Inhibitory effect of p-nitrothiophenol in the light on the Photosystem II activity of spinach chloroplasts

The treatment of spinach chloroplasts with p-nitrothiophenol in the light at acidic and neutral pH's caused specific inhibition of the Photosystem II activity, whereas the same treatment in the dark did not affect the activity at all. The photosystem I activity was not inhibited by p-nitrothiophenol both in the light and in the dark. The inhibition was accompanied by changes of fluorescence from chloroplasts. As observed at room temperature, the 685-nm band was lowered by the p-nitrothiophenol treatment in the light and, at liquid nitrogen temperature, the relative height of the 695-nm band to the 685-nm band increased and the 695-nm band shifted to longer wavelengths. The action spectra for these effects of p-nitrothiophenol on the activity and fluorescence showed a peak at 670 nm with a red drop at longer wavelengths. It was concluded that the light absorbed by Photosystem II is responsible for the chemical modification of chloroplasts with p-nitrothiophenol to causing the specific inhibition of Photosystem II.     

Covalent modification of chloroplast Photosystem II polypeptides by p-nitrothiophenol

Illumination of the chlorophyll $\text{a}\text{b}$ light-harvesting complex in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); $\text{1}\text{2}$ maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of $\text{p-}\text{nitrothio}\text{[}{}^{14}\text{C]phenol}$ caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

Some Factors Concerning the Centripetal Disposition of Bundle Sheath Chloroplasts during the Leaf Development of Eleusine coracana

Factors concerning the chloroplast disposition in bundle sheathcells were investigated in finger millet (Eleusine coracanaGaertn.), and NAD malic enzyme type C₄ plant with the centripetalarrangement of bundle sheath chloroplasts. Segments were cutfrom immature regions of emerging leaves in which the centripetalarrangement of bundle sheath chloroplasts had not yet been established.The leaf segments were floated on solutions with or withoutreagents. Sections were made of the segments at time intervalsand the distribution of bundle sheath chloroplasts was observedby light microscopy. The bundle sheath chloroplasts migratedto the vascular bundle and established a centripetal arrangementby 12-16 h in control solutions. Auxins, cycloheximide and cytochalasinB inhibited the disposition of bundle sheath chloroplasts whilechloramphenicol and colchicine had no effect. The inhibitoryeffect of auxins appeared only at early stages of chloroplastmigration while cycloheximide and cytochalasin B were effectiveeven at later stages. Cessation of elongation growth, cytoplasmicprotein synthesis and microfilaments seemed to be associatedwith the centripetal disposition of bundle sheath chloroplasts.Copyright1993, 1999 Academic Press Bundle sheath chloroplast, C₄ plant, chloroplast orientation, Eleusine coracana, finger millet     

Methyl Viologen-Dependent Cyclic Electron Transport in Spinach Chloroplasts in the Absence of Oxygen

The methyl viologen (MV)-dependent, linear electron flow fromPS II to PS I was severely blocked in intact or broken, uncoupledchloroplasts when oxygen was removed from the suspension medium,as revealed by measurements of chlorophyll fluorescence andthe rate of photoreduction of MV. Kinetics of the reductionof pre-oxidized P700 by a saturating light pulse showed thatreduced MV in the absence of oxygen re-reduces P700⁺ via theintersystem electron transport chain. Since the re-reductionof P700⁺ was inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone,the MV-mediated cyclic electron flow, in contrast to the phenazinemethosulphate-catalyzed one, involves the plastoquinone pool.However, 2-n-heptyl-4-hydro-xyquinoline-N-oxide, 2-n-nonyl-4-hydroxyquinoline-N-oxideand antimycin A did not inhibit the MV-mediated flow. Thus,the inhibition of the linear electron flow in chloroplasts underanaerobic conditions suggested the overreduction of the plastoquinonepool as a result of the MV-mediated cyclic flow (Received February 13, 1990; Accepted March 31, 1990)     

Effects of Deuterium Oxide and Free Radical Scavengers on Carotenoid Photobleaching in Spinach Chloroplasts

