Ferredoxin-Dependent Photoreduction of the Monodehydroascorbate Radical in Spinach Thylakoids

Thylakoid-bound and stromal ascorbate peroxidases scavenge thehydrogen peroxide that is photoproduced in PSI of chloroplastthylakoids. The primary oxidation product of ascorbate in thereaction catalyzed by ascorbate peroxidase, the monodehydroascorbate(MDA) radical, is photoreduced by thylakoids [Miyake and Asada(1992) Plant Cell Physiol. 33: 541]. We have now shown thatthe photoreduction of MDA radical in spinach thylakoids is largelydependent on ferredoxin (Fd), as determined by the monitoringthe MDA radical by electron paramagnetic resonance. Further,the reduced Fd generated by NADPH and Fd-NADP reductase couldreduce the MDA radical at a rate of over 10⁶ M^–1 s^–1,indicating that the photoreduced Fd in PSI directly reducesthe MDA radical to ascorbate. Photoreduction of NADP⁺ by spinach thylakoids was suppressedby the MDA radical and conversely that of MDA radical was suppressedby NADP⁺, indicating a competition between the MDA radical andNADP⁺ for the photoreduced Fd in PSI. The ratio of the rateconstant for the photoreduction of MDA radical to that for thephotoreduction of NADP⁺ was estimated to be more than 30 to1. Thus, MDA radical is preferentially photoreduced as comparedto NADP⁺. From these results, we propose that the thylakoid-boundascorbate peroxidase and the Fd-dependent photoreduction ofMDA radical in PSI are the primary system for the scavengingof the hydrogen peroxide that is photoproduced in the thylakoids. (Received December 9, 1993; Accepted February 16, 1994)     

Purification and Molecular Properties of the Thylakoid-Bound Ascorbate Peroxidase in Spinach Chloroplasts

The hydrogen peroxide that is photoproduced in thylakoids isscavenged by the thylakoid-bound ascorbate peroxidase (tAPX)[Miyake and Asada (1992) Plant Cell Physiol. 33: 541]. tAPXwas purified from spinach thylakoids to homogeneity as judgedby SDS-polyacrylamide gel electrophoresis, and its molecularproperties were studied. Spinach tAPX was a monomer with a molecularweight of 40,000, which is about 10,000 higher than that ofthe stromal ascorbate peroxidase (sAPX) from spinach chloroplasts.tAPX cross-reacted with the antibody raised against sAPX fromtea leaves, as determined by Western blotting, which also providedevidence for the higher molecular weight of tAPX from spinachthylakoids than that of tea sAPX. The amino acid sequence ofthe amino-terminal region of tAPX showed a low degree of homologyto those of cytosolic APXs from spinach, pea and Arabidopsisthaliana, but a high degree of homology to that of stromal APXfrom tea. Thus, the amino-terminal region of tAPX seems notto be a domain required for binding of the enzyme to the thylakoidmembranes. tAPX contained protoheme IX, as identified by itspyridine hemochromogen, and gave a Soret peak at 403 nm and433 nm with an a band at 555 nm in its oxidized and reducedforms, respectively. Resembling sAPX but differing from cytosolicAPX, tAPX showed high specificity for ascorbate as the electrondonor. tAPX was inhibited by cyanide, thiol-modifying reagents,thiols and several suicide inhibitors, such as hydroxyurea andp-aminophenol. ¹Present address: Beijing Vegetable Research Centre, PO Box2443, Beijing, China.     

Thylakoid Membrane-Bound, NADPH-Specific Pyridine Nucleotide Dehydrogenase Complex Mediates Cyclic Electron Transport in the Cyanobacterium Synechocystis sp. PCC 6803

