Effects of Leaf Chilling on Thylakoid Functions, Measured at Room Temperature, in Cucumis sativus L. and Oryza sativa L.

Photosynthetic functions in leaves of cucumber (Cucumis sativusL.) and rice (Oryza sativa L.) were examined before and aftervarious chilling treatments. Cucumber leaves lost the capacityfor the photosynthetic oxygen evolution after chilling at 0°Cin the dark for 48 h. Thyla-koids isolated from such leaveswere not able to reduce dichloroindophenol (DCIP), but the additionof diphenylcarbazide (DPC), an electron donor to PS II, restoredthe ability to reduce DCIP, indicating that the site of damageis in the water-splitting machinery of PS II. In moderate light (500 jumol quanta m^–2s^–1), chillingof cucumber leaves at 5°C for 5 h was sufficient to inducethe complete loss of the capacity for photosynthetic oxygenevolution. Electron transport rates measured in thylakoids wereunaltered, but thylakoids were totally permeable to protons.Since the addition of dicyclohexylcarbodiimide (DCCD) restoredcoupling and the capacity for proton uptake, the primary siteof damage was deduced to be in the ATPase. In rice, both chilling treatments had barely any effect on thylakoidfunctions, although some negative effects was apparent in photosynthesisin leaves. ¹Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. ²Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received January 11, 1989; Accepted June 12, 1989)     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

A His-tagged PSII core complex was purified from recombinantChlamydomonas reinhardtii D2-H thylakoids by single-step Ni²⁺-affinitycolumn chromatography and its properties were partially characterizedin terms of their PSII functions and chemical compositions.The PSII core complex that has a His-tag extension at the C-terminusof the D2 protein evolved oxygen at a high rate of 2,400 µmol(mg Chl)^–1h^–1 at the optimum pH of 6.5 with ferricyanideand 2,6-dichlorobenzoquinone as electron acceptors in the presenceof Ca²⁺ as an essential cofactor, and approximately 90% of theactivity was blocked by 10 µM DCMU. The core complex exhibitedthe thermoluminescence Q-band but not the B-band regardlessof the presence or absence of DCMU, although both bands wereobserved in the His-tagged thylakoids. The core complex wasfree from PSI and contained one Y_D, Tyr 160 of the D2 protein,four Mn atoms, two cytochrome b-559, about 46 Chl a molecules,and probably one Q_A, the primary acceptor quinone of PSII. Itwas inferred from these results that His-tagging at the C-terminusof the D2 protein does not affect the functional and structuralintegrity of the PSII core complex, and that the ‘His-tagstrategy’ is highly useful for biochemical, physicochemical,and structural studies of Chlamydomonas PSII. (Received October 22, 1998; Accepted December 25, 1998)     

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.     

Regulation of Synthesis of PSI in the Cyanophytes Synechocystis PCC6714 and Plectonema boryanum during the Acclimation of the Photosystem Stoichiometry to the Light Quality

The effects of light quality on the formation of the PSI complexwere examined in Synechocystis PCC6714 and in Plectonema boryanum.The rate of increase in levels of core polypeptides of PSI,PsaA/B, doubled upon shift from Chl a-absorbed light (PSI light)to phycobilisome-ab-sorbed light (PSII light). The elevatedrate was decreased upon the reverse shift. Half time of theacceleration was approximately 10 min, and that of the decreasewas approximately 4 min. The rate of degradation of the polypeptideswas far lower than the rate of the increase under either lightregime. Neither synthesis nor degradation of the PsbA and PsbCpolypeptides of PSII was significantly altered by the lightquality. We conclude that synthesis of the PSI complex is chromaticallyregulated to allow adjustments in photosystem stoichiometry.The level of mRNA for PsaA/B was not altered by the light regime.Anomalous inhibition by chloramphenicol suggested that the regulationoccurs at a step(s) other than the peptide elongation step,perhaps at the initiation of the ribosome cycle or at the insertionof Chl a for the stabilization of the polypeptides. The pho-toreductionof protochlorophyllide (Pchlide) was compared with the synthesisof the polypeptides in a mutant of Plectonema boryanum thatlacked Pchlide dark reductase (YFC1004). The results indicatedthat the synthesis of stable PsaA/B polypeptides was not limitedby the reduction of Pchlide, although the synthesis did dependon a supply of Chl a. ¹Present address: Department of Plant Biology, University ofMaryland at College Park, MD 20742, U.S.A. ²Present address: Department of Marine Bioscience, Fukui Pre-fecturalUniversity, Obama, Fukui, 917 Japan     

