NAD(P)H Dehydrogenase-Dependent Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechocystis PCC 6803: a Study of Dark-Starved Cells and Spheroplasts

Electron donation to P700⁺ through plastoquinone in the intersystemchain from both respiratory substrates and the photoreductantsin PSI has been shown to be mediated by the NAD(P)H-dehydrogenasecomplex (NDH) in Synechocystis PCC 6803 cells [Mi et al. (1992)Plant Cell Physiol. 33: 1233]. To confirm the participationof NDH in the cyclic electron flow around PSI, the redox kineticsof P700 and Chi fluorescence were analyzed in cells rendereddeficient in respiratory substrates by dark starvation and inspheroplasts. Dark-starved cells showed a high steady-state level of P700⁺under far-red (FR) illumination and the plastoquinone pool wasin a highly oxidized state. An NDH-defective mutant consistentlyshowed a high level of P700 oxidation under FR before and afterthe dark starvation. Donation of electrons either from exogenousNADPH or from photoreduced NADPH⁺ to the intersystem chain viaplastoquinone was demonstrated using spheroplasts from wild-typecells, but not those from the NDH-defective mutant, as monitoredby following changes in the kinetics of Chi fluorescence andthe redox state of P700. The electron flow to PSI via plastoquinone,mediated by NADPH, was sensitive to rotenone, Hg²⁺ ions and2-thenoyltrifluoroacetone, inhibitors of mitochondrial NDH andsuccinate dehydrogenase, but not to antimycin A. The pool sizeof electrons that can be donated to P700⁺ from the cytosol throughthe intersystem chain increased with increasing duration ofillumination time by actinic light and was sensitive to rotenonein both wild-type cells and spheroplasts, but no such resultswere obtained in the NDH-defective mutant of Synechocystis 6803.The results support our previous conclusion that NDH is a mediatorof both respiratory electron flow and cyclic electron flow aroundPSI to the intersystem chain in the cyanobacterium Synechocystis. (Received August 20, 1993; Accepted November 22, 1993)     

Suppression of Quantum Yield of Photosystem II by Hyperosmotic Stress in Chlamydomonas reinhardtii

Addition of ethylene glycol (EG) or NaCl to cells of Chlamydomonasreinhardtii induced transient non-photochemical quenching ofChl fluorescence correlated with the inhibition of photosyntheticoxygen evolution. The induction of the quenching and subsequentrecovery proceeded not only in the light but also in the dark.The quenching was almost unaffected by the protonophore nigericin,suggesting the involvement of a type of non-photochemical quenchingattributable to a state 2 transition. Higher concentrationsof EG or NaCl caused a delay of the recovery of the maximumfluorescence yield (Fm'). Dark reduction rate of P700⁺ afterthe application of a flash light in the presence of DCMU wasenhanced by the hyperosmotic shock, suggesting a stimulatedreduction of the intersystem electron carriers. It is proposedthat the osmotic stress stimulates electron donation from stromalcomponents via the NAD(P)H dehydrogenase, which results in thereduction of the intersystem chain and triggering of a state2 transition leading to stimulated cyclic PSI activity. (Received May 16, 1995; Accepted July 26, 1995)     

Electron Donation from Cyclic and Respiratory Flows to the Photosynthetic Intersystem Chain is Mediated by Pyridine Nucleotide Dehydrogenase in the Cyanobacterium Synechocystis PCC 6803

The redox kinetics of P700 induced by far-red light and a pulseof strong white light in wild type cells were compared withthose in NAD(P)H dehydrogenase (NDH)-defective mutants of thecyanobacterium Synechocystis PCC 6803. The wild type cells showedthe electron donation from the respiratory donor and the photoreductantgenerated in PS I to P700⁺ through the plastoquinone, whichis mediated by a Hg²⁺-sensitive enzyme. The NDH-defective mutantcells, however, did not show the electron transfer to P700⁺through the plastoquinone from both the photoreductant in PSI and cytosolic electron donors using pyndine nucleotides asan intermediate. Thus, NDH appears to be the site of main entryof electrons into the plastoquinone pool in the NAD(P)H-mediatedcyclic electron flow and the respiratory electron flow in Synechocystis. (Received August 31, 1992; Accepted October 1, 1992)     

Isolation and Characterization of Sodium Chloride-Resistant Callus Culture of Vigna radiata (L.) Wilczek var. radiata

