Effect of linolenate on photosynthesis by intact spinach chloroplasts II. Influence of preillumination of leaves on the inhibition of photosynthesis by linolenate

The inhibitory effect of linolenate on intact spinach chloroplastsdepends on the level of the internal pool of metabolites. Chloroplastsfrom preilluminated leaves or chloroplasts artificially loadedwith 3-phosphoglyceric acid required higher concentrations oforthophosphate for maximal rates of CO₂ dependent O₂ evolutionthan untreated chloroplasts. The loaded chloroplasts were moresensitive to linolenate, and in the presence of linolenate theoptimal phosphate concentration was shifted toward lower values.We propose that the inhibition of photosynthesis by linolenateis due to inhibition of the "phosphate translocator". ¹ Part of this work has been published in the Book of Abstracts,4th International Congress on Photosynthesis, Reading, U.K.,1977, p. 265–266. ² This work is part of a doctoral programme carried out by L.Mv6 Akamba in this laboratory. ³ To whom reprint requests should be adressed. (Received October 14, 1978; )     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

{beta}-Naphthyl oligophosphates: Inhibitors of photophosphorylation and H+-ATPase of spinach chloroplasts

ß-Naphthyl di-, tri- or tetraphosphate inhibits photophosphorylationof spinach chloroplasts competitively with ADP, whereas ß-naphthylmonophosphate inhibits it competitively with Pi. The apparentKi of ß-naphthyl diphosphate for the ADP site was300 µM and that of ß-naphthyl monophosphatefor the Pi site was 1.45 mM. At 10 mM, both of these two organicphosphates inhibited photophosphorylation more than 90%. Noneof the above four ß-naphthyl phosphates were phosphorylatedby chloroplasts. ß-Naphthyl di-, tri- or tetraphosphateinhibits ATPase activity of isolated chloroplast coupling factor1 (CF₁) (EC 3.6.1.3 [EC] ) and light-triggered ATPase activity ofchloroplasts competitively with ATP, whereas ß-naphthylmonophosphate acts non-competitively. None of the four ß-naphthylphosphates were hydrolyzed by these two ATPase activities. Atconcentrations equal to ADP or ATP, ß-naphthyl di-,tri- or tetraphosphate inhibited these three reactions in theorder; ATPase of isolated CF₁> photophosphorylation>light-triggeredATPase of chloroplasts. The results suggest that the effect of the monophosphate isprincipally on the Pi site(s) and that of the di-, tri- or tetraphosphateis on the adenine nucleotide site(s) on the active center ofCF₁. ¹Part of this work was reported at the 1979 Annual Meeting ofthe Japanese Society of Plant Physiologists (Nagoya, April 7,1979) and the 52nd Annual Meeting of the Japanese BiochemicalSociety (Tokyo, October 7, 1979). This work was supported inpart by Grants-in-Aid for Scientific Research from the Ministryof Education, Science and Culture, Japan (311808 and 311909). (Received November 14, 1979; )     

Studies on photophosphorylation II. Uncoupling of chloroplasts by anions at low temperatures and at acidic pH values

Photophosphorylation of spinach chloroplasts was uncoupled bypreincubation at 0°C in the presence of a neutral salt atpH 6.0 to 6.5 ("cold-anion uncoupling"). Preincubation at 20°Ccaused some depression in both photophosphorylation and theHill reaction, but the efficiency of photophosphorylation wasnot depressed much. Low pH values accelerated uncoupling. Theeffectiveness of anions tested as sodium salts in inducing uncouplingwas of the order: SCN->>NO₃^–>Cl^–>SO₄^2–There was little difference in effectiveness among monovalentcations; LiCl, NaCl, KCl, RbCl and CsCl. 10^–4M ATP orADP largely protected chloroplasts from cold-anion uncoupling.Addition of EDTA-extract or dicyclohexylcarbodiimide to uncoupledchloroplasts partially restored photophosphorylation. Theseobservations suggest that inactivation of chloroplast ATPaseis one cause of cold-anion uncoupling. At low light intensities, the time lag and the depression ofthe efficiency of photophosphorylation were more pronouncedin cold-anion uncoupled chloroplasts than in the control chloroplasts. (Received February 15, 1972; )     

