Intensity and Decay Half-Time of the Electron Paramagnetic Resonance Signal I in Bean Chloroplasts at Various Stages of Greening

The size of EPR signal I and its decay half-life were measuredon chloroplasts isolated from primary bean leaves after illuminationof 7 d old dark-grown seedlings. The results indicate that the ratio of P700/total chlorophylldecreased approximately 2-fold only between the 6th and 24thhour of illumination. The relative size of the signal increasedwith increasing light intensity up to 360 W m^–2 in allstages of greening studied. Addition of HgCl₂ (ImM) caused anincrease in the magnitude of the signal, especially at lowerlight intensities. Treatment of isolated chloroplasts with DCMU(20 µM) did not have any effect on the intensity of thesignal at the early stages of greening, that is 6 or 12 h ofillumination, but it caused a slight enhancement after 24 or48 h of Illumination. The higher intensity used for the signalproduction the smaller the enhancement observed. The addition of HgCl₂ caused an increase in decay half-lifeof the signal 20-fold or 35-fold after 6 h or 48 h of greening,respectively. In DCMU-treated chloroplasts, an enhancing effectbecomes visible after 12 h of greening and subsequently it graduallyincreased. The data obtained were interpreted in terms of an alterationin efficiency of the re-reduction of P700⁺ under different conditionsand different stages of greening.     

Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

The effects of copper on photosynthetic electron transfer systemsin isolated spinach chloroplasts were studied. Two differentinhibitions were observed. First, copper markedly inhibitedferredoxin-catalyzed reactions such as NADP⁺ photoreduction.The concentration required for 50% inhibition was about 2 µMof cupric sulfate. However, electron flow from reduced 2,6-dichloroindophenol(DCIP) to methyl viologen was not affected. The dissociationconstant between ferredoxin and ferredoxin-NADP⁺ reductase wasunchanged in the presence of 2.5 µM of cupric sulfate.In enzymic reaction systems, the ferredoxin-dependent electronflow from NADPH to cytochrome c was also strongly inhibitedin the presence of cupric sulfate, while DCIP reduction withNADPH as the electron donor was not affected. Second, DCIP photoreductionwas weakly blocked by copper and the lost activity could notbe recovered by adding 1,5-diphenylcarbazide (DPC). It can be concluded that copper directly interacted with ferredoxincausing inhibition of ferredoxin-dependent reactions. Further,copper caused weak inactivation between the oxidizing side ofthe reaction center of photosystem II and the electron donatingsite of DPC. (Received August 8, 1977; )     

Photodestruction of Pigments in Higher Plants by Herbicide Action: I. THE EFFECT OF DCMU (DIURON) ON ISOLATED CHLOROPLASTS

3-(3',4'-Dichlorophenyl)-1',1'-dimethyl urea (DCMU) inducedthe photobleaching of chlorophylls and carotenoids in isolatedchloroplasts of Hordeum vulgare. In chloroplasts illuminatedin both the absence and presence of DCMU (5.0 mmol m^–3),the destruction of carotenoid preceded that of the chlorophylls.The rate of photodestruction was accelerated by the presenceof DCMU. After only 2 h illumination the rates of loss of ß-caroteneand of the epoxyxanthophylls, neoxanthin and violaxanthin, weresimilar (approximately 40–50% loss in the presence of5–0 mmol m^–3 DCMU) but weremuch greater than thatof lutein (25% loss). Analysis of the individual pigment-proteincomplexes, isolated from chloroplasts following such treatment,showed that whilst pigment destruction had occurred in all complexes,the relative content of the LHCP2/CPa complexes (containingthe PSII core) had fallen to the greatest extent. Further illuminationof the chloroplasts, for up to 22 h, resulted in far greaterbleaching but showed a similar pattern of pigment loss, withDCMU again accelerating the rate at which this loss occurred.ß-Carotene-5,6-epoxide was identified as a productof such photo-oxidative conditions. Key words: DCMU, carotenoids, chlorophylls, photobleaching, ß-carotene-5,6-expoxide     

The Effect of Cadmium on Photosystem II in Spinach Chloroplasts

Cadmium ions, as an environmental pollution factor, significantly inhibited the photosynthesis especially, photosystem Ⅱ activity in isolated spinach chloroplasts. The presence of 5 mmol/l Cd2+ inhibited the O2-evolution to 53%. Cd2+ reduced the activity of photoreduction of DCIP and the variable fluorescence of chloroplasts and PSⅡ preparation. The inhibited DCIP photoreduction activity could only be restored slightly by the addition of an artificial electron donor of PSII, DPC, and the inhibited variable fluorescence could not be obviously recovered by the addition of NH2OH, another artificial electron donor of PSⅡ. It is considered that, besides the oxidizing side of PSI1, Cd2+ could also inhibit directly the PSⅡ reaction center. The inhibitory effect of Cd2+ on the whole chain electron transport (H2O→MV) was more serious than on O2-evolution (H2O→DCMU). It is suggested that the oxidizing side of PSⅡ is not the only site for Cd2+ action. There may be another site inhibited by Cd2+ in the electron transport chain between PSⅠ and PSⅡ.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )     

