Oxygenic photosynthesis and the distribution of chloroplasts

The integrated functioning of two photosystems (I and II) whether in cyanobacteria or in chloroplasts is the outstanding sign of a common ancestral origin. Many variations on the basic theme are currently evident in oxygenic photosynthetic organisms whether they are prokaryotes, unicellular, or multicellular. By conservative estimates, oxygenic photosynthesis has been around for at least ca. 2.2–2.7 billions years, consistent with cyanobacteria-type microfossils, biomarkers, and an atmospheric rise in oxygen to less than 1.0% of the present concentration. The presumptions of chloroplast formation by the cyanobacterial uptake into a eukaryote prior to 1.6 BYa ago are confounded by assumptions of host type(s) and potential tolerance of oxygen toxicity. The attempted dating and interrelationships of particular chloroplasts in various plant or animal lineages has relied heavily on phylogenomic analysis and evaluations that have been difficult to confirm separately. Many variations occur in algal groups, involving the type and number of accessory pigments, and the number(s) of membranes (2–4) enclosing a chloroplast, which can both help and complicate inferences made about early or late origins of chloroplasts. Integration of updated phylogenomics with physiological and cytological observations remains a special challenge, but could lead to more accurate assumptions of initial and extant endosymbiotic event(s) leading toward stable chloroplast associations.     

Structure and activity of chloroplasts of sunflower leaves having various water potentials

Summary Changes in membrane integrity, conformation and configuration, and in photosystem II (PS II) activity (measured as dichloroindophenol photoreduction) of sunflower (Helianthus annuus L.) chloroplasts were studied after leaf tissue had been desiccated to various water potentials ( _w ). Fixatives for electron microscopy were adjusted osmotically to within 1 bar of the _w of the tissue to prevent rehydration during fixation. PS II activity decreased to 50% of the control activity at a _w of-26 bar. At this _w , leaf viability was being lost but there was virtually no loss of integrity of the thylakoid lamellar system. Even at extreme _w (below-100 bar), thylakoids retained much structural detail but were less stained. At-26 bar, intrathylakoid spacing (configuration) and lamellar thickness (conformation) were decreased in vivo. Upon isolation of the plastids, the differences in configuration disappeared but the differences in conformation remained. The decreases in membrane conformation and PS II activity both, in vivo and in vitro suggest that alterations in conformation may cause decreases in chloroplast activity at _w as low as-26 bar. Since structural detail was maintained, however, previous observations of altered membrane integrity, which involved tissue fixed without osmotic support, may have been affected by tissue rehydration during fixation.Abbreviations DCIP sodium 2,6-dichloroindophenol - PS II photosystem II - _w leaf water potential     

Reconstitution of amino acid synthesis by combining spinach chloroplasts with other leaf organelles

By adding leaf peroxisomes to purified intact chloroplasts, glycine synthesis was reconstituted. On adding leaf mitochondria, serine synthesis was also reconstituted. However, aromatic amino acid synthesis which was effected by purified chloroplasts was not enhanced on adding peroxisomes or mitochondria although the rate in whole leaves was considerably higher.     

Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts

Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Biochemical differentiation of plastids and other organelles in rye leaves with a high-temperature-induced deficiency of plastid ribosomes

