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Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N(2) fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N(2)-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO(3) (-) and NH(4) (+) (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N(2)-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N(2)-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N(2)-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K(m) values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N(2)-fixing as well as N-supplemented plants, and G1,6BP levels in N(2)-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N(2)-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N(2)-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO(3) (-) and NH(4) (+), the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N(2)-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N(2)-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis.

Leaf area, chlorophyll content, net CO(2) photoassimilation, and the partitioning of fixed carbon between leaf sucrose and starch and soluble protein were examined in Glycine max (L) Merr. cv Williams grown under three different nitrogen regimes. One group (Nod+/+) was inoculated with Bradyrhizobium and watered daily with a nutrient solution containing 6 millimolar NH(4)NO(3). A second set (Nod+/-) was inoculated and had N(2) fixation as its sole source of nitrogen. A third group (Nod(-)) was not inoculated and was watered daily with a nutrient solution containing 6 millimolar NH(4)NO(3). The mean net micromole CO(2) uptake per square decimeter per hour of the most recently matured source leaves was similar among the three groups of plants, being about 310. Mean leaf area of the source leaves, monitored for net photosynthesis was also similar. However, the mean milligram of chlorophyll per square decimeter of Nod+/- test leaves was about 50% lower than the other groups' leaves and indicated nitrogen deficiency. Thus, Nod+/- utilized their chlorophyll more efficiently for photosynthetic CO(2) uptake than the plants of the other treatments. The ratio of foliar carbohydrate:protein content was high in Nod+/- but low in the plants from the other two treatments. This inverse relationship between foliar protein and carbohydrate content suggests that more fixed carbon is diverted to the synthesis of protein when nitrogen availability is high. It was also found that Nod+/- sequestered more storage protein in their paraveinal mesophyll than plants of the other treatments. This study indicates that when inorganic nitrogen regimes are used to control photosynthate partitioning, then both leaf carbohydrate and leaf protein must be considered as end products of carbon assimilate allocation.

It was hypothesized previously that an O(2) inhibition of NO(2) (-) photoreduction would reflect a competition between O(2) and NO(2) (-) for electrons from ferredoxin at the site of plastid nitrite reductase. In order to test this in vivo, intact spinach (Spinacia oleracea L.) leaf chloroplast and mesophyll cell isolates held in high light were aerated with streams of 20% O(2)/80% N(2) (250 micromolar O(2) in aqueous solution) or, alternatively, streams of 100% N(2). Bicarbonate plus CO(2) and NO(2) (-) were supplied to reaction mixtures at levels just sufficient to promote maximal assimilations of CO(2) and NO(2) (-). In chloroplast isolates, there was a 9 to 30% O(2) inhibition of NO(2) (-) reduction while there were high rates of CO(2) fixation. In spinach and soybean (Glycine max) leaf cell isolates, NO(2) (-) photoreduction rates were 10 to 55% inhibited by O(2) at near ambient levels. It is possible that O(2) may compete, albeit weakly, with NO(2) (-) (nitrite reductase) for equivalents derived from reduced ferredoxin. Also, O(2) may oxidize sulfhydryl groups on nitrite reductase which are involved in substrate binding and/or activation.

Steady state rates of NADP reduction decline upon commencement of nitrite reduction in reconstituted chloroplast preparations. Similarly, steady state rates of nitrite reduction are lower, but not zero, during concurrent NADP reduction. These results imply that competition for substrate occurs and suggest that nitrite reduction can successfully compete for reduced ferredoxin, even at high rates of NADP reduction.

The light-dependent synthesis of glycolate derived from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate was studied in the intact spinach (Spinacia oleracea) chloroplasts in the absence of CO(2). Glycolate yield increased with an elevation of O(2), pH, and the concentration of the phosphorylated compound supplied. No pH optimum was observed as the pH was increased from 7.4 to 8.5. The average maximal rate of glycolate synthesis was 50 mumoles per milligram chlorophyll per hour while the highest rate observed was 92 with 2.5 mm fructose 1,6-diphosphate in 100% O(2). The highest yields of glycolate synthesized from fructose 1,6-diphosphate, ribose 5-phosphate, or glycerate 3-phosphate were 0.14, 0.24, and 0.30, respectively, on a molar basis.

