Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Viability of Chlamydomonas Mutants with Amino Acid Substitutions in the Precursor D1 Protein at the Carboxyl-Terminal Processing Site: an Analysis by Mixed-Culture Growth Experiments

Department of Biology, Faculty of Science, Okayama University,3-1-1, Tsushima-naka, Okayama, 700-8530 Japan In order to analyzethe influence of amino acid substitutions at the carboxyl-terminalprocessing site of the D1 precursor protein, mixed-culture growthexperiments were conducted for psbA directed mutants of Chlamydomonasreinhardtii. Wild type and D1 mutants were mixed in the sameculture and their viability was compared. Replacement of Ser-345by Gly or Val at the cleavage site markedly affected the relativegrowth rate of the mutant in the high intensity light, but notin a dim light or the darkness. This was consistent with theprevious result obtained by in vitro analysis using substitutedcarboxyl-terminal oligopeptides as substrates [Taguchi et al.(1995) J Biol. Chem. 270: 10711], This is a clear indicationthat the rate of carboxyl-terminal processing of the D1 precursorin the photosystem II reaction center is a rate-limiting stepfor growth under some environmental stress conditions. (Received June 9, 1998; Accepted September 25, 1998)     

Localisation and processing of the precursor form of photosystem II protein D1 inSynechocystis 6803

Pure plasma membrane and thylakoid membrane fractions from Synechocystis 6803 were isolated to study the localisation and processing of the precursor form of the D1 protein (pD1) of photosystem II (PSII). PSII core proteins (D1, D2 and cytb559) were localised both to plasma and thylakoid membrane fractions, the majority in thylakoids. pD1 was found only in the thylakoid membrane where active PSII is known to function. Membrane fatty acid unsaturation was shown to be critical in processing of pD1 into mature D1 protein. This was concluded from pulse-labelling experiments at low temperature using wild type and a mutant Synechocystis 6803 with a low level of membrane fatty acid unsaturation. Further, pD1 was identified as two distinct bands, an indication of two cleavage sites in the precursor peptide or, alternatively, two different conformations of pD1. Our results provide evidence for thylakoid membranes being a primary synthesis site for D1 protein during its light-activated turnover. The existence of the PSII core proteins in the plasma membrane, on the other hand, may be related to the biosynthesis of new PSII complexes in these membranes.     

Photoinhibition and light-induced cyclic electron transport in ndhB(-) and psaE(-) mutants of Synechocystis sp. PCC 6803

The ndhB^– and psaE^– mutants of the cyanobacteriumSynechocystis sp. PCC 6803 are partly deficient in PSI-drivencyclic electron transport. We compared photoinhibition in thesemutants to the wild type to test the hypothesis that PSI cyclicelectron transport protects against photoinhibition. Photoinhibitorytreatment greatly accelerated PSI cyclic electron transportin the wild type and also in both the mutants. The psaE^–mutant showed rates of PSI cyclic electron transport similarto the wild type under all conditions tested. The ndhB^–mutant showed much lower rates of PSI cyclic electron transportthan the wild type following brief dark adaptation but exceededwild type rates after exposure to photoinhibitory light. Thewild type and both mutants showed similar rates of photoinhibitiondamage and photoinhibition repair at PSII. Photoinhibition atPSI was much slower than at PSII and was also similar betweenthe wild type and both mutants, despite the known instabilityof PSI in the psaE^– mutant. We conclude that photoinhibitorylight induces sufficient PSI-driven cyclic electron transportin both the ndhB^– and psaE^– mutants to fulfill anyrole that cyclic electron transport plays in protection againstphotoinhibition. ⁴ Corresponding author: E-mail, sherbert@uwyo.edu; Fax, +1-307-766-2851;Phone, +1-307-766-4353.     

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)     

A role of the C-terminal extension of the photosystem II D1 protein in sensitivity of the cyanobacterium Synechocystis PCC 6803 to photoinhibition.