The effect of D₂O on carotenoid photobleaching was examinedin spinach chloroplasts poisoned by carbonylcyanide m-chlorophenylhydrazone.D₂O, which prolongs a life time of singlet molecular oxygen,stimulated carotenoid photobleaching under aerobic conditions,but not under anaerobic conditions. The stimulation became smalleras the intensity of actinic light was lowered. Propyl gallateand (+)-catechin, radical scavengers, suppressed photobleaching.The suppression was greater at a low actinic light intensity.These results suggest that cartoenoid is photobleached by singletmolecular oxygen and radical chain reactions. (Received July 17, 1982; Accepted January 13, 1983)     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Regeneration of Ribulose 1,5-bisphosphate and Ribulose 1,5-bisphosphate carboxylase/oxygenase Activity Associated with Lack of Oxygen Inhibition of Photosynthesis at Low Temperature

The nature of the lack of oxygen inhibition of C₃-photosynthesisat low temperature was investigated in white clover (Trifoliumrepens L.). Detached leaves were brought to steady-state photosynthesisin air (34 Pa p(CO²), 21 kPa p(O₂), balance N₂) at temperaturesof 20°C and 8°C, respectively. Net photosynthesis, ribulose1,5-bisphosphate (RuBP) and ATP contents, and ribulose 1,5-bisphosphatecarboxylase/oxygenase (RuBPCO) activities were followed beforeand after changing to 2·0 kPa p(O₂). At 20°C, lowering p(O₂) increased net photosynthesis by37%. This increase corresponded closely with the increase expectedfrom the effect on the kinetic properties of RuBPCO. Conversely,at 8°C net photosynthesis rapidly decreased following adecrease in p(O₂) and then increased again reaching a steady-statelevel which was only 7% higher than at 21 kPa p(O₂). The steady-staterates of RuBP and associated ATP consumption were both estimatedto have decreased. ATP and RuBP contents decreased by 18% and33% respectively, immediately after the change in p(O₂) suggestingthat RuBP regeneration was reduced at low p(O₂) due to reducedphotophosphorylation. Subsequently, RuBP content increased again.Steady-state RuBP content at 2·0 kPa p(O₂) was 24% higherthan at 21 kPa p(O₂). RuBPCO activity decreased by 22%, indicatingcontrol of steady-state RuBP consumption by RuBPCO activity. It is suggested that lack of oxygen inhibition of photosynthesisat low temperature is due to decreased photophosphorylationat low temperature and low p(O₂). This may be due to assimilateaccumulation within the chloroplasts. Decreased photophosphorylationseems to decrease RuBP synthesis and RuBPCO activity, possiblydue to an acidification of the chloroplast stroma. Key words: Oxygen inhibition, photosynthesis, ribulose bisphosphate carboxylase/oxygenase     

Role of pulvinar chloroplasts in light-driven leaf movements of the trifoliate leaf of bean (Phaseolus vulgaris L.)

Laminar pulvini of bean (Phaseolus vulgaris L.) contain numerouschloroplasts in cells of their motor tissue. The quantitativerelationships of the chloroplast pigments, chlorophyll a andb, ß-carotene, lutein, neoxanthin as well as the xanthophyllcycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin)were similar to those of mesophyll chloroplasts from leafletlaminae. Exposure of pulvinules to light caused deepoxidationof violaxanthin to zeaxanthin, showing that the xanthophyllcycle is functioning. Chlorophyll fluorescence analysis of pulvinulesconfirmed that their chloroplasts are capable of both photosyntheticelectron transport and non-photochemical fluorescence quenching,showing that they build up a considerable transthylakoid protongradient in the light. Application of DCMU to excised pulvinulesand laminar discs, as well as to pulvinules of intact, attachedterminal leaflets blocked electron transport and fluorescencequenching. Application of the uncoupler CCCP to intact pulvinulesalso prevented non-photochemical fluorescence quenching. Therate of movement of the low-light-adapted terminal leaflet inresponse to exposure of its pulvinule to overhead red light(500 µmol m^–2 s^–1) was reduced when the pulvinulewas pretreated with DCMU. The pulvinar response to overheadblue light (50 µmol ^–2 s^–1), which is morepronounced than to red light, was not affected by similar pretreatmentwith DCMU. Pretreatment with CCCP caused a short lag in theresponse to red light, but did not affect its subsequent rate.The results suggest that the pulvinar response to red, but notto blue light, requires non-cyclic electron transport and theresulting generation of ATP Key words: Leaf movements, light, non-cyclic electron transport, Phaseolus, pulvinar chloroplasts     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Photoactivation of the Electron Flow from NADPH to Plastoquinone in Spinach Chloroplasts

