Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts.

Intense illumination isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg2+ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowing in the dark and is inhibited by N,N'-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.     

Light-induced Mg2+ ATPase activity of coupling factor in intact chloroplasts

Intense illumination of isolated, intact, spinach chloroplasts triggers the well known proton-pumping Mg²⁺ ATPase activity of coupling factor, which can be assayed in subsequently lysed chloroplasts by monitoring ATP-driven quenching of 9-aminoacridine fluorescence. The light-triggered ATPase activity decays slowly in the dark and is inhibited by N,N′-dicyclohexylcarbodiimide. After osmotic lysis and washing of the chloroplasts, preillumination no longer triggers maximal proton-pumping ATPase until methylviologen and dithiothreitol are added to the medium. It is suggested that intact organelles contain soluble or loosely bound cofactors necessary for light-triggering of coupling factor ATPase. On osmotic lysis, these endogenous cofactors are diluted or inactivated and must be replaced by addition of a dithiol reagent and an electron acceptor.     

Interactions of inorganic phosphate with spinach coupling factor 1. Effects on ATPase and ADP binding activities

Illumination of chloroplast thylakoid membranes results in both the release of adenine nucleotides from the tight nucleotide binding site(s) on chloroplast coupling factor 1 (CF1) and the activation of a light-triggered ATPase activity of CF1. Because inorganic phosphate stabilizes the light-triggered ATPase activity of CF1 in the dark, the effects of Pi on the rebinding of ADP to CF1 and on the light-triggered ATPase activity have been studied. Pi appears to be a partial noncompetitive inhibitor, with respect to ADP, of adenine nucleotide binding to the tight nucleotide binding site(s) on CF1 and induces negative cooperativity. The latter result suggests the existence of heterogeneous ADP binding sites in the presence of Pi. However, even under conditions where Pi causes a 50% reduction of tightly bound ADP, the ADP-induced dark decay of the ATPase activity is still complete. It was found that Pi inhibition of the light-induced dark binding of ADP can be reversed by the removal of the Pi. Removal of Pi also induces a small but significant ATPase activity. A model for the roles of the adenine nucleotide tight binding site(s) and Pi in the modulation of the spinach CF1 ATPase activity is proposed.     

Development of Photochemical Activity and the Appearance of the High Potential Form of Cytochrome b-559 in Greening Barley Seedlings

The development of photochemical activity during the greening of dark-grown barley seedlings (Hordeum vulgare L. cv. Svalöfs Bonus) was studied in relation to the formation of the high potential form of cytochrome b-559 (cytochrome b-559_HP). Photosynthetic oxygen evolution from leaves was detected at 30 minutes of illumination. The rate of oxygen evolution per gram fresh weight of leaf was as high at 2 to 2.5 hours of greening as at 24 hours or in fully greened leaves. On a chlorophyll basis, the photosynthetic rate at 90 minutes of greening was 80-fold greater than the rate at 45 hours. It is concluded that the majority of photosynthetic units are functional at an early stage of greening, and that chlorophyll synthesis during greening serves to increase the size of the units.     

Interconversion of two distinct states of active CF0-CF1 (chloroplast ATPase complex) in chloroplasts

Chloroplast ATPase complex is activated by illumination in the presence or absence of dithiothreitol. ATPase complex which has been activated without dithiothreitol catalyzes ATP hydrolysis which is insensitive to stimulation by NH4Cl and is highly sensitive to medium pH. Addition of dithiothreitol during illumination results in an increase in the stimulating effect of NH4Cl on ATP hydrolysis and a decrease in pH sensitivity of ATP hydrolysis. With increasing time in the dark, the ability of NH4Cl to stimulate ATP hydrolysis decreases and the effect of pH on the ATP hydrolysis increases. The onset of resistance to NH4Cl stimulation and the increase in sensitivity to pH are accelerated by ADP and the acceleration is inhibited by Pi. ATP hydrolysis restores NH4Cl sensitivity and renders the activity more resistant to pH. These results suggest that active chloroplast ATPase complex converts its state reversibly from the NH4Cl-insensitive and highly pH-sensitive one to the NH4Cl-sensitive and relatively pH-insensitive one. The conversion from the former to the latter requires both sulfhydryl compound and energy.     