Light-dependent O(2) reduction concomitant with O(2) evolution, ATP formation, and NADP reduction were determined in isolated spinach (Spinacia oleracea L. var. America) chloroplast lamellae fortified with NADP and ferredoxin. These reactions were investigated in the presence or absence of catalase, providing a tool to estimate the reduction of O(2) to H(2)O(2) (Mehler reaction) concomitant with NADP reduction. In the presence of 250 micromolar O(2), O(2) photoreduction, simultaneous with NADP photoreduction, was dependent upon light intensity, ferredoxin, Mn(2+), NADP, and the extent of coupling of phosphorylation to electron flow.In the presence of an uncoupling concentration of NH(4) (+), saturating light intensity (>500 watts/square meter), saturating ferredoxin (10 micromolarity) rate-limiting to saturating NADP (0.2-0.9 millimolarity), and Mn(2+) (50-1000 micromolarity), the maxium rates of O(2) reduction were 13-25 micromoles/milligram chlorophyll per hour, while concomitant rates of O(2) evolution and NADP reduction were 69 to 96 and 134 to 192 micromoles/milligram chlorophyll per hour, respectively. Catalase did not affect the rate of NADPH or ATP formation but decreased the NADPH:O(2) ratios from 2.3-2.8 to 1.9-2.1 in the presence of rate-limiting as well as saturating concentrations of NADP.Photosynthetic electron flow at a rate of 31 micromoles O(2) evolved/milligram chlorophyll per hour was coupled to the synthesis of 91 micromoles ATP/milligram chlorophyll per hour, while the concomitant rate of O(2) reduction was 0.6 micromoles/milligram chlorophyll per hour and was calculated to be associated with an apparent ATP formation of only 2 micromoles/milligram chlorophyll per hour. Thus, electron flow from H(2)O to O(2) did not result in ATP formation significantly above that produced during NADP reduction.

Spinach leaf fructose 2,6-bisphosphate levels increase rapidly during the first 15 minutes of a normal dark period followed by a gradual decline during the next 5 hours. The regulatory mechanism responsible for the dark-induced rise in fructose 2,6-bisphosphate levels can be counteracted by a brief exposure to light intensities greater than 1 microeinstein per square meter per second.

During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO(2) per liter air, was 1.45 milligrams CO(2) evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO(2) per liter air). There was a higher foliar nonstructural carbohydrate level (e.g. sucrose and starch) in the CO(2) enriched compared with CO(2) normal plants. For example, leaves of enriched plants displayed levels of nonstructural carbohydrate equivalent to 174 milligrams glucose per gram dry weight compared to the 84 milligrams glucose per gram dry weight found in control plant leaves. As the leaves of CO(2) enriched plants approached full expansion, both the foliar respiration rate and carbohydrate content of the CO(2) enriched leaves decreased until they were equivalent with those same parameters in the leaves of control plants. A strong positive correlation between respiration rate and carbohydrate content was seen in high CO(2) adapted plants, but not in the control plants.Mitochondria, isolated simultaneously from the leaves of CO(2) enriched and control plants, showed no difference in NADH or malate-glutamate dependent O(2) uptake, and there were no observed differences in the specific activities of NAD(+) linked isocitrate dehydrogenase and cytochrome c oxidase. Since the mitochondrial O(2) uptake and total enzyme activities were not greater in young enriched leaves, the increase in leaf respiration rate was not caused by metabolic adaptations in the leaf mitochondria as a response to long term CO(2) enrichment. It was concluded, that the higher respiration rate in the enriched plant's foliage was attributable, in part, to a higher carbohydrate status.

The influence of pH upon the O(2) inhibition of (14)CO(2) photoassimilation (Warburg effect) was examined in intact spinach (Spinacia oleracea) chloroplasts. With conditions which favored the Warburg effect, i.e. rate-limiting CO(2) and 100% O(2), O(2) inhibition was greater at pH 8.4 to 8.5 than at pH 7.5 to 7.8. At pH 8.5, as compared with 7.8, there was an enhanced (14)C-labeling of glycolate, and a decrease of isotope in some phosphorylated Calvin cycle intermediates, particularly triose-phosphate. The (14)C-labeling of starch was also more inhibited by O(2) at higher pH. The enhanced synthesis of glycolate during (14)CO(2) assimilation at higher pH resulted in a diminution in the level of phosphorylated intermediates of the Calvin cycle, and this was apparently a causal factor of the increased severity of the Warburg effect.The (14)C-labeling profiles have been interpreted in terms of a "CO(2)"-sensitive as well as a "CO(2)"-insensitive mechanism for glycolate synthesis. Both mechanisms functioned optimally at the higher pH and both responded to O(2).

A technique to isolate mitochondria from chamber-grown soybeans (Glycine max cv Williams) was developed. The mitochondria were isolated by centrifugation on discontinuous Percoll gradients which yielded a sharp band of mitochondria contaminated by only 4% of the total chlorophyll in the gradient. Contamination by peroxisomes was also slight. The isolated mitochondria oxidized malate plus glutamate, NADH, and malate with respiratory control. They also showed cyanide-insensitive, alternative pathway activity which was inhibited by salicylhydroxamic acid.