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; )     

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)     

Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

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; )     

Identification of the Carboxyl-Terminal Processing Protease for the D1 Precursor Protein of the Photosystem II Reaction Center of Spinach

The enzyme involved in the carboxyl-terminal processing of theD1 precursor protein (pD1) of the photosystem II reaction centerwas purified from extracts of sonicated spinach thylakoids bya method that included chromatography on quaternary aminoethylanion-exchange, hydroxylapatite, copper-chelating affinity andgel-filtration columns. The enzyme was identified, from itschromatographic behavior, to be a monomeric protein of about45 kDa. The sequence of the amino-terminal 27 amino acids ofthis protein was determined directly, which exhibited low butappreciable (37%) homology to that deduced from a gene (ctpA)in Synechocystis sp. PCC 6803 that was proposed recently toencode the processing protease from results of genetic complementationanalysis. ³Present address: Asahi Kasei Chem. Co.     

Cloning of the Gene Encoding a Protochlorophyllide Reductase: the Physiological Significance of the Co-Existence of Light-Dependent and -Independent Protochlorophyllide Reduction Systems in the Cyanobacterium Plectonema boryanum

Cyanobacteria have two protochlorophyllide (Pchlide) reductasescatalyzing the conversion of Pchlide to chloro-phyllide, a keystep in the biosynthetic pathway of chlorophylls (Chls); a light-dependent(LPOR) and a light-independent (DPOR) reductase. We found anopen reading frame (ORF322) in a 2,131-bp EcoRI fragment fromthe genomic DNA of the cyanobacterium Plectonema boryanum. Becausethe deduced amino acid sequence showed a high similarity tothose of various plant LPORs and the LPOR activity was detectedin the soluble fraction of Esche-richia coli cells over-expressingthe ORF322 protein, ORF322 was defined as the por gene encodingLPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolatedby targeted mutagenesiss to investigate the physiological importanceof LPOR. YFP12 grew as well as wild type under low light conditions(10-25 µE m^–2 S^–1). However, its growth wassignificantly retarded as a result of a significant decreasein its Chl content under higher light conditions (85-130 µEm^–2 s^–1). Furthermore, YFP12 stopped growing andsuffered from photobleaching under the highest light intensity(170 µE m^–2 s^–1). In contrast, a chlL-dis-rupted(DPOR-less) mutant YFC2 grew as well as wild type irrespectiveof light intensity. From these phenotypic characteristics, weconcluded that, although both LPOR and DPOR contribute to Chlsynthesis in the cells growing in the light, the extent of thecontribution by LPOR increases with increasing light intensity;without it, the cells are unable to grow under light intensitiesof more than 130 µ Em^–2s-^–. (Received September 26, 1997; Accepted November 21, 1997)     

Joint field experiments for comparisons of measuring methods of photosynthetic production

During the 1st GAP Workshop at Konstanz in April 1982 comparativemeasurements of phytoplankton primary production by severaltechniques were conducted simultaneously at an offshore stationin Lake Konstanz and an experimental algal pond. Suspended glassbottle exposure techniques using ¹⁴C and ¹³C uptake gave P_z(mg C m^–3 h^–1) values which varied considerablynear-surface, but estimates of areal rates for the euphoticzone P_cu(mg C m^–3 h^–1) which were reasonably close.In the lake, P_z, from a vertical tube exposure (with ¹⁴C uptake)was greater than rates derived for integrated bottle samples.The oxygen bottle method permitted a good estimate of compensationdepth, corresponding to in situ growth studies. There were difficultiesin direct comparison between O₂ and carbon methods. Correlationbetween them for P_z was good in the lake but poor in the pond,both for suspended bottle and vertical tube methods. This seriesdemonstrates that despite reasonable overall estimates, comparativelyminor methodological differences in experimental technique cancause large variation. ⁺ Coordinator of the group for comparative measurements of photosyntheticproduction at the GAP Workshop, Konstanz, April 1982. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP) First International Workshop heldat the Limnological Institute, University of Konstanz, in April1982.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta II. Aspects of Photoregulative Reaction