The donation of electrons from NADPH to the intersystem chain,as monitored by an increase in Chl fluorescence, occurred inthe isolated thylakoid membranes of Synechocystis PCC 6803.The stimulation by NADPH of the methyl viologen-dependent photoreductionof dioxygen and of the reduction of P700⁺ after photooxidationin the presence of DCMU also confirmed the donation of electronsfrom NADPH to the electron carriers in the intersystem. Thesereactions were sensitive to rotenone, capsaicin, l-(2-thenoyl)-3,3,3-trifluoroacetoneand HgCl₂ but not to antimycin A or flavone. In contrast tothe thylakoid membranes from the wild type, those from a mutant,designated M55, in which a gene of a subunit of the pyridinenucleotide dehydrogenase complex (NDH) had been inactivated,did not show evidence of such reactions. These results supportour previous hypothesis that the transport of electrons fromNADPH to the intersystem chain is mediated by NDH [Mi et al.(1994) Plant Cell Physiol. 35: 163] and indicate the bindingof an NADPH-specific NDH to the thylakoid membranes. The Chlfluorescence was quenched transiently by addition of ferredoxinand NADP₊ to the thylakoid membranes but showed a subsequentincrease. This result suggests the reduction of plastoquinoneby the photoreduced NADP₊ and initiation of the NADPH-mediatedcyclic flow of electrons around PSI. Furthermore, a similarresponse of Chl fluorescence was observed upon the additionof ferredoxin only, demonstrating the ferredoxin-dependent cyclicflow of electrons. Both pathways of cyclic electron transportwere inhibited by rotenone, and were not detected in the NDH-defectedthylakoid membranes from M55, indicating the participation ofthe NDH complex. These results confirm that, in Synechocystis,the thylakoid-bound NDH complex mediates the ferredoxin-dependentcyclic electron flow, as well as the NADPH-dependent cyclicelectron flow. (Received November 24, 1994; Accepted March 16, 1995)     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

Inactivation of Ascorbate Peroxidase in Spinach Chloroplasts on Dark Addition of Hydrogen Peroxide: Its Protection by Ascorbate

Dark addition of hydrogen peroxide to intact spinach chloroplastsresulted in the inactivation of ascorbate peroxidase accompaniedby a decrease in ascorbate contents. This was also the casein reconstituted chloroplasts containing ascorbate, NADP⁺, NAD⁺and ferredoxin. The addition of hydrogen peroxide during light,however, showed little effect on ascorbate contents and ascorbateperoxidase activity in either the intact or reconstituted chloroplasts.In contrast to ascorbate peroxidase, the enzymes participatingin the regeneration of ascorbate in chloroplasts (monodehydroascorbatereductase, dehydroascorbate reductase and glutathione reductase)were not affected by the dark addition of hydrogen peroxide.Ascorbate contents increased again by illumination of the chloroplastsafter the dark addition of hydrogen peroxide. These resultsshow that the inactivation of the hydrogen peroxide scavengingsystem on dark addition of hydrogen peroxide [Anderson et al.(1983) Biochim. Biophys. Acta 724: 69, Asada and Badger (1984)Plant & Cell Physiol. 25: 1169] is caused by the loss ofascorbate peroxidase activity. Ascorbate peroxidase activitywas rapidly lost in ascorbate-depleted medium, and protectedby its electron donors, ascorbate, isoascorbate, guaiacol andpyrogallol, but not by GSH, NAD(P)H and ferredoxin. (Received June 14, 1984; Accepted August 15, 1984)     

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)     

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

Proton/Pyruvate Cotransport into Mesophyll Chloroplasts of C4 Plants

Light-enhanced active pyruvate uptake into mesophyll chloroplastsof C₄ plants was reported to be mimicked by either of the twotypes of cation jump: H⁺-jump in maize and phylogenically relatedspecies (H⁺-type) and Na⁺-jump in all the other C₄ species tested(Na⁺-type) [Aoki, N., Ohnishi, J. and Kanai, R. (1992) PlantCell Physiol. 33: 805]. In this study, medium and stromal pH was monitored in the suspensionof C₄ mesophyll chloroplasts. Medium alkalization lasting for5 to 10 seconds after pyruvate addition was detected by a pHelectrode and observed only in the light and only in mesophyllchloroplasts from H⁺-type species, Zea mays L. and Coix lacryma-jobiL., but not in those from Na⁺-type species Panicum miliaceumL., Setaria italica (L.) Beauv. and Panicum maximum Jacq. Theinitial rate of H⁺ consumption showed good correlation with[¹⁴C]pyruvate uptake measured by silicone oil filtering centrifugation,both being inhibited by N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole to the same degree. The ratio of the rate of H⁺ uptaketo that of pyruvate uptake was always about 1. Pyruvate-inducedacidification of the stroma was observed in maize mesophyllchloroplasts. These results show one to one cotransport of H⁺and pyruvate anion into mesophyll chloroplasts of H⁺-type C₄species in the light. (Received January 5, 1994; Accepted May 6, 1994)     