LOCALIZATION OF LIGHT-INDUCED STRONTIUM ACCUMULATION IN SPINACH CHLOROPLASTS

Electron-microscopic evidence showing the location of strontiumand calcium deposits on the lamellae of spinach chloroplastsis presented. The chloroplast limiting membrane did not actas a barrier for this ion accumulation, since the chloroplastswere ruptured during the isolation and incubation, especiallyin the light. The grana stacks also were extensively swollen.The number of electrondense deposits attached on the lamellaewas greatly increased by light under conditions favoring thehydrolysis of ATP. The average diameter of the deposits aftera 6 minute illumination with 1 mM SrCl₂ was 14 mµ, after15 minutes with 1 mM SrCl₂ was 21 mµ, and after 15 minuteswith 20 µM SrCl₂ was 13 mµ. The findings are inharmony with the results of ion-uptake experiments using radioactivestrontium and calcium. ¹ United States National Science Foundation Postdoctoral Fellow.     

Energy conversion and changes in physical parameters in chloroplasts and subchloroplast particles II. Energy conversion in chloroplasts and different types of subchloroplast particles

The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH₄Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH₄Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC₄Cl, but in subchloroplast particles the additionalinhibitory effect of NH₄Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH₄Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH₄Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH₄Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation. ¹Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan. ²Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; )     

Anion-dependent changes of energy transfer in chloroplasts I. Effects of Tris salts on electron flow and phosphorylation

Washing spinach chloroplasts with high-concentration Tris-saltbuffers induced various types of anion-dependent changes inthe electron flow and photophosphorylation in chloroplasts. Tris-HCl buffer caused enhancement of NADP photoreduction andinhibition of phosphorylation. Tris-HNO₃ buffer, on the otherhand, caused inhibition of both electron flow and phosphorylationand decreased trypsin-activated Ca²+-dependent ATPase activity.Tris-H₂SO⁴ and Tris-H₃PO⁴ buffers, however, had no effect onthe rates of electron flow and photophosphorylation. Determination of the presence of the coupling factor (as measuredby ATPase activity) revealed a normal enzyme activity levelin chloroplasts washed with Tris-HCl or Tris-H₂SO⁴ buffer. Removalof the coupling factor by EDTA from chloroplasts washed withTris salts inhibited phosphorylation severely. Phosphorylationactivity could be partially restored by reconstitution withthe coupling factor in die presence of Mg²+. In addition to their different effects on electron flow, Tris-HCland Tris-HNO₃ induced a marked decrease in phosphorylative activityitself. The much decreased rate of phosphorylation can be explainedby the release of the coupling factor and by damage to the high-energystate generating mechanism by Tris-HNO₃-washing and by modificationof the coupling factor in the case of Tris-HCl-washing. ¹Present address: Biology Department, College of Science andEngineering, Ryukyu University, Naha, Okinawa. Japan. (Received June 27, 1972; )     

Light-enhanced carbon dioxide fixation in isolated chloroplasts

Preillumination of leaves of spinach, soybean and maize in theabsence of CO₂ greatly enhanced the capacity for fixing CO₂in an immediately following dark period. Lightenhanced darkCO₂-fixation was further observed in isolated chloroplasts ofspinach and soybean. When isolated chloroplasts were illuminated,CO₂-fixing capacity in the subsequent dark period increasedrapidly at first and later more slowly attaining a stationaryvalue in about 20 min. When the light was turned off at thisstage, the capacity decreased very rapidly becoming zero inabout 10 min. The magnitude of the enhanced dark fixation andits decay in the dark were not influenced by the presence orabsence of atmospheric oxygen. In both leaves and isolated chloroplasts,no significant change in oxygen (21%) occurred in distributionpatterns of radioactivity in products fixed by photosynthetic,or light-enhanced, dark, ¹⁴CO₂-fixation. In preilluminated leaves¹⁴C was incorporated into sucrose in the subsequent dark period,indicating that the photosynthetic carbon reduction cycle isoperating in light-enhanced dark fixation in higher plants. ¹Present address: Noda Institute for Scientific Research, Noda,Chiba Prefecture (Received August 10, 1970; )     