The photoregulation of carotenogenesis in Rhodotorula minutawas found to consist of tow phases, a temperature-independentphotochemical reaction (light process) and temperature-dependentbut light-independent biochemical reactions (dark process).These processes were separately examined by regulating the temperatureand were characterized as follows: 1) The quantity of carotenoid produced [C (µg g^–1)]and the rate of carotenoid production [Vc (µg g^–1hr^–1)] in the dark process were regulated by the lightdose [D (erg cm^–2)] to which cells were exposed in thelight process. These relationships were expressed by the equations:C=9.1 log D–62.0 and Vc=0.81 log D–5.60. This photoresponsefollowed the Roscoe-Bunsen reciprocity law. 2) The induced state toward carotenogenesis, once acquired inthe light process, was very stable, suggesting that the proposedphotochemical product is stable as an inducer of carotenogenesisand decreases only in conjunction with carotenoid biosynthesis. 3) The photochemical reaction was oxygen-independent, but subsequentdark reactions were completely dependent on oxygen. 4) Postulated compounds related to the photochemical reactionwere not metabolized in vivo. (Received September 12, 1981; Accepted February 20, 1982)     

Characterization and Translation of Poly(A)+RNA from Wounded and Crown Gall Tissues of Potato Tubers

Poly(A)⁺ and poly(A)^–RNA from wounded potato tuber tissuesand crown gall tumors were separated from total RNA by oligodeoxythymidylicacid-cellulose affinity chromatography. The poly(A)⁺RNA wascharacterized by sucrose density gradient centrifugation, hybridizationwith ³(H)polyuridylic acid [Poly(U)] and in vitro translationin a rabbit reticulocyte lysate system. The tumor poly(A)⁺RNAwas a heterodisperse mixture from 3.5S to 35S. Upon poly(U)hybridization of the gradient fractions two major hybridizationpeaks at 7S and 21S and two peaks at 11S and 16S appeared. Inan in vitro translation system the poly(A)⁺RNA programmed thesynthesis of 23 different polypeptides of 9,000 to 79,800 daltonsmolecular weight as determined by SDS-polyacrylamide gel electrophoresis.The 21S poly(A)+RNA was about 5 times more active in in vitroprotein synthesis than the 7S poly(A)⁺RNA. The poly(A)⁺RNA from wounded tissues was also heterodisperse(from 4.5S to 31S) with a modal peak at 18S. This RNA codedfor at least 28 polypeptides, which were different from thoseof crown gall tumor tissues. On a per unit poly(A)⁺RNA basis the tumor RNA was slightly moreactive in translation than that from wounded tissues. The translationof tumor poly(A)⁺RNA was completely blocked by 0.5 mM 7-methylguanosine5'-phosphate, but not by 7-methylguanosine, suggesting the presenceof a 5'-cap structure. (Received May 15, 1982; Accepted June 30, 1982)     

Requirements for the light-stimulated degradation of stromal proteins in isolated pea (Pisum sativum L.) chloroplasts

Chloroplasts from 17-d-old pea leaves (Pisum sativum L.) wereisolated to elucidate the requirements for the light-induceddegradation of stromal proteins. The influence of electron transportthrough the thylakoids and the influence of ATP on protein degradationwere investigated. When chloroplasts were incubated in the light(45 µmol m^–2s^–1), glutamine synthetase, thelarge subunit of ribulose-1,5-bisphosphate carboxylase and glutamatesynthase were degraded, whereas phosphoribulokinase, ferredoxin-NADP⁺reductase and the 33 kDa protein of photosystem II remainedmore stable. Major protein degradation was not observed over240 mm in darkness. The electron transport inhibitor dichlorophenyldimethylureareduced protein degradation in the light over several hours,whereas dibromothymoquinone was less effective. Inhibiting theproduction of ATP with tentoxin or by destroying the     