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)     

Chloroplast ultrastructure of sugar beet (Beta vulgaris L.) cultivated in normal and elevated CO2 concentrations with two contrasted nitrogen supplies

Sugar beet (Beta vulgaris L., cultivar Celt) plants were grownunder simulated field conditions in pots and supplied with adequateor deficient nitrogen (HN and LN, respectively) combined withtwo CO₂ concentrations, ambient (c. 350µmol mol^–1C0₂—AC), or elevated CO₂ (c. 600 µmol mol^–1CO₂—HC). Chloroplast structure in mesophyll palisade cellsof mature leaves (leaf number 19 in HN and 9 in LN), sampledat midday on 16 August 1993 was studied by transmission electronmicroscopy and quantified stereologically. The ultrastructureof palisade parenchyma chloroplasts was affected by the elevatedCO₂ concentration and strikingly affected by nitrogen supply.Chloroplast diameter (cross-sectional length) was slightly,but not significantly, greater in HC than AC treatments withinan N treatment, but was smaller in LN than HN; chloroplast cross-sectionalarea also increased with HC in both N treatments, but only significantlyso in LN. Elevated CO₂ reduced the proportion of total thylakoids(significant at 5% and 0.1% in HN and LN, respectively) dueto decreased granal thylakoids, but the proportion of inter-granal(stromal) thylakoid membranes was not affected compared to chloroplastsfrom plants grown with ambient CO₂. Chloroplast stroma increasedas a proportion of chloroplast volume with elevated comparedto ambient CO₂ with HN but not LN. Starch inclusions were notsignificantly different with elevated compared to ambient CO₂at HN, but the proportion of starch increased considerably atelevated compared to ambient C0₂ at LN, indicating an over-productionof assimilates. Plastoglobuli in chloroplasts increased withdeficient N, but decreased with elevated CO₂. Larger chloroplastswith a greater proportion of stroma, but a smaller proportionof granal thylakoids, suggest increased CO² assimilating capacityand decreased light harvesting/PSII capacity with elevated CO₂. Key words: Chloroplast, ultrastructure, elevated CO₂ concentration, nitrogen deficiency, sugar beet, Beta vulgaris     

Cyt b-559 (Fd) Participating in Cyclic Electron Transport in Spinach Chloroplasts: Evidence for Kinetic Connection with the Cyt b6/f Complex

A small fraction of low potential Cyt b-559, amounting to only13% of total Cyt b-559 in spinach chloroplasts, is analyzedwith the help of a highly selective, computer-controlled spectrophotometer,which simultaneously applies 16 pulse modulated narrow bandmeasuring beams with wavelengths in the cytochrome -band (500–600nm) for recordings of time resolved difference spectra. ThisCyt b-559 fraction remains oxidized upon dark incubation withascorbate and is reduced upon illumination. It can be reducedby cyclic PSI in an antimycin A-sensitive reaction or in thecourse of antimycin A-insensitive linear electron transportvia the Cyt b₆/f complex. Reduction by NADPH in the dark requiresferredoxin. Simultaneous recordings of Cyt b-563 and Cyt f revealclose kinetic connection between this Cyt b-559 fraction andthe low potential chain of the Cyt b₆/f complex. These resultsconfirm and extend previous observations of Miyake et al. 1995(Plant Cell Physiol. 36: 743) in maize mesophyll thylakoids,which led to the hypothesis that Cyt b-559 (Fd) occupies theposition of the postulated ferredoxin-plastoquinone reductase(FQR) in cyclic electron transport. (Received March 9, 1999; Accepted May 21, 1999)     

Changes in Photosystem Stoichiometry in Response to Environmental Conditions for Cell Growth Observed with the Cyanophyte Synechocystis PCC 6714

