Kinetic study of oxygen evolution parameters in tris-washed,reactivated chloroplasts

Tris-washed chloroplasts which have lost the ability to evolve oxygen can be reactivated by the procedure of Yamashita. T., Tsuji, J. and Tomita, G. ((1971)Plant Cell Physiol. 12, 117–126) [7] to give 100% of the rate of control chloroplasts in continuous illumination. Furthermore, in flashing light the reactivated chloroplasts exhibit oxygen-yield oscillations of period four that are characteristic of the control. Similar kinetic parameters for intermediate steps in the water-splitting process are observed for the two preparations. We conclude that the reactivation procedure restores the native oxygen evolution mechanism to Tris-washed chloroplasts.A relatively rapid and reversible (0.5 s decay) light-induced component of EPR Signal II is observed upon inhibition of O₂ evolution by Tris washing (Babcock G. T. and Sauer, K. (1975) Biochim. Biophys. Acta 376, 315–328) [10]. Reactivated chloroplasts are similar to untreated chloroplasts in that this Signal II transient is not observed. Manganese, which is released by Tris treatment to the interior of the thylakoid membrane in an EPR-detectable state, is returned to an EPR-undetectable state by reactivation. The reactivation procedure does not require light to restore O₂ evolution and EDTA has no effect on the extent of reactivation. These results are discussed in terms of possible mechanisms for manganese incorporation into photosynthetic membranes.     

Comparative study of the reactivation of oxygen evolution in chloroplasts inhibited by various treatments

Reactivation of photosynthetic oxygen-evolution was investigatedwith chloroplasts inhibited by 0.8 M Tris-, 0.8 M Tris-20% acetone-,0.8 M KCl-, 0.5 M NaClO₄- or 1 mM NH₂OH-washing, and with heat-treatedor aged chloroplasts. These chloroplasts restored oxygen evolvingactivity by two successive treatments; incubation of chloroplastswith reduced DPIP, then with Mn^2$, Ca^2$, dithiothreitol andbovine serum albumin under weak illumination (light-reactivation). Some factors required for light-reactivation could be omitteddepending on the inhibition treatment. For example, Mn^2$, Ca^2$and dithiothreitol were not necessary for (1 mM NH₂OH-STN (pH7.0)-washed)-DPIP-treated chloroplasts, and dithiothreitol for(Tris-acetone (pH 8.4)-washed)-DPIP-treated chloroplasts. Uncouplers, such as atebrin, CCCP, DCCD and NH₄Cl, inhibitedthe lightreactivation. The Mn and Ca contents of the chloroplasts were determined withinhibited and DPIP-treated chloroplasts. The Mn content of thechloroplasts tended to decrease with increasing pH of the washingmedium for inhibition. The Ca content decreased when chloroplastswere washed with 0.8 M KCl. (Received November 22, 1974; )     

Inhibition of the phosphate transporter during isolation of intact chloroplasts from leaves of sunflower

Chloroplasts isolated from spinach leaves by the mechanical method were intact and exhibited high rates of CO₂-dependent oxygen evolution whereas chloroplasts isolated from sunflower leaves by the same technique were also intact but showed only low rates of oxygen evolution. The rate of uptake of orthophosphate (Pi) from the suspending medium with sunflower chloroplasts was less than 20% of that in spinach chloroplasts. The apparent Km for Pi transport was lower in sunflower chloroplasts but uptake was competitively inhibited by 3-phosphoglycerate in chloroplasts from both species. Uptake of malate (via the dicarboxylate transporter) and of ATP (via the adenine nucleotide transporter) was also reduced in sunflower chloroplasts compared to spinach chloroplasts. The endogenous Pi content and total exchangeable phosphate pool of sunflower chloroplasts were less than half that in spinach chloroplasts.Addition of a number of possible protective agents to the grinding medium failed to prevent the loss of photosynthetic activity during mechanical isolation of sunflower chloroplasts. Grinding mixtures of spinach and sunflower leaves together indicated that spinach chloroplasts were not inhibited by the sunflower leaf extract. Chloroplasts isolated from sunflower leaves via protoplasts had high rates of CO₂-dependent oxygen evolution. The Vmax and Km for Pi uptake, endogenous Pi content and total exchangeable phosphate pool of chloroplasts isolated from sunflower protoplasts were all similar to spinach chloroplasts. It is concluded that inner envelope membrane proteins are damaged during mechanical isolation of sunflower chloroplasts. The decrease in activity of the phosphate transporter and loss of endogenous phosphate may contribute to the low rates of photosynthesis observed in chloroplasts isolated by the mechanical method from leaves of sunflower and possibly other species.Abbreviations PGA 3-phosphoglyceric acid     