Anion-dependent changes of energy transfer in chloroplasts II. Relationships of the coupling factor to light-induced proton transport and absorbance change at 515 nm

Roles of the coupling factor in light-induced proton transportand 515-nm absorption change were investigated in chloroplastswashed with high concentrations of Tris salts (pH 7.2). Washingthe chloroplasts with Tris-HCl and Tris-HNO₃ buffers diminishedboth the light-induced pH rise and absorbance change at 515-nm,while Tris-H₂SO₄ buffer was much less effective. Inhibited activitiescould be restored by replacement of the coupling factor afterextraction with EDTA. N,N'-dicyclohexylcarbodiimide also restoredboth activities. Effects of various anions on the proton pumpand 515-nm shift were also investigated. The order of effectivenesswas NO₃^–>Cl^–>SO₄^2–. The role of thecoupling factor and its mode of action; the action mechanismsof Tris and anionsn energy transducing processes in chloroplasts,photophosphorylation, proton transport and absorbance changeat 515 nm, are discussed. ¹Present address: Biology Department, College of Science andEngineering, Ryukyu University, Naha, Okinawa, Japan. (Received June 27, 1972; )     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

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; )     

Inhibition of photosystem I and photosystem II in chloroplasts by UV radiation

The effects of UV radiation on the low temperature fluorescenceand primary photochemistry of PSII and PSI of spinach chloroplastswere studied. Fluorescence induction curves at –196°Cwere measured at 695 nm for PSII fluorescence and at 730 nmfor PSI fluorescence to determine both the initial F_o and finalF_M levels. The primary photochemistry of PSII was measured asthe rate of photoreduction of C-550 at – 196°C, thatof PSI as the rate of photooxidation of P₇₀₀ at –196°C.The results were analyzed in terms of a model for the photosyntheticapparatus which accounts for the yields of fluorescence andprimary photochemistry. According to this analysis UV radiationincreases nonradiative decay processes at the reaction centerchlorophyll of PSII. However, the effect of UV radiation isnot uniform throughout the sample during irradiation so thataccount must be taken of the fraction of PSII reaction centerswhich have been irradiated at any given time. UV radiation alsoinactivates P700 and causes a slight increase in nonradiativedecay in the antenna chlorophyll of PSI. All fluorescence ofvariable yield, F_V = F_M–F_o, at 730 nm is due to energytransfer from PSII to PSI so that the sensitivity of Fv to UVradiation is the same at 730 and 695 nm. ¹Present address: Department of Biology, Faculty of Science,Toho University, Narashino, Chiba 275, Japan. ²Present address: Central Research Laboratories, Fuji PhotoFilm Co., Ltd., 105 Mizonuma, Asaka-Shi, Saitama 351, Japan. (Received September 10, 1975; )     

Specific inhibition of photosystem II activity in chloroplasts by fumigation of spinach leaves with SO2

The phytotoxic effects of sulfur dioxide (SO₂) were investigatedby fumigating spinach plants with SO₂. Inhibition of 2,6-dichloroindophenol(DCIP) photoreduction was observed in spinach chloroplasts isolatedfrom fumigated leaves. NADP and DCIP photoreductions were inhibitedto a similar extent by fumigation with 2.0 ppm SO₂ but electronflow from reduced DCIP to NADP was not affected. When electronflow from H₂O to NADP was inhibited by 36%, a 39% inhibitionof non-cyclic photophosphorylation was observed. However, phenazinemethosulfate(PMS)-catalyzed cyclic photophosphorylation wasas active as in the control chloroplasts. Moreover, in the presenceof PMS, no significant suppression was observed in the extentof light-induced H⁺ uptake or in the rate of H⁺ efflux in chloroplasts.From these results, it can be concluded that SO₂ inhibits theelectron flow driven by photosystem II when plants have beenfumigated with SO₂. In spinach leaves fumigated with SO₂, the rate of photosyntheticO₂ evolution was reduced under light-limited conditions, whilethe rate of respiratory O₂ uptake changed slightly. (Received February 8, 1979; )     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Light-induced ATP Formation from Acetyl Phosphate and ADP by Broken Spinach Chloroplasts

