Antenna Sizes of Photosystem I and Photosystem II in Chlorophyll b-Deficient Mutants of Rice. Evidence for an Antenna Function of Photosystem II Centers That are Inactive in Electron Transport

Light-harvesting capacities of photosystem I (PSI) and photosystemII (PSII) in a wild-type and three chlorophyll b-deficient mutantstrains of rice were determined by measuring the initial slopeof light-response curve of PSI and PSII electron transport andkinetics of light-induced redox changes of P-700 and Q_A, respectively.The light-harvesting capacity of PSI determined by the two methodswas only moderately reduced by chlorophyll b-deficiency. Analysisof the fluorescence induction that monitors time course of Q_Aphotoreduction showed that both relative abundance and antennasize of PSII_a decrease with increasing deficiency of chlorophyllb and there is only PSII_rß in chlorina 2 which totallylacks chlorophyll b. The numbers of antenna chlorophyll moleculesassociated with the mutant PSII centers were, therefore, threeto five times smaller than that of PSII_a in the wild type rice.Rates of PSII electron transport determined on the basis ofPSII centers in the three mutants were 60–70% of thatin the normal plant at all photon flux densities examined, indicatingthat substantial portions of the mutant PSII centers are inactivein electron transport. The initial slopes of light-responsecurves of PSII electron transport revealed that the functionalantenna sizes of the active populations of PSII centers in themutants correspond to about half that of PSII in the wild typerice. Thus, the numbers of chlorophyll molecules that serveas antenna of the oxygen-evolving PSII centers in the mutantsare significantly larger than those that are actually associatedwith each PSII center. It is proposed that the inactive PSIIserves as an antenna of the active PSII in the three chlorophyllb-deficient mutants of rice. In spite of the reduced antennasize of PSII, therefore, the total light-harvesting capacityof PSII approximately matches that of PSI in the mutants. (Received July 29, 1994; Accepted February 7, 1996)     

The Biological Properties of Cyclopenin and Cyclopenol

Cyclopenin (C₁₇H₁₄O₃N₂) and cyclopenol (C₁₇H₁₄O₄N₂), isolatedfrom an abberent strain of Penicillium cyclopium (NRRL 6233),significantly inhibited the growth of etiolated wheat (Triticumaestivum) coleoptile segments. The former inhibited at 10^–3and 10^–4 M, the latter at 10^–3 M. Cyclopenin producedmalformation of the first set of trifoliate leaves in bean (Phaseolusvulgaris) at 10^–2 M and necrosis and stunting in corn(Zea mays) at 10^–2 M. Cyclopenol induced no apparent effectsin bean or corn plants. Neither compound changed the growthor morphology of tobacco (Nicotiana tabacum) plants. Cyclopenininduced intoxication, prostration and ataxia in day-old chicksat 500 mg/kg, but they recovered within 18 hours. Cyclopenolwas inactive against chicks when dosed at levels up to 500 mg/kg. (Received October 11, 1983; Accepted December 15, 1983)     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) II. Effects of Inhibitors, Uncouplers and an Artificial Electron Mediator on the Inhibition of 14CO2 Fixation by Anaerobiosis