EFFECT OF LIPASE-DIGESTION ON PHOTOCHEMICAL ACTIVITIES OF ISOLATED CHLOROPLASTS

In order to elucidate the role of lipids in photosynthesis,chloroplasts were digested with lipase, and the effect of lipase-digestionon some photochemical activities was studied. The HILL reactionwas sensitive to the digestion, but chloroplasts having intactmembrane were somewhat resistant to the action of lipase. Theinactivation by lipase digestion seems to be due to the destructionof a component necessary for the Hill reaction to proceed. Thechloroplasts treated with lipase showed the following activities. (1) Active photooxidation of reduced cytochrome c and menadione. (2) Photooxidation of ascorbate, which was enhanced in the presenceof DPIP, and retarded in the absence of the dye. (3) NADP-photoreduction in the presence of the DPIP-ascorbatecouple, as the electron donor. These facts suggested that the site attacked with lipase wasresponsible for the photochemical oxygen evolution. The decrease in the fluorescence intensity of chlorophyll awas also observed during the digestion. ¹Present address : Biological Laboratory, General EducationDeparment, Kyushu University, Otsubo-machi, Fukuoka.     

Effect of Thiolactomycin on Lipid Synthesis in Higher Plants

The effect of thiolactomycin (TLM), an inhibitor of type IIfatty acid synthase, on lipid synthesis in greening tissueswas examined. Pulse-chase experiments with Na[1-¹⁴C]acetatefor greening Avena leaves showed that continuous administrationof TLM (100µg/ml) decisively reduced phosphatidylcholine(PC) synthesis from acetate and blocked the subsequent conversionof PC to monogalactocyldiacylglycerol (MGDG), whereas temporaladministration of TLM (100 µ/ml) reduced PC synthesisfrom acetate by only 50% and did not block the conversion ofPC to MGDG. In the reduced PC synthesis, the ratio of oleicto palmitic acid decreased at earlier stages of greening, reflectingmore suppression of oleic acid synthesis. In later greeningstages the modulated fatty acid composition recovered to thenormal composition. In further steps, the fatty acid compositionwas not affected by TLM throughout the greening stages. Greeningof either etiolated Avena leaves or etiolated Brassica cotyledonsin the presence of TLM led to a marked decrease in the contentsof MGDG, digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol(PG), but only a small change in the fatty acid compositionof their lipids. The only inhibition characteristic of TLM wasthe desaturation of palmitic to 3-trans-hexadecenoic acid inAvena leaf PG. These results suggest the presence of a mechanismby which the modulated fatty acid composition of lipids is normalizedin the flow of the synthesis. Electron microscopic observationsshowed that Avena chloroplasts developed into round forms ratherthan normal ellipse forms and the thylakoid membranes of Brassicachloroplasts were abnormally swollen everywhere in the presenceof TLM. Photosynthetic oxygen evolution in both tissues wasnot inhibited. (Received December 26, 1986; Accepted April 24, 1987)     

Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

Analysis of Rapid Light-Induced Potential Change in Cells of Chara corallina

Perfused Chara cells were used to measure the rapid light-inducedpotential change (rapid LPC) caused by activation of a K⁺ channelin the plasma membrane through photosynthesis in the presenceof various photosynthetic inhibitors. The rapid LPC was inhibitedby DCMU but recovered on addition of phenazinemethosulfate (PMS)in the presence of DCMU. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated the rapid LPC. DCCD partially inhibited therapid LPC with a partial inhibition of oxygen evolution. Itis concluded that both cyclic and noncyclic electron flows arecoupled with the rapid LPC. To understand the mechanism of K⁺ channel activation by photosyntheticelectron flow, the rapid LPC was measured under continuous internalperfusion. It was suggested that a diffusible substance wasnot released from chloroplasts, since vigorous continuous perfusiondid not inhibit the rapid LPC. The suggestion that the rapid LPC is caused by changes in surfacecharge density of chloroplasts was supported by the fact thatthe rapid LPC was inhibited by increasing the ionic strengthof the perfusion medium. (Received February 28, 1986; Accepted April 30, 1986)     

On the functional site of manganese in photosynthetic electron transport system

Normal Euglena chloroplasts contained 1 atom of Mn per 47±8chlorophyll molecules. The manganese content of chloroplastswas decreased by heat treatment. After complete removal of manganeseby incubation at 45°C for 5 min, Hill activity with DPIPas electron acceptor was abolished, but the activity of DPIPphotoreduction with diphenylcarbazide as electron donor wasunaffected. Hill activity was inactivated by incubating Euglena chloroplastsat alkaline pH. The presence of a high concentration of Trisduring incubation of chloroplasts at an alkaline pH had no additionaleffect on the activity drop. Donor-supported DPIP photoreduction in heated Euglena chloroplasts,as well as the normal Hill reaction in untreated chloroplasts,was inhibited by DCMU, HOQNO and ioxynil which block electrontransport at the reducing side of system II. These reactionswere also inhibited by another group of inhibitors; CCCP, salicylaldoxime,antimycin A and azide, which block electron transport at a sitebetween the electron carriers, Y₁ and Y₂ located on the oxidizingside of system II. Possible sites of inhibition by heat treatment and by inhibitorsand sites for entry of electrons from artificial electron donorsin the photosynthetic electron transport chain, especially inrelation to the functional site of endogenous manganese in chloroplasts,were proposed. (Received October 30, 1971; )     

Relationship between Photosynthesis and Chlorophyll Content during Leaf Senescence of Rice Seedlings

Photosynthetic oxygen evolution, chlorophyll contents and chlorophylla /b ratios of 3rd to 6th leaves of rice seedlings were measuredto examine whether or not inactivation of photosynthesis duringsenescence is related to loss of chlorophyll. Photosyntheticactivity decreased more rapidly than chlorophyll content duringleaf senescence; as a result, the lower the leaf position, thelower was the rate of oxygen evolution determined on the basisof chlorophyll. Chlorophyll ab ratio also decreased with advancingsenescence. Electrophoretic analysis revealed that the declinein chlorophyll ab ratio is due to more rapid degradation ofthe reaction center complexes than light-harvesting chlorophyllab proteins of photosystem II and that the photosystem I reactioncenter disappears in parallel with the inactivation of photosynthesis.A simple method was developed to estimate the amounts of chlorophylla associated with the reaction center complexes of the two photosystemsfrom the total chlorophyll contents and chlorophyll ab ratiosof leaves. Rates of oxygen evolution, determined on the basisof chlorophyll a bound to the reaction center complexes, remainedconstant throughout the course of senescence. Thus, inactivationof photosynthesis is closely related with loss of the reactioncenter complexes during leaf senescence of rice seedlings. Theresults suggest that loss of photosynthesis is mainly causedby loss of a functional unit of photosynthesis or by a decreasein the number of whole chloroplasts. (Received April 22, 1987; Accepted August 13, 1987)     

Kinetic study of oxygen evolution parameters in Triswashed, reactivated chloroplasts.

