Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: I. Initial Products of CO(2) Assimilation and Levels of Whole Leaf C(4) Metabolites

Labeling patterns from ¹⁴CO₂ pulses to leaves and whole leaf metabolite contents were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. During the first one to two minutes of illumination, malate was the primary initial product of ¹⁴CO₂ assimiltion (about 77% of total ¹⁴C incorporated). After about 5 minutes of illumination, the proportion of initial label to aspartate increased from 16 to 66%, and then gradually declined during the following 7 to 10 minutes of illumination. Nutrition experiments showed that the increase in ¹⁴CO₂ partitioning to aspartate was delayed about 2.5 minutes in plants grown with limiting N, and was highly dampened in plants previously treated 10 to 12 days with ammonia as the sole N source. Measurements of C₄ leaf metabolites revealed several transients in metabolite pools during the first few minutes of illumination, and subsequently, more gradual adjustments in pool sizes. These include a large initial decrease in malate (about 1.6 micromoles per milligram chlorophyll) and a small initial decrease in pyruvate. There was a transient increase in alanine levels after 1 minute of illumination, which was followed by a gradual, prolonged decrease during the remainder of the induction period. Total leaf aspartate decreased initially, but temporarily doubled in amount between 5 and 10 minutes of illumination (after its surge as a primary product). These results are discussed in terms of a plausible sequence of metabolic events which lead to the formation of the intercellular metabolite gradients required in C₄ photosynthesis.     

Mechanism of c(4) photosynthesis: the size and composition of the inorganic carbon pool in bundle sheath cells

We sought to characterize the inorganic carbon pool (CO₂ plus HCO₃⁻) formed in the leaves of C₄ plants when C₄ acids derived from CO₂ assimilation in mesophyll cells are decarboxylated in bundle sheath cells. The size and kinetics of labeling of this pool was determined in six species representative of the three metabolic subgroups of C₄ plants. The kinetics of labeling of the inorganic carbon pool of leaves photosynthesizing under steady state conditions in ¹⁴CO₂ closely paralleled those for the C-4 carboxyl of C₄ acids for all species tested. The inorganic carbon pool size, determined from its ¹⁴C content at radioactivity saturation, ranged between 15 and 97 nanomoles per milligram of leaf chlorophyll, giving estimated concentrations in bundle sheath cells of between 160 and 990 micromolar. The size of the pool decreased, together with photosynthesis, as light was reduced from 900 to 95 microeinsteins per square meter per second or as external CO₂ was reduced from 400 to 98 microliters per liter. A model is developed which suggests that the inorganic carbon pool existing in the bundle sheath cells of C₄ plants during steady state photosynthesis will comprise largely of CO₂; that is, CO₂ will only partially equlibrate with bicarbonate. This predominance of CO₂ is believed to be vital for the proper functioning of the C₄ pathway.     

Mechanism of C4 photosynthesis in Chloris gayana: pool sizes and kinetics of 14CO2 incorporation into 4-carbon and 3-carbon intermediates.

In one group of C₄ species, including Chloris gayana, C₄ acids are decarboxylated via phosphoenolpyruvate carboxykinase to give phosphoenolpyruvate as the initial C₃ product. This paper presents an analysis of the kinetics of labeling of various photosynthetic intermediates in Chloris gayana leaves exposed to ¹⁴CO₂, and the pool sizes of these intermediates, primarily to provide information about the subsequent metabolism of phosphoenolpyruvate. Saturation labeling of the C-4 of aspartate and malate, and the C-1 of 3-phosphoglycerate, indicated photosynthetically active pools of 0.45, 0.22, and 0.95 μol/mg chlorophyll, respectively. For aspartate and 3-phosphoglycerate, the total leaf pools and the photosynthetic pools were of similar size, but the total pool of malate was about 100 times larger than the photosynthetically active pool. From the relative rates of labeling of phosphoenolpyruvate, pyruvate, alanine, and C-1, C-2 plus C-3 of aspartate, during steady-state ¹⁴CO₂ assimilation, relative pool sizes were calculated to be about 10:11:78:100, respectively. Pulse/chase labeling of leaves provided estimates of relative photosynthetic pool sizes in the ratio of about 6:15:90:100, respectively, where aspartate is arbitrarily assigned a value of 100 in both cases. Notably, labeling of alanine was consistent with its derivation from the C-1, C-2 plus C-3 carbons of aspartate, and the alanine pool was at least eight times larger than the phosphoenolpyruvate pool that showed similar labeling kinetics. Results were consistent with the view that at least most of the phosphoenolpyruvate produced by C₄ acid decarboxylation is metabolized via alanine.     

