Initial ATP Formation, NADP Reduction, CO(2) Fixation, and Chloroplast Flattening Upon Illuminating Pea Leaves

Chloroplasts in living cells of detached and sectioned leaves of Pisum sativum had a thickness of 2.68 ± 0.04 μ in the dark as determined from photographs made using a phase contrast microscope. Upon illumination with 4000 lux for 10 min, the chloroplasts flattened to 2.15 ± 0.04 μ. There was a short lag period of about 11 sec at 1000 lux and 2 sec at 4000 lux before appreciable light-induced flattening occurred. Both ATP and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in detached pea leaves increased upon illumination and then fell during the initial 60 sec. The maximum ATP level was attained in 16 sec at 1000 lux and 10 sec at 4000 lux, while NADPH required about twice as long to reach a maximum. A sustained rate of carbon dioxide fixation occurred after a lag period coinciding in time with the drop in the NADPH level. ATP appeared to be involved not only with carbon dioxide fixation, but also with some reaction beginning sooner, perhaps the light-induced chloroplast flattening. Considering the initial photophosphorylation and the sustained CO₂ fixation rates, the ATP formation rate in vivo apparently increased after the leaves had been in the light for a few min.     

Light-induced Changes in the Ultrastructure of Pea Chloroplasts in Vivo: Relationship to Development and Photosynthesis

Light-induced structural changes of chloroplasts and their lamellae were studied in leaves of Pisum sativum L., cv. Blue Bantam, using electron microscopy. Upon illumination of 14-day-old plants with 2000 lux, the chloroplasts decreased in thickness by about 23% with an accompanying increase in electron scattering by the stroma. Concomitantly, the average thickness of granal lamellae (thylakoids) decreased from 195 ± 4 angstroms in the dark to 152 ± 4 angstroms in the light, and this change was half-saturated at only 50 lux. Lamellar flattening at 50 lux and its reversal in the dark both had half-times of a minute or less. The thickness of a partition (a pair of apposed lamellar membranes) was 140 ± 9 angstroms in both the light and the dark, indicating that the observed light-induced change was in the volume enclosed within the thylakoid. The effect of illumination could be inhibited by various uncouplers of photophosphorylation but not by 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea, suggesting that it depended on ATP (or its precursor). In the presence of 0.5 micromolar nigericin, the thickness of the granal lamellae increased in the light to 213 ± 3 angstroms; this may reflect an uptake of K⁺ into an osmotically responding space within the thylakoids.     

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.     

Increased CO2 fixation by Pisum sativum chloroplasts in vitro reflecting a change in coupling caused by illuminating the plants

Illumination of pea plants caused a doubling in the rate ofCO₂ fixation by the subsequently isolated chloroplasts comparedwith the rate obtained for chloroplasts from plants in the dark.This enhancement in the CO₂ fixation rate was half-maximal for800 lux incident on the plants and was 90% light saturated at2000 lux. The half-time for the enhancement of the CO₂ fixationrate following illumination of the plants was about 4 min andthe half-time for its reversal when the plants were placed backin the dark was 5 min. Illuminating the plants had relativelylittle effect on the O₂ evolution rate of the subsequently isolatedchloroplasts. Moreover, the ferricyanide reduction rate by theisolated chloroplasts was also essentially unaffected by theillumination condition of the plants from which the chloroplastswere isolated. Consequently, light on the plant apparently causesa doubling in the CO₂ fixed per electron moving in the photosyntheticelectron transport pathway. This enhanced coupling is discussedin terms of a concomitant increase in endogenous photophosphorylationand flattening of the chloroplasts in vivo, other changes causedby light incident on the plant. (Received January 16, 1970; )     

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; )     

