Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Mechanism of glycolate transport in spinach leaf chloroplasts

The incorporation of ¹⁴CO₂ into glycolate by intact spinach leaf (Spinacia oleracea L. var. Kyoho) chloroplasts exposed to ¹⁴CO₂ (NaH¹⁴CO₃, 1 millimolar) in the light was determined as a function of O₂ concentrations in the reaction media. A hyperbolic saturation curve was obtained, apparent K_m (O₂) of 0.28 millimolar, indicating that glycolate is produced predominantly by ribulose-1,5-bisphosphate carboxylase/oxygenase. A concentration gradient of glycolate was invariably observed between chloroplast stroma and the outside media surrounding chloroplasts during photosynthetic ¹⁴CO₂ fixation under an O₂ atmosphere.     

Maintenance of High Photosynthetic Rates in Mesophyll Cells Isolated from Papaver somniferum

The establishment and maintenance of high rates of photosynthetic CO₂ incorporation in mesophyll cells of Papaver somniferum (opium poppy) depend on a regime of dark and light periods immediately following isolation, as well as carefully adjusted conditions of isolation. Analysis of the incorporation pattern of ¹⁴CO₂ by the isolated cells indicates an initial “stress-response” period of approximately 20 hours characterized by increased respiratory-type metabolism and diminished photosynthesis. Under the favorable regime, this period is followed by rapid recovery and the reinstatement of a metabolic state strikingly similar to that of intact leaves in which the initial rate of CO₂ incorporation is between 110 and 175 μmoles CO₂ fixed per mg chlorophyll per hour. The photosynthetic viability of these cells can be maintained for up to 80 hours.     

Biosynthesis of plastoquinone and β-carotene in isolated chloroplasts

Intact, isolated spinach chloroplasts incorporated ¹⁴C from ¹⁴CO₂ into plastoquinone and β-carotene under photosynthetic conditions. Addition of unlabelled l-tyrosine, p-hydroxyphenylpyruvate, or homogentisate increased the incorporation of ¹⁴C into plastoquinone, but decreased that into β-carotene.     

Effect of Triacontanol on Chlamydomonas: I. Stimulation of Growth and Photosynthetic CO(2) Assimilation

Treatment of Chlamydomonas reinhardtii cells, cultured at 5% CO₂, with 1 to 1000 micrograms triacontanol (TRIA) per liter resulted in 21 to 35% increases in cell density, 7 to 31% increases in total chlorophyll, and 20 to 100% increases in photosynthetic CO₂ assimilation. The increase in CO₂ fixation with TRIA treatment occurred before, and was independent of, increases in total chlorophyll or cell number. Chlamydomonas cells responded to a broad range of TRIA concentrations that were at least one order of magnitude above the optimum concentration established for higher plants. The necessity for larger concentrations of TRIA may be due to destabilizing effects of Ca²⁺ and K⁺ present in the Chlamydomonas growth medium. These ions caused flocculation of the colloidally dispersed TRIA in apparent competition with binding of [¹⁴C]TRIA to Chlamydomonas cells. Octacosanol inhibited the effect of TRIA on photosynthetic CO₂ assimilation. TRIA treatment did not alter the distribution of ¹⁴C-label among photosynthetic products. The effect of TRIA on photosynthetic CO₂ assimilation increased with time after treatment up to 3 days. Chlamydomonas cells that had been grown at low-CO₂ (air) did not respond to TRIA, and transfer of high-CO₂ (5%) grown cells that had responded to TRIA to a low-CO₂ atmosphere resulted in a loss of the effect of TRIA. The effect of pH on photosynthetic CO₂ assimilation indicated that CO₂ is probably the species of inorganic carbon utilized by control and TRIA-treated Chlamydomonas cells.     