Studies on the mechanism of glycerate 3-phosphate synthesis in tomato and maize leaves

The net carbon incorporation in maize (Zea mays) and tomato (Lycopersicum esculentum) leaves was mainly the result of the carboxylation of ribulose 1,5-diphosphate. In both of these organisms synthesis of glycerate 3-phosphate was studied during short chase experiments (2 or 3 seconds in ¹⁴CO₂ then 8 to 27 seconds in unlabeled CO₂). Changes in the radioactivity in the individual carbon atoms of glycerate 3-phosphate, malate, and aspartate are consistent with the formation, in both leaves, of 2 molecules of glycerate 3-phosphate for each CO₂ molecule incorporated. The CO₂, before reacting with ribulose 1,5-diphosphate, is first incorporated in an intracellular CO₂ pool which has a different composition according to the species. This pool is constituted in tomato by volatile compounds (50 nanomoles per gram of fresh weight) more or less in equilibrium with atmospheric CO₂. In maize the pool consists of carbon atoms 4 of malate and aspartate (for at least 80% of the pool) and volatile compounds which correspond, in all, to 540 nanomoles per gram of fresh weight where atmospheric CO₂ enters through an irreversible reaction.     

Photosynthetic carbon metabolism of isolated corn chloroplasts

Chloroplasts have been isolated from 4- to 6-day-old corn (Zea mays) leaves capable of assimilating 45 micromoles CO₂ per milligram chlorophyll per hour. The effects of various factors such as inorganic phosphate, reducing agents, inhibitors, intermediates of the photosynthetic carbon reduction cycle, organic acids, and oxygen on the photosynthetic rate and on the distribution of ¹⁴C within the products by these chloroplasts were determined. The photosynthetic carbon metabolism of the corn plastids appeared to be similar to that already observed in spinach and pea chloroplasts. It was concluded that the corn plastids can fix CO₂ at meaningful rates via the photosynthetic carbon reduction cycle of Calvin without the operation of a cycle involving the C-4 compounds, malate and aspartate.     

Effect of nitrate limitation on the photosynthetically active pools of aspartate and malate in maize, a NADP malic enzyme C4 plant

After two weeks of moderate N restriction, growth of 3-week-old Zea mays L. plants was less than half that of the control and aspartate and malate levels in the leaves were severely suppressed (45 and 65% decrease, respectively). Since in NADP malic enzyme type C₄ plants, such as maize, malate and aspartate are intermediates in the C₄ photosynthetic pathway, the operation of the latter was investigated. Moderate nitrogen deficiency had only a small effect on the rate of photosynthesis (20% decrease) measured under 1000 umol m^?2 s^?1 irradiance. ¹⁴CO₂ pulse-¹²CO₂ chase experiments combined with measurements of in vitro photosynthetic enzyme activities demonstrated the operation of a typical C⁴ photosynthetic pathway in N-restricted plants. The turnover rates of malate and aspartate molecules involved in the C₄ cycle were determined by the loss of label in the carbon 4 moiety of these molecules during the chase period. It is shown that N restriction did not alter the turnover of malate but greatly accelerated that of aspartate. The amounts of malate and aspartate moving through photosynthetically active pools were estimated using a kinetic model. For malate, the size of this pool appeared to be only slightly diminished whereas for aspartate the size of the corresponding pool decreased by a factor of 3. It is proposed that under moderate NO₃^? deficiency, despite deviations in malate metabolism leading to a pronounced decrease in the size of its cellular pool, a large amount of malate remained in the operation of the C₄ pathway. By contrast, the participation of aspartate in the operation of the C₄ pathway was greatly reduced.     