PROTONATION AND CHLOROPLAST MEMBRANE STRUCTURE

Light changes the structure of chloroplasts. This effect was investigated by high resolution electron microscopy, photometric methods, and chemical modification. (a) A reversible contraction of chloroplast membrane occurs upon illumination, dark titration with H⁺, or increasing osmolarity. These gross structural changes arise from a flattening of the thylakoids, with a corresponding decrease in the spacing between membranes. Microdensitometry showed that illumination or dark addition of H⁺ resulted in a 13–23% decrease in membrane thickness. Osmotically contracted chloroplasts do not show this effect. (b) Rapid glutaraldehyde fixation during actual experiments revealed that transmission changes are closely correlated with the spacing changes and therefore reflect an osmotic mechanism, whereas the light scattering changes have kinetics most similar to changes in membrane thickness or conformation. (c) Kinetic analysis of light scattering and transmission changes with the changes in fluorescence of anilinonaphthalene sulfonic acid bound to membranes revealed that fluorescence preceded light scattering or transmission changes. (d) It is concluded that the temporal sequence of events following illumination probably are protonation, changes in the environment within the membrane, change in membrane thickness, change in internal osmolarity accompanying ion movements with consequent collapse and flattening of thylakoid, change in the gross morphology of the inner chloroplast membrane system, and change in the gross morphology of whole chloroplasts.     

LIGHT-INDUCED VOLUME CHANGES IN SPINACH CHLOROPLASTS

A light-dependent mechanism that results in a slow, high-amplitude swelling of spinach chloroplasts in vitro has been discovered. The swelling is readily observed by optical and gravimetric methods, and by the use of an electronic particle counter; all show a 100 per cent increase of chloroplast volume in the light with an approximately 10-minute half-time. The existence of an osmotic mechanism for chloroplast swelling in the dark is confirmed. The volume of illuminated chloroplasts versus NaCl concentration represents the addition of osmotic and light effects. The action of light is enhanced by electron flow cofactors, such as phenazine methosulfate (PMS). However, neither conditions for ATP hydrolysis or synthesis nor NH₄Cl influence the time course and extent of swelling. Hence, high-amplitude chloroplast swelling is light- (or electron flow), but not energy-dependent. A remarkable inhibitory effect of inorganic phosphate on chloroplast swelling is observed in the light, but not in the dark. Another action of light on chloroplasts is known to result in a shrinkage of chloroplasts which is rapid, reversible, energy-dependent, and requires phosphate. Thus phosphate determines the action of light on chloroplast volume. Since shrinkage is reversible, but swelling is not, it may be that they reflect physiological and deteriorative processes, respectively. Chloroplasts and mitochondria appear to control their volume by similar mechanisms.     

Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume

Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, −1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at −1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, −2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to −1.8 megapascals and the chloroplasts had higher rates of CO₂-dependent O₂ evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.

Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20°C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20°C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20°C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [¹⁴C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [¹⁴C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.

It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology.

     

Different kinetics of membrane potential formation in dark-adapted and preilluminated chloroplasts

The effects of varying dark interval on the kinetics of light-induced formation of the membrane potential were studied on individual chloroplasts of Anthoceros with the use of capillary microelectrodes. Illumination of the chloroplast with 1 s light pulse after 3 min dark period induced the photoelectrical response with two peaks of the potential that were located at 20 and 500 ms after the onset of illumination. The position of the second peak was shifted along the time-scale depending on the preceding dark interval. The repeated illumination of the chloroplast with 1 s light pulse after 30 s dark interval induced the electrical response with only one maximum and a monotonous decay of the potential in the light. Distinctions in the electrical responses induced by the first and the second light pulses were eliminated by the addition of 50 μM dicyclohexylcarbodiimide (DCCD). The results show that the photoinduction kinetics of the membrane potential in chloroplasts is affected by functioning of H⁺-ATPase. The delayed peak of the membrane potential in the photoinduction kinetics is interpreted as a consequence of the photoactivated electron transport supported by Photosystem I.     