Identification, expression, and functional analyses of a thylakoid ATP/ADP carrier from Arabidopsis

In plants the chloroplast thylakoid membrane is the site of light-dependent photosynthetic reactions coupled to ATP synthesis. The ability of the plant cell to build and alter this membrane system is essential for efficient photosynthesis. A nucleotide translocator homologous to the bovine mitochondrial ADP/ATP carrier (AAC) was previously found in spinach thylakoids. Here we have identified and characterized a thylakoid ATP/ADP carrier (TAAC) from Arabidopsis.(i) Sequence homology with the bovine AAC and the prediction of chloroplast transit peptides indicated a putative carrier encoded by the At5g01500 gene, as a TAAC. (ii) Transiently expressed TAAC-green fluorescent protein fusion construct was targeted to the chloroplast. Western blotting using a peptide-specific antibody together with immunogold electron microscopy revealed a major location of TAAC in the thylakoid membrane. Previous proteomic analyses identified this protein in chloroplast envelope preparations. (iii) Recombinant TAAC protein specifically imports ATP in exchange for ADP across the cytoplasmic membrane of Escherichia coli. Studies on isolated thylakoids from Arabidopsis confirmed these observations. (iv) The lack of TAAC in an Arabidopsis T-DNA insertion mutant caused a 30-40% reduction in the thylakoid ATP transport and metabolism. (v) TAAC is readily expressed in dark-grown Arabidopsis seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. We propose that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover in plants.     

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts.

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinites for fructose diphosphate and Mg-2+ and were similarly activated by EDTA, dithiothreitol and cystamine. Mg-2+, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Development of Photosynthetic Structure and Function during Light-Induced Greening in Photomixotrophic Cells of Arachis hypogaea L.

The synthesis of chlorophyll and development of photochemicalactivities were complete within 70–80 h in greening leaves,whereas these processes continued for 8 days with an initiallag of 8 h for pigment synthesis in greening Arachis hypogaeaL cells. The activity of photosystem I in cultured Arachis cellswas detected earlier (24–36 h after illumination) thanthat of photosystem II (42–54 h after illumination) andthe development of the latter coincided with the synthesis ofa 46,000 dalton polypeptide of the thylakoid membranes. Experimentalstudies with cultured cells have the advantage in that the temporalsequence of the assembly of membrane components and associatedfunctions are determined easily because of longer developmentalperiod of chloroplast. (Received January 29, 1982; Accepted April 13, 1983)     

Characterization and Effect of Light on the Plasma Membrane H+-ATPase of Bean Leaves

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the K_m was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, K_m, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.     

Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease

The formation of zeaxanthin (Zea) from violaxanthin (Vio) in chloroplasts of leaves and algae upon strong illumination is currently suggested to play a role in the photoprotection of plants. Properties and location of the enzyme Vio de-epoxidase, which is responsible for the transformation of Vio to Zea, were studied using thylakoid membrane vesicles isolated from leaves of Spinacia oleracea L. Without using detergents a repeated freeze-thaw treatment of thylakoid vesicles was sufficient to release the enzyme into the medium. With the same procedure the mobile electron carrier plastocyanin, known to occur in the thylakoid lumen, was also released. The enzyme was demonstrated by its activity in the supernatant of the pelleted thylakoid vesicles in the presence of the added substrates Vio and ascorbic acid, as well as by staining of the released proteins after polyacrylamide gel electrophoresis. The release of the deepoxidase from the vesicles was pH-dependent, declined below pH 6.5 and ceased in the pH range around 5, which corresponds to the pH optimum of the enzyme activity. By using thylakoid vesicles isolated from pre-illuminated and therefore Zea-containing leaves the release by freeze-thaw cycles of both the de-epoxidase and plastocyanin was diminished compared with the dark control. However, the reason for this effect was not the Zea content but an unknown effect of the illumination on the thylakoid membrane properties. The de-epoxidase collected at pH 7 was able to re-bind to thylakoid membranes at pH 5.5 and to transform intrinsic Vio to Zea in the presence of ascorbate. The isolated de-epoxidase, as well as the endogenous membrane-bound de-epoxidase, was inhibited by dithiothreitol. From these results it is concluded that Vio de-epoxidase, like plastocyanin, is mobile within the thylakoid lumen at neutral pH values which occur under in-vivo conditions in the dark. However, upon strong illumination, when the lumen pH drops (pH < 6.5) due to the formation of a proton gradient, the properties of the de-epoxidase are altered and the enzyme becomes tightly bound to the membrane (in contrast to plastocyanin) thus gaining access to its substrate Vio. These findings corroborate the assumption of a transmembrane opposite location of the two enzymes of the xanthophyll cycle, the ascorbate-dependent Vio deepoxidase at the lumenal side and the NADPH-dependent Zea epoxidase at the stromal side. Indications in favour of a location of Vio within the lipid bilayer of the thylakoid membrane and of a binding of the active deepoxidase to these areas are discussed.     

THE EFFECT OF GROWTH CONDITIONS AND THE STAGE OF LEAF DEVELOPMENT ON THE HILL REACTION IN HOMOGENATES OF PISUM SATIVUM LEAVES

Miller, John H. (Yale U., New Haven, Conn.) The effect of growth conditions and the stage of leaf development on the Hill reaction in homogenates of Pisum sativum leaves. Amer. Jour. Bot. 47(7): 532–540. Illus. 1960.—With plants grown under short-day conditions (8 hr. light and 16 hr. dark), crude chloroplast suspensions from young leaves have a higher Hill-reaction activity between 1 and 3 hr. after the beginning of illumination than suspensions from older leaves, while after 5–7 hr. of illumination, this activity difference is not found. These differences result from a marked diurnal rise and fall in the Hill reaction. The magnitude of the rise depends on the age of the leaf from which the chloroplast suspension is prepared. Peak activity occurs after the plants have received between 3 and 4 hr. of light and is highest in suspensions prepared from young leaves. Suspensions from the oldest leaves show no diurnal change in activity. No diurnal changes in activity are found in chloroplast suspensions from plants which are grown under continuous light, and the diurnal rise and fall is dependent on the plant receiving an alternation of light-dark-light.     

A light- and cysteine-activated ATP-Pi exchange reaction in chloroplasts and its relationship to ATPase and photophosphorylation

1. ‘Broken’ chloroplasts from spinach if illuminated for a period in the presence of cysteine and phenazine methosulphate develop an ATP-P_i exchange activity which can be observed in the dark. The conditions giving rise to ATP-P_i exchange activity are similar to those giving rise to the thiol-activated light-triggered ATPase.

2. ATP is not necessary during illumination for development of ATP-P_i exchange activity, but the activity declines if a period of time elapses between illumination and addition of ATP. This is accompanied by a similar decline in the cysteine-activated light-triggered ATPase.

3. The ATP-P_i exchange and ATPase show the same dependence on ATP concentration and are both inhibited by added ADP.

4. Both reactions are inhibited by Dio-9.

5. Desaspidin, 4-octyl-2,6-dinitrophenol and carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, added immediately after illumination, inhibit the ATP-P_i exchange. The ATPase is initially stimulated under these conditions and then inhibited. If present during illumination, desaspidin and octyldinitrophenol inhibit the ATPase.