A Manganese Protein from Thylakoids of a Cyanobacterium, Plectonema boryanum; Inhibition of Oxygen Evolution with Its Antibody

A manganese protein was solubilized from the thylakoids of thecyanobacterium Plectonema boryanum with a cholate-deoxycholatemixture, and purified to homogeneity by gel-filtration. Isolatedmanganese protein had a molecular weight of 13,000 and showedcatalase activity, which was insensitive to 3-aminotriazole.The antibody raised against the manganese protein inhibitedthe oxygen evolution using dichlorophenol indophenol (DCIP)as the electron acceptor by P. boryanum thylakoids, but notthe diphenylcarbazide-supported photoreduction of DCIP and ascorbate-supportedphotoreduction of methyl viologen in the presence of DCMU. Theseobservations suggest that isolated manganese protein is a componentof the oxygen evolving enzyme, water dehydrogenase. ¹ Dedicated to the late Dr. Joji Ashida, the first presidentof the Japanese Society of Plant Physiologists. (Received August 7, 1982; Accepted December 10, 1982)     

Electron transfer and energy dependent reactions in glutaraldehyde-fixed chloroplasts

The effects of GA fixation on electron transfers in photosystemsI and II in photosynthesis and energy dependent reactions ofchloroplasts, such as changes in light scattering, H⁺ uptakeand 515-nm absorbance, were investigated. Fixation of chloroplastswith GA resulted in a lowering of the DCIP and MV photoreductions.DCIP photoreduction activity in fixed chloroplasts was not restoredin the presence of DPC, an electron donor to photosystem II,but was significantly stimulated by DPC when chloroplasts werefixed after aging. The results suggest that the inhibitory effectof GA fixation on photosystem II differs in its mechanism fromthose of treatments such as heating, Tris-washing and aging.The oxidation-reduction reaction of P700 was depressed by GAfixation. Energy dependent reactions in fixed chloroplasts were more markedlydepressed than were electron transfers. Fixed chloroplasts showeda slight conformational response in the presence of PMS. Analysis of the emission spectrum and the induction of chlorophylla fluorescence in fixed chloroplasts suggested that the twopigment systems were partially disordered and that the correspondingprimary photochemical processes were inhibited. (Received November 21, 1972; )     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Effect of {alpha}-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Respiration in isolated mung bean shoot mitochondria was notaffected by either of the tomatine treatments (10^–3 M,pH 5, and 10^–5 M, pH 8) but was reduced in the excisedshoots by both treatments, although only at the higher of thetwo temperatures (5 °C and 25 °C). Inhibition was gradualand took at least 2 h. Tomatine treatment of excised shootsalso resulted in an increased leakage of K+. PS II activityin isolated spinach leaf chloroplasts was reduced only by thehigh pH tomatine treatment at 25 °C. Again, about 2 h treatmentwas required before significant effects were observed but thealkaloid did not cause disintegration of the chloroplast asmeasured by pigment release. Disruption of lysosomes isolatedfrom cauliflower inflorescence tissue and release of acid phosphatasewas enhanced by tomatine. Initially only 10^–5 M tomatineat pH 8 was effective but, later, effects could only be obtainedwith 10^–3 M alkaloid at pH 5. The differential susceptibilityof these organelles to tomatine is discussed in relation tomembrane structure and to the mode of toxicity of the alkaloid.     

Multiple sites of DCIP reduction by sonicated Oat chloroplasts: Role of plastocyanin

1. Oat chloroplasts, in the presence of 0.02 M methylamine, reduce 2,6 dichlorophenolindophenol (DCIP) at a rate of 350–500 μmoles/mg chl per h, in saturating light. Brief sonication for approx. 1 min lowers the rate to approx. 50 μmoles/mg chl per h; longer sonication does not reduce activity further. During brief sonication, plastocyanin is lost from the chloroplasts. When plastocyanin is added back to sonicated fragments, DCIP reduction is approximately doubled to 100 μmoles/mg chl per h.