Stromal free calcium concentration and light-mediated activation of chloroplast fructose-1,6-bisphosphatase

Light-mediated activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) in intact spinach chloroplasts (Spinacia oleracea L.) is enhanced in the presence of 10⁻⁵ molar external free Ca²⁺. The most pronounced effect is observed during the first minutes of illumination. Ruthenium red, an inhibitor of light-induced Ca²⁺ influx, inhibits this Ca²⁺ stimulated activation. In isolated stromal preparations, the activation of fructose-1,6-bisphosphatase is already enhanced by 2 minutes of exposure to elevated Ca²⁺ concentrations in the presence of physiological concentrations of Mg²⁺ and fructose-1,6-bisphosphate. Maximal activation of the enzyme is achieved between 0.34 and 0.51 millimolar Ca²⁺. The Ca²⁺ mediated activation decreases with increasing fructose-1,6-bisphosphate concentration and with increasing pH. The data are consistent with the proposal that the illumination of chloroplasts leads to a transient increase of free stromal Ca²⁺. In dark-kept chloroplasts the steady-state concentration of free stromal Ca²⁺ is 2.4 to 6.3 micromolar as determined by null point titration. These observations support our previous proposal that light-induced Ca²⁺ influx into chloroplasts does not only influence the cytosolic concentration of free Ca²⁺ but also regulates enzymatic processes inside the chloroplast.     

The high-energy state of the thylakoid system as indicated by chlorophyll fluorescence and chloroplast shrinkage

Certain long-term fluorescence phenomena observed in intact leaves of higher plants and in isolated chloroplasts show a reverse relationship to light-induced absorbance changes at 535 nm (“chloroplast shrinkage”).

1. 1. In isolated chloroplasts with intact envelopes strong fluorescence quenching upon prolonged illumination with red light is accompanied by an absorbance increase. Both effects are reversed by uncoupling with cyclohexylammonium chloride.

2. 2. The fluorescence quenching is reversed in the dark with kinetics very similar to those of the dark decay of chloroplast shrinkage.

3. 3. In intact leaves under strong illumination with red light in CO₂-free air a low level of variable fluorescence and a strong shrinkage response are observed. Carbon dioxide was found to increase fluorescence and to inhibit shrinkage.

4. 4. Under nitrogen, CO₂ caused fluorescence quenching and shrinkage increase at low concentrations. At higher CO₂ levels fluorescence was increased and shrinkage decreased.

5. 5. In the presence of CO₂, the steady-state yield of fluorescence was lower under nitrogen than under air, whereas chloroplast shrinkage was stimulated in nitrogen and suppressed in air.

6. 6. These results demonstrate that the fluorescence yield does not only depend on the redox state of the quencher Q, but to a large degree also on the high-energy state of the thylakoid system associated with photophosphorylation.

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Salinity Promotes Accumulation of 3-Dimethylsulfoniopropionate and Its Precursor S-Methylmethionine in Chloroplasts

Wollastonia biflora (L.) DC. plants accumulate the osmoprotectant 3-dimethylsulfoniopropionate (DMSP), particularly when salinized. DMSP is known to be synthesized in the chloroplast from S-methylmethionine (SMM) imported from the cytosol, but the sizes of the chloroplastic and extrachloroplastic pools of these compounds are unknown. We therefore determined DMSP and SMM in mesophyll protoplasts and chloroplasts. Salinization with 30% (v/v) artificial seawater increased protoplast DMSP levels from 4.6 to 6.0 μmol mg⁻¹ chlorophyll (Chl), and chloroplast levels from 0.9 to 1.9 μmol mg⁻¹ Chl. The latter are minimum values because intact chloroplasts leaked DMSP during isolation. Correcting for this leakage, it was estimated that in vivo about one-half of the DMSP is chloroplastic and that stromal DMSP concentrations in control and salinized plants are about 60 and 130 mm, respectively. Such concentrations would contribute significantly to chloroplast osmoregulation and could protect photosynthetic processes from stress injury. SMM levels were measured using a novel mass-spectrometric method. About 40% of the SMM was located in the chloroplast in unsalinized W. biflora plants, as was about 80% in salinized plants; the chloroplastic pool in both cases was approximately 0.1 μmol mg⁻¹ Chl. In contrast, ≥85% of the SMM was extrachloroplastic in pea (Pisum sativum L.) and spinach (Spinacia oleracea L.), which lack DMSP. DMSP synthesis may be associated with enhanced accumulation of SMM in the chloroplast.     

Pb-Induced Avoidance-Like Chloroplast Movements in Fronds of Lemna trisulca L.