Changes in photosystem stoichiometry in response to shift ofenvironments for cell growth other than light regime were studiedwith the cyanophyte Synechocystis PCC 6714 in relation to thechange induced by light-quality shift. Following two environment-shiftswere examined: the shift of molecular form of inorganic carbonsource for photosynthesis from CO₂ to HCO₃^– (CO₂ stress)and the increase in salinity of the medium with NaCl (0.5 M)(Na⁺ stress). Both CO₂ and Na⁺ stresses induced the increasein PSI abundance resulting in a higher PSI/PSII stoichiometry.CO₂ stress was found to elevate simultaneously Cyt c oxidaseactivity (V_max). The feature was the same as that caused bylight-quality shift from preferential excitation of PSI to PSII(light stress) though the enhancement by either stress was smallerthan that by light stress. Under our experimental conditions,PSI/PSII stoichiometry appeared to increase at a fairly constantrate to the basal level even when the basal level had been differentlydetermined by the light-quality. Enhancing rates for PSI/PSIIstoichiometry and for Cyt c oxidase activity were also similarto each other. Since the two stresses affect the thylakoid electrontransport similarly to the shift of light-quality, we interpretedour results as follows: three environmental stresses, CO₂, Na⁺,and light stresses, cause changes in electron turnover capacityof PSI and Cyt c oxidase under a similar, probably a common,mechanism for monitoring redox state of thylakoid electron transportsystem. ¹On leave from Department of Biology, College of Natural Science,Kyngpook National University, Taegu 702-701, Korea. ₂Present address: Department of Marine Bioscience, Fukui Pre-fecturalUniversity, Obama, Fukui, 917 Japan.     

Regulation of the Activity of Ribulose-1,5-Bisphosphate Carboxylase in Response to Changes in the Photosynthetic Source-Sink Balance in Intact Soybean Plants

Sink-limited conditions cause a reduction in the rate of photosyntheticfixation of CO₂ in single-rooted soybean leaves (Glycine max.Merr.). We suggested previously that this reduction is due tothe deactivation of ribulose-1,5-bisphosphate carboxylase (RuBPcase;EC 4.1.1.39 [EC] ) that is caused by a decrease in the level of freeP_i via a decrease in the rate of conversion of phosphorylatedintermediates to the end-product (sucrose) in sink-limited leaves[Sawada et al. (1989) Plant Cell Physiol. 30: 691, Sawada etal. (1990) Plant Cell Physiol. 31: 697, Sawada et al. (1992)Plant Cell Physiol. 33: 943]. In the present study, we investigatedwhether, in intact soybean plants, sink-limited conditions wouldalso cause a reduction in the rate of photosynthesis and whethersuch a reduction might be due to the deactivation of RuBPcasevia the same regulatory mechanism as that suggested from previousstudies with single-rooted leaves. Continuous removal of flowerbuds from intact plants brought a large decrease in ratio ofthe dry weight of sink organs (stem, roots, pods) to sourceorgan (leaves) as a result of the absence of pod formation.Pods are likely to function as the major sink at the reproductivestage. Upon continuous removal of flower buds, the treated (sink-limited)plants showed a large decrease in the rate of photosyntheticfixation of CO₂ as compared to control plants. RuBPcase in theleaves of treated plants was continuously inactivated with thedecrease in photosynthetic activity. However, the inactivatedenzyme was totally reactivated upon incubation in the presenceof 10 mM NaHCO₃ and 5 mM MgCl₂. The levels of sucrose and ribulose-1,5-bisphosphatein leaves of the treated plants increased significantly. Allthese results coincide exactly with those obtained in previousstudies of single-rooted leaves under the sink-limited conditions. (Received July 14, 1994; Accepted February 21, 1995)     

Involvement of the Binuclear Copper Site in the Proteolytic Activity of Polyphenol Oxidase

Plant polyphenol oxidase (PPO) is apt to degrade during andeven after purification. We developed a method to stabilizePPO by 0.3 M NaCl, 0.1% (w/v) Tween 20, and 50% (w/v) ethyleneglycol at pH 6.5. The protein slowly degraded by itself whenthe stabilizing reagents were removed. Ascorbate and/or H₂O₂accelerated the degradation. The ascorbate-induced degradationwas inhibited by catalase, suggesting that H₂O₂ is generatedthrough reduction of PPO by ascorbate. It is likely that dissolvedoxygen is converted to peroxide through two-electron reductionby the reaction center of PPO, binuclear Cu site, and a Fenton-typereaction occurred on it. This understanding was supported bythe finding that the H₂O₂-induced degradation was inhibitedby metal-chelators as well as by polyphenolic substrate of PPO.Considering the postulated mechanism of the self-degradationof PPO, we re-examined the degradation of the 23-kDa proteinof PSII by PPO [Kuwabara et al. (1997) Plant Cell Physiol. 38:179]. The obtained results suggested that the 23-kDa proteintriggers the active oxygen production by the binuclear Cu site,probably as reductant, and receives the radical species preferentiallyto the polypeptide moiety of PPO. (Received April 15, 1999; Accepted July 21, 1999)     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Changes in Stoichiometry among PSI, PSII and Cyt b6-f Complexes in Response to Chromatic Light for Cell Growth Observed with the Red Alga Porphyra yezoensis