Callus cultures were initiated from seedling root segments ofmungbean (Vigna radiata (L.) Wilczek var. radiata) cv. K 851on modified PC-L2 basal medium. Growing cells were exposed toincreasing concentrations of NaCl in the medium. A concentrationof 300 mol m^–3 NaCl proved completely inhibitory to growthof the calli. On incubation for 25 d, cells which could toleratethis concentration of NaCl grew to form cell clones. Selectedclones were characterized with regard to their growth behaviour,K⁺, Na⁺ and free proline content when grown under stress aswell as on normal media and were compared with the normal sensitivecallus. The selected callus was capable of growing on mediumcontaining NaCl at the inhibitory concentration. The K⁺ contentof the selected callus was lower in the case of the NaCl mediumthan for the normal medium. However, the selected clones maintainedhigher K⁺ and Na⁺ levels, with increased salinization comparedwith the wild-type cells. Salt-selected cells accumulated higherlevels of free proline under NaCl stress compared to wild-typecells. Under normal conditions, however, the amounts of freeproline in selected and non-selected calli were comparable. Key words: Vigna radiata, callus culture, NaCl stress     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

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)     

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.     

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.     

Phosphate Absorption, Fluxes, and Symplasmic Transport in Osmotically-Shocked Zea mays Roots

Osmotic shock with sequential 30 min treatments in ice-coldsaline solutions and distilled water inhibited both the subsequentuptake of orthophosphate (Pi) and its transport into the xylemof excised corn (Zea mays L.) roots. Measurements of Pi fluxeswith ³²P indicated that the decrease in net Pi uptake over a24 h period caused by osmotic shock was due primarily to delayedrecovery of Pi influx rather than to increasing efflux. Despitecomplete recovery of Pi absorption within 2–6 h aftershocking with 150–200 mM NaCl, transport to the xylemduring the subsequent 24 h only partially recovered. Leucineuptake and incorporation into protein was also markedly inhibitedby osmotic shock but both almost completely resumed controlrates within 24 h after shocking with up to 150 mM NaCl. Tetracyclineinhibited recovery of Pi uptake after NaCl treatment whereaspuromycin did not. These results with corn roots are consistentwith the hypothesis that recovery of Pi uptake activity aftermoderate osmotic shock requires de novo synthesis of membraneproteins. Incomplete recovery of Pi transport to the xylem suggeststhat osmotic shock may damage plasmodesmata. Key words: Corn, Ion uptake, Leucine uptake, NaCl, Puromycin, Tetracycline     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Effect of Sudden Salt Stress on Ion Fluxes in Intact Wheat Suspension Cells

Although salinity is one of the major problems limiting agriculturalproduction around the world, the underlying mechanisms of highNaCl perception and tolerance are still poorly understood. Theeffects of different bathing solutions and fusicoccin (FC),a known activator of plasma membrane ATPase, on plasma membranepotential (E_m) and net fluxes of Na⁺, K⁺and H⁺were studied inwheat suspension cells (Triticum aestivum) in response to differentNaCl treatments. E_mof cells in Murashige and Skoog (MS) mediumwas less negative than in cells exposed to a medium containing10 mM KCl + 0.1 m M CaCl₂(KSM) and to a basic salt medium (BSM),containing 1 m M KCl and 0.1 m M CaCl₂. Multiphasic Na⁺accumulationin cells was observed, peaking at 13 min after addition of 120m M NaCl to MS medium. This time scale was in good agreementwith net Na⁺flux changes measured non-invasively by moving ion-selectivemicroelectrodes (the MIFE system). When 120 m M NaCl was addedto all media studied, a quick rise of Na⁺influx was reversedwithin the first 20 min. In both 120 and 20 m M NaCl treatmentsin MS medium, net Na⁺efflux was observed, indicating that activeNa⁺transporters function in the plant cell response to saltstress. Lower external K⁺concentrations (KSM and BSM) and FCpre-treatment caused shifts in Na⁺fluxes towards net influxat 120 m M NaCl stress. Copyright 2000 Annals of Botany Company Sodium, potassium, proton, membrane potential, fusicoccin, salt stress, wheat, Triticum aestivum     

NOX5 NAD(P)H oxidase regulates growth and apoptosis in DU 145 prostate cancer cells