The D1 protein, a key protein subunit of Photosystem II complex (PSII), is synthesised as a precursor (pD1) with a carboxyl-terminal extension. In the cyanobacterium Synechocystis sp. PCC 6803, this extension consists of 16 amino acid residues and it is cleaved by a specific protease in two putative steps with the final cleavage after the residue Ala344. In order to define the importance of the extension for the functioning of PSII, we constructed and characterized several site-directed mutants of Synechocystis that differ in the length and amino acid sequence of this extension. The mutant lacking the entire C-terminal extension exhibited slightly increased sensitivity to photoinhibition. Analysis of the PSII assembly in the mutant by the blue-native electrophoresis in combination with radioactive labelling revealed an increased level of the unassembled D1 protein in this strain. Replacement of the amino acid residue Asn359 by His or Asp also led to the higher vulnerability to photoinhibition of both mutants. In the Asn359His mutant, this vulnerability was accompanied by an increased level of the PSII core lacking CP43 indicating limitation of the repair cycle in the CP43 reassembly step.     

Competitive inhibition analysis of the enzyme-substrate interaction in the carboxy-terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides

A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic C-terminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems.     

2-Trans-Abscisic Acid Biosynthesis and Metabolism of ABA-Aldehyde and Xanthoxin in Wild Type and the aba Mutant of Arabidopsis thaliana

Abscisic acid (ABA) and 2-trans-ABA (t-ABA) biosynthesis werestudied in wild type Landsberg erecta and the three allelicaba mutants of Arabidopsis thaliana (L.) Heynh., which are impairedin epoxy-carotenoid biosynthesis. Labelling experiments with¹⁸O₂and mass spectrometric analysis of [¹⁸O]ABA and its catabolitesABA-glucose ester (ABA-GE) and phaseic acid (PA), and t- ABAand t-ABA-GE, showed that t-ABA biosynthesis was less affectedthan ABA biosynthesis by mutations at the ABA locus. The aba-4allele caused the most severe impairment of ABA biosynthesiscompared with the other two mutant alleles aba-1 and aba-3,yet aba-4 plants synthesized as much t-ABA as wild type Landsbergerecta plants. Feeding experiments with RS- [²H₆]ABA-aldehydeisomers and unlabelled xanthoxin isomers suggest that t-xanthoxinand t-ABA-aldehyde are precursors to ABA and t-ABA in Arabidopsis Key words: ABA-alcohol, ABA-aldehyde, ABA-glucose ester, ¹⁸O₂ labelling, phaseic acid     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Molecular cloning and nucleotide sequence analysis of psbA from the dinoflagellates: Origin of the dinoflagellate plastid

Cloning and sequencing of psbA, the gene encoding D1 protein of photosystem II, from six species of dinoflagellates harboring a peridinin type plastid [Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra Stein, Lingulodinium polyedra (Dodge) Stein, Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech] is reported. Using the polymerase chain reaction technique, the psbA gene was detected in a satellite DNA band isolated from total DNA of A. catenella by CsCl-Hoechst 33258 gradient ultracentrifugation. This finding suggests that in dinoflagellates psbA is encoded in the plastid genome. The deduced amino acid sequences of D1 from the dinoflagellates did not reveal a typical ‘C-terminus extension’, which should be removed by proteolytic cleavage from the D1 precursor. Molecular phylogenetic analysis based on the deduced amino acid sequences of D1 revealed that the six species of dinoflagellates are monophyletic and also showed that dinoflagellates cluster with rhodophytes, a cryptophyte and heterokonts. These results support the hypothesis that the peridinin type plastid in dinoflagellates originated from an engulfed red alga.     