In "air-grown" Chroomonas sp. cells, low concentrations of DCMU(less than 0.1 µM) could prevent the inhibition of ¹⁴CO₂fixation by anaerobiosis under light-saturating conditions (morethan 40 W.m^–2), with phenazine methosulfate showing asimilar effect. Antimycin A, carbonyl cyanide m-chlorophenylhydrazone(CCCP), and N,N'-dicyclohexylcarbodiimide strongly inhibitedanaerobic photosynthesis at concentrations which did not significantlyinhibit the rate under 2% O₂ at high light intensity (200 W.m^–2),although 0.2 µM CCCP stimulated the rate under 2% O₂ tosome extent. On the other hand, KCN inhibited the rate muchmore strongly under 2% O₂ than N₂, although it inhibited therate very strongly at concentrations above 5 µM both underN₂ and 2% O₂. These results suggest that the inhibition of photosynthetic¹⁴CO₂ fixation by anaerobiosis in this alga result from ATPdeficiency caused by over-reduction of electron carriers ofthe cyclic electron flow and that oxygen can prevent the over-reduction.Cyclic electron flow seems to be necessary to provide additionalATP for CO₂ reduction under anaerobic conditions, although itseems to be less necessary under aerobic conditions. (Received July 21, 1983; Accepted January 23, 1984)     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated in Chlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtiicells was increased by treatment with poly-L-lysine or dimethylsulphoxideas indicated by 3-phosphoglyceric acid dependent O₂ evolution.These treatments decreased the ability of the cells to accumulateinorganic carbon internally and hence their photosynthetic affinityfor inorganic carbon in the medium. With saturating light andinorganic carbon, the photosynthetic rate was less affectedby the poly-L-lysine and dimethylsulphoxide treatments. Thusthe poly-L-lysine and dimethylsulphoxide did not alter the activityof the chloroplasts but rather made the intracellular inorganiccarbon pool more freely exchangeable with the medium. It isconcluded that the transporting system for inorganic carbonis located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, didnot affect photosynthetic rate and accumulation of inorganiccarbon when CO₂ was supplied but strongly inhibited both parameterswhen HCO^–₃ was supplied. In a mutant of Chlamydomonasreinhardtii lacking a cell wall, carbonic anhydrase leaks tothe medium and uptake of inorganic carbon is much faster whenCO₂ is supplied than when HCO^–₃ is supplied. These resultssuggest that CO₂ rather than HCO^–₃ is the inorganic carbonspecies that is actively translocated across the plasmalemma. Key words: Chlamydomonas, Inorganic carbon uptake     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

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)     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

New fava bean guard cell signaling mutant impaired in ABA-induced stomatal closure

We isolated a mutant from Vicia faba L. cv. House Ryousai. Itwilts easily under strong light and high temperature conditions,suggesting that its stomatal movement may be disturbed. We determinedresponses of mutant guard cells to some environmental stimuli.Mutant guard cells demonstrated an impaired ability to respondto ABA in 0.1 mM CaCl₂ and stomata did not close in thepresence of up to 1 mM ABA, whereas wild-type stomata closedwhen exposed to 10 µM ABA. Elevating external Ca²⁺caused a similar degree of stomatal closure in the wild typeand the mutant. A high concentration of CO₂ (700 µlliter^–1) induced stomatal closure in the wild type, butnot in the mutant. On the basis of these results, we proposethe working hypothesis that the mutation occurs in the regiondownstream of CO₂ and ABA sensing and in the region upstreamof Ca²⁺ elevation. The mutant is named fia (fava bean impairedin ABA-induced stomatal closure). ³ Corresponding author: E-mail, smoiwai{at}agri.kagoshima-u.ac.jp;Fax, +81-99-285-8556.     

Weak Light-Induced Oxygen Consumption Observed during Photoreactivation Is Coupled to the Recovery of Oxygen Evolving Activity

During photoreactivation of the O₂-evolving center in Tris-inactivated/Mn-depletedthylakoids, a slow O₂-consumption occurred. This O₂-consumptionbecame detectable when the O₂-evolving activity of thylakoidswas inactivated by Tris-treatment and decreased as photoreactivationproceeded. The O₂-consumption and photoreactivation similarlyrequired Mn²⁺ at µM levels in addition to PSII electrondonors and shared severa common characteristics. Stimulationof O₂-consumption and photoreactivation by these cofactors werealways accompanied by enhancement in chlorophyll fluorescenceinduction, suggesting the involvement of a Mehler-type reactionin photoreactivation. Although the electron transport due tothis O₂-consumption was rapid enough to oxidize 4 Mn²⁺ ionsto reconstitute the tetranuclear Mn-cluster in each O₂-evolvingcenter in a few seconds, actual recovery of O₂-evolving activityoccurred more slowly in a few minutes. It was inferred thatphotoreactivation in Tris-inactivated thylakoids is not a simplephotooxidation of Mn2²⁺ but involves more complicated processeswhich are coupled to the Mehlertype electron transport fromPSII to oxygen via PSI. (Received July 11, 1994; Accepted August 23, 1996)     