Reactive oxygen species (ROS) appear to play an important role in regulating growth and survival of prostate cancer. However, the sources for ROS production in prostate cancer cells have not been determined. We report that ROS are generated by intact American Type Culture Collection DU 145 cells and by their membranes through a mechanism blocked by NAD(P)H oxidase inhibitors. ROS are critical for growth in these cells, because NAD(P)H oxidase inhibitors and antioxidants blocked proliferation. Components of the human phagocyte NAD(P)H oxidase, p22^phox and gp91^phox, as well as the Ca²⁺ concentration-responsive gp91^phox homolog NOX5 were demonstrated in DU 145 cells by RT-PCR and sequencing. Although the protein product for p22^phox was not detectable, both gp91^phox and NOX5 were identified throughout the cell by immunostaining and confocal microscopy and NOX5 immunostaining was enhanced in a perinuclear location, corresponding to enhanced ROS production adjacent to the nuclear membrane imaged by 2',7'-dichlorofluorescin diacetate oxidation. The calcium ionophore ionomycin dramatically stimulated ferricytochrome c reduction in cell media, further supporting the importance of NOX5 for ROS production. Antisense oligonucleotides for NOX5 inhibited ROS production and cell proliferation in DU 145 cells. In contrast, antisense oligonucleotides to p22^phox or gp91^phox did not impair cell growth. Inhibition of ROS generation with antioxidants or NAD(P)H oxidase inhibitors increased apoptosis in cells. These results indicate that ROS generated by the newly described NOX5 oxidase are essential for prostate cancer growth, possibly by providing trophic intracellular oxidant tone that retards programmed cell death. superoxide anion; diphenylene iodonium; p22^phox; gp91^phox; adenosine 3',5'-cyclic monophosphate response element; caspases     

Carbon Economy of Sour Orange in Relation to Mycorrhizal Colonization and Phosphorus Status

The effects of plant phosphorus (P) status and the mycorrhizal(M) fungus, Glomus intraradices Schenck & Smith, on thecarbon (C) economy of sour orange (Citrus aurantium L.) weredetermined during and following active M colonization. Therewere four treatments: M seedlings grown at standard-strength(1 mM) P (M1) and nonmycorrhizal (NM) plants grown at one, twoand five times standard-strength P (NM1, NM2 and NM5). Mycorrhizalcolonization, tissue dry mass, P content, root length and leafarea were determined in five harvests from 6 to 15 weeks ofage. Rate of C assimilation (A) was determined at 7, 8 and 12weeks by gas exchange. Partitioning of ¹⁴ C was determined from7 to 15 weeks using a 10-min pulse followed by a 24-h chaseperiod. For a given attribute, M1 plants were compared to thecurve defining the NM response as a function of tissue P concentration.In contrast to the large effects of P nutrition on C economyof sour orange, M effects were generally subtle. Mycorrhizaeincreased the root biomass fraction, the root length/leaf arearatio and the percentage of ¹⁴C recovered from below-groundcomponents. A higher percentage of below-ground ¹⁴ C was inthe respiration and soil fractions in M than NM plants of equivalentP status. Mycorrhizal plants tended to enhance A only for abrief period. Mycorrhizal plants had lower relative growth ratesthan NM plants of equivalent P status, suggesting that the temporarilyenhance A of M plants did not fully compensate for their greaterbelow-ground carbon expenditure. Problems of interpreting thedynamic effects of mycorrhizae on C economy that are independentof P nutrition are discussed.Copyright 1993, 1999 Academic Press Citrus aurantium L., sour orange, carbon economy, ¹⁴carbon, CO₂ assimilation, vesicular-arbuscular mycorrhizae, phosphorus fertilization, phosphorus nutrition     

Targeted inactivation of the gene psaK encoding a subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Mutant strains of the unicellular cyanobacterium Synechocystissp. PCC 6803, in which the psaK gene was insertionally inactivatedby targeted mutagenesis, were constructed. The gene is one ofthe two potential PsaK-coding genes which have been found asa result of the genome project with this cyanobacterium. Oneof the mutants was characterized in detail. A monocistronic,480-nucleotide mRNA of psaK was absent in total RNA from themutant cells. Inactivation of psaK had little effect on theaccumulation of polypeptides in the isolated PSI complexes exceptfor a polypeptide with an apparent molecular mass of 4.6 kDawhich was absent in the mutant. The amino-terminal amino acidsequence of the 4.6-kDa polypeptide confirmed that it was thetranslation product of psaK and further revealed a presequenceof PsaK. Characteristics of photoautotrophic growth at differenttemperatures, the amount of chlorophyll per cell, photosyntheticelectron transport rates with various electron acceptors, thekinetics of charge recombination between P700⁺ and reduced F_A/F_B,and the molar ratio of chlorophyll to P700, of the mutant werenot significantly different from those of the wild type. Furthermore,the trimer to monomer ratio of the PSI complexes isolated fromthe mutant was similar to that isolated from the wild type. (Received July 27, 1998; Accepted October 13, 1998)     

Effects of Salinity on Growth, Water Relations and Ion Accumulation of the Subtropical Perennial Halophyte, Atriplex griffithii var. stocksii

