Photophosphorylation and related properties of reaggregated vesicles from spinach photosystem I particles.

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH+4 are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

Photophosphorylation and related properties of reaggregated vesicles from spinach Photosystem I particles

The small Photosystem I particles prepared from spinach chloroplasts by the action of Triton X-100 (TSF 1 particles) reaggregate into membrane structures when they are incubated with soybean phospholipids and cholate and then subjected to a slow dialysis. The membranes so formed are vesicular in nature and show the capability of catalyzing phenazine methosulfate-mediated cyclic photophosphorylalation at rates which are usually about 20% of those observed with chloroplasts, but higher rates have been obtained. When coupling factor is removed from the chloroplasts by treatment with EDTA, a requirement for coupling factor can be shown for the subsequent ATP formation. The uncouplers carbonylcyanide 3-chlorophenyl-hydrazone, valinomycin, Triton X-100 and NH⁺₄ are effective with the reformed vesicles, which do not show the typical light-induced pH gradient observed with chloroplasts. Incubation of the TSF 1 particles with phospholipids alone allows for the formation of membrane vesicles, but such vesicles are only slightly active in ATP formation. In most properties investigated, the reformed membrane vesicles resemble the original chloroplast membrane so far as phenazine methosulfate-mediated cyclic photophosphorylation is concerned, which indicates a high degree of selectivity in the reaggregation process. The major difference between chloroplasts and the reformed vesicles is the failure of the latter to show a light-induced pH gradient.     

Ultrastructural and Photometric Evidence for Light-Induced Changes in Chloroplast Structure in vivo

Photometric evidence for a reversible, red-light induced transmission decrease in excised leaf tissue or the thalli of certain marine algae has been obtained under conditions which correspond to the occurrence of a light-induced shrinkage of chloroplasts within the cells. Evidence supporting this conclusion is: A) The kinetics of the nonspecific transmission changes are similar to those observed in chloroplasts in vitro. B) The magnitude of the response is larger than could be accounted for by any known pigment which absorbs at 546 mμ. C) The light-induced transmission changes are optimal at pH 5.5 to 6.5 in the presence of electron flow cofactors and weak acid anions, conditions which are optimal for light-induced chloroplast shrinkage in isolated chloroplasts. D) Examination of chloroplast ultrastructure in dark incubated and illuminated chloroplasts reveals a flattening of the chloroplast structure and shrinkage.     

Sensitivity of light-grown and dark-grown Euglena cells to gamma-irradiation.

Light-grown cells which contain fully developed chloroplasts were found to be more resistant to gamma-irradiation than dark-grown cells which are devoid of chloroplasts. The radio-resistance of dark-grown cells progressively increased during light-induced development of chloroplasts and, conversely, radio-resistance of light-grown cells decreased progressively with chloroplast de-development during growth in the dark. The presence of chloroplasts seemed to play a major role in the capacity of cells to recover from radiation damage, the efficiency of cellular recovery being correlatable with the degree of chloroplast development.     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.     

Light-induced chloroplast movements in Lemna trisulca. Identification of the motile system

In mesophyll cells of the water plant Lemna trisulca L. chloroplasts redistribute in response to blue light. In the present study it is shown that an actin depolymerizing agent cytochalasin D, a crosslinker of actin subunits in F-actin m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) as well as N-ethylmaleimide (NEM)—a sulfhydryl group reagent, are potent inhibitors of these blue light-induced chloroplast movements in Lemna. Extraction with cold, buffered glycerol solution preserves light-induced chloroplast arrangements within cells producing permeabilized cell models. ‘Reactivation’ of these cell models by Mg-ATP results in remarkable movements which can be inhibited by treatment with NEM and cytochalasin D. Immunofluorescence microscopy demonstrates that a component which is associated with isolated Lemna chloroplasts cross-reacts with antibodies directed against bovine myosin. These results indicate that a contractile actomyosin system is involved in blue light-induced chloroplast movements in Lemna and a putative motor protein, similar to myosin, is associated with the surface of Lemna chloroplasts.     

Light-Induced Chloroplast Shrinkage in vivo Detectable After Rapid Isolation of Chloroplasts From Pisum sativum

A light-induced shrinkage of chloroplasts in vivo could be detected with chloroplasts isolated within 2 minutes of harvesting pea plants. As determined both by packed volume and Coulter counter, the mean volume of chloroplasts from plants in the dark was 39 μ³, whereas it was 31 μ³ for chloroplasts from plants in the light. Upon illumination of the plants, the half-time for the chloroplast shrinkage in vivo was about 3 minutes, and the half-time for the reversal in the dark was about 5 minutes. A plant growth temperature of 20° was optimal for the volume change. The chloroplast shrinkage was half-maximal for a light intensity of 400 lux incident on the plants and was light-saturated near 2000 lux. The light-absorbing pigment responsible for the volume change was chlorophyll. This light-induced shrinkage resulted in a flattening and slight indenting of the chloroplasts. This chloroplast flattening upon illumination of the plants may accompany an increase in the photosynthetic efficiency of chloroplasts.     