Intact chloroplasts from spinach showed a transient increasein Chl fluorescence after saturating illumination with actiniclight and its yield depended on the duration of illuminationand the intensity of the actinic light (AL). The increase waspartially suppressed when antimycin A was added immediatelyafter termination of the AL. The inhibited fluorescence increase,therefore, reflected the electron flow from the reductant(s)that had accumulated during the actinic illumination to theplastoquinone (PQ) pool via ferredoxin and the antimycin A-sensitiveCyt b-559 [Miyake et al. (1995) Plant Cell Physiol. 36: 743].Addition of dihydroxyacetone phosphate (DHAP) to chloroplastscaused the enhancement of the increase in fluorescence afterAL, which was inhibited by antimycin A. Decay of the transientlyraised fluorescence was retarded by 2-heptyl-4-hydroxyquinolineN-oxide and stigmatellin, suggesting that re-oxidation of thereduced PQ pool is coupled with the operation of Q-cycle. Althoughthe activity of the stromal enzyme system that supplies NADPHon addition of DHAP was constant irrespective of light or darkness,the capacity of the intact chloroplasts to show a DHAP-dependentfluorescence increase had a limited lifetime after AL was turnedoff. This result suggests that the antimycin A-sensitive Cytb-559 or ferredoxin-NADP reductase is activated by light anddeactivated in the dark. In ruptured chloroplasts, the additionof NADPH increased the dark fluorescence yield only in the presenceof Fd, which also was inhibited by antimycin A. Thus the photoregulatorymechanism of Cyt b-559 (Fd) in intact chloroplasts appearedto be lost when chloroplasts were ruptured. (Received June 21, 1995; Accepted September 25, 1995)     

Polarotropism and Photomovement of Chloroplasts in the Protonemata of the Ferns Pteris and Adiantum: Evidence for the Possible Lack of Dichroic Phytochrome in Pteris

The actions of red and blue light in the photomovement of chloroplastsand the polarotropic response were studied in the protonemataof the homosporous ferns Pteris vittata L. and Adiantum capillus-venerisL. In Pteris, polarotropism could be induced with blue lightbut not with red light, while both colors of light were effectivein Adiantum protonemata. The photomovement of chloroplasts inthe two species studied by both polarized light and microbeamirradiation, also revealed similar responses to red and bluelight as the polarotropism; i.e. both colors of light were effectivein Adiantum but only blue light was active in Pteris. The resultsin Adiantum were consistent with previous results, which ledto the conclusion that both phytochrome and a blue light-absorbingpigment are involved in the two responses (Kadota et al. 1982,1984, Hayami et al. 1986, Yatsuhashi et al. 1985). By contrast,phytochrome is not involved in either polarotropism or chloroplastmovement in Pteris. Since the phytochrome system is evidentlyactive in every other photoresponses so far investigated inPteris as well as in Adiantum, the present study suggests thata phytochrome system specific to polarotropism and to photomovementof chloroplasts is absent in Pteris. Discussions are presentedon the possible involvement of two phytochrome populations ina fern gametophyte cell and on the possible lack of dichroicphytochrome in Pteris. (Received October 7, 1988; Accepted March 8, 1989)     

Light-Dependent Uptake of Pyruvate by Mesophyll Chloroplasts of a C4 Plant, Panicum miliaceum L.