The effects of salinity on growth, water relations, glycinebetainecontent, and ion accumulation in the perennial halophyte Atriplexgriffithii var. stocksii were determined. The following questionswere addressed: (1) What effect does salinity have on growthresponses at different ages? (2) Is A. griffithii an ion accumulator?(3) Does A. griffithii accumulate glycinebetaine in responseto salinity? Atriplex griffithii plants were grown in pots at0, 90, 180 and 360  m M NaCl in sand culture in a plantgrowth chamber and plants were harvested after 30, 60 and 90d. Plant total dry weight was significantly inhibited at 360m M NaCl. Root growth showed a substantial promotion at 90 mM NaCl. The water potential and osmotic potential of shootsbecame more negative with increasing salinity and time of growth.The Na⁺and Cl^-content in both shoots and roots increased withincreases in salinity. Increased treatment levels of NaCl induceddecreases in Ca⁺, K⁺and Mg²⁺in plants. Atriplex griffithii accumulateda large quantity of ions, with the ash content reaching 39%of the dry weight in leaves. Inorganic ion accumulation is significantin osmotic adjustment and facilitates water uptake along a soil-plantgradient. Glycinebetaine concentration was low in roots, andin stems it increased with increases in salinity. Total amountsof glycinebetaine in leaves increased with increases in salinity,and its concentration increased substantially at 360 m M NaCl.Copyright 2000 Annals of Botany Company Atriplex griffithii, glycinebetaine, growth, ions, water relations.     

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     

Malate Decarboxylation by Mitochondria of the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum

Mitochondria isolated from leaves of Mesembryanthemum crystallinumoxidized malate by both NAD malic enzyme and NAD malate dehydrogenase.Rates of malate oxidation were higher in mitochondria from plantsgrown at 400 mil NaCl in the rooting medium and performing Crassulaceanacid metabolism (CAM) than in mitochondria from plants grownat 20 mM NaCl and exhibiting C₃-photosynthetic CO₂ fixation.The mitochondria isolated from plants both in the CAM and C₃modes were tightly coupled and gave high respiratory control.At optimum pH for malate oxidation (pH 7.0), pyruvate was themajor product in mitochondria from CAM-M. crystallinum, whereasmitochondria from C₃-M. crystallinum produced predominantlyoxaloacetate. Both the extracted NAD malic enzyme in the presenceof CoA and the oxidation of malate to pyruvate by the mitochondriafrom plants in the CAM mode had a pH optimum around 7.0 withactivity declining markedly above this pH. The activity of NAD-malicenzyme, expressed on a cytochrome c oxidase activity basis,was much higher in mitochondria from the CAM mode than the C₃mode. The results indicate that mitochondria of this speciesare adapted to decarboxylate malate at high rates during CAM. ¹Current address: Lehrstuhl für Botanik II, UniversitätWurzburg, Mittlerer Dallenbergweg 64, 8700 Würzburg, WestGermany. ²Current address: KD 120, Chemical Research Division, OntarioHydro, 800 Kipling Avenue, Toronto, Ontario M8Z5S4, Canada. ³Current address: Department of Botany, Washington State University,Pullman, Washington 99164-4230, U.S.A. (Received March 13, 1986; Accepted September 18, 1986)     

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.     

Simultaneous CO2- and 16O2/18O2-gas exchange and fluorescence measurements indicate differences in light energy dissipation between the wild type and the phytochrome-deficient aurea mutant of tomato during water stress

The CO₂-, H₂O- and ¹⁶O₂/¹⁸O₂ isotopic-gas exchange and the fluorescencequenching by attached leaves of the wild-type and of the phytochrome-deficienttomato aurea mutant was compared in relation to water stressand the photon fluence rate. The chlorophyll content of aurealeaves was reduced and the ultra-structure of the chloroplastswas altered. Nevertheless, the maximum rate of net CO₂ uptakein air by the yellow-green leaves of the aurea mutant was similarto that by the dark-green wild-type leaves. However, less O₂was produced by the leaves of the aurea mutant than by leavesof the wild-type. This result indicates a reduced rate of photosyntheticelectron flux in aurea mutant leaves. No difference in bothphotochemical and non-photochemical fluorescence quenching wasfound between wild-type and aurea mutant leaves. Water stresswas correlated with a reversible decrease in the rates of bothnet CO₂ uptake and transpiration by wild-type and aurea mutantleaves. The rate of gross ¹⁶O₂ evolution by both wild-type andaurea mutant leaves was fairly unaffected by water stress. Thisresult shows that in both wild-type and aurea leaves, the photochemicalprocesses are highly resistant to water stress. The rate ofgross ¹⁸O₂ uptake by wild-type leaves increased during waterstress when the photon fluence rate was high. Under the sameconditions, the rate of gross ¹⁸O₂ uptake by ^aurea mutant leavesremained unchanged. The physiological significane of this differencewith respect to the