Role of Phospholipid Transfer Protein in the Exchange of Phospholipids between Microsomes and Chloroplasts

When microsomes containing phosphatidylcholine labelled with[1-¹⁴C]-linoleate were incubated with pea or spinach chloroplasts,active transfer of this phospholipid took place in the presenceof phospholipid transfer protein. This transfer also was demonstratedby incubating unlabelled microsomes, chloroplasts and the phospholipidtransfer protein in the presence of [1-¹⁴C]-acetate. The reconstitutedsystems could synthesize fatty acids which were acylated inmicrosomal phosphatidylcholine. The transfer of this phospholipidto chloroplasts is mediated by the transfer protein. Our resultssuggest a role for phospholipid transfer protein in the synthesisof chloroplast lipids. (Received October 25, 1983; Accepted July 18, 1984)     

Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. II. Alternative spectral forms and inhibitor effects on kinetics of Signal II in flashing light

Linewidth and hyperfine structure measurements of the EPR spectrum of Signal II in spinach chloroplasts show that the signal reflects two alternative states. One state is characterized by a 16-G linewidth and four partially resolved hyperfine components. The other state has 19 G linewidth and five partially resolved hyperfine components. It is possible to interconvert these two states by changing the ionic strength of the chloroplast suspension. Both states of Signal II show similar light-induced increases in dark-adapted chloroplasts and respond to 10-μs white light flashes with identical kinetics.

In chloroplasts at room temperature, Signal II dark decays to 50% of its total light-induced level in about 1 h. Single flashes increase the spin concentration in these aged chloroplasts but with decreased effectiveness compared with fresh, dark-adapted chloroplasts. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) decreases the decay time of Signal II from hours to seconds without appreciably altering the level of Signal II formed in saturating continuous light. However, both the formation time constant and the extent of Signal II increase stimulated by a single saturating flash are decreased in CCCP-treated chloroplasts.

These results are interpreted in terms of the model, proposed in the preceding paper, in which Signal II is generated by oxidation-reduction reactions on the water side of Photosystem II.     

Light-induced movement of magnesium ions in intact chloroplasts. Spectroscopic determination with Eriochrome Blue SE

The metallochromic indicator Eriochrome Blue SE was used to measure light-induced internal movement of Mg²⁺ in intact chloroplasts. By dual-wavelength spectroscopy (measuring wavelength 554 nm, reference 592 nm) a light-induced, dark-reversible absorbance increase of Eriochrome Blue in samples of isolated intact chloroplasts was observed. The light/dark difference spectrum of Eriochrome Blue between 550 and 590 nm (reference wavelength 562 nm) indicated that this absorbance increase was caused by an increased concentration of free Mg²⁺ in a neutral or slightly alkaline chloroplast compartment.

The signal was seen only with intact, but not with broken, envelope-free chloroplasts, which had lost most of their divalent cations. This is interpreted to show that the indicator responds to an increase of Mg²⁺ concentration in the chloroplast stroma, which represents an efflux of Mg²⁺ from the intra-thylakoid space caused by light-dependent proton pumping.

As calculated from corrected values of the absorbance increase of Eriochrome Blue, the light-induced internal release of Mg²⁺ was close to 100 nequiv per mg chlorophyll at pH 7.6 and 250 nequiv at pH 7.1. This corresponds to a light-dependent increase in the concentration of free Mg²⁺ in the stroma of about 2 and 5 mM, respectively.     

Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris

In gametophytic cells (prothalli) of the fern Adiantum capillus-veneris, nuclei as well as chloroplasts change their position according to light conditions. Nuclei reside on anticlinal walls in darkness and move to periclinal or anticlinal walls under weak or strong light conditions, respectively. Here we reveal that red light-induced nuclear movement is mediated by neochrome1 (neo1), blue light-induced movement is redundantly mediated by neo1, phototropin2 (phot2) and possibly phot1, and dark positioning of both nuclei and chloroplasts is mediated by phot2. Thus, both the nuclear and chloroplast photorelocation movements share common photoreceptor systems.     