2. When oxidized plastocyanin is added, a transient is observed when light is first turned on: this is due to a reduction of the plastocyanin before DCIP reduction begins. When reduced plastocyanin is added, a different transient occurs: this is due to a fast photoreduction of DCIP by the plastocyanin and is followed by the slower steady state reduction of DCIP by water. When light is turned off before complete reduction of DCIP, a transient reduction of oxidized plastocyanin by reduced DCIP is seen. Insensitivity of these transients to 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the greater effectiveness of 710 nm light, along with the known capacity of plastocyanin to mediate electron transfer to System I, prove that an intrinsically fast reduction of DCIP occurs at a site close to the primary photoreduced product of System I.

3. After brief sonication and washing, no residual plastocyanin was detected in chloroplast fragments, and the rate of the slow DCIP reduction (about 50μmoles/mg chl per h) sustained by such fragments was essentially identical to that maintained by fragments of mutants lacking System I activity. Following et al.⁹, the simplest explanation for this slow DCIP reduction is that is occurs at a site close to System II and the system I is not involved.

4. A very slow transient reduction of DCIP occurs after extinguishing light; this presumably involves another reduction site close to System II, as suggested by ⁹.     

Effects of Hydrostatic Pressure on Photosynthetic Systems. I. Preferential Destruction of the O2-Evolving Center

The effects of hydrostatic pressure treatments on the photosyntheticactivities of isolated thylakoids and PSII membranes were studied,and the following results were obtained. (1) The O₂-evolvingactivity was selectively inhibited by treatment at 200 MPa andabove, while the electron transport in the PSII reaction center,as measured by the photoreduction of 2,6-dichlorophenolindophenol(DCIP) with 1,5-diphenylcarbazide (DPC), was markedly enhanced.(2) The activity of PSI, as measured by the photoreduction ofmethylviologen with reduced DCIP, was not much affected evenafter treatment at 500 MPa, whereas this electron transportbecame uncoupled from phosphorylation at 200 MPa and above.(3) In pressure-treated PSII membranes, the EPR signal of Y⁺_zbecame photoinducible with the concomitant appearance of anEPR signal for Mn²⁺. (4) The 33-kDa extrinsic protein was retainedin inhibited PSII membranes, but the 17- and 23-kDa extrinsicproteins were lost. (5) Cross-linking of PSII proteins by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) suppressed the pressure-induced inactivation of the evolutionof O₂. (6) In pressure-treated PSII membranes, higher concentrationsof DCMU were required to inhibit the photoreduction of DCIP.Features of the pressure-induced inhibition of various reactionsin photosynthetic membranes are discussed on the basis of theseresults. ¹On leave from Advanced Research Laboratory, Hitachi Ltd., Hatoyama,Saitama, 350-03 Japan.     

Requirement of Manganese for Electron Donation of Hydrogen Peroxide in Photosystem II Reaction Center Complex

Mn²⁺ was required for the electron donating reaction from H₂O₂,but not for that from diphenylcarbazide (DPC), in the PS IIreaction center complex which was prepared from spinach chloroplastsby Triton X-100 extraction. The reaction center complex showeda high activity of 2,6-dichloroindophenol (DCIP) photoreductionin the presence of DPC, but a low activity with H₂O₂. The H₂O₂-supportedDCIP photoreduction was suppressed by EDTA and enhanced by asmall amount of Mn²⁺. Ca²⁺ and Mg²⁺ could not replace Mn²⁺.The activation by Mn²⁺ and its binding showed two binding sitesof Mn²⁺ in the reaction center complex, with high (1.5?10⁷ M^–1)and low (1 ? 10⁶ M^–1) binding constants. (Received November 8, 1986; Accepted April 10, 1987)     

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)     

Inhibition of Hill Reaction and Photophosphorylation by l,l,l-Trichloro-2,2-bis(p-chlorophenyl)ethane

The effect of DDT on DCIP and Fe(CN)₆^3– photoreductions,and cyclic and non-cyclic photo-phosphorylations, in some 30varieties of barley from widely different parts of the worldis reported. Whereas resistant barleys were not affected byDDT treatment, chloroplasts from treated susceptible barleysshowed parallel inhibitions of all the investigated aspectsof photosynthesis. However, in a few susceptible varieties inhibitionsof Fe(CN)₆^3– photoreduction or non-cyclic photophosphorylationwere not so pronounced. Possible reasons for these anomaliesare discussed; in particular earlier reports that DDT had noeffect on these latter photosynthetic activities may have beendue to the use of hypotonic media during chloroplast isolation.     

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; )     

Photosynthesis-Dependent Volume Regulation in Guard Cell Protoplasts from Vicia faba L.