Spinach chloroplasts catalyzed ATP formation from acetyl phosphateand ADP when exposed to light. No ATP formation was detectablein the dark. In the absence of ADP, chloroplasts did not hydrolyzeacetyl phosphate in the light or dark. Neither high-energy phosphatessuch as creatine phosphate and phosphoenol pyruvate nor inhibitorsof photophosphorylation competitive with P_i, such as ß-naphthylmonophosphate, phenyl phosphate and pyridoxal 5-phosphate, couldsubstitute for acetyl phosphate as a P_i donor. The apparentK_m values for acetyl phosphate and P_i were 0.81 mM and 0.25mM, respectively. The maximal rate of ATP formation with acetylphosphate and P_i were 331 and 521 µmol ATP formed mg chl^–1hr^–1, respectively. The optimum pH value for acetyl phosphate-dependentATP formation was about 8.0. NH₄Cl, dicyclohexylcarbodiimideand triphenyltin chloride inhibited the acetyl phosphate-dependentATP formation. Acid-base transition also could induce subsequentATP formation from acetyl phosphate and ADP. These results suggestthat the acetyl phosphate-dependent ATP formation requires theformation and the utilization of a proton-motive force as ordinaryphotophosphorylation does. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H. S. Part of this work was reported at the 1981 AnnualMeeting of the Japanese Society of Plant Physiologists (Sapporo,May 8, 1981). (Received August 25, 1981; Accepted November 1, 1981)     

Photoproduction and Detoxification of Hydroxyl radicals in Chloroplasts and Leaves and Relation to Photoinactivation of Photosystems I and II

The light-dependent production of hydroxyl radicals (HO{dot})by thylakoids, chloroplasts and leaves of Spinacia oleraceawas investigated using dimethylsulfoxide as HO{dot} trappingagent. Maximum rates of HO{dot} production by thylakoids asindicated by the formation of methane sulfinic acid were observedunder aerobic conditions in the absence of added electron acceptors.They were higher than 2 µmol (mg Chl h)^–1. Saturationof HO{dot} production occurred at the low photon flux densityof 100 µmol m^–2 s^–1. Trapping of HO{dot} bydimethylsulfoxide suppressed, but did not eliminate light-dependentinactivation of PSI and II suggesting that HO{dot} formationcontributed to the photosensitivity of isolated thylakoids.DCMU inhibited HO{dot} formation. Importantly, methylviologendecreased HO{dot} formation in the absence, but stimulated itin the presence of Fe³⁺. In intact chloroplasts, HO{dot} formation became appreciableonly after KCN had been added to inhibit effective H₂O₂ scavengingby ascorbate peroxidase. It was stimulated by ferrisulfate,but not by ferricyanide which does not penetrate the chloroplastenvelope. Infiltrated spinach leaves behaved similar in principleto intact chloroplasts in regard to HO{dot} formation but HO{dot}production was very slow if detectable at all by the formationof methylsulfinic acid indicating effective radical detoxification. HO{dot} formation is interpreted to be the result of a Fenton-typereaction which produces HO{dot} in chloroplasts from H₂O₂ andreduced ferredoxin, when O₂ is electron acceptor in the Mehlerreaction and radical detoxification reactions are inhibited. (Received November 13, 1996; Accepted April 23, 1996)     