Growth of grana from "primary" thylakoids in Phaseolus vulgaris

The plastids of young dark-grown bean leaves, exposed to periodiclight are agranal, devoid of chlorophyll b and contain primarythylakoids and chlorophyll a. Transfer of these plants to continuousillumination results in synthesis of new chlorophyll a, chlorophyllb and grana. This study was done in order to study whether andhow the grana are formed from preexisting primary thylakoids.¹⁴C--aminolevulinic acid was used to label the chlorophyll aof the primary thylakoids, and its fate was studied after transferof the plants to continuous light. It was found that chlorophyll b and grana become ¹⁴C-labelled.The total radioactivity of chlorophyll b per bean increasedwith the parallel decrease of that of chlorophyll a. All subchloroplastfractions, obtained after digitonin disruption of chloroplasts,contained chlorophyll a of equal specific radioactivity. Thespecific radioactivity of chlorophyll b was lower than thatof chlorophyll a, and, in addition, it was lower in the granathan in the stroma lamellae fraction. The data suggest that chlorophyll b is formed from chlorophylla; the grana are formed by stacking of preexisting primary thylakoids;chlorophyll b is synthesized faster in the grana than the stromalamellae; the newly formed chlorophyll a molecules are distributedat random throughout the developing photosynthetic membraneand not on specific growing sites. (Received April 24, 1976; )     

Response to Waterlogging and Differing Sensitivity to Divalent Iron and Manganese in Clones of Dactylis glomerata L. derived from Well-drained and Poorly-drained Soils

Clonally propagated plants of Dactylis glomerata derived froma well-drained, heavily grazed cliff habitat (clone L) and froman undergrazed poorly-drained soil (clone A) were tested forwaterlogging tolerance in soil-culture. Water-logging did notaffect the two clones differentially, a result, which contrastedstrongly with that of a previous experiment in which simulatedgrazing (clipping to 20 cm) unexpectedly caused clone A to beless tolerant of waterlogging than clone L. Maximum leaf andleaf sheath length was reduced more by water-logging in cloneL than in clone A (P < 0.05). In solution-culture when providedwith factorial combinations of 0.5, 5 and 50 mg dm^–2 ofFe²⁺ and Mn²⁺ the shoot dry weight yield of the dry-soil clonewas reduced more than that of the wet-soil clone by 50 mg Fedm^–3 irrespective of Mn²⁺ concentration (P < 0.01)but the reduction of growth was less at higher Mn²⁺ concentrations.Fifty milligrams of Mn²⁺ dm^–3 reduced the growth of thedry soil clone but increased the growth of the wet soil clonewith Fe²⁺ at 5 mg dm^–2 (P < 0.05). Iron at 0.5 mg dm^–2was suboptimal for shoot growth of both clones at any levelof Mn²⁺ and caused more severe leaf chlorosis in the wet soilclone. Leaf tissue of clone L contained more iron than thatof clone A after waterlogging (P < 0.01) but in solutionculture, though increasing iron from 0.5 to 50 mg dm^–3almost doubled leaf iron content (P < 0.001), the interactionClones x Mn x Fe just failed to reach significance at P <0.05. The manganese content of leaf tissue from the two clonesvaried differently in response to solution manganese (Clonesx Mn P < 0.01), clone A showing a slightly greater increaseof manganese content at high solution concentration. Iron at50 mg dm^–3 suppressed Mn uptake (Mn x Fe, P < 0.001)in both clones. The two clones thus show marked environmentaladaptation to the chemistry of wet and dry soils. Dactylis glomerata, Cocksfoot grass, Orchard grass, waterlogging, iron, manganese, toxicity, deficiency, ecotypes     

Identification of a nifDK-Like Gene (ORF467) Involved in the Biosynthesis of Chlorophyll in the Cyanobacterium Plectonema boryanum