Regeneration of Ribulose 1,5-bisphosphate and Ribulose 1,5-bisphosphate carboxylase/oxygenase Activity Associated with Lack of Oxygen Inhibition of Photosynthesis at Low Temperature

The nature of the lack of oxygen inhibition of C₃-photosynthesisat low temperature was investigated in white clover (Trifoliumrepens L.). Detached leaves were brought to steady-state photosynthesisin air (34 Pa p(CO²), 21 kPa p(O₂), balance N₂) at temperaturesof 20°C and 8°C, respectively. Net photosynthesis, ribulose1,5-bisphosphate (RuBP) and ATP contents, and ribulose 1,5-bisphosphatecarboxylase/oxygenase (RuBPCO) activities were followed beforeand after changing to 2·0 kPa p(O₂). At 20°C, lowering p(O₂) increased net photosynthesis by37%. This increase corresponded closely with the increase expectedfrom the effect on the kinetic properties of RuBPCO. Conversely,at 8°C net photosynthesis rapidly decreased following adecrease in p(O₂) and then increased again reaching a steady-statelevel which was only 7% higher than at 21 kPa p(O₂). The steady-staterates of RuBP and associated ATP consumption were both estimatedto have decreased. ATP and RuBP contents decreased by 18% and33% respectively, immediately after the change in p(O₂) suggestingthat RuBP regeneration was reduced at low p(O₂) due to reducedphotophosphorylation. Subsequently, RuBP content increased again.Steady-state RuBP content at 2·0 kPa p(O₂) was 24% higherthan at 21 kPa p(O₂). RuBPCO activity decreased by 22%, indicatingcontrol of steady-state RuBP consumption by RuBPCO activity. It is suggested that lack of oxygen inhibition of photosynthesisat low temperature is due to decreased photophosphorylationat low temperature and low p(O₂). This may be due to assimilateaccumulation within the chloroplasts. Decreased photophosphorylationseems to decrease RuBP synthesis and RuBPCO activity, possiblydue to an acidification of the chloroplast stroma. Key words: Oxygen inhibition, photosynthesis, ribulose bisphosphate carboxylase/oxygenase     

Thylakoid Membrane Development and Differentiation: Assembly of the Chlorophyll a-b Light-Harvesting Complex and Evidence for the Origin of Mr=19, 17.5 and 13.4 kDa Proteins

Pea plants were grown under intermittent illumination (ImL)conditions. The low dosage of light given to ImL plastids limitedthe rate of chlorophyll (Chl) a and Chl b biosynthesis and,therefore, it retarded the rate of photosynthetic unit formationand thylakoid membrane development. Depending on the developmentalstage of the photosynthetic unit, ImL plastids had variableChl a/Chl b ratios (2.7 <Chl a/Chlb<20) and showed distinctintermediates in the assembly of the chlorophyll a–b light-harvestingcomplex (LHC) of photosystem-II (PSII). The results are consistentwith a step-wise increment in the PSII antenna size involvingthree distinct forms of the PSII unit: (i) a PSII-core formwith about 37 Chl a molecules; (ii) a PSIL_ß form containingthe PSII-core and the LHC-II-inner antenna with a total of about130 Chl (a + b) molecules, and (iii) the mature PSII_a form containingPSII_ß and the LHC-II-peripheral antenna with a totalof 210–300 Chl (a + b) molecules. The thylakoid membranecontained polypeptide subunits b, c and d (the Lhcb1, 2 and3 gene products, respectively) when only the LHC-II-inner waspresent. Polypeptide subunit a, (the apoprotein of the chlorophyll-proteinknown as CP29), along with increased amounts of b and c appearedlater in the development of thylakoids, concomitant with theassembly of the LHC-II-peripheral. The results suggest thatpolypeptide subunit d has priority of assembly over subunita. It is implied that, of all LHC-II constituent proteins, subunitd is most proximal to the PSII-core complex and that it servesas a linker in the transfer of excitation energy from the bulkLHC-II (subunits b and c) to the PSII-core. The work also addressesthe origin of low-molecular-weight proteins (M_r = 19, 17.5 and13.4 kDa) which co-isolate with intact developing plastids andwhose abundance decreases during plastid development. Aminoacid compositional and immunoblot analyses show a nuclear histoneorigin for these low-molecular-weight proteins and suggest co-isolationof histone-containing nuclear vesicles along with intact developingplastids. ¹Present address: Plant Physiology Research Group, The Universityof Calgary, Department of Biological Sciences, 2500 UniversityDrive N.W., Calgary, Alberta CANADA T2N 1N4.     