Summary 1. In developing rye (Secale cereale L.) leaves the formation of plastidic ribosomes was selectively prevented in light as well as in darkness, when the seedlings were grown at an elevated temperature of 32° instead of 22° where normal development ocurred. Plastid ribosome deficient parts of lightgrown leaves were chlorotic at 32°. — 2. At both temperatures the leaves contained under all conditions (light or dark, on H₂O or nutrient solution) equal or very similar amounts of total amino nitrogen. In light, the contents of total protein and dry weight were lower at 32° than at 22°, especially when the plants were grown on nutrient solution. — 3. Mitochondrial marker enzymes had normal or even higher activities in 32°-grown leaves. Respiration rates were similar for segments of leaves grown on water in light either at 32° or at 22° but by 20–30% lower for 32°-grown plants when they had been raised in darkness or on nutrient solution. In contrast to 22°-grown tissue, respiration of 32°-grown leaf segments was rather insensitive to KCN. Comparative inhibitor studies indicated the presence of both the cyanide-sensitive and the cyanide-insensitive pathway of respiration in 32°-grown leaves. — 4. Leaf microbody marker enzymes were present in leaves grown at 32°. From chlorotic parts of 32°-light-grown leaves a typical microbody fraction was isolated on sucrose densitygradients. — 5. Leaves of seedlings grown at 32° contained only very low levels of ribulosediphosphate carboxylase activity and of fraction I protein. Photosynthetic ¹⁴CO₂-fixation of such leaves was only a few per cent of that observed in normal leaves, and no photosynthetic oxygen evolution was observed in chlorotic leaf segments. However, ten other soluble enzymes which are exclusively or partially localized in chloroplasts reached high activities under all conditions at 32° (Table 4). — 6. From chlorotic parts of 32°-light-grown leaves as well as from etiolated 32°-grown leaves a fraction of intact plastids was isolated and purified by sucrose gradient centrifugation which contained several soluble chloroplast enzymes. From the results we conclude that cytoplasmic protein synthesis must contribute a functional chloroplast envelope including the mechanism for the recognition and uptake of chloroplast proteins which are synthesized on cytoplasmic ribosomes.     

Effect of powdery mildew infection on photosynthesis by leaves and chloroplasts of sugar beets

Chloroplasts isolated from powdery mildew-infected (Erysiphe polygoni DC) sugar beet leaves (Beta vulgaris L) showed a reduction in the rate of electron transport and in the accompanying ATP formation in noncyclic photophosphorylation (water as electron donor, NADP as electron acceptor) and little or no change in the rate of ATP formation in cyclic photophosphorylation catalyzed by phenazine methosulfate. The inhibition of noncyclic photophosphorylation appeared to lead in the parent leaves to a decreased rate of photosynthetic CO₂ assimilation and a shift in products resulting in a relative increase of amino acids. These changes were accompanied by alterations in chloroplast ultrastructure and by a reduction in the activity of enzymes necessary for the formation of organic acids (phosphoenolpyruvate carboxylase and malate dehydrogenase). These results are similar to the findings of Montalbini and Buchanan (1974 Physiol. Plant Pathol. 4: 191-196) with chloroplasts from rust-infected Vicia faba leaves.     

Effects of pH and other factors on the phosphate dependence of photosynthesis in spinach chloroplasts

Chloroplast stromal volume and pH influenced the phosphate (Pi)-dependence of photosynthesis of spinach (Spinacia oleracea L.) chloroplasts. Decreasing the sorbitol concentration in the reaction mixture from 0.35 to 0.25 M, or decreasing the external pH from 8.3 to 7.3, extended the induction period of photosynthesis and decreased both the optimal [Pi] and the minimal [Pi] required to inhibit O₂ evolution completely. At least part of the effect of external pH was attributable to changes in stromal pH on the basis of effects of NH₄Cl and sodium acetate at a constant external pH. When the external pH was increased from 7.3 to 8.3, the stromal pH changed only about 0.6 pH units. Hence, the pH gradient across the envelope was diminished and the efflux of phosphoglycerate relative to dihydroxyacetone phosphate was enhanced.Calvin-cycle activity, varied with light intesity or electron transport inhibitors, affected the rate of photosynthesis but not the induction period or the Pi optimum for photosynthesis. Relatively low Calvin-cycle activity was apparently sufficient to fill metabolite pools and thus terminate the induction period. The results indicate that pH does not affect the Pi dependence of photosynthesis by reducing Calvin-cycle activity. Rather, it is postulated that at low stromal pH, larger metabolic pools are required to maintain maximum rates of photosynthesis because of changes in substrate affinity of some Calvin-cycle enzymes. Consequently, chloroplast photosynthesis would be more sensitive to exogenous Pi.Abbreviations DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - Pi inorganic phosphate Cooperative investigations of the North Carolina Agricultural Research Service and Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Raleigh, N.C. Paper No. 6391 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N.C., USA     