A rapid and convenient procedure was developed for isolatingguard cell protoplasts (GCPs) from epidermal strips of Viciafaba L. The mean rates of O₂ uptake in the dark and evolutionin light of the isolated GCPs were 200 and 290 µmol O₂mg^–1 Chl h^–1, respectively, showing net O₂ evolutionin light. Photosynthetic O₂ evolution was suppressed completelyby 5 µM DCMU. Addition of 5 µM DCMU to the incubationmedium after 30 min of light exposure also suppressed the light-inducedswelling of GCP, indicating possible participation of PS IIin volume regulation in GCP. ⁴Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Yatabe machi, Tsukuba,Ibaraki 305, Japan. (Received December 17, 1983; Accepted March 21, 1984)     

The Photoinhibition Site of Photosystem I in Isolated Chloroplasts under Extremely Reducing Conditions

Under anaerobic atmosphere where the gas phase was simply replacedby N₂, photo-inhibition of PS I of isolated spinach chloroplastswas insignificant. However, when dithionite was included inthe irradiation mixture, severe photoinhibition of the NADP⁺and the MV photo-reduction occurred. Neither P700 determinedby continuous illumination-induced difference spectroscopy,Fe-S centers determined by EPR under cryogenic temperatures,nor vitamin K-l determined by HPLC analysis were significantlydecreased under these photoinhibition conditions. Although photobleachingof antenna chlorophylls occurred to more or less extent, NADP⁺photoreduction activities were markedly depressed even undersaturating actinic light. The maximal amplitude of the flashinduced absorbance changes of P700 in ms range decreased almostin parallel with the loss of NADP⁺ photoreduction activity.These results indicate that although the Fe-S centers of thephotoinhibited chloroplasts were reducible by continuous illumination,to almost the same extents as that of the control chloroplasts,the efficiency of reduction by each flash was much lower thanthat of the control chloroplasts. The site of photoinhibitionin PS I under extremely reducing conditions is between A₀ andFe-S X. (Received July 28, 1988; Accepted October 31, 1988)     

Discrimination and characterization of photosystem I- and photosystem II-induced 515-nm absorbance change in chloroplasts I. Dissipation of electric field and related processes

Distinctive characteristics of the photosystem I-induced 515-nmabsorbance change and the photosystem II-induced change wereanalyzed in spinach chloroplasts in the absence of added salt.Two types of changes were distinguished by 3-(3,4-dichloro-phenyl)-1,1-dimethylurea(DCMU), carbonylcyanide m-chlorophenylhydrazone (CCCP) and illuminationwith red or far-red light. Half-recovery time of the photosystem I-induced absorbance changewas shorter than that of over-all absorbance change and wasinsensitive to a low concentration (<0.50 µM) of CCCP. In the presence of DCMU, the 515-nm absorbance change decayedin parallel with the rapid protonation of reduced 2,6-dichloroindophenol(DCIP) or methyl viologen. This indicates that the photosystemI-induced local field is dissipated in the electron transferfrom photosystem I to an electron acceptor. Thus the mechanismin dissipation of electric field formed by photosystem I isdifferent from that induced by photosystem II where rapid protonationof plastosemiquinone anion may be directly involved in fielddissipation (Yamamoto, Y. and M. Nishimura: Plant & CellPhysiol. 18: 293–301 (1977)). (Received December 9, 1977; )     

Appearance of Photochemical Activity in Isolated Chloroplasts from Far-red-illuminated Leaves of Phaseolus vulgaris

Chloroplast development was followed in intact bean leaves illuminatedwith far-red light by extracting chloroplasts at various timesto assay photosynthetic activities. Photochemical activity wasdetected in isolated chloroplasts prior to the times which werepreviously reported for intact leaf discs. Cyclic phosphorylationwas observed in isolated chloroplasts after 8 h of far-red illuminationwhile non-cyclic electron transport and phosphorylation weremeasurable after 12 and 16 h of illumination respectively. TheP/2e ratios were less than 0.5 after 24 h of far-red exposurebut approached a value of 1.0 by 60 h of illumination. Ammoniumchloride (10^–3 M) had little effect on electron transportin isolated chloroplasts until after 24 h of far-red illumination.Chlorophyll a accumulated slowly from the onset of far-red illuminationwhile chlorophyll b was not detected until after 48 h of far-redexposure. Leaf fresh weight increased four-fold over the 60h illumination period. Electron microscopy of isolated chloroplasts from far-red-illuminatedleaves indicated the presence of unfused primary thylakoidsby 12 h of exposure and prolamellar bodies throughout the entire60 h illumination period. Grana were not observed in isolatedchloroplasts nor were they induced by a 2 min exposure of thechloroplasts to 172 000 lx of white light. O₂ evolution in leaf discs of far-red-illuminated plants wasmeasurable after 16 h of illumination, attained a maximum valueby 36 h of far-red exposure, and then declined. Net CO₂ fixationwas observed in leaf discs after 8 h of far-red illuminationand the rates remained constant for an additional 16 h, beforeincreasing at least two-fold.