Stoichiometry among 3 thylakoid components, PSI and PSII andCyt b6-f complexes, was determined with the red alga Porphyrayezoensis with special reference to the regulation of PSI/PSIIstoichiometry in response to light regime. The ratio of PSIto PSII abundance was four times greater in thalli grown underorange light which excites mainly phycobilisome, thus PSII,than that under red light which excites preferentially Chl a,thus PSI. Cyt b₆-f abundance remained almost constant. The PSIand PSII content was regulated separately under the two growthlight conditions as was also observed with the red alga Porphyridiumcruentum by Cunningham et al. [(1990) Plant Physiol. 93: 888].This differs from the cyanophyte Synechocystis PCC 6714 whereadjustment occurs only in the PSI content [(1987) Plant CellPhysiol. 28: 1547]. However, results on the marine cyanophyteSynechococcus NIBB 1071 indicate that changes in the PSI/PSIIsoichiometry is similar to red algae. In this species, as inthe red algae, more than one PSII is associated with each phycobilisome.The light regime also induced changes in the phycobiliproteincomposition in Porphyra yezoensis. Under PSII light, phycoerythrinincreased, and phycocyanin decreased, while under PSI lightthe response was reversed. The change suggests an occurrenceof complementary chromatic adaptation. (Received April 8, 1994; Accepted June 1, 1994)     

Reaction of nitric oxide with superoxide inhibits basolateral K+ channels in the rat CCD

We previously demonstrated that nitric oxide (NO) stimulates thebasolateral small-conductance K⁺channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 (Cell Physiol. 39): C1336-C1342,1996]. Because NO at high concentration has been shown to reactwith superoxide (O₂) to formperoxynitrite (OONO)[W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol.Physiol. 12): L699-L722, 1995 and M. S. Wolin.Microcirculation 3: 1-17,1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors[100-200 µMS-nitroso-N-acetyl-penicillamine(SNAP) or sodium nitroprusside (SNP)] reduced channel activity,defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect ofNO was completely abolished in the presence of 10 mM Tiron, anintracellular scavenger of O₂. NOdonors, 10 µM SNAP or SNP, which stimulate channel activity undercontrol conditions, can also inhibit SK in the presence of anO₂ donor, pyrogallol, or in thepresence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence ofexogenous cGMP, suggesting that the NO-induced inhibition is not theresult of decreased cGMP production. We conclude that the inhibitoryeffect of NO on channel activity results from an interaction between NOand O₂.

     

Maximum H+/h{nu}PSI Stoichiometry of Proton Transport during Cyclic Electron Flow in Intact Chloroplasts Is at Least Two, but Probably Higher than Two

Effects of antimycin A on 9-aminoacridine (9AA) fluorescencequenching by intact chloroplasts during light-dependent electronflow to different electron acceptors indicated that considerablecyclic electron flow occurs concurrently with linear electrontransport already at low PFDs, when oxygen supported electronflow, but not, when nitrite or methylviologen (MV) were present.Quantum efficiencies of the use of 696 and 675 nm light werecalculated for oxygen-, nitrite- and MV-dependent linear electronflows. Since H⁺/e=3 during linear electron transport [Ivanov(1993) Photosynthesis, p. 111; Kobayashi et al. (1995) PlantCell Physiol. 36: 1613] and comparable 9AA fluorescence quenchingindicates comparable transthylakoid proton gradients, totalproton transport could be calculated and part of it could beassigned to linear and the remainder to cyclic electron transportwhen oxygen was electron acceptor. Quanta of 696 nm light notused to support linear electron flow to oxygen at h/e=2 wereassumed to be available for coupled proton transport duringcyclic electron flow. H⁺/h ratios for cyclic electron transportobtained on this basis were consistently higher than 1 and occasionallyapproached 3. No allowance was made in these calculations foroxidized P700 in the reaction center of PSI, which could notdonate electrons to the cyclic pathway, and for reduced Q_A inthe reaction center of PSII. It therefore appears likely thatmaximum H⁺/h ratios in cyclic electron transport are higherthan values calculated in this work. Our observations with intactchloroplasts agree in principle with those of [Heath (1972)Biochim. Biophys. Acta 256: 645] with thylakoids, who also reportedhigh H⁺/ e ratios in cyclic electron transport. These ratiosare briefly discussed in relation to the H⁺/ATP stoichiometryof ATP production during carbon assimilation of leaves and toprotection of chloroplasts against photoinactivation. ²Present address: Timiriasev Institute of Plant Physiology,Russian Academy of Sciences, Botanicheskaya, 35, Moscow, Russia ³Present address: Department of Forestry, Faculty of Agriculture,Kyushu University, Hakozaki, Higashi-ku, Fukuoka, 812 Japan     