Energetic basis of the light-induced chloroplast shrinkage in vivo

A light-induced chloroplast shrinkage occurring in vivo wasmeasured with a Coulter counter and a packed weight techniqueusing chloroplasts isolated within two minutes of harvestingpea plants. Introduction of the photosynthetic inhibitor DCMU(5 µM) into the plant either by bathing the cut stem orinjection through a fine hypodermic needle decreased the light-inducedchloroplast shrinkage in vivo 11 to 20%. The uncoupler tri-Fl-CCP(5 µM) inhibited the light-induced shrinkage 80 % , whilenigericin (0.5 µM) completely abolished it. An actionspectrum for the chloroplast volume decrease in vivo had a shoulderat 700-715 mµ. These results are considered in terms ofthe various forms of energy available during photosynthesis.A consistent interpretation is that the lightinduced chloroplastshrinkage in vivo depends either on a high energy state createdby electron flow or on ATP. This chloroplast volume change uponillumination of the plants may increase the photosynthetic efficiency. (Received April 15, 1968; )     

Gustav Senn(1875–1945): The pioneer of chloroplast movement research

Gustav Senn analyzed for the first time light-induced movement and arrangement of chloroplasts. Using many plant species he performed physiological analyses of chloroplast migration in response to exte...     

Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase.

In Pisum sativum, distinct chloroplast and cytosolic forms of glutamine synthetase (GS) are encoded by homologous nuclear genes that are differentially expressed in vivo (Tingey, S. V., Tsai, F.-Y., Edwards, J. W., Walker, E. L., and Coruzzi, G. M. [1988]. J. Biol. Chem. 263, 9651-9657). In leaves, light selectively affects the expression of the nuclear gene for chloroplast GS2. Differences in the maximal levels of GS2 mRNA in etiolated plants treated with red or white light indicate that only part of the white-light-induced accumulation of GS2 mRNA is due to a phytochrome-mediated response. The kinetics of GS2 mRNA accumulation in response to white-light illumination of etiolated or dark-adapted green plants indicates that GS2 mRNA accumulates more rapidly in plants containing mature, photosynthetically competent chloroplasts. Other evidence that GS2 mRNA levels are affected by the metabolic status of chloroplasts concerns the selective induction of GS2 mRNA in plants grown under conditions that result in the production of photorespiratory ammonia. These results indicate that the light-induced accumulation of GS2 mRNA in leaves results from the action of phytochrome as well as light-induced changes in chloroplast metabolism.     

Chlorophyll Formation and Photosynthetic Competence in Euglena During Light-Induced Chloroplast Development in the Presence of 3, (3,4-dichlorophenyl) 1,1-Dimethyl Urea (DCMU)

The possibility that photosynthetic competence is gratuitous for light-induced chloroplast development in Euglena gracilis var. bacillaris was examined by incubating dark-grown resting cells in the light with DCMU, an inhibitor of photosynthesis. Under these conditions photosynthetic carbon dioxide fixation was inhibited essentially completely at all times during chloroplast development, but about 70% of the chlorophyll was formed with essentially the same pattern of accumulation found for cells incubated in the absence of the inhibitor. Electron microscopy of cells incubated with DCMU in the light revealed the formation of morphologically recognizable chloroplasts having comparable overall dimensions and structural elements to those found in normally developed chloroplasts, but frequently lacking a readily detectable pyrenoid with paramylum sheaths, and often containing increased numbers of discs per lamella. Such abnormalities are considered minor since upon removal of DCMU by centrifugation, the cells usually regained almost full photosynthetic competence on a chlorophyll basis.

It is concluded that photosynthetic competence is not necessary for chloroplast development in Euglena and supports the hypothesis, already suggested from other evidence, that light induction results in activation of synthetic machinery external to the developing chloroplast.

     

Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT

Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O₂ evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light-induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II.     

Light-induced increase in free Mg2+ concentration in spinach chloroplasts: measurement of free Mg2+ by using a fluorescent probe and necessity of stromal alkalinization

Free Mg(2+) in chloroplasts may contribute to the regulation of photosynthetic enzymes, but adequate methodology for the determination of free Mg(2+) concentration ([Mg(2+)]) in chloroplasts has been lacking. We measured internal chloroplast [Mg(2+)] by using a Mg-sensitive fluorescent indicator, mag-fura-2. In intact, dark-kept spinach chloroplasts, internal [Mg(2+)] was estimated to be 0.50 mM, and illumination caused an increase in [Mg(2+)] to 2.0mM in the stroma. The light-induced increase in [Mg(2+)] was inhibited by a blocker of driven electron transport and uncouplers. The K(+)-specific ionophore valinomycin inhibited the [Mg(2+)] increase in the absence of external K(+), and addition of KCl restored the [Mg(2+)] increase. NH(4)Cl, which induces stromal alkalinization, enhanced the [Mg(2+)] increase. A Ca(2+)-channel blocker, ruthenium red, inhibited the [Mg(2+)] increase, but LaCl(3) had no effect. These results indicate that stromal alkalinization is essential for light-induced increase in [Mg(2+)]. This system for measuring internal chloroplast [Mg(2+)] might provide a suitable system for assay of Mg(2+) transport activity of chloroplast membranes.