Effects of calcium on the photosynthesis of intact leaves and isolated chloroplasts of sugar beets

Effects of calcium on photosynthesis in sugar beets (Beta vulgaris L. cv. F58-554H1) were studied by inducing calcium deficiency and determining changes in CO₂ uptake by attached leaves, electron transport, and photophosphorylation by isolated chloroplasts, and CO₂ assimilation by ribulose diphosphate carboxylase extracts. Calcium deficiency had no significant effect on leaf CO₂ uptake, photoreduction of ferricyanide, cyclic or noncyclic ATP formation of isolated chloroplasts, or on ribulose diphosphate carboxylase CO₂ assimilation, when the rates were expressed per unit chlorophyll. When expressed per unit leaf area CO₂ uptake increased by about 15% in low calcium leaves. The most noticeable effect of calcium deficiency was reduction in leaf area: low calcium had no effect on dark respiratory CO₂ evolution, on leaf diffusion resistance, or on mesophyll resistance to CO₂. We concluded that only small amounts of calcium are required for normal photosynthetic activity of sugar beet leaves.     

Effects of sulfur on the photosynthesis of intact leaves and isolated chloroplasts of sugar beets

Effects of sulfur on photosynthesis in sugar beets (Beta vulgaris L. cv. F58-554H1) were studied by inducing sulfur deficiency and determining changes in the photosynthesis of whole attached leaves and of isolated chloroplasts. The rates of photosynthetic CO₂ uptake by intact leaves, photoreduction of ferricyanide, cyclic and noncyclic photophosphorylation of isolated chloroplasts, and the rate of CO₂ assimilation by ribulose diphosphate carboxylase, decreased with decrease in total leaf sulfur from 2500 to about 500 μg g⁻¹ dry weight. Sulfur deficiency reduced photosynthesis through an effect on chlorophyll content, which decreased linearly with leaf sulfur, and by decreasing the rate of photosynthesis per unit chlorophyll. There was only a small effect of sulfur deficiency on stomatal diffusion resistance to CO₂ until leaf sulfur decreased below 1000 μg g⁻¹ when stomatal resistance became a more significant proportion of the total diffusion resistance to CO₂. Light respiration rates were positively correlated with photosynthesis rates and dark respiration was unchanged as leaf sulfur concentrations declined.     

Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the watersplitting activity in photosynthesis

Plant materials (intact leaves, chloroplasts or subchloroplast particles) preilluminated at a low temperature (e.g. −60°C) were rapidly cooled to −196°C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as Z_v, A, B₁, B₂ and C bands. The A, B₁, B₂ and C bands appeared at constant temperatures, −10, +25, +40 and +55°C, respectively, being independent of the illumination temperature, but the Z_v band appeared at a variable temperature slightly higher than the illumination temperature. The B₁ and B₂ bands were absent in the thermoluminescence profiles of samples devoid of the oxygenevolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

探究非绿叶植物的光合作用

光合作用是高中生物学中重要的一部分,现行各个版本的高中教材均将绿叶中色素的提取作为一个重要的实验。自然界中非绿叶植物虽然只占很小的比例,但是由于叶片的颜色与常见的绿色叶片不同,使其光合作用很容易引起学生的注意,作为学生课外探究实验非常具有可行性。本实验通过提取、分离非绿叶植物叶片中的色素及测定其色素的作用光谱来探究非绿叶植物的光合作用,并以此为契机引入探究实验的一般模式．引导学生掌握科学探究的过程。     

Interaction of cadmium with glutathione and photosynthesis in developing leaves and chloroplasts of Phragmites australis (Cav.) Trin. ex Steudel