Tris-washed chloroplasts which have lost the ability to evolve oxygen can be reactivated by the procedure of Yamashita T., Tsuji, J. and Tomita G. (1971) Plant Cell Physiol. 12, 117-126) [7] to give 100 percent of the rate of control chloroplasts in continuous illumination. Furthermore, in flashing light the reactivated chloroplasts exhibit oxygen-yield oscillations of period four that are characteristic of the control. Similar kinetic parameters for intermediate steps in the water-splitting process are observed for the two preparations. We conclude that the reactivation procedure restores the native oxygen evolution mechanism to Tris-washed chloroplasts. A relatively rapid and reversible (0.5 s decay) light-induced component of EPR Signal II is observed upon inhibition of O2 evolution by Tris washing (Babcock G. T. and Sauer, K. (1975) Biochim. Biophys. Acta 376, 315-328) [10]. Reactivated chloroplasts are similar to untreated chloroplasts in that this Signal IItransient is not observed. Manganese, which is released by Tris treatment to the interior of the thylakoid membrane in an EPR-detectable state, is returned to an EPR-undetectable state by reactivation. The reactivation procedure does not require light to restore O2 evolution and EDTA has no effect on the extent of reactivation. These results are discussed in terms of possible mechanisms for manganese incorporation into photosynthetic membranes.     

Ageing of Euglena chloroplasts in vitro I. Variations in pigment pattern and in morphology

Isolated chloroplasts of Euglena gracilis Klebs were kept for10 d in complete darkness at 4 C in a maintenance buffer (pH7.5) without shaking. During incubation, the qualitative andquantitative changes in the pattern of photosynthetic pigmentswere evaluated by the combined use of spectrophotometry in thevisible range of whole chloroplasts and their acetone extracts,of in vivo spectrofluorimetry and of reversed-phase HPLC. Microscopicand submicroscopic modifications were also followed by UV andtransmission electron microscopy. The main findings were as follows: (1) a fast decay of all photosyntheticpigments, chiefly chlorophylls, not accompanied by evident signsof alteration of the thylakoid system during the first 5 d;(2) a higher stability of PSII compared to PSI and of antennacomplexes compared to the relative reaction centres during thefirst 24–48 h; (3) a low accumulation of phaeoderivativecompounds in spite of the marked decrease of chlorophyll content;(4) a lack of dephytylated compounds; (5) a quicker decay ofthe intensity of fluorescence emission with respect to the decreasingchlorophyll a content; and (6) a fast degradation of xanthophyllsand ß-carotene with the consequent lack of defencefrom the ageing oxidative stresses. This accounts for the rapidloss of pigments, although the lack of other antioxidant defencemechanisms is not excluded. The characterization of some of the steps involved in plastiddegradation may render this experimental model viable for furtherstudies on plastid senescence, a multifactorial process stillawaiting definite answers. Key words: Euglena gracilis, ageing, isolated chloroplasts, morphological changes, pigment degradation     

The roles of weak light,oxygen and dithiothreitol in the photoreactivation of the oxygen evolving center of tris-washed and 2, 6-dichlorophenol indophenol-treated chloroplasts

The inactivated O₂-evolving center of Tris-washed chloroplasts was reactivated by DCPIP-treatment and photoreactivation in the presence of Mn²⁺, Ca²⁺, DTT and weak light. Many electron donors (Asc and reduced DCPIP, etc.) were found to be suitable substitutes for DTT. By studying the anaerobic inhibition of the reactivation, the electron acceptors O₂, NADP⁺, etc. were also found to be essential factors in photoreactivation. Weak light stimulated the chloroplast electron transport from the above-mentioned electron donors to the electron acceptor and effected the photoreactivation. More than 280 electrons were transported to NADP⁺ in the anaerobic photoreactivation of one unit of an O₂-evolving center with 400 Chl. Electron transport in the reactivation was inhibited by omitting DTT or Mn²⁺ ion, and by adding DCMU. The photoreactivated chloroplasts incorporated about 2 Mn by 400 Chl. Omission of DTT in the reactivation caused chloroplasts in the weak light to bind large amounts of excess Mn.Abbreviations Asc ascorbate - Chl chlorophyll - DCPIP 2, 6-dichlorophenol indophenol - DPC diphenyl carbazide - DTT dithiothreitol - Fd ferredoxin - STN a chloroplast preparation medium, containing 0.4 M sucrose, 0.05 M Tris-Cl and 0.01 M NaCl (pH 7.8 and 8.0) - TMPD tetramethyl-p-phenylenediamine     

