KN1803. The second assay measures excreted nitrite resulting from nitrate reduction under anaerobic conditions. Nitrite production in this anaerobic, intact-tissue assay was dependent upon the presence of phosphate (pH 7.5) and was increased by ethanol and bisulfite.After ten hours of nitrate induction, nitrate reductase activities measured by the KN1803 assay are one-sixth, and those measured by the anaerobic intacttissue assay are one-third, of those observed in cell-free extracts of aleurone layers. Addition of ethanol to the anaerobic intact-tissue medium increased the rate of nitrate reduction to a level greater than that found in the cell-free assay.
A method has been devised for the detection and measurement of nitrite reductase activity in intact barley (Hordeum vulgare L. cv. Himalaya) aleurone layers. The technique involves feeding aleurone layers nitrite and measuring nitrite disappearance after a given time period. The method also allows simultaneous determination of nitrite uptake by the tissue. Quantitative recovery of nitrite is obtained by rapid heating of tissue in the presence of dimethyl sulfoxide.Using the procedure described, nitrite reductase activity in intact barley aleurone layers was determined. Enzyme activity was increased by prior incubation of the tissue with nitrate, but considerable activity was present in tissue incubated without nitrate. Nitrate-induced activity was inhibited by cycloheximide but not by actinomycin D. Enzyme activity in induced layers was inhibited by 2,4.dinitrophenol, and partially by antimycin A and 2-n-heptyl4-hydroxyquinoline Noxide. Activity in noninduced tissue appeared to be less sensitive to these respiratory inhibitors. In contrast, both activities were inhibited more than 90% by anaerobiosis; but nitrateinduced and noninduced aleurone layers were able to reduce nitrite anaerobically when the concentration of substrate in the assay medium was reduced from 250 pM to 25 pM. Nitrite uptake was relatively insensitive to anaerobiosis and to the inhibitors tested.Nitrite depletion from the medium by aleurone layers was rapid at pH 4.5 and negligible at pH 7.5. Nitrite accumulated at pH 4.5 under anaerobic conditions was rapidly released when the tissue was transferred to medium at pH 7.5. Nitrite release at pH 7.5 occurred whether the tissue was maintained under anaerobic or aerobic conditions. The reduction of nitrate by intact plant tissues and the release of the reduced product, nitrite, occurs under anaerobic conditions (4,6,9). A similar reponse can be observed aerobically in the presence of respiratory inhibitors (4 ,ug of chloramphenicol in a total volume of 5 ml were stoppered with cotton plugs and placed at 23 C in a metabolic water bath shaker at 200 rpm. Tissue preparation and incubation were done under sterile conditions. After 6 to 12 hr of incubation, the tissue was rinsed with approximately 50 ml of distilled water, and the effect of various conditions on enzyme activity was determined.Assay of Nitrite Reductase Activity. Unless otherwise indicated, nitrite reductase activity was assayed by measuring nitrite disappearance after placing 10 aleurone layers in 2 ml of medium containing 0.1 M potassium phosphate, pH 4.5, chloramphenicol (20 ug), nitrite, and the treatment solution as indicated in the text. Nitrite (final NaNO, concentration, 2.5 X 10-4 M) was added to start the assay. Two 0.1-ml aliquots were immediately removed to provide an accurate measure of the initial nitrite concentration of the medium with tissue present. After 40 min, two 0.1-ml aliquots of the medium were again removed for nitrite determination. The difference between the final and initial nitrite concentration of...
Pollen derived from cabbage, broccoli, and collards (Brassica oleracea, var. capitata, Italica and acephala, respectively) will germinate in a synthetic medium consisting of sucrose, H3BO4, CaC12 and purified polyethylene glycol 20,000. The optimum levels of constituents varied from genotype to genotype. With one genotype of cabbage, a germination percentage of 84~,, and tube lengths of at least 3.5 mm long were obtained. Whereas pollen from broccoli germinated in 1 to 2 hours, cabbage pollen required a 3 to 4 hour incubation period. Optimum germination occurred at pH 5.8 and at a pollen density of 0.5 2.0 mg/ml for cabbage. The effect of flower age on pollen germination varied from genotype to genotype. Of 7 cabbage genotypes tested, pollen germination either increased, decreased or remained the same as flower age increased. Pollen germination in vitro required polyethylene glycol purified by dialysis or passage through Sephadex G-25. Cabbage stigmas release a heat-labile substance capable of inhibiting germination of selfpollen, but stigma extracts from a cross plant have no effect.
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