Photo-inhibition and the Effects of Protein Phosphorylation on Electron Transport in Wheat Thylakoids

The kinetics of changes in photosystem I (PSI), photosystemII (PSII), and whole chain (PSII and PSI) electron transport,chlorophyll fluorescence parameters, the capacity to bind atrazineand the polypeptide profiles of thylakoids isolated from wheatleaves on exposure to a photon flux density of 2000 µmolm^–2 s^–1 were determined. Severe and similar levelsof photo-inhibitory damage to both PSII and whole chain electrontransport occurred and were correlated with decreases in theratio of variable to maximal fluorescence, the proportionalcontribution of the rapid a phase of the fluorescence kineticsand the capacity to bind atrazine. Severe photo-inhibition ofelectron transport was not associated with a major loss of chlorophyllor total thylakoid protein. However, a small decrease in a 70kDa polypeptide together with increases in a number of low molecularmass polypeptides (8–24 kDa) occurred. Phosphorylation of thylakoid polypeptides alleviated photo-inhibitionof PSII electron transport but stimulated photoinhibitory damageto whole chain electron transport. The consequences of suchphosphorylation-induced effects on photoinhibition in vivo areconsidered. Key words: Chlorophyll fluorescence, electron transport, photo-inhibition, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Location and effects of long-term NaCl stress on superoxide dismutase and ascorbate peroxidase isoenzymes of pea (Pisum sativum cv. Puget) chloroplasts

The present work describes the intrachloroplast localization and the changes that took place in the thylakoid and stroma-located superoxide dismutases (SOD, EC 1.15.1.1) and ascorbate peroxidases (APX, EC 1.11.1.11), in response to long-term NaCl stress in Pisum sativum L. cv. Puget plants. Native PAGE using high chloroplast protein concentrations pointed to the presence of the two main Fe-SODs, together with CuZn-SODs, both in thylakoids and in the stroma. Western blot and immunogold labelling using the antibodies against chloroplastic Fe-SOD from Nuphar luteum also confirmed the chloroplastic localization of a Fe-SOD. Thylakoidal Fe-SOD activity was induced by a NaCl concentration as low as 70 mM, while CuZn-SOD was induced at 90 mM, although in severe stress conditions (110 mM) both activities were similar to the levels at 90 mM NaCl. NaCl stress also induced stromatic Fe-SOD and CuZn-SOD activities, although these inductions only started at higher NaCl concentration (90 mM) and were significant at 110 mM NaCl. The increase in activity of both Fe-SODs was matched by an increase in Fe-SOD protein. Chloroplastic APX isoenzymes behaved differently in thylakoids and stroma in response to NaCl. A significant increase of stromal APX occurred at 70 mM, whereas the thylakoidal APX activity was significantly and progressively lost in response to NaCl stress (70-110 mM). A significant increase in the H2O2 content of chloroplasts during stress and a reduction in the ascorbate level at 90 mM NaCl also took place, although the oxidized ascorbate pool at the highest NaCl concentration did not show significant changes. These results suggest that the loss of thylakoidal APX may be an important factor in the increase in chloroplastic H2O2, which also results from the increased thylakoid and stroma-located Fe-SOD and CuZn-SOD activities. This H2O2 may be involved in the induction of stromal APX. The up-regulation of the above enzymes in the described stress conditions would contribute to the adaptation of cv. Puget plants to moderate NaCl stress.