Inhibition of Photosynthesis by Sulfite in Mesophyll Protoplasts Isolated from Vicia faba L. in Relation to Intracellular Sulfite Accumulation

Inhibition of photosynthesis by Na₂SO₃ in mesophyll protoplastsisolated from Vicia faba leaves and uptake of sulfite by theprotoplasts were examined at various pH values of the incubationmedium containing Na₂SO₃. As the pH of the incubation mediumlowered, the rate of photosynthesis in the protoplasts decreasedand the amount of sulfite taken up by the protoplasts increased.Most of sulfite accumulated in the protoplasts was not metabolizedduring the dark incubation, as measured with an ion chromatograph.Photosynthetic O₂ evolution by the chloroplasts isolated fromVicia mesophyll protoplasts was inhibited by exogenously-appliedNa₂SO₃ over pH region examined (7.4–9.0). The sulfiteconcentration required for a half inhibition of photosynthesisby the isolated chloroplasts was similar to the intracellularsulfite level required for that by the protoplasts. These resultsindicate that the intracellular sulfite accumulated in the protoplastsin an unmetabolized state is responsible for the inhibitionof protoplast photosynthesis. (Received January 24, 1985; Accepted May 29, 1985)     

Ontogenetic Change of the PSI- and PSII-Distribution in the Thylakoid System of Euglena gracilis

The thylakoid membranes of isolated Euglena chloroplasts wereseparated into two fractions by aqueous two-phase-partitioning(mixture of dextran 500 and poly(ethylene glycol) 4000) followingpress disruption. These two fractions differ in many respectsduring most of the cell cycle of this alga in comparison withthe thylakoid characteristics of higher plants or green algae.The amount of thylakoid membranes with separation characteristicscomparable with inside-out-vesicles of higher plant chloroplastschanges depending on the cell cycle stage of Euglena gracilis.Photosystems II and I are not restricted to one fraction. Boththylakoid membrane fractions evolve oxygen photosynthetically.When chloroplast differentiation in Euglena gracilis is complete(i.e. at the end of the light-time) the composition and thephotosynthetic efficiency of the two thylakoid fractions aregenerally equal. Photosystem I-related LHCI is present in bothfractions. Photosystem II-related CP29, however, was only detectedin unfractionated thylakoid membranes. The implications forthylakoid organization in Euglena chloroplasts are discussed. Key words: Euglena gracilis, photosystem I, photosystem II, stacking, thylakoids     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

Oxygen evolution and the permeability of the outer envelope of isolated whole chloroplasts

A rapid oxygraph method of studying the permeability of the envelope of isolated chloroplasts was used. The outer envelope of aqueously isolated whole spinach (Spinacia oleracea L.) chloroplasts in buffer is readily permeable to 3-phosphoglyceric acid, which induces an immediate light dependent oxygen evolution. This light dependent oxygen evolution was completely eliminated by swelling these plastids in an osmotically dilute solution. Exogenous adenosine diphosphate, but not inorganic phosphate, strongly stimulated this oxygen evolution. This indicated that the chloroplast envelope is relatively permeable to adenosine diphosphate.

Oxygen evolution and swelling studies indicated that the chloroplast envelope is relatively impermeable to NADP and to ferredoxin.

A method is described whereby the percent of whole chloroplasts present in a chloroplast preparation may be rapidly estimated.