We investigated how the presence of cadmium (Cd) at the emergence of Phragmites australis Trin. (Cav.) ex Steudel plants from rhizomes interacted with leaf and chloroplast physiological and biochemical processes. About 8.5 nmol Cd mg-1 chlorophyll was found in leaves, and 0.83 nmol Cd mg-1 chlorophyll was found in chloroplasts of plants treated with 50 microm Cd. As a result, a 30% loss of chlorophyll was measured concomitantly with a comparable percentage reduction in light-saturated photosynthesis. Rubisco content and activity were lowered by 10% and 60%, respectively. Antioxidant activity was stimulated by Cd treatment and was associated with an increase in the glutathione and pyridine pools, and with a larger pool of reduced glutathione. It is suggested that the glutathione pool and its predominance in the reduced state protected the activity of many key photosynthetic enzymes against the thiophilic binding of Cd. Chloroplast ultrastructure was not significantly altered with 50 microm treatment and the efficiency of photosystem II, measured as the fluorescence ratio Fv/Fm, remained high because F0 and Fm were proportionally decreased. In plants treated with 100 microm Cd, all effects were exacerbated, but Fv/Fm remained close to that of control leaves and the glutathione and pyridine nucleotides pools were lowered. The results suggest that glutathione exerted a direct important protective role on photosynthesis in the presence of Cd.     

Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the water-splitting activity in photosynthesis.

Plant materials (intact leaves, chloroplasts or subchloroplast particles) pre-illuminated at a low temperature (e.g. -60 degrees C) were rapidly cooled to -196 degrees C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as ZV, A, B1, B2 and C bands. The A, B1, B2 and C bands appeared at constant temperatures, -10, +25, +40 and +55 degrees C, respectively, being independent of the illumination temperature, but the ZV band appeared at a variable temperature slightly higher than the illumination temperature. The B1 and B2 bands were absent in the thermoluminescence profiles of samples devoid of the oxygen-evolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

Effect of 3-formylchromones substituted with 4-aminosalicylic acid and some other aniline derivatives on photosynthesis inhibition in spinach chloroplasts

The inhibitory effect was investigated of 16 different 3-formylchromone derivatives, the condensation products of 6-R¹-3-formylchromone with 4-aminosalicylic acid and of the adducts of 6-R¹-3-formylchromone withn-alcohols and aminosalicylic acids or some other derivatives of aniline, on the photochemical activity of spinach chloroplasts. The inhibitory activity of the compounds studied correlated with the lipophilicity of the R¹ and R² (alkoxy) substituents. Using fluorescence study it was found that the site of action of the studied effectors is photosystem (PS) 2. By EPR spectroscopy it was confirmed that the studied effectors interact with the intermediates Z⁺/Y⁺ which are situated in D 1 and D 2 proteins on the donor side of PS 2 which is reflected in a partial decrease in the photosynthetic electron flow through PS 2 to PS 1. It was found that the core of PS 2 is not damaged.     

Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts

Starch synthesis in leaves was increased by phosphate starvation or by treatments which decreased cytoplasmic orthophosphate levels (such as mannose feeding). Usually less than 30% of the total carbon fixed during CO₂ assimilation was incorporated into starch in spinach (Spinacia oleracea L.), spinach beet (Beta vulgaris), and tobacco (Nicotiana tabacum) leaves.     

Effect of linolenate on photosynthesis by intact spinach chloroplasts II. Influence of preillumination of leaves on the inhibition of photosynthesis by linolenate

The inhibitory effect of linolenate on intact spinach chloroplastsdepends on the level of the internal pool of metabolites. Chloroplastsfrom preilluminated leaves or chloroplasts artificially loadedwith 3-phosphoglyceric acid required higher concentrations oforthophosphate for maximal rates of CO₂ dependent O₂ evolutionthan untreated chloroplasts. The loaded chloroplasts were moresensitive to linolenate, and in the presence of linolenate theoptimal phosphate concentration was shifted toward lower values.We propose that the inhibition of photosynthesis by linolenateis due to inhibition of the "phosphate translocator". ¹ Part of this work has been published in the Book of Abstracts,4th International Congress on Photosynthesis, Reading, U.K.,1977, p. 265–266. ² This work is part of a doctoral programme carried out by L.Mv6 Akamba in this laboratory. ³ To whom reprint requests should be adressed. (Received October 14, 1978; )