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb²⁺, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H₂O₂). In addition, we detected an enhanced accumulation of endogenous H₂O₂ after treatment of plants with lead. Interestingly, H₂O₂-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H₂O₂ can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic effect of lead on chloroplasts can include disturbances in their movement and distribution pattern.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana

Background

The mechanism of the light-dependent movements of chloroplasts is based on actin and myosin but its details are largely unknown. The movements are activated by blue light in terrestrial angiosperms. The aim of the present study was to determine the role of myosin associated with the chloroplast surface in the light-induced chloroplast responses in Arabidopsis thaliana. The localization of myosins was investigated under blue light intensities generating avoidance and accumulation responses of chloroplasts. The localization was compared in wild type plants and in phot2 mutant lacking the avoidance response.

Results

Wild type and phot2 mutant plants were irradiated with strong (36 µEm⁻²s⁻¹) and/or weak (0.8 µEm⁻²s⁻¹) blue light. The leaf tissue was immunolabeled with antimyosin antibodies. Different arrangements of myosins were observed in the mesophyll depending on the fluence rate in wild type plants. In tissue irradiated with weak blue light myosins were associated with chloroplast envelopes. In contrast, in tissue irradiated with strong blue light chloroplasts were almost myosin-free. The effect did not occur in red light and in the phot2 mutant.

Conclusions

Myosin displacement is blue light specific, i.e., it is associated with the activation of a specific blue-light photoreceptor. We suggest that the reorganization of myosins is essential for chloroplast movement. Myosins appear to be the final step of the signal transduction pathway starting with phototropin2 and leading to chloroplast movements.Key Words: Arabidopsis, blue light, chloroplast movements, myosins, phototropins     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Effect of 4-Thiouridine on Photosystems of a Higher Plant

Effect of 4-thiouridine, which was proved to inhibit selectively and “light-reversibly” the synthesis of chloroplast ribosomal RNAs in radish cotyledons, on the photo-induced development of photosystem I, II and a complete electron transport chain was investigated with plastids obtained from 4-thiouridine treated dark-grown radish cotyledons after various times of development in the light. It was demonstrated that the 4-thioridine treated chloroplasts showed a higher activity of photoreduction than the control untreated chloroplasts in every system on a chlorophyll basis during the development after 24 hr illumination. This specific activity decreased in both chloroplasts, as the chloroplasts matured with the time of illumination. The activity per g of fresh cotyledons treated with 4-thiouridine, especially in the early stage of development, was lower than that of ones untreated with the drug because total chlorophyll content was poor, but the activity of the former was enhanced with the increase of total chlorophyll content upon illumination while the activity of the latter decreased on 24 hr illumination. Moreover, Hill reaction measurements showed that 4-thiouridine treated chloroplasts were saturated at lower light intensity than untreated ones inspite of the same content of chlorophyll in both the chloroplasts: photoreduction of NADP⁺ was saturated at 3000 lux for the former and at 5000 lux for the latter. Based upon these results, specific development of the chloroplast is discussed.     

Cation-induced inhibition of the 515-nm absorption change in chloroplasts: relation to structural changes

Divalent cations were found to inhibit the light-induced 515-nm absorption change in chloroplasts with half-maximal effects occurring between 0.3 and 0.7 mm. Monovalent cations were also effective but higher concentrations (~ 30–40 mm) were required for half-maximal effects. Divalent and monovalent cations also caused absorption changes of chloroplasts in the dark which superficially resemble 515-nm absorption changes. However, they can be correlated with volume changes and represent a combination of turbidity and pigment-absorption changes (flattening) which result from shrinkage. Half-maximal effects occurred at 0.8–1.2 mm for divalent cations and between 15 and 20 mm for monovalent cations. The relationship between salt-induced and osmotic-induced structural changes is also discussed.     

Regulation of 5-aminolevulinic Acid synthesis in developing chloroplasts : I. Effect of light/dark treatments in vivo and in organello

Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were returned to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALA-synthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from dark-treated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feed-back inhibitor on ALA synthesis. Chloroplasts isolated from dark-treated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not turn over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment.