Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO₂ reduction and were insensitive to additions of photosynthetic intermediates when CO₂ was available at saturating concentrations. Photosynthetic rates were measured polarographically as O₂ evolution, and the extent of the reduction of substrate was estimated from the amount of O₂ evolved. With CO₂ as substrate, inhibition of photosynthesis by O₂ was dependent on pH. At pH values above 8, rates of O₂ evolution were strongly inhibited by O₂ and only a fraction of the added bicarbonate was reduced before O₂ evolution ceased. The extent of O₂ evolution declined with increasing O₂ concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O₂ levels, but the extent of O₂ evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O₂ evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O₂ concentration. In contrast, there was little effect of O₂ and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O₂. The results are discussed in terms of a balance between photosynthetic O₂ evolution and O₂ consumption by the ribulose diphosphate oxygenase reaction.     

pH Dependence and Kinetics of Glycolate Uptake by Intact Pea Chloroplasts

Experiments in which [1-¹⁴C]glycolate uptake is carried out in conjunction with measurements of stromal pH indicate that only glycolic acid and not the glycolate anion is crossing the pea (Pisum sativum var. Progress No. 9, Agway) chloroplast envelope. This mechanism of glycolate transport appears to be too slow to account for observed photorespiratory carbon fluxes in C₃ plants.     

Photosynthetic Production of Hydrogen Peroxide by Anacystis nidulans

A sensitive assay based upon fluorescence of scopoletin allowed continuous recording of H₂O₂ production in illuminated intact cells of Anacytis nidulans. Onset of illumination was followed by a 5 to 10 second lag, a burst of very rapid production continuing for up to 5 minutes, and finally a slow and continuing steady rate of H₂O₂ production. Size of the H₂O₂ burst was decreased by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, by low O₂, and by certain Calvin cycle intermediates; it was increased by high light intensity, CO₂ depletion, Calvin cycle inhibitors (as iodoacetamide), cold shock, carbonyl cyanide m-chlorophenylhydrazone, and certain organic acids as glycolate). The H₂O₂ burst was explained by the following hypothesis: a low potential reductant is produced more rapidly than it can be used in the normal pathway to CO₂ reduction and, instead, reacts with oxygen. H₂O₂ production is regarded as a metabolic defect observable in Anacystis most dramatically during the transition from a very low rate of oxidative dark metabolism to a high rate of photosynthetic metabolism.     

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

Intact spinach chloroplasts scavenge hydrogen peroxide witha peroxidase that uses a photoreductant as the electron donor,but the activity of ruptured chloroplasts is very low [Nakanoand Asada (1980) Plant & Cell Physiol. 21 : 1295]. Rupturedspinach chloroplasts recovered their ability to photoreducehydrogen peroxide with the concomitant evolution of oxygen afterthe addition of glutathione and dehydroascorbate (DHA). In rupturedchloroplasts, DHA was photoreduced to ascorbate and oxygen wasevolved in the process in the presence of glutathione. DHA reductase(EC 1.8.5.1 [EC] ) and a peroxidase whose electron donor is specificto L-ascorbate are localized in chloroplast stroma. These observationsconfirm that the electron donor for the scavenging of hydrogenperoxide in chloroplasts is L-ascorbate and that the L-ascorbateis regenerated from DHA by the system: photosystem IferredoxinNADPglutathione.A preliminary characterization of the chloroplast peroxidaseis given. (Received April 16, 1981; Accepted June 3, 1981)     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

CO(2) Assimilation and Malate Decarboxylation by Isolated Bundle Sheath Chloroplasts from Zea mays

Conditions for optimal CO₂ fixation and malate decarboxylation by isolated bundle sheath chloroplasts from Zea mays were examined. The relative rates of these processes varied according to the photosynthetic carbon reduction cycle intermediate provided. Highest rates of malate decarboxylation, measured as pyruvate formation, were seen in the presence of 3-phosphoglycerate, while carbon fixation was highest in the presence of dihydroxyacetone phosphate; only low rates were measured with added ribose-5-phosphate. Chloroplasts exhibited a distinct phosphate requirement and this was optimal at a level of 2 millimolar inorganic phosphate in the presence of 2.5 millimolar 3-phosphoglycerate, dihydroxyacetone phosphate, or ribose-5-phosphate. Malate decarboxylation and CO₂ fixation were stimulated by additions of AMP, ADP, or ATP with half-maximal stimulation occurring at external adenylate concentrations of about 0.15 millimolar. High concentrations (>1 millimolar) of AMP were inhibitory. Aspartate included in the incubation medium stimulated malate decarboxylation and CO₂ assimilation. In the presence of aspartate, the apparent Michaelis constant (malate) for malate decarboxylation to pyruvate by chloroplasts decreased from 6 to 0.67 millimolar while the calculated V_max for this process increased from 1.3 to 3.3 micromoles per milligram chlorophyll. Aspartate itself was not metabolized. It was concluded that the processes mediating the transport of phosphate, 3-phosphoglycerate, and dihydroxyacetone phosphate transport on the one hand, and also of malate might differ from those previously described for chloroplasts from C₃ plants.     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO₂-free buffer at 21% O₂, showed a decrease (30-70%) in CO₂-dependent O₂ evolution and ¹⁴CO₂ uptake. This photoinhibition was observed only when the O₂ concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO₂ concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO₂ concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O₂ evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO₂ concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O₂ evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O₂-dependent photoinhibition of light saturated CO₂ fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O₂ for photoinhibition of CO₂ fixation capacity in isolated chloroplasts may be explained by an effect of O₂ in allowing metabolic depletion of Calvin cycle intermediates.     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