Flag Leaf Photosynthesis of Triticum aestivum and Related Diploid and Tetraploid Species

Rates of net photosynthesis of the flag leaves of 15 genotypesof wheat and related species were measured throughout theirlife, using intact leaves on plants grown in the field. At thestage when rates were maximal, they were in general highestfor the diploid species, intermediate for the tetraploidspeciesand lowest for Triticum aestivum (means of 38, 32 and 28 mgCO₂ dm^–2 h^–1 respectively). Rates were stronglynegatively correlated with leaf area, leaf width and the meanplan area per mesophyll cell and positvely correlated with stomatalfrequency and number of veins per mm of leaf width. The differencesamong species in these attributes were mainly related to ploidylevel. It was not possible to determine the relative importanceof each anatomical feature, though the changes in stomatal frequencyhad only slight effects on stomatal conductance and the observeddifferences in rates of photosynthesis were much greater thanwould be expected from those in stomatal conductance alone. There was genetic variation in rates of light dependent oxygenevolution of isolated protoplasts and intact chloroplasts butno difference attributable to ploidy. The mean rate, 91 µmolO₂ mg^–1 chlorophyll h^–1, equivalent to 3.9 mg CO₂mg^-1chlorophyll h^-1 was considerably less than the rate of photosynthesisin comparable intact leaves, which was 7.2 mg CO₂ mg^–1chlorophyll h^–1. The total above-ground dry matter yields were least for thewild diploids T. urartu and T. thauodar and the wild tetraploidT. dicoccoides, but the other wild diploids produced as muchdry matter as the hexaploids. The prospects of exploiting differences in photosynthetic ratein the breeding of higher yielding varieties are discussed. Triticum aestivum L., wheat, Aegilops spp, photosynthesis, stomatal conductance, stomatal frequency, polyploidy     

Can the 15N Dilution Technique be used to Study N2 Fixation in Tropical Tree Symbioses as Affected by Water Deficit?

Three methods were used to study N₂ fixation and effects ofwater deficit on N₂ fixation: C₂H₂ reduction assay (ARA), ¹⁵Ndilution technique and accumulated N content. In addition, ¹⁵Ndilution was calculated both in a traditional way and in a modifiedway, which takes into consideration N and ¹⁵N content for theplants before the experiment started. The three methods wereapplied on the following Rhizobium-symbioses: Acacia albidaDel (Faidherbia albida (Del) A. Chev.) and Leucaena leucocephala(Lam) de Wit., and the Frankia-symbiosis Casuarina equisetifoliaL. The plants wereabout 4-months-old when they were harvested. Nitrogen derived from N₂ fixation in control plants of Acaciaalbida was 54·2 mg as measured with ARA, while it was28·5 mg as measured with the ¹⁵N dilution technique,compared to 30·7 mg calculated as accumulated N. In comparison,L. leucocephala fixed 41·6 mg N (ARA), 53·5 mgN(15N dilution technique) and 56·3 mg N (accumulatedN). The Frankia-symbiosis had fixed 27·4 mg N as measuredby ARA, 8·1 mg N as measured by ¹⁵N dilution techniqueand 12·3 mg N as accumulated N. There were no differencesbetween the estimates based ontraditional and modified waysof calculating ¹⁵N dilution. The immediate effect of water deficit treatment on N₂ fixationwas continuously measured inall species with ARA, which startedto decrease approximately 10 d after the initiation of the treatment,and declined to less than 5% of the initial level after 21–28d. The decrease in the amount of N derived from N₂ fixation wasstudied in L. leucocephala during the period of treatment. Therewas a 26% decrease in amount of N derived from N₂ fixation asresult of water deficit (as measured with ARA), while the decreasewas 23% when measured withboth the ¹⁵N dilution method and asaccumulated N. The three different methods for measuring N₂ fixation and effectsof water deficit on N₂ fixation are discussed. Key words: Acacia albida, ARA, Casuarina equisetifolia, Leucaena leucocephala, ¹⁵N dilution, N₂N fixation, water deficit     