Protoplasts as a tool for isolating functional chloroplasts from leaves

Leaf protoplasts from various grasses can be used for isolatingchloroplasts with high photosyndietic activity. The protoplastswere stable for more than 20 hr during which time chloroplastscould be isolated from protoplasts without any loss of originalCO₂ fixation capacity (100–157 µmoles/mg chl-hr).Using Triticum aestiuum to optimize assay conditions, the pHoptimum for CO₂ fixation by the chloroplasts isolated from protoplastswas between 8.2 and 8.6. Magnesium (0.75 mM) was required formaximum CO₂ fixation by the isolated chloroplasts and sodiumascorbate in the medium allowed a more linear increase in CO₂fixation with time. Based upon ¹⁴CO₂ fixation and ferricyanide-dependentoxygen evolution as criteria of intactness, chloroplasts fromprotoplasts exhibited a high degree of intactness compared tothose obtained by mechanical grinding. Chloroplasts isolatedfrom grass leaves by mechanical grinding had a relatively lowcapacity for endogenous CO₂ fixation and required addition ofribose-5-phosphate and ADP for maximum activity. (Received September 8, 1975; )     

Photosynthesis by Guard Cell Chloroplasts of Vicia faba L.: Effects of Factors Associated with Stomatal Movement

The properties of photosynthetic O₂ evolution by mesophyll cellchloroplasts (MCC) and guard cell chloroplasts (GCC) isolatedfrom protoplasts of Vicia faba L. have been studied and effectson O₂ evolution of factors known to regulate stomatal movementshave been compared. The O₂ evolution of GCC was CO₂-dependent.The saturating light intensity for O₂ evolution was between150 and 200 µmol m^–2s^–1 for MCC and was between400 and 1,000µmol m^–2s^–1 for GCC. Light quality(red vs. blue) had no significant effect on O₂ evolution byeither MCC or GCC. The O₂ evolution rate of MCC was stronglydependent on external K⁺ concentration, but GCC did not respondsignificantly to variations in external K⁺ concentration between0 and 250 mM. The optimal external pH for O₂ evolution by MCCwas approximately 7.5, and either higher or lower external pHsignificantly inhibited O₂ evolution. However, O₂ evolutionby GCC was only slightly enhanced when external pH was increasedfrom 6.0 to 8.0. Our observation of differential sensitivityof MCC and GCC to light intensity and to variations of cytoplasmicK⁺ and pH may indicate differential regulation of photosynthesisin MCC and GCC. ¹Current address: Biology Department, Pennsylvania State University,208 Mueller Laboratory, University Park, PA 16802, U.S.A.     

In Vitro NADH Oxidation as an Early Indicator for Growth Reduction in Spinach Exposed to H2S in the Ambient Air

H₂S reduced growth of spinach at levels higher than 0.075 µl/literand inhibited NADH oxidation by shoot extracts. Inhibition ofNADH oxidation was maximum after a 48-h exposure. Oxidationof NADH was equally sensitive to sulfide and cyanide. NADH oxidationshowed promise as an early indicator for growth reduction byH₂S. ¹Present address: Centre for Agrobiological Research, P.O. Box14, 6700 AA Wageningen, The Netherlands. (Received May 18, 1987; Accepted February 9, 1988)     

Minor carotenoid components in the chloroplasts of higher plants

One-dimensional thin layer chromatography with microcrystallinecellulose was used for the separation of minor carotenoid componentsin spinach, parsley and Brassicachloroplasts. It was revealedthat chloroplasts of these plants contain two minor xanthophyllcomponents besides carotenes, lutein, violaxanthin and neoxanthin.These minor components, designated as xanthophyll–443and –439, were different in spectral properties from antheraxanthinand zeaxanthin which are known to be present in the chloroplastsof some higher plants, and evidence was obtained showing thatxanthophyll–439 has an epoxy group. Spectral propertiesof these xanthophylls in benzene, ethanol, H-hexane and carbondisulfide, as well as their contents relative to other carotenoids,are presented in this paper. ¹Postal address