     

Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

Studies on the manganese of the chloroplast

Manganese deficiency of green plants is known to affect preferentially the activity of the oxygen evolving system in the photosynthetic apparatus. Our studies showed that the time needed to reactivate photosynthesis in Mn-deficient algae varies with each culture, and is often very short when Mn is added not before illumination but during the light period. The recent finding by Cheniae and Martin that the reactivation requires light, is confirmed. The plain incorporation of ⁵⁴Mn into deficient algae as distinguished from reactivation was barely affected by light, yet was inhibited by uncouplers of phosphorylation. Higher plants responded to manganese deficiency either by adjusting the number of chloroplasts per cell to the limited Mn supply, or by forming disorganized chloroplasts with low chlorophyll content. These 2 types of responses produced chlorotic plants which had either a few photosynthetically active or many disabled chloroplasts. Photosystem I mediated photophosphorylation turned out to be much more sensitive to manganese deficiency than the system I dependent photoreduction of NADP⁺.     

Uptake of ATP analogs by isolated pea chloroplasts and their effect on CO2 fixation and electron transport

1. The ATP analog, adenylyl-imidodiphosphate rapidly inhibited CO₂-dependent oxygen evolution by isolated pea chloroplasts. Both α, β- and β, γ-methylene adenosine triphosphate also inhibited oxygen evolution. The inhibition was relieved by ATP but only partially relieved by 3-phosphoglycerate. Oxygen evolution with 3-phosphoglycerate as substrate was inhibited by adenylyl-imidodiphosphate to a lesser extent than CO₂-dependent oxygen evolution. The concentration of adenylyl-imidodiphosphate required for 50% inhibition of CO₂-dependent oxygen evolution was 50 μM.2. Although non-cyclic photophosphorylation by broken chloroplasts was not significantly affected by adenylyl-imidodiphosphate, electron transport in the absence of ADP was inhibited by adenylyl-imidodiphosphate to the same extent as by ATP, suggesting binding of the ATP analog to the coupling factor of phosphorylation.3. The endogenous adenine nucleotides of a chloroplast suspension were labelled by incubation with [¹⁴C]ATP and subsequent washing. Addition of adenylyl-imidodiphosphate to the labelled chloroplasts resulted in a rapid efflux of adenine nucleotides suggesting that the ATP analog was transported into the chloroplasts via the adenine nucleotide translocator.4. It was concluded that uptake of ATP analogs in exchange for endogenous adenine nucleotides decreased the internal ATP concentration and thus inhibited CO₂ fixation. Oxygen evolution was inhibited to a lesser extent in spinach chloroplasts which apparently have lower rates of adenine nucleotide transport than pea chloroplasts.     

Stoichiometries of Photosystem I and Photosystem II in Rice Mutants Differently Deficient in Chlorophyll b

Stoichiometries of photosystem I (PSI) and photosystem II (PSII)reaction centers in a cultivar of rice, Norin No. 8, and threechlorophyll b-deficient mutants derived from the cultivar wereinvestigated. Quantitation of PSI by photooxidation of P-700and chromatographic assay of vitamin K₁ showed that, on thebasis of chlorophyll, the mutants have higher concentrationsof PSI than the wildtype rice. Greater increases were observedin the PSII contents measured by photoreduction of Q_A, bindingof a radioactive herbicide and atomic absorption spectroscopyof Mn. Consequently, the PSII to PSI ratio increased from 1.1–1.3in the wild-type rice to 1.8 in chlorina 2, which contains noChl b, and to 2.0–3.3 in chlorina 11 and chlorina 14,which have chlorophyll a/b ratios of 9 and 13, respectively.Measurement of oxygen evolution with saturating single-turnoverflashes revealed that, whereas at most 20% of PSII centers areinactive in oxygen evolution in the wildtype rice, the non-functionalPSII centers amount to about 50% in the three mutant strains.The fluorescence induction kinetics was also analyzed to estimateproportions of the inactive PSII in the mutants. The data obtainedsuggest that plants have an ability to adjust the stoichiometryof the two photosystems and the functional organization of PSIIin response to the genetically induced deficiency of chlorophyllb. (Received July 29, 1994; Accepted February 7, 1996)