Photosynthetic CO(2) Fixation Products and Activities of Enzymes Related to Photosynthesis in Bermudagrass and Other Plants

After a 5-second exposure of illuminated bermudagrass (Cynodon dactylon L. var. `Coastal') leaves to ¹⁴CO₂, 84% of the incorporated ¹⁴C was recovered as aspartate and malate. After transfer from ¹⁴CO₂-air to ¹²CO₂-air under continuous illumination, total radioactivity decreased in aspartate, increased in 3-phosphoglyceric acid and alanine, and remained relatively constant in malate. Carbon atom 1 of alanine was labeled predominantly, which was interpreted to indicate that alanine was derived from 3-phosphoglyceric acid. The activity of phosphoenolpyruvate carboxylase, alkaline pyrophosphatase, adenylate kinase, pyruvate-phosphate dikinase, and malic enzyme in bermudagrass leaf extracts was distinctly higher than those in fescue (Festuca arundinacea Schreb.), a reductive pentose phosphate cycle plant. Assays of malic enzyme activity indicated that the decarboxylation of malate was favored. Both malic enzyme and NADP⁺-specific malic dehydrogenase activity were low in bermudagrass compared to sugarcane (Saccharum officinarum L.). The activities of NAD⁺-specific malic dehydrogenase and acidic pyrophosphatase in leaf extracts were similar among the plant species examined, irrespective of the predominant cycle of photosynthesis. Ribulose-1, 5-diphosphate carboxylase in C₄-dicarboxylic acid cycle plant leaf extracts was about 60%, on a chlorophyll basis, of that in reductive pentose phosphate cycle plants.     

Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea L.

Summary The photosynthetic products of Portulaca oleracea differ greatly depending on leaf age and length of exposure to ¹⁴CO₂. Mature leaves of P. oleracea fix ¹⁴CO₂ primarily into organic and amino acids during a 10-s exposure period. Less than 2% of the ¹⁴CO₂ fixed appears in phosphorylated compounds. In contrast, incorporation into amino acids can account for over 60% of the total ¹⁴CO₂ fixed by young leaves in an equal time period, and incorporation into alanine alone can account for up to one half of this amount. Senescent leaves display a quantitative shift of primary products toward phosphorylated compounds with a concomitant reduction of the label residing in malate and asparate. About 8 times more phosphoglyceric acid is produced in senescent leaves than in mature leaves. The aspartate/ malate ratio is not constant and depends on the length of time the leaves are exposed to ¹⁴CO₂ and the age of the leaves under study. It appears as if the stage of leaf development is one of the most important factors determining the operation of a particular enzyme system in C₄ plants.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway under Various Light Intensities in Maize Leaves