The frxC gene, which is found in chloroplast DNA (ctDNA) andin cyanobacteria, encodes a protein that is required for thelight-independent reduction of protochlorophyllide (Pchlide)to chlorophyllide a (Chlide). A DNA fragment downstream of frxCin the filamentous cyanobacterium Plectonema boryanum was clonedand analyzed. Sequencing of the DNA fragment revealed an openreading frame (ORF) that encoded a protein of 467 amino acidresidues (designated ORF467), which showed extensive homologyto the proteins encoded by genes on ctDNAs (ORF465 in liverwort,gidA in pine and chlN in Chlamydomonas reinhardtii) and to ORF469protein of the cyanobacterium Synechocystis sp. strain PCC 6803.We isolated a targeted mutant YFM6D-3 in which ORF467 was inactivatedby the insertion of a kanamycin-resistance gene into the codingregion. YFM6D-3 exhibited a phenotype similar to that of YFC1004,an frxC-disrupted mutant, which did not synthesize chlorophyll(Chl) and accumulated Pchlide, a precursor to Chl, in the dark.These phenotypic characteristics of YFM6D-3 indicate that thelight-independent reduction of Pchlide requires not only theFrxC protein but also the ORF467 protein. The amino acid sequencesof the homologues of ORF467 exhibit low but significant similarityto those of the and ß subunits of nitrogenase MoFe-protein,suggesting a phylogenetic relationship between the light-independentPchlide reductase and nitrogenase, as is observed between theFrxC protein and the Fe-protein of nitrogenase. ¹Institute for Protein Research, Osaka University, Suita, Osaka,565 Japan     

Salinity and in vitro Ageing Effects on Primary Photosynthetic Processes of Thylakoids Isolated from Pisum sativum and Spinacia oleracea

The effects of in vitro ageing and salinity of the reactionmedium on the primary photochemistry of photosystem II and thepattern of energy distribution within the photochemical apparatusof thylakoids isolated from Pisum sativum and Spinacia oleraceaare described. Analyses of the low temperature induction curvesof fluorescence emission at 695 and 735 nm and of low temperatureabsorption and fluorescence emission spectra were used to examinethese processes. In vitro ageing over short periods reducedthe photochemical activity and changed the energy distributionwithin thylakoids of P. sativum, but had little effect on thylakoidsof S. oleracea. A synergistic effect of in vitro ageing andsalinity of the reaction medium was observed for P. sativumthylakoids. Ageing effects could be minimized by addition of100 mM NaCl to the resuspension medium. Changes in NaCl concentrationin the reaction medium produced large and similar changes inthe primary photochemical functioning of thylakoids from P.sativum and S. oleracea, which could be attributed mainly tothe cation species, Na^$; however, experiments using mannitolto produce osmotic stress indicated some small osmotically inducedchanges in photofunction of the thylakoids. Optimal primaryphotochemical activity of photosystem II, for both species,was observed with 200 mM NaCl. Cation-induced regulation ofexciton distribution appears to be facilitated by controllingthe degree of energy coupling between the light-harvesting chlorophylla/b complex and the two photosystems, I and II, and not by regulationof coupling between photosystems I and II.     

Photoregulation of Respiratory Activity in the Cyanophyte Synechocystis PCC 6714: the Possibility of the Simultaneous Regulation of the Amount of PS I Complex and the Activity of Respiratory Terminal Oxidase in Thylakoids

Respiration of the cyanophyte Synechocystis PCC 6714 was studiedin relation to conditions for cell growth. Under our experimentalconditions, the KCN-sensitive O₂-uptake observed with intactcells was found to be limited at the step catalyzed by the terminaloxidase in thylakoids, indicating that the activity of O₂-uptakeby intact cells corresponds to that of the terminal oxidasein thylakoids. The activity was found to be variable dependingon the growth conditions; it was higher under conditions wherethe level of PS I, another terminal component of the thylakoidelectron transport system (ETS) was elevated, whereas it waslower under conditions where the level of PS I was reduced.Changes in the activity did not occur when protein synthesiswas suppressed by chloramphenicol. The results suggest that,similarly to the regulation of levels of PS I, the activityor the amount of terminal oxidase in thylakoids is regulatedin response to the redox steady-state of intermediate component(s)of ETS, in order to maintain a balance between the efflux ofelectrons from the ETS and the influx to the ETS. ¹Present address: P.G. Department of Botany, Utkal University,Bhubaneswar-751004, Orissa, Keonjhar, India (Received September 27, 1989; Accepted March 22, 1990)