Photosynthetic products of division synchronized cultures of euglena

Rates and products of photosynthetic ¹⁴CO₂ fixation by division synchronized cultures of Euglena gracilis strain Z were determined over the cycle. Rate of ¹⁴CO₂ fixation doubled in a continuous manner throughout the light phase followed by a slight reduction of photosynthetic capacity in the dark phase. Greater ¹⁴C incorporation into the nucleic acid-polysaccharide fraction occurred with mature cells. Products of ¹⁴CO₂ fixation varied markedly over the cycle: although with mature cells ¹⁴C-labeled sucrose was not detected, with dividing cells this was the main sugar labeled; in young cells ¹⁴C maltose was formed. Cells removed at end of dark phase accumulated ¹⁴C in glycolate, whereas at other stages over the cycle less ¹⁴C was present in glycolate, and this was accompanied by a rapid incorporation of ¹⁴C into glycine and serine. Glycerate was an early and major product of photosynthesis with cells at the mature stage of the cycle.     

Studies on Effects of Certain Quinones: II. Photosynthetic Incorporation of CO(2) by Chlorella

The effects of various quinone herbicides and fungicides on the photosynthetic ¹⁴CO₂ fixation and the incorporation of ¹⁴C among the products of photosynthesis in Chlorella pyrenoidosa was investigated. Addition of 30 μm 2,3-dichloro-1,4-naphthoquinone (dichlone), 2-amino-3-chloro-1,4-naphthoquinone (06K-quinone), or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil) inhibited CO₂ fixation, whereas 1,4-benzoquinone had no effect. Treatment with 3 μm or higher concentrations of dichlone, 06K-quinone or 1,4-benzoquinone also produced marked changes in the pattern of ¹⁴C distribution. A noticeable effect was an increase in the proportion of ¹⁴C in sucrose and glycine accompanied by a reduction in ¹⁴C lipids and glutamic acid. These changes appear to occur as a result of shifts in the flow of carbon along various biosynthetic pathways of photosynthetic CO₂ fixation. It is suggested that inactivation of coenzyme A and shortage of reduced triphosphopyridine nucleotide in the quinone-treated cells inhibited the synthesis of lipids and glutamic acid, thereby diverting more carbon into sucrose and glycine.     

Influence of Nutrient Stress on the Relationships between PAM Measurements and Carbon Incorporation in Four Phytoplankton Species

Two methods of measuring primary production, modulated fluorimetry (PAM) and the traditional carbon incorporation method (¹³C), were compared in four phytoplankton species, two diatoms (Pseudo-nitzschia pungens and Asterionellopsis glacialis), and two dinoflagellates (Heterocapsa sp and Karenia mikimotoï), under N (nitrogen), P (phosphorus) and Si (silicon) limited semi-continuous culture. N and Si-limited cultures showed relatively high quantum efficiency of the PSII (F_v/F_m) values, confirming that F_v/F_m is not a good proxy for nutrient stress in balanced systems, whereas P limitation had a drastic effect on many physiological parameters. In all species, the physiological capacity of phytoplankton cells to acclimate to nutrient limitations led to changes in the cellular biochemical composition and the structure of the photosynthetic apparatus. The observed physiological responses were species and nutrient specific. The values of the chlorophyll-specific absorption cross section (a*) increased with nutrient limitation due to package effect, while the carbon/Chl a ratio was higher under N and P limitations. In diatoms, Si limitation did not affect photosynthesis confirming the uncoupling between Si and carbon metabolisms. In all four species and under all treatments, significant relationships were found between photosynthetic activities, ETR^Chl (electron transport rate) and P^Chl (carbon fixation rate) estimated using PAM measurements and ¹³C incorporation, showing that the fluorescence technique can reliably be used to estimate carbon fixation by phytoplankton. The relationship between ETR^Chl and P^Chl can be described by the shape and the slope of the curve (Φ_C.e). Linear relationships were found for dinoflagellates and P. pungens under all treatments. A decrease in Φ_C.e was observed under N and P limitation probably due to structural damage to the photosynthetic apparatus. A. glacialis showed a logarithmic relationship in N and P limited conditions, due to the alternative electron flow which takes place to optimise photosynthetic performances under high light and/or nutrient stress.     