The rate of CO₂ assimilation and levels of metabolites of the C₄ cycle and reductive pentose phosphate pathway in attached leaves of maize (Zea mays L.) were measured over a range of light intensity from 0 to 1,900 microEinsteins per square meter per second under a saturated CO₂ concentration of 350 microliters per liter and a limiting CO₂ concentration of 133 microliters per liter. The level of ribulose 1,5-bisphosphate (RuBP) stayed almost constant (around 60 nanomoles per milligram chlorophyll [Chl]) from low to high light intensities under 350 microliters per liter. Levels of 3-phosphoglycerate (PGA) increased from 100 to 650 nanomoles per milligram Chl under 350 microliters per liter CO₂ with increasing light intensity. The calculated RuBP concentration of 6 millimolar (corresponded to 60 nanomoles per milligram Chl) was about two times above the estimated RuBP binding-site concentration on ribulose bisphosphate carboxylase-oxygenase (Rubisco) of ~2.6 millimolar in maize bundle sheath chloroplasts in the light. The ratio of RuBP/PGA increased with decreasing light intensity under 350 microliters per liter CO₂. These results suggest that RuBP carboxylation is under control of light intensity possibly due to a limited supply of CO₂ to Rubisco through the C₄ cycle whose activity is highly dependent on light intensity. Pyruvate level increased with increasing light intensity as long as photosynthesis rate increased. A positive relationship between levels of PGA and those of pyruvate during steady-state photosynthesis under various conditions suggests that an elevated concentration of PGA increases the carbon input into the C₄ cycle through the conversion of PGA to PEP and consequently the level of total intermediates of the C₄ cycle can be raised to mediate higher photosynthesis rate.     

beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism

Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form β-carotene from NaH¹⁴CO₃ or [U-¹⁴C]-3-phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-¹⁴C]-3-PGA or 3 to 6 millimolar NaH¹⁴CO₃ was fully sufficient to supply β-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles ¹⁴C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the β-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO₂ to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute β-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis.     

Photosynthetic Characteristics of the C(3)-C(4) Intermediate Parthenium hysterophorus

The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO₂ compensation concentration (20-25 microliters per liter at 30°C and 21% O₂) and by a reduced sensitivity of net photosynthesis to 21% O₂. In contrast, the related C₃ species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO₂ compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O₂. Furthermore, in P. hysterophorus the CO₂ compensation concentration is relatively less sensitive to changes in O₂ concentrations and shows a biphasic response to changing O₂, with a transition point at about 11% O₂. Based on these results, P. hysterophorus is classified as a C₃-C₄ intermediate. The activities of diagnostic enzymes of C₄ photosynthesis in P. hysterophorus were very low, comparable to those observed in the C₃ species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10-29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to ¹⁴CO₂ (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial ¹⁴C products (77-84%), with ≤4% of the ¹⁴C-label in malate plus aspartate. These results indicate that in the C₃-C₄ intermediate P. hysterophorus, the reduction in leaf photorespiration cannot be attributed to C₄ photosynthesis.     

Isolation of Bundle Sheath Cell Chloroplasts from the NADP-ME Type C(4) Plant Zea mays: Capacities for CO(2) Assimilation and Malate Decarboxylation

Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO₂ assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30°C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO₂ assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C₄ photosynthesis by species of the NADP-malic enzyme type.     

Utilization of Photosynthetically Fixed 14CO2 into Alkaloids in Relation to Primary Metabolites in Developing Leaves of Catharanthus roseus

Partitioning of current photosynthates towards primary metabolites and its simultaneous incorporation in leaf alkaloids was investigated in developing leaves of medicinally important Catharanthus roseus. Of the total ¹⁴CO₂ assimilated, the leaves at positions 1–6 fixed 8, 22, 25, 19, 13, and 8 %, respectively, and stem 3 %. Leaf fresh mass, chlorophyll content, and CO₂ exchange rate increased up to the third leaf. The total alkaloid content was highest in young actively growing leaves, which declined with age. Total ¹⁴C fixed and its content in ethanol soluble fraction increased up to the third leaf and then declined. The ¹⁴C content in primary metabolites such as sugars and organic acids was also highest in the 3^rd leaf. The utilization of ¹⁴C assimilates into alkaloids was maximum in youngest leaf which declined with leaf age. Hence the capacity to synthesize alkaloids was highest in young growing leaves and metabolites from photosynthetic pathway were most efficiently utilized and incorporated into alkaloid biosynthetic pathway by young growing leaves.     