The flow of carbon into sucrose and starch was investigated in fully expanded primary leaves of spinach using the long to short day transition and partial defoliation as tools to manipulate sucrose/starch synthesis. Transfer from 12 hour to 7 hour photosynthetic periods resulted in a 4-fold increase in the initial rate of starch synthesis, a 50% increase in the initial rate of sucrose synthesis, a 30% increase in leaf sucrose, and a 40% decrease in fructose, 2,6-biphosphate. In addition, sucrose synthesis rates in cells isolated from shortened daylength plants are 80% higher than in cells isolated from control plants. These results show that, in spinach, an increase in the rates of both sucrose and starch synthesis can occur under short day conditions. In contrast, when short day plants are partially defoliated, starch levels remain high, fructose 2,6-biphosphate levels remain low, but the level of leaf sucrose drops by 50%. Thus, when demand exceeds supply, starch synthesis has priority over filling of leaf sucrose pools in the short day plant.

Potential competition between CO(2) and NO(2) (-) photoassimilation for photogenerated reductant (e.g. reduced ferredoxin and NADPH) was examined employing isolates of mesophyll cells and intact chloroplasts derived from mature ;source' spinach leaves. Variations in the magnitude of incident light energy were used to manipulate the supply of reductant in situ within chloroplasts. Leaf cell and plastid isolates were fed with saturating CO(2) and/or NO(2) (-) to produce the highest demand for reductant by CO(2) and/or NO(2) (-) assimilatory processes (enzymes). Even in the presence of CO(2) fixation, NO(2) (-) reduction in intact leaf cell isolates as well as plastid isolates was maximal at light energies as low as 50 to 200 microeinsteins per second per square meter. Simultaneously, 500 to 800 microeinsteins per second per square meter were required to support maximal CO(2) assimilation. Regardless of the magnitude of the incident light energy, CO(2) assimilation did not repress NO(2) (-) reduction, nor were these two processes mutually repressed. These observations have been interpreted to mean that reduced ferredoxin levels in situ in the plastids of mature source leaf mesophyll cells were adequate to supply the concurrent maximal demands exerted by enzymes associated with CO(2) as well as with inorganic nitrogen photoassimilation.

Increasing levels of CO(2) have been shown to stimulate the rate of photosynthesis, eliminate the oxygen inhibition of photosynthesis (Warburg effect), and decrease glycolate formation in isolated spinach chloroplasts. Ribose 5-phosphate and fructose 1,6-diphosphate at concentrations of 5 to 10 mum also stimulate the rate of plastid photosynthesis and eliminate the Warburg effect. In contrast to the effect of high CO(2) levels, these sugar phosphates have little effect on glycolate formation. Evidence is presented to show that the level of intermediates of the photosynthetic carbon reduction cycle may influence the Warburg effect in vivo. It is postulated that the formation of glycolate is not the causal factor of the Warburg effect.

Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12-h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any ;feedback' inhibition of PS. The higher 7-hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates.

Previously, C Baysdorfer and JM Robinson (1985 Plant Physiol 77: 318-320) demonstrated that, in a reconstituted spinach chloroplast system, NADP photoreduction functioning at most maximal rate and reductant demand, was the successful competitor with NO(2) (-) photoreduction for reduced ferredoxin. This resulted in a repression of NO(2) (-) reduction until all NADP available had been almost totally reduced. Further experiments, employing isolated, intact spinach leaf plastids and soybean leaf mesophyll cells, were conducted to examine competition for reductant between CO(2) and NO(2) (-) photoassimilation, in situ. In isolated, intact plastid preparations, regardless of whether the demand for reductant by CO(2) photoassimilation was high (5 millimolar ;CO(2)') with rates of CO(2) fixation in the range 40 to 90 micromoles CO(2) fixed per hour per milligram chlorophyll, low (0.5 millimolar ;CO(2)') with rates in the range 5 to 8 micromoles CO(2) per hour per milligram chlorophyll, or zero (no ;CO(2)'), NO(2) (-) photoreduction displayed equal rates in the range of 8 to 22 micromoles per hour per milligram chlorophyll. In the absence of ;CO(2)', but in the presence of saturating white light, 3-phosphoglycerate photoreduction at rates of 82 to 127 micromoles per hour per milligram chlorophyll did not repress, and occasionally stimulated concomitant rates of NO(2) (-) reduction which ranged from 23.4 to 38.5. Conversely, in plastid preparations, NO(2) (-) at levels of 50 to 100 micromolar, stimulated plastid CO(2) fixation when ;CO(2)' was saturating with respect to carboxylation. Further, levels of NO(2) (-) in the range 250 to 2500 micromolar, stimulated soybean leaf mesophyll cell net CO(2) fixation as much as 1.5-fold if ;CO(2)' was saturating with respect to CO(2) fixation. It appeared likely that, in high light in vivo, CO(2) and NO(2) (-) photoassimilatory processes are not forced to intercompete for reduced ferredoxin in the intact chloroplast.