An Evidence Indicating That a Weak Orange Light Absorbed by Phycobilisomes Causes Inactivation of PSII in Cells of the Red Alga Porphyridium cruentum Grown under a Weak Red Light Preferentially Exciting Chl a

Changes in the PSII fluorescence upon shift of light qualitywere studied with the red alga Porphyridium cruentum IAM R-1and supplementarily with P. cruentum ATCC 50161, the cyanophytesSynechocystis spp. PCC6714 and PCC6803 and Synechococcus sp.NIBB1071. When Porphyridium cruentum grown under a weak redlight (PSI light) preferentially absorbed by Chl a was illuminatedwith a weak orange light (PSII light) mainly absorbed by phycobilisomes(PBS), a change of PSII fluorescence at room temperature wasinduced. The ratio of Fvm (Fm— Fo) to Fm was reduced rapidlyaccompanying the increase in Fo (T^1/2 ca. 3 min). The effectsof DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneindicated that the fluorescence change is induced when plastoquinonepool is highly reduced. The fluorescence change after a shortPSII light illumination was reversible; it rapidly recoveredin the dark (T ^1/2 ca. 3 min). The reversibility was graduallyreduced and disappeared after 40 h under PSII light accompanyingdecrease in PSII activity per PBS down to almost 50%. Sincethe pattern of the fluorescence change resembles that observablewhen PSII is photoinactivated, PSII light probably induces thephotoinactivation of PSII, possibly reversibly at first andirreversibly after prolonged illumination. Such a rapid fluorescencechange was insignificant in Synechocystis sp. either PCC6714or PCC6803. Only a slow and small decrease in Fvm/Fm level appearedafter prolonged PSII light illumination (the reduction of PSIIactivity per PBS was around 20%). In Porphyridium, shift fromPSII light to PSI light caused a rapid and chloramphenicol-sensitiveFvm/Fm elevation during the first 10 h while the increase inPSH activity per PBS was only 10% of that before the light shift.Then, a gradual elevation followed up to the level at the steadystate under PSI light. A similar rapid increase in Fvm/Fm wasobserved with Synechocystis PCC6714, in which the synthesisof PSII is not regulated, suggesting that a rapid increase inFvm/Fm does not reflect the acceleration of the synthesis ofPSII. Results were interpreted as that (1) PSII light causesphotoinactivation of PSII. Such a photoinactivation is markedin Prophyridium cells grown under PSI light. (2) In Porphyridium,changes in the abundance of PSII upon shift of light qualityare largely attributed to the photoinactivation of this type. (Received February 19, 1999; Accepted June 14, 1999)     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

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.     

The cyanobacterial homologue of HCF136/YCF48 is a component of an early photosystem II assembly complex and is important for both the efficient assembly and repair of photosystem II in Synechocystis sp. PCC 6803

The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair.     

Interaction between lysine 102 and aspartate 338 in the insect amino acid cotransporter KAAT1

KAAT1 is a lepidopteran neutral amino acid transporter belonging to the NSS super family (SLC6), which has an unusual cation selectivity, being activated by K⁺ and Li⁺ in addition to Na⁺. We have previously demonstrated that Asp338 is essential for KAAT1 activation by K⁺ and for the coupling of amino acid and driver ion fluxes. By comparing sequences of NSS family members, site-directed mutagenesis, and expression in Xenopus laevis oocytes, we identified Lys102 as a residue likely to interact with Asp338. Compared with wild type, the single mutants K102V and D338E each showed altered leucine uptake and transport-associated currents in the presence of both Na⁺ and K⁺. However, in K102V/D338E double mutant, the K102V mutation reversed both the inhibition of Na⁺-dependent transport and the block in K⁺-dependent transport that characterize the D338E mutant. K⁺-dependent leucine currents were not observed in any mutants with D338E. In the presence of the oxidant Cu(II) (1,10-phenanthroline)₃, we observed specific and reversible inhibition of K102C/D338C mutant, but not of the corresponding single cysteine mutants, suggesting that these residues are sufficiently close to form a disulfide bond. Thus both structural and functional evidence suggests that these two residues interact. Similar results have been obtained mutating the bacterial transporter homolog TnaT. Asp338 corresponds to Asn286, a residue located in the Na⁺ binding site in the recently solved crystal structure of the NSS transporter LeuT_Aa (41). Our results suggest that Lys102, interacting with Asp338, could contribute to the spatial organization of KAAT1 cation binding site and permeation pathway. residue interaction; oxidants; tertiary structure     