Photosynthesis of Lipids from CO(2) in Spinacia oleracea

Young expanding spinach leaves exposed to ¹⁴CO₂ under physiological conditions for up to 20 minutes assimilated CO₂ into lipids at a mean rate of 7.6 micromoles per milligram chlorophyll per hour following a lag period of 5 minutes. Label entered into all parts of the lipid molecule and only 28% of the ¹⁴C fixed into lipids was found in the fatty acid moieties, i.e. fatty acids were synthesized from CO₂in vivo at a mean rate of 2.1 micromoles per milligram chlorophyll per hour. Intact spinach chloroplasts isolated from these leaves incorporated H¹⁴CO₃ into fatty acids at a maximal rate of 0.6 micromole per milligram chlorophyll per hour, but were unable to synthesize either the polar moieties of their lipids or polyunsaturated fatty acids. Since isolated chloroplasts will only synthesize fatty acids at rates similar to the one obtained with intact leaves in vivo if acetate is used as a precursor, it is suggested that acetate derived from leaf mitochondria is the physiological fatty acid precursor.     

Boron Deficiency Induced Changes in Translocation of 14CO2-Photosynthate Into Primary Metabolites in Relation to Essential Oil and Curcumin Accumulation in Turmeric (Curcuma longa L.)

Changes in leaf growth, net photosynthetic rate (P _N), incorporation pattern of photosynthetically fixed ¹⁴CO₂ in leaves 1–4 from top, roots, and rhizome, and in essential oil and curcumin contents were studied in turmeric plants grown in nutrient solution at boron (B) concentrations of 0 and 0.5 g m^-3. B deficiency resulted in decrease in leaf area, fresh and dry mass, chlorophyll (Chl) content, and P _N and total ¹⁴CO₂ incorporated at all leaf positions, the maximum effect being in young growing leaves. The incorporation of ¹⁴CO₂ declined with leaf position being maximal in the youngest leaf. B deficiency resulted in reduced accumulation of sugars, amino acids, and organic acids at all leaf positions. Translocation of the metabolites towards rhizome and roots decreased. In rhizome, the amount of amino acids increased but content of organic acids did not show any change, whereas in roots there was decrease in contents of these metabolites as a result of B deficiency. Photoassimilate partitioning to essential oil in leaf and to curcumin in rhizome decreased. Although the curcumin content of rhizome increased due to B deficiency, the overall rhizome yield and curcumin yield decreased. The influence of B deficiency on leaf area, fresh and dry masses, CO₂ exchange rate, oil content, and rhizome and curcumin yields can be ascribed to reduced photosynthate formation and translocation.     

Inorganic Carbon Diffusion between C(4) Mesophyll and Bundle Sheath Cells: Direct Bundle Sheath CO(2) Assimilation in Intact Leaves in the Presence of an Inhibitor of the C(4) Pathway

Photosynthesis rates of detached Panicum miliaceum leaves were measured, by either CO₂ assimilation or oxygen evolution, over a wide range of CO₂ concentrations before and after supplying the phosphoenolpyruvate (PEP) carboxylase inhibitor, 3,3-dichloro-2-(dihydroxyphosphinoyl-methyl)-propenoate (DCDP). At a concentration of CO₂ near ambient, net photosynthesis was completely inhibited by DCDP, but could be largely restored by elevating the CO₂ concentration to about 0.8% (v/v) and above. Inhibition of isolated PEP carboxylase by DCDP was not competitive with respect to HCO₃⁻, indicating that the recovery was not due to reversal of enzyme inhibition. The kinetics of ¹⁴C-incorporation from ¹⁴CO₂ into early labeled products indicated that photosynthesis in DCDP-treated P. miliaceum leaves at 1% (v/v) CO₂ occurs predominantly by direct CO₂ fixation by ribulose 1,5-bisphosphate carboxylase. From the photosynthesis rates of DCDP-treated leaves at elevated CO₂ concentrations, permeability coefficients for CO₂ flux into bundle sheath cells were determined for a range of C₄ species. These values (6-21 micromoles per minute per milligram chlorophyll per millimolar, or 0.0016-0.0056 centimeter per second) were found to be about 100-fold lower than published values for mesophyll cells of C₃ plants. These results support the concept that a CO₂ permeability barrier exists to allow the development of high CO₂ concentrations in bundle sheath cells during C₄ photosynthesis.     

The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In ¹⁴CO₂ pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 μmoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 μmoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279).     

Effects of Light and Elevated Atmospheric CO(2) on the Ribulose Bisphosphate Carboxylase Activity and Ribulose Bisphosphate Level of Soybean Leaves

Soybean (Glycine max L. Merr. cv Bragg) was grown throughout its life cycle at 330, 450, and 800 microliters CO₂ per liter in outdoor controlled-environment chambers under solar irradiance. Leaf ribulose-1,5-bisphosphate carboxylase (RuBPCase) activities and ribulose-1,5-bisphosphate (RuBP) levels were measured at selected times after planting. Growth under the high CO₂ levels reduced the extractable RuBPCase activity by up to 22%, but increased the daytime RuBP levels by up to 20%.

Diurnal measurements of RuBPCase (expressed in micromoles CO₂ per milligram chlorophyll per hour) showed that the enzyme values were low (230) when sampled before sunrise, even when activated in vitro with saturating HCO₃⁻ and Mg²⁺, but increased to 590 during the day as the solar quantum irradiance (photosynthetically active radiation or PAR, in micromoles per square meter per second) rose to 600. The nonactivated RuBPCase values, which averaged 20% lower than the corresponding HCO₃⁻ and Mg²⁺-activated values, increased in a similar manner with increasing solar PAR. The per cent RuBPCase activation (the ratio of nonactivated to maximum-activated values) increased from 40% before dawn to 80% during the day. Leaf RuBP levels (expressed in nanomoles per milligram chlorophyll) were close to zero before sunrise but increased to a maximum of 220 as the solar PAR rose beyond 1200. In a chamber kept dark throughout the morning, leaf RuBPCase activities and RuBP levels remained at the predawn values. Upon removal of the cover at noon, the HCO₃⁻ and Mg²⁺-activated RuBPCase values and the RuBP levels rose to 465 and 122, respectively, after only 5 minutes of leaf exposure to solar PAR at 1500.

These results indicate that, in soybean leaves, light may exert a regulatory effect on extractable RuBPCase in addition to the well-established activation by CO₂ and Mg²⁺.

     

Biosynthesis of Sulfoquinovosyldiacylglycerol in Higher Plants: The Incorporation of SO(4) by Intact Chloroplasts in Darkness

Intact spinach chloroplasts incorporated ³⁵SO₄²⁻ into sulfoquinovosyldiacylglycerol in the dark at rates equivalent to those previously reported for illuminated chloroplasts provided that either ATP itself or an ATP-generating system was added. No additional reductant was necessary for SQDG synthesis by chloroplasts. The optimal concentration of ATP was between 2 and 3 millimolar. Rates of synthesis up to 2.6 nanomoles per milligram chlorophyll per hour were observed. UTP, GTP, and CTP could not substitute for ATP. Incubation of UTP with ATP (1:1) stimulated synthesis of sulfoquinovosyldiacylglycerol. No additional stimulation of the reaction was observed upon addition of other nucleoside triphosphates with ATP. For the generation of ATP in the chloroplast, addition of dihydroxyacetone phosphate alone did not promote synthesis of sulfoquinovosyldiacylglycerol, but in combination with inorganic phosphate and oxaloacetate, rates of synthesis up to 3.2 nanomoles per milligram chlorophyll per hour were observed. Dark synthesis was optimal in the presence of 2 millimolar dihydroxyacetone phosphate, 2 millimolar oxaloacetate, and 1 millimolar KH₂PO₄.