Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO(2) (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO(2) concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO(2) incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O(2) evolution could be detected although at high pH and light intensity O(2) evolution was recorded.

Light dependency of nitrate and nitrite assimilation to reduced-N in leaves remains a controversial issue in the literature. With the objective of resoling this controversy, the lght requirement for nitrate and nitrite assimilation was investigated in several plant species. Dark and light assimilation of I'5Nlnitrate and 1"Nlnitrite to a ium and am_io-N was determined with leaves of wheat, corn, soybean, sunflower, and tobacco. In dark aerobic conditions, assimilation of I1Nlnitrate as a percentage of the lght rate was 16 to 34% for wheat, 9 to 16% for tobacco, 26% for corn, 35 to 76% for soybean, and 55 to 63% for sunflower. In dark aerobic conditions, assimlation of I'5Ninitrite as a percentage of the lght rate was 11% for wheat, 7% for tobacco, 13% for corn, 28 to 36% for soybeans, and 12% for sunflower. It Is concluded that variation among plant species in the light requirement for nitrate and nitrite assimilation explain some of the contradictory results in the literature, but ad i explanations must be sought to fuliy resolve the controversy.In dark anaerobic conditons, the assimilation of [l'Nlnitrate to ammonium and amino-N in leaves of wheat, corn, and soybean was 43 to 58% of the dark aerobic rate while dark anaerobic assimilation of l"Nlnitrite for the same species was 31 to 41% of the dark aerobic rate. In contrast, accumulation of nitrite in leaves of the same species in the dark was 2.5-to 20-fold higher under anaerobic than aerobic conditions. Therefore, dark assimilation of nitrite cannot alone account for the absence of nitrite accumulation in the in vivo nitrate reductase assay under aerobic conditons. Oxygen apparently inhibits nitrate reduction in the dark even in leaves of plant species that exhibit a relatively high dark rate of I15Nlnitrite assimilation.In the in vivo NR3 assay (12) leaf tissue accumulates N02-when incubated under dark anaerobic conditions but N02-accumulation is strongly suppressed by low concentrations of 02 (2,3,6,13,17 In contrast to the results of these studies, substantial rates of 15N03-and 15N02-assimilation to NH4' and amino-N in the dark have been reported for leaves of barley (1), sunflower (10), citrus (11), and several plant species (21). Such results have led to uncertainty of the light dependency of N03 and N02 assimilation and of the interpretation of 02 inhibition of N02 accumulation in the dark. With the objective of resolving these contradictions in the literature, the present study examines the light and 02 requirements for 15N03-and '5N02-assimilation in leaves of several plant species. Similar experiments were conducted independently in two separate laboratories to both confirm the results and to eliminate experimental procedure as a causal factor in the controversies. MATERL4,S AND METHODSThe experiments presented in this paper were conducted at Queen's University, Kingston, Ontario (Experiment A) and at the University of Illinois, Urbana (Experiment B).Plant Material. At Kingston, plant material comprised wheat (Triticum aestivum L. cv Freder...

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Experiments were carried out to clarify problems encountered in measuring metabolic and storage pool sizes of nitrate in wheat leaf sections with an in vivo nitrate reductase assay. The leaf sections were from seedlings grown on 15 millilar nitrate. Data obtained show that the cessation of nitrite accumulation, used as an index of the active nitrate pool size, could be caused by lack of anaerobiosis in the assay system, the lack of energy for nitrate reduction, or a loss of nitrate reductase activity. Availability of nitrate was never the limiting factor in this system. It is concluded that pool sizes of nitrate cannot be determined in wheat leaves with the in vivo assays employed. In 1973, Ferrari et al. (6) proposed that the in vivo assay for NR,3 with slight modifications, could be used to estimate the size of a metabolic (active) pool and a storage pool of nitrate in plant cells and tissues. Subsequently, other workers (2, 3) used this procedure to measure pool sizes in plant organs. Because NR is substrate-inducible, the pool sizes should affect the level of the enzyme and be a useful adjunct in attempts to use measurements of NRA in identifying crop cultivars with high production potential (7).The original objective of the work was to measure NRA and nitrate pool sizes in the leaves of growth chamber-grown seedlings of Australian wheats of commercial importance and to compare these values with their known field yield performance. Because our attempts to measure pool sizes with the in vivo method of Ferrari et al. (6) were unsuccessful, identification of the cause of this problem was attempted. These investigations led to a series of observations and conclusions concerning the proposed method for measuring pool sizes and factors that affect the in vivo assay. MATERIALS AND METHODS PLANT MATERIALIn Australia, seedlings of Triticum aestivum L., var. Olympic, were grown as described (19 Method A. Leaf sections (0.2 g) were added to vials or flasks (20-to 30-ml capacity) containing 10 ml 2 mm CaSO4. An initial volume of 8 ml CaSO4 was used when 2.0 ml of nitrate, malate etc. were to be added during the assay. All media were purged with N2 for 10 min prior to adding to the assay vials. N2 was bubbled through the medium of each vial for 3 min after the leaf sections had been added and just prior to sealing the vials with a rubber stopper. The samples were repurged with N2 for 3 min after vials were opened to supply additives. Incubation was in the dark at 30 C. Reactions were stopped at the desired time by transferring the vials (rubber stopper replaced with a marble or serum cap) to boiling water for 5 min. After cooling, aliquots of the liquid were removed for determination of nitrite (18). The CaS04 was used instead of phosphate because Ca2+ should help maintain membrane integrity.Method A-1 was similar to method A in all details except that nitrite accumulation was followed by sequential removal of aliquots of the medium at hourly intervals from the same assay vial, and after each sampling the conten...