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)     

Different Localization of Two Ca2+ in Spinach Oxygen-Evolving Photosystem II Membranes. Evidence for Involvement of Only One Ca2+ in Oxygen Evolution

Localization of the two Ca²⁺ bound to oxygen-evolving photosystemII (PSII) membranes from spinach was investigated by fractionatingthe membranes into the PSII reaction center core complexes andperipheral antenna Chl a/b-proteins after solubilization withn-heptylthioglucoside. The core complex fraction contained oneCa²⁺ per PSII, while another Ca²⁺ was found in the solubilizedmajor light-harvesting Chl a/b-proteins (LHCII). LHCII isolatedwith Triton X-100 or dodecylmaltoside also contained Ca²⁺ inan amount corresponding to one per PSII. The Ca²⁺ bound to LHCIIcould not be removed by treatment with Chelex 100, which effectivelysequestered extraneous Ca²⁺ bound to LHCII, or by preparationof LHCII in the presence of 40 mM citrate. Localization of thetwo Ca²⁺ in different functional domain of PSII membranes conclusivelyindicates that the number of the bound Ca²⁺ that can functionin oxygen evolution is one per PSII. The results also suggestthat one Ca²⁺ has a structural role in the peripheral antennaassembly. (Received July 21, 1992; Accepted March 9, 1993)     

Effects of Light Stress on Redox Potential Forms of Cyt b-559 in Photosystem II Membranes Depleted of Water-Oxidizing Complex

Effects of photoinhibition on the redox properties of Cyt b-559were studied with NH₂OH treated PSII membranes, which are depletedof the water-oxidizing complex. The membranes contained threeredox forms (HP-, IP- and LP-forms) of Cyt b-559, with E_m valuesof +435, +237 and +45 mV, respectively. A novel intermediate-potentialform of Cyt b-559 was generated during photoinhibition on thedonor side of PSII: photoinhibitory illumination (7,000 µEm^–2 s^–1) for 1 min induced a 30% decrease in thelevel of the HP-form, with concomitant generation of the intermediate-potential(IP-) form whose E_m value was about +350mV. Prolonged illumination(10 min) resulted in complete loss of the HP-form and an apparentincrease in the level of the IPform. After further photoinhibitorytreatment (60 min), complete loss of the IP'-form was observedand levels of the IP- and LP-forms each increased to about 50%of the total amount of Cyt b-559. Kinetic analysis of thesedata led to the conclusion that the HP-form is converted tothe LP-form via two intermediate-potential forms (IP' and IP),and that IP'-form appears only at the early phase of photoinhibition. (Received March 30, 1994; Accepted February 27, 1995)     

Ammonium tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 and the role of the psbA multigene family

Ammonium is one of the major nutrients for plants, and a ubiquitous intermediate in plant metabolism, but it is also known to be toxic to many organisms, in particular to plants and oxygenic photosynthetic microorganisms. Although previous studies revealed a link between ammonium toxicity and photodamage in cyanobacteria under in vivo conditions, ammonium‐induced photodamage of photosystem II (PSII) has not yet been investigated with isolated thylakoid membranes. We show here that ammonium directly accelerated photodamage of PSII in Synechocystis sp. strain PCC6803, rather than affecting the repair of photodamaged PSII. Using isolated thylakoid membranes, it could be demonstrated that ammonium‐induced photodamage of PSII primarily occurred at the oxygen evolution complex, which has a known binding site for ammonium. Wild‐type Synechocystis PCC6803 cells can tolerate relatively high concentrations of ammonium because of efficient PSII repair. Ammonium tolerance requires all three psbA genes since mutants of any of the three single psbA genes are more sensitive to ammonium than wild‐type cells. Even the poorly expressed psbA1 gene, whose expression was studied in some detail, plays a detectable role in ammonium tolerance.     

Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

cDNA encoding the plasma membrane H⁺-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 79–85% identical in terms ofamino acid sequence to other plant H⁺-ATPases. High levels ofmRNA explain the high H⁺-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995)