Photosynthetic CO2-fixing activity in dark-grown spruce seedlings

Bicarbonate-dependent O₂-evolving activity in dark-grown cotyledonsof Picea abies was measured with an oxygen electrode with differentpreillumination times. The activity showed a slight linear increasewith increasing preillumination time. On the other hand, O₂-evolvingactivity (Hill activity) of chloroplasts prepared from preilluminateddark-grown cotyledons exhibited a characteristic change of asteep rise followed by a gradual increase with increasing preilluminationtime. The results obtained were discussed in connection withthe light activation of the latent, inactive O₂-evolving centerin dark-grown cotyledons. (Received December 8, 1978; )     

Reduced Phosphorus Requirement of a Mutant Azolla-Anabaena Symbiotic N2-fixing Complex

The use of the photo-autotrophic nitrogen-fixing water fernAzolla as an effective source of organic nitrogen in tropicalpaddy fields has been limited by a high phosphorus requirement.Azolla species with a minimum of 1.5 to 2.0 mM phosphate (P)requirement, under controlled conditions, are known. A local Azolla species requiring at least 1.5 mM sodium phosphatefor a normal rate of multiplication and N₂ fixation was exposedto N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The resultingmutant population had a significantly lower P requirement, butwas auxotrophic for glutamine with an extremely reduced glutaminesynthetase (GS) activity. An L-methionine-DL-sulphoximine (MSX)-resistant(MSX^r) Azolla population, having an approximately 1.5 timeshigher GS activity than that of the wild type (WT) parent organism,was cultured and subjected to MNNG-induced mutation for lowP requirement while putting MSX as a control in the mutant selectionmedium. The resulting population of mutant Azolla was a normalprototroph with a P requirement as low as 0.75 mM for its ‘WTparent-like’ usual growth and N₂ fixation. Key words: Azolla, phosphorus requirement, mutation     

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

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

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn² and Asn¹⁵. The predominant glycan (60% of total) had thestructure GlcNAcß1–2Man1–3(Man1–6)Manß1–4GlcNAcß1–4GlcNAc-(Asn),with the remaining structures containing 1–3 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn² site. FAB-MS analysis of oligosaccharides releasedfrom the Asn² site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc₁Hex₆HexNAc₃(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin     

Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata

Raphidophycean flagellates, Chattonella marina and C. ovata,are harmful red tide phytoplankters; blooms of these phytoplanktersoften cause severe damage to fish farming. Previous studieshave demonstrated that C. marina and C. ovata continuously producereactive oxygen species (ROS) such as superoxide anion (O₂^–)hydrogen peroxide (H₂O₂) under normal growth conditions, andan ROS-mediated toxic mechanism against fish and other marineorganisms has been proposed. Although the exact mechanism ofROS generation in these phytoplankters still remains to be clarified,our previous study suggested that NADPH oxidase-like enzymelocated on the cell surface of C. marina may be involved inO₂^– generation. To investigate the localization of O₂^–and H₂O₂ generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-oneand 5-(and-6)-carboxy-2',7'-dichlorodihydrodihydrofluoresceindictate, acetyl ester, which are specific fluorescent probefor detecting O₂^– and H₂O₂, respectively. Observationby fluorescence microscopy of live phytoplankters incubatedwith each probe revealed that O₂^– is mainly generatedon the cell surface, whereas H₂O₂ is generated in the intracellularcompartment in these phytoplankters. When the cells were rupturedby ultrasonic treatment, O₂^– levels of C. marina and C.ovata decreased significantly, whereas a few times higher levelsof H₂O₂ were detected in the ruptured cell suspensions whencompared with the levels of the live cell suspension. In immunoblottinganalysis, the protein recognized by anti-human gp91 phox wasdetected in both species. These results suggest that, in bothphytoplankters, the underlying mechanisms of O₂^– and H₂O₂generation may be distinct and such systems are independentlyoperating in the cells.