Light-dependent active uptake of pyruvate was reported in mesophyllchloroplasts of a C₄ plant, Panicum miliaceum [Ohnishi and Kanai(1987) Plant Cell Physiol. 28: 1]. The present study tried toclarify the energy source of this active uptake. Preilluminationof the mesophyll chloroplasts increased over tenfold their pyruvateuptake in the light and dark. This indicates that light itselfis not essential for the enhancement. The pyruvate uptake capacity(the initial uptake rate) of the mesophyll chloroplasts increasedon illumination and reached a steady-state level after a fewminutes; this rise was faster under higher light intensities.When the chloroplasts were returned to darkness, the uptakecapacity decayed with a half-life of about 1 min; this was independentof the light intensity of preillumination. Illumination of thechloroplasts also increased the stromal pH from about 7 to 8and the stromal ATP level from about 5 to 15–25 nmol.(mg chl)^–1. The change of the former during dark-to-lightand light-to-dark transitions occurred within 2 to 5 min, whilethe change of the latter took place much faster within 1 min.The steady-state levels of the pyruvate uptake capacity andstromal pH were saturated at a light intensity of 3 µE.m^–2.s^–1,while the ATP level increased with a further increase in thelight intensity. The former two parameters also showed similarsensitivity to the inhibition by carbonylcyanide-m-chlorophenylhydrazone,while a higher concentration of the inhibitor was needed toreduce the ATP level. Nitrite at 4 mM inhibited the light-dependentpyruvate uptake and stromal alkalization but had little effecton the stromal ATP level, while 2 mM arsenate decreased thestromal ATP without significant effects on pyruvate uptake andstromal pH. The good correlation of pyruvate uptake and stromalpH suggests that the active pyruvate uptake by the mesophyllchloroplasts is primarily driven by the pH gradient across theenvelope. (Received August 15, 1986; Accepted December 8, 1986)     

Chlorophyll a forms in mesophyll and bundle sheath chloroplasts of Zea mays leaves grown at low light intensity

Mesophyll and bundle sheath chloroplasts were prepared fromleaves of Zea mays grown at light intensities of 1.1 and 240µW/cm², respectively. The mesophyll chloroplasts thatdeveloped at the low intensity and bundle sheath chloroplatsthat developed at both low and high intensities showed higherratios of chlorophyll a/b and P700/chlorophylls compared withthe normal ratios found for the mesophyll chloroplasts thathad developed at the high intensity. Derivative absorption spectrophotometryat 77?K revealed that the low intensity mesophyll chloroplastscontained more of chlorophyll a forms with longer wavelengthred bands than high intensity mesophyll chloroplasts. More ofthe longer wavelength forms of chlorophyll a were also presentin the bundle sheath chloroplasts that had developed at lowand high intensities. All these four types of chloroplasts showedtwo peaks of fluorescence, one at 687 hra and the other at 733or 738 nm. In addition to these peaks, the high intensity mesophyllchloroplasts showed a shoulder at 697 nm, and the two typesof bundle sheath chloroplasts showed a shoulder at 680 nm. (Received June 17, 1974; )     

Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: The site of light-dependent regulation of electron transport

Dark-adapted intact spinach chloroplasts exhibited two peaks,P and M₁, at the early phase of fluorescence induction and atransient reduction of cytochrome f shortly after its initialphotooxidation and in parallel to the appearance of P. Analysisof the peak P and the transient reduction of cytochrome f indicatedthat electron transport in intact spinach chloroplasts was regulatedby light: electron transport was inactivated at the reducingside of photosystem I in the dark-adapted chloroplasts but rapidlyreactivated by illumination. The fluorescence peak M₁ was correlatedto the proton gradient formed across the thylakoid membrane. Effects on P and transient reduction of cytochromef of NO₂^–,3-phosphoglycerate (PGA) and oxalacetate (OAA), which can penetrateinto intact chloroplasts and accept electrons at different sitesafter photosystem I, were studied to determine the site of thelight regulation. NC₂^–, which receives electrons fromreduced ferredoxin, markedly diminished both P and the transientreduction of cytochrome.f, whereas PGA and OAA, the reductionsof which are NADP-dependent, failed to affect the two transients.The ineffectiveness of PGA and OAA could not be attributed tothe dark inactivation of glyceraldehyde-3-phosphate and malicdehydrogenases, because dark-adapted chloroplasts still retainedsufficiently high levels of the enzyme activities. The resultsindicate that electron transport in intact spinach chloroplastsis regulated by light after ferredoxin but before NADP, i.e.,at the reducing terminal of the electron transport chain. (Received May 29, 1980; )     

Differential Positioning of C4 Mesophyll and Bundle Sheath Chloroplasts: Recovery of Chloroplast Positioning Requires the Actomyosin System