Photosynthesis in c(4) plant tissue cultures: significance of kranz anatomy to c(4) Acid metabolism in c(4) plants

The pattern of photosynthetic carbon metabolism was determined in tissue cultures of Portulaca oleracea. Four-carbon acids are the most heavily labeled photosynthetic products during short term exposure to ¹⁴CO₂, containing greater than 40% of the total radioactivity incorporated. Phosphoglyceric acid and sugars account for only 10% of the label after equal exposure times. Other features of the CO₂ assimilation pattern in Portulaca callus tissue include a relatively large percentage of label located in various minor products throughout the time course studied, and a greater incorporation of ¹⁴C into sugars in tissue cultures than occurs in leaves. Ultrastructurally, the chloroplasts and cells of the callus are like those in the mesophyll cells of Portulaca leaves. The requirement for Kranz anatomy for operation of functional C₄ physiology is discussed.     

Inhibition of coral and algal photosynthesis by ca-antagonist phenothiazine drugs

The effects of various calcium ion antagonists and ion transport inhibitors on photosynthetic O₂ evolution of corals, isolated zooxanthellae, sea anemone tentacles, and Chlorococcum oleofaciens were measured. Only the phenothiazine drugs were effective at inhibiting photosynthesis. Trifluoperazine, a calcium ion antagonist drug, inhibited at low concentrations, with 10⁻⁴ molar and 8 × 10⁻⁶ molar completely abolishing photosynthesis in the intact corals and isolated zooxanthellae, respectively. Net photosynthetic O₂ evolution of C. oleofaciens was eliminated by concentrations of trifluoperazine as low as 2.8 × 10⁻⁵ molar.     

Feasibility of 55Fe Autoradiography as Performed on N2-Fixing Anabaena spp. Populations and Associated Bacteria

⁵⁵Fe emits low-energy X rays and Auger electrons by electron capture decay. Auger electrons are useful for autoradiographic examination of ⁵⁵Fe incorporation among microbial communities. Attainable resolution, in terms of silver grain deposition, is excellent and comparable to ³H. Two known Fe-demanding processes, photosynthetic CO₂ fixation and N₂ fixation, were examined by autoradiography of Anabaena populations. During photosynthetically active (illuminated) N₂-fixing periods, biological incorporation of ⁵⁵FeCl₃ by vegetative cells and heterocysts was evident. When N₂ fixation was suppressed by NH₄⁺ additions, heterocysts revealed no incorporation of ⁵⁵Fe. Conversely, when N₂-fixing Anabaena filaments were placed in darkness, ⁵⁵Fe incorporation decreased in vegetative cells, whereas heterocysts showed sustained rates of ⁵⁵Fe incorporation. Bacteria actively incorporated ⁵⁵Fe under both light and dark conditions. The chelated (by Na₂-ethylenediaminetetraacetate) form of ⁵⁵FeCl₃ was more readily incorporated than the nonchelated form. Furthermore, abiotic adsorption of ⁵⁵Fe to filters and nonliving particles proved lower when chelated ⁵⁵Fe was used in experiments. ⁵⁵Fe autoradiography is useful for observing the fate and cellular distribution of various forms of Fe among aquatic microbial communities.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

Carbonic Anhydrase-Dependent Inorganic Carbon Uptake by the Red Macroalga, Chondrus crispus

The rate of photosynthetic carbon uptake of Chondrus crispus Stack-house plants, at various CO₂ concentrations and pretreated with carbonic anhydrase (CA) inhibitors, was determined using an air-suspension, differential infra-red gas analyzer technique. It was found that the CA inhibitors, acetazolamide, dextran-bound acetazolamide (DBI, which does not permeate cell membranes), and subtilisin (a protease that attacks the cell surface) inhibit photosynthetic carbon uptake in C. crispus. Inhibition was greatest at low CO₂ concentrations, and decreased at CO₂ saturation. Acetazolamide inhibited carbon uptake to a greater extent than DBI. The data support the conclusion that C. crispus plants utilize HCO₃⁻ for photosynthesis, and that both cell-surface and internal CA are involved in the photosynthetic uptake of inorganic carbon.     

Chemical inhibition of the glycolate pathway in soybean leaf cells

Isolated soybean (Glycine max [L.] Merr.) leaf cells were treated with three inhibitors of the glycolate pathway in order to evaluate the potential of such inhibitors for increasing photosynthetic efficiency. Preincubation of cells under acid conditions in α-hydroxypyridinemethanesulfonic acid increased ¹⁴CO₂ incorporation into glycolate, but severely inhibited photosynthesis. Isonicotinic acid hydrazide (INH) increased the incorporation of ¹⁴CO₂ into glycine and reduced label in serine, glycerate, and starch. Butyl 2-hydroxy-3-butynoate (BHB) completely and irreversibly inhibited glycolate oxidase and increased the accumulation of ¹⁴C into glycolate. Concomitant with glycolate accumulation was the reduction of label in serine, glycerate, and starch, and the elimination of label in glycine. The inhibitors INH and BHB did not eliminate serine synthesis, suggesting that some serine is synthesized by an alternate pathway. The per cent incorporation of ¹⁴CO₂ into glycolate by BHB-treated cells or glycine by INH-treated cells was determined by the O₂/CO₂ ratio present during assay. Photosynthesis rate was not affected by INH or BHB in the absence of O₂, but these compounds increased the O₂ inhibition of photosynthesis. This finding suggests that the function of the photorespiratory pathway is to recycle glycolate carbon back into the Calvin cycle, so if glycolate metabolism is inhibited, Calvin cycle intermediates become depleted and photosynthesis is decreased. Thus, chemicals which inhibit glycolate metabolism do not reduce photorespiration and increase photosynthetic efficiency, but rather exacerbate the problem of photorespiration.     

Photosynthesis in Rhodospirillum rubrum. I. Autotrophic Carbon Dioxide Fixation

The incorporation and distribution of activity from ¹⁴CO₂ was investigated under autotrophic conditions in the facultative photoautotroph, Rhodospirillum rubrum, with cells cultured on hydrogen, carbon dioxide, and ammonium sulfate. In 1 second ¹⁴CO₂ fixation experiments essentially all of the activity was found in 3-phosphoglyceric acid: plotted against time percent incorporation into phosphate esters has a strikingly negative slope. These results suggest that under autotrophic conditions the reductive pentose phosphate cycle or the key reactions of the cycle play a major role in carbon metabolism in this photosynthetic bacterium. Incorporation into amino acids and into intermediates of the tricarboxylic acid cycle was quite low.     

Effect of Abscisic Acid on the Activities of Photosynthetic Enzymes and 14CO2 Fixation Products in Leaves of Pennisetum typhoides Seedlings

Abscisic acid inhibited the rate of ¹⁴CO₂ fixation in leaves of Pennisetum typhoides (Burm. f.) Stapf & Hubbard seedlings, but increased the activities of phosphoenol-pyruvate-carboxylase and malic enzyme. The leaves of the seedlings grown in the presence of abscisic acid incorporated, in comparison to the control, more radioactivity in the fraction of organic acids, but less radioactivity was recorded in the amino acid fraction. On the other hand, gibberellic acid which also inhibits photosynthetic ¹⁴CO₂ assimilation and decreases the activities of photosynthetic enzymes, favours greater incorporation in alanine, and reduces that in malate. It is deduced that bio-regulants can greatly influence the flow of ¹⁴C into individual photosynthetic products. As in growth, abscisic and gibberellic acids in combination tended to antagonize each other in their effects on enzyme activity as well as in incorporation of ¹⁴CO₂ into photosynthetic products.     

Quantitative analysis of photosynthetic carbon metabolism in protoplasts and intact leaves of barley. Determination of carbon fluxes and pool sizes of metabolites in different cellular compartments

Rates of carbon fluxes and pool sizes of photosynthetic metabolites in different cellular compartments of barley protoplasts were calculated from the time curves of their labeling in the medium of ¹⁴CO₂. Using membrane filtration procedure, kinetics of ¹⁴C incorporation into the products of steady-state photosynthesis was determined separately in chloroplasts, mitochondria and cytosol of barley protoplasts illuminated for different periods in the air containing ¹⁴CO₂. To extract the quantitative information, analytical labeling functions P(t) describing the dependence of ¹⁴C content in the primary, intermediate and end products of a linear reaction chain upon the duration of tracer feeding have been derived. The parameters of these functions represent pool sizes of metabolites and rates of carbon fluxes. The values of these parameters were determined by fitting the experimental labeling curves to the functions P(t) by means of non-linear regression procedure. To elucidate the possible effects of fractionation on the photosynthetic carbon metabolism, the parameters of protoplasts were compared with corresponding values in intact leaves of barley.     

A possible mechanism of ammonium ion regulation of photosynthetic carbon flow in higher plants

Addition of NH₄⁺ to the photosynthesizing leaf cells of Dolichos lab lab L. var. Lignosis Prain and leaf discs of Vigna sinensis L. savi ex Hassk caused a significant increase in the flow of photosynthetic carbon toward amino acids with a concomitant decrease toward sugars without affecting the over-all photosynthetic rate. Similar diversion of photosynthetic carbon away from sugars was also observed in the photosynthesizing isolated chloroplasts of V. sinensis, but the latter differed in that they accumulated organic acids rather than amino acids. In an effort to understand the mechanism of NH₄⁺-mediated regulation, the specific and total activities of NAD(P)-glutamate dehydrogenase, glutamine synthetase, pyruvate kinase, alkaline fructose 1,6-bisphosphatase, and NAD(P)-glyceraldehyde-3-phosphate dehydrogenase of the cells of D. lab lab were checked but none was affected by the added ammonium salts even after prolonged incubation. At certain concentrations, ammonium ions abolished the light activation of NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose 1,6-bisphosphatase in isolated chloroplasts from dark-adapted Vigna leaves without interfering with the basal dark activity of these enzymes. Based on these observations, a possible mechanism of action of NH₄⁺ in regulating the photosynthetic carbon flow is postulated.     

Growth and Development of Soybean (Glycine max [L.] Merr.) Pods: CO(2) Exchange and Enzyme Studies

The rates of CO₂ exchange and ¹⁴CO₂ incorporation in the light and dark and the activities of several photosynthetic, photorespiratory, and respiratory enzymes of soybean (Glycine max [L.] Merr. cv. Wye) reproductive structures were determined at weekly intervals from anthesis to pod maturity. At all stages of pod development soybean reproductive structures were found to be incapable of net photosynthesis under the experimental conditions employed, but capable of gross photosynthesis and light-induced ¹⁴CO₂ uptake. Consistent with the lack of net photosynthesis throughout the development of the reproductive structure, the maximum in vitro activity of ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) in pod tissue was only 3% of that in leaf extracts when expressed on a fresh weight basis. We concluded that the major role of the reproductive structure of the soybean with respect to photosynthetic carbon metabolism is the reassimilation of its respiratory CO₂.     

Stachyose Synthesis in Source Leaf Tissues of the CAM Plant Xerosicyos danguyi H. Humb

Leaf tissues from Xerosicyos danguyi H. Humb., a succulent member of the Cucurbitaceae, were found to possess both galactinol synthase activity and the capacity for photosynthetic production of stachyose, the phloem transport oligosaccharide common to other nonsucculent cucurbits. The amounts of stachyose isolated from leaf tissues, and the extractable activity of galactinol synthase, were somewhat higher in leaf tissues obtained from plants operating in the Crassulacean acid metabolism (CAM) mode (well watered plants) compared to leaf tissues from plants operating in the CAM-idling mode (water-stressed plants). In contrast, in leaf discs, the photosynthetic incorporation of label into stachyose following pulse labeling with ¹⁴CO₂ was similar for stressed and for nonstressed tissues. Stachyose could be extracted from, and was synthesized photosynthetically by, leaf discs which contained no vascular tissues, indicating that synthesis of stachyose can occur in photosynthetic mesophyll cells of Xerosicyos.