     

Uptake of l-Ascorbate by Intact Spinach Chloroplasts

Uptake of l-[1-¹⁴C]ascorbate by intact ascorbate-free spinach (Spinacia oleracea L. cv Vital^r) chloroplasts has been investigated using the technique of silicone oil filtering. Rates greater than 100 micromoles per milligram chlorophyll per hour (external concentration, 10 millimolar) of ascorbate transport were observed. Ascorbate uptake into the sorbitol-impermeable space (stroma) followed the Michaelis-Menten-type characteristic for substrate saturation. A K_m of 18 to 40 millimolar was determined. Transport of ascorbate across the chloroplast envelope resulted in an equilibrium of the ascorbate concentrations between stroma and medium. A pH optimum of 7.0 to 7.5 and the lack of alkalization of the medium upon ascorbate uptake suggest that only the monovalent ascorbate anion is able to cross the chloroplast envelope. The activation energy of ascorbate uptake was determined to be 65.8 kilojoules (16 kilocalories) per mole (8 to 20°C). Interference of ascorbate transport with substrates of the phosphate or dicarboxylate translocator could not be detected, but didehydroascorbate was a competitive inhibitor. Preloading of chloroplasts with didehydroascorbate resulted in an increase of V_max but did not change the K_m for ascorbate. Millimolar concentrations of the sulfhydryl reagent p-chloromercuriphenyl sulfonate inhibited ascorbate uptake. The data are interpreted in terms of ascorbate uptake into chloroplasts by the mechanism of facilitated diffusion mediated by a specific translocator.     

Na2CO3-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics

Alkali-salinity exerts severe osmotic, ionic, and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na₂CO₃ stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na₂CO₃-responsive proteins. A total of 104 and 102 Na₂CO₃-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na₂CO₃-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction, and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na₂CO₃ tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na₂CO₃ stress, which help to improve our understanding of the Na₂CO₃-responsive mechanisms in halophytes.     

Luminol-Dependent Chemiluminescence Analysis of Chitooligosaccharide-Induced Rapid Production of Hydrogen Peroxide by Intact Wheat Seedlings

The feasibility of a luminol-dependent chemiluminescence analysis of hydrogen peroxide production by intact wheat seedlings using a KhL-003 chemiluminometer was determined. It was shown that the minimal H₂O₂ concentration that can be detected in a 0.5-ml sample with this instrument is 0.125 µM. Analysis of biological activity of a mixture of chitooligosaccharides with molecular masses from 5 to 10 kD and acetylation degree of 65% demonstrated that, at a concentration of 1 µg/ml, they induce rapid overproduction of H₂O₂ in roots of 3-day-old wheat seedlings.     

Photosynthetic Assimilation of NO(3) by Intact Cells of the Cyanobacterium Anacystis nidulans: Influence of NO(3) and NH(4) Assimilation on CO(2) Fixation

Illuminated suspensions of Anacystis nidulans, supplied with saturating concentrations of CO₂ evolved O₂ at a greater rate when nitrate was simultaneously present. The extent of the stimulation of noncyclic electron flow induced by nitrate was dependent on light intensity, being maximal under light saturating conditions. Accordingly, nitrate depressed the rate of CO₂ fixation at limiting but not at saturating light, this depression reflecting the competition between both processes for assimilatory power. In contrast, ammonium stimulated CO₂ fixation at any light intensity assayed, the stimulation being dependent on the incorporation of ammonium to carbon skeletons. The positive effect of ammonium on CO₂ fixation also appeared to occur when nitrate was the nitrogen source, since with either nitrogen source an increase in the incorporation of newly fixed carbon into acid-soluble metabolites took place. From these results, the in vivo partitioning of assimilatory power between photosynthetic nitrogen and carbon assimilation and the quantitative and qualitative effects of inorganic nitrogen assimilation on CO₂ fixation are discussed.     

Formation of Hydrogen Peroxide by a Polyvinyl Alcohol Degrading Enzyme

A method for purification and crystallization, and some properties of ribonuclease from Aspergillus sp. [EC 2.7.7.17] (RNase L) are reported. The purification procedure consisted of six steps, including acetone precipitation, column chromatographies on Duolite A–2 and DEAE-cellulose, repeated chromatography on DEAE-Sephadex A–50 column and affinity chromatography on 5’-AMP-Sepharose 4B column. Crystallization was performed by the dialysis against ammonium sulfate solution at 60% saturation.

The crystalline enzyme was shown to be homogeneous by polyacrylamide disc electrophoresis and ultracentrifugation. Svedberg value of the crystalline enzyme was 4.2. The enzyme was the most active at pH 3.5 and 60~65°C, and it was inhibited markedly with Fe.³⁺

RNase L has no absolute base specificity, and produces four kinds of 3’-mononucleotides from yeast RNA. However, the susceptibility of four nucleotide residues to RNase L increases in the order; G < A < C < U and is quite different from those of RNase T₂, RNase M and RNase R.