In C₄ plants, bundle sheath (BS) chloroplasts are arranged inthe centripetal position or in the centrifugal position, althoughmesophyll (M) chloroplasts are evenly distributed along cellmembranes. To examine the molecular mechanism for the intracellulardisposition of these chloroplasts, we observed the distributionof actin filaments in BS and M cells of the C₄ plants fingermillet (Eleusine coracana) and maize (Zea mays) using immunofluorescence.Fine actin filaments encircled chloroplasts in both cell types,and an actin network was observed adjacent to plasma membranes.The intracellular disposition of both chloroplasts in fingermillet was disrupted by centrifugal force but recovered within2 h in the dark. Actin filaments remained associated with chloroplastsduring recovery. We also examined the effects of inhibitorson the rearrangement of chloroplasts. Inhibitors of actin polymerization,myosin-based activities and cytosolic protein synthesis blockedmigration of chloroplasts. In contrast, a microtubule-depolymerizingdrug had no effect. These results show that C₄ plants possessa mechanism for keeping chloroplasts in the home position whichis dependent on the actomyosin system and cytosolic proteinsynthesis but not tubulin or light.     

Studies of pesticides effects on Chlorella metabolism II. Effect of DCMU on galactolipid metabolism

DCMU (N'-(3,4-dichlorophenyl)-N, N-dimethylurea) was testedfor effects on the metabolism of galactolipids in Chlorellaand chloroplasts isolated from higher plants. In Chlorella,DCMU affected galactolipid synthesis in the light more thanthat of other lipids, but it showed no effect on lipid synthesisin the dark. DCMU did not affect the turnover of galactolipidsin the light. In vitro studies using ¹⁴C-acetate or ¹⁴C-UDP-galactoseas a precursor showed that DCMU had no effect on the synthesisof gross lipid or galactolipids in chloroplasts isolated fromhigher plants. The significance of these observations are discussed. (Received September 21, 1974; )     

Ethylene-induced changes of chloroplast structure in Satsuma mandarin (Citrus unshiu Marc.)

Ethylene-induced changes of chloroplast structure in Satsumamandarin (Citrus unshiu Marc.) were examined using light andelectron microscopy. In ethylene-treated fruits, the numberof chloroplasts decreased; this was especially remarkable incells distant from the epidermis. Rapid reduction in chloroplastsize was a characteristic feature. The inner membrane system of the chloroplasts of ethylene-treatedfruits disintegrated prior to the disintegration of other cellstructures. The disintegration of the membranes within the chloroplastswas expressed by the word "melt". The double-layered structureof lamellar and granal membranes was degraded and the membranelayers became separated. Another interesting feature was the appearance of finger-likeprotuberances and peripheral reticula in the chloroplasts ofthe ethylene-treated fruits. (Received June 13, 1977; )     

The Level of Stromal ATP Regulates Translation of the D1 Protein in Isolated Chloroplasts

The synthesis of the D1 subunit of the reaction center of photosystemII is light-dependent in isolated chloroplasts. The mechanismof the regulation by light was analyzed using spinach chloroplasts.The light-regulated synthesis of the D1 protein was preventedby the addition of atrazine and the dependence on the concentrationof atrazine of the inhibition was practically identical withthat of the inhibition of photosynthetic electron transportin photosystem II, as measured by the photoreduction of 2,6-dichlorophenolindophenol. Inhibitors of photosynthetic phosphorylation, suchas phloridzin, nigericin and carbonyl cyanide m-chlorophenylhydrazone,also inhibited the light-dependent synthesis of the D1 protein.Determination of the levels of ATP in chloroplasts and the ratesof synthesis of D1 protein under the various degrees of inhibitioncaused by these reagents suggested that the level of ATP inthe soluble, stromal fraction can control the synthesis of theD1 protein. The level of stromal ATP in chloroplasts was furthermanipulated, either by modulating the intensity of actinic lightor by the addition of metabolites, such as glycerate, whichwas used to decrease the level of ATP in the light, and dihydroxyacetonephosphate/oxaloacetate, which was used to raise the level ofATP in the dark. The results definitely support the hypothesisthat the light-induced level of ATP is an essential determinantin the regulation of the synthesis of the D1 protein in isolatedchloroplasts. (Received July 25, 1991; Accepted October 22, 1991)