Photosynthesis and the Warburg effect (O(2) inhibition of photosynthesis) were evaluated in preparations of intact spinach chloroplasts enriched with varying amounts of lysed chloroplasts. Increasing the ratio of broken to intact plastids resulted in decreased rates of CO(2) assimilation.Hydrogen peroxide when added at 10 or more micromolar also inhibited photosynthesis in these preparations. Inhibition of the photosynthetic rate by both factors was eliminated by addition of catalase. These findings indicate that H(2)O(2) presumably generated by the broken chloroplasts was the causal agent of this inhibition.The Warburg effect also became more pronounced by increasing the level of broken to intact chloroplasts. This effect was examined as a function of added catalase, pH, and O(2) concentration. At 21% O(2) and 0.44 to 0.68 millimolar CO(2), catalase relieved the effect almost completely at pH 7.5, but at pH 8.3, the rate was restored only to about half or less of the control. At pH 7.6, 0.44 millimolar CO(2), and 100% O(2), the effect was only slightly overcome by catalase.A rise in glycolate synthesis by the isolated spinach chloroplast has been shown previously to be coupled to an increase in pH and O(2) (Kow, Robinson, Gibbs 1977 Plant Physiol 60: 492-495; Robinson, Gibbs, Cotler 1977 Plant Physiol 59: 530-534). At 21% O(2), glycolate synthesis was not affected by the addition of catalase at pH 7.5 or 8.3. It is proposed that at 21% O(2) and without some means of removing H(2)O(2), that portion of the Warburg effect attributed to glycolate synthesis has been overestimated at pH values in the order of 7.5. In contrast, that portion of the Warburg effect which was, at alkaline pH, insensitive to catalase represented the stress placed upon the photosynthetic carbon reduction cycle which resulted from an enhanced synthesis of glycolate. At 100% O(2) aeration and pH 7.5 to 8.5, the Warburg effect may also represent O(2)-mediated inhibition of a Calvin cycle enzyme within the intact plastid.

Disalicylidenepropanediamine (DSPD) at 0.1 to 1 mm levels inhibited light-dependent (14)CO(2) assimilation in intact spinach chloroplasts about 50 to 80%, and this inhibition was accompanied by an increased ratio of (14)C-glycerate 3-phosphate to (14)C-glyceraldehyde 3-phosphate. Enzymatic analysis established that DSPD also inhibited the light-dependent reduction of glycerate 3-phosphate in intact spinach chloroplasts. DSPD at 0.5 mm did not inhibit ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, glycerate 3-phosphate kinase, NADP(+)-linked glyceraldehyde 3-phosphate dehydrogenase or ribulose 1,5-diphosphate carboxylase. The inhibition of chloroplast (14)CO(2) assimilation by DSPD appeared to be related to the inhibition of the photosynthetic electron transport chain. These observations are consistent with experimental results which demonstrated that DSPD inhibited directly the chloroplast lamellar membrane-mediated, light-dependent reduction of ferredoxin (Trebst, A. and M. Burba, 1967, Z. Pflanzenphysiol. 57: 419-433 and Ben-Amotz, A. and M. Avron, 1972, Plant Physiol. 49: 244-248).

A rapid oxygraph method of studying the permeability of the envelope of isolated chloroplasts was used. The outer envelope of aqueously isolated whole spinach (Spinacia oleracea L.) chloroplasts in buffer is readily permeable to 3-phosphoglyceric acid, which induces an immediate light dependent oxygen evolution. This light dependent oxygen evolution was completely eliminated by swelling these plastids in an osmotically dilute solution. Exogenous adenosine diphosphate, but not inorganic phosphate, strongly stimulated this oxygen evolution. This indicated that the chloroplast envelope is relatively permeable to adenosine diphosphate.Oxygen evolution and swelling studies indicated that the chloroplast envelope is relatively impermeable to NADP and to ferredoxin.A method is described whereby the percent of whole chloroplasts present in a chloroplast preparation may be rapidly estimated.