6. It is concluded that the ATP-P_i exchange reaction and the ATPase are activities of the same enzyme complex in the chloroplast and that this is probably part of the terminal enzyme system of photophosphorylation.     

Independent effects of leaf growth and light on the development of the plastid and its DNA content in Zea species

In maize (Zea mays L.), chloroplast development progresses from the basal meristem to the mature leaf tip, and light is required for maturation to photosynthetic competence. During chloroplast greening, it was found that chloroplast DNA (cpDNA) is extensively degraded, falling to undetectable levels in many individual chloroplasts for three maize cultivars, as well as Zea mexicana (the ancestor of cultivated maize) and the perennial species Zea diploperennis. In dark-grown maize seedlings, the proplastid-to-etioplast transition is characterized by plastid enlargement, cpDNA replication, and the retention of high levels of cpDNA. When dark-grown seedlings are transferred to white light, the DNA content per plastid increases slightly during the first 4 h of illumination and then declines rapidly to a minimum at 24 h during the etioplast-to-chloroplast transition. Plastid autofluorescence (from chlorophyll) continues to increase as cpDNA declines, whereas plastid size remains constant. It is concluded that the increase in cpDNA that accompanies plastid enlargement is a consequence of cell and leaf growth, rather than illumination, whereas light stimulates photosynthetic capacity and cpDNA instability. When cpDNA from total tissue was monitored by blot hybridization and real-time quantitative PCR, no decline following transfer from dark to light was observed. The lack of agreement between DNA per plastid and cpDNA per cell may be attributed to nupts (nuclear sequences of plastid origin).     

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinities for fructose diphosphate and Mg²⁺ and were similarly activated by EDTA, dithiothreitol and cystamine.Mg²⁺, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Plastid Development in Primary Leaves of Phaseolus vulgaris: FREE NUCLEOTIDE CONCENTRATIONS IN GREENING LEAVES

The free nucleotides of 14-day-old dark-grown bean leaves havebeen extracted by perchloric acid and fractionated by ion-exchangechromatography. AMP, ATP, CMP, GMP, GDP, ADP-ribosephosphate(the acid-breakdown product of NADPH), NADP⁺, UMP, UDP-glucose,and UDP-xylose were the main components identified. During 45h continuous illumination greening occured and there was a 192per cent increase in the total free nucleotide per leaf. Mostof the constituent nucleotides showed rises during greening,the most notable increases being of substances concerned withphotosynthesis, namely NADPH, and UDP-glucose. A rise in theNADPH/NADP⁺ ratio after 15 h illumination appeared to signifythe onset of photosynthesis.     

Light-dependent development of thermoluminescence, delayed emission and fluorescence variation in dark-grown spruce leaves

Thermoluminescence profiles of spruce leaves grown under various light or dark conditions were measured after excitation at a low temperature (−70 to −20 °C) by 1-min illumination with red light, and the following results were obtained. Mature spruce leaves showed five thermoluminescence bands at −30, −5, +20, +40 (or +35) and +70 °C (denoted as Z_v, A, B₁, B₂ and C bands, respectively), but dark-grown spruce leaves with a similar chlorophyll content showed only two bands, at −30 and +70 °C (the Z_v and C bands) and were devoid of the three other bands (the A, B₁ and B₂ bands). On exposure of the dark-grown leaves to continuous red light, the A, B₁ and B₂ bands were rapidly developed, and the development was accompanied by enhancement of delayed emission, fluorescence variation and the Hill activity (photoreduction of 2,6-dichlorophenolindophenol with water as electron donor). It was demonstrated that the dark-grown spruce leaves are devoid of the water-splitting system in Photosystem II, and that the latent water-splitting activity is rapidly photoactivated by exposure of the leaves to continuous red light. These results on the gymnosperm spruce leaves, in which greening proceeds in complete darkness, being independent of the development of the water-splitting system in light, were discussed in relation to previous observations on angiosperm leaves, in which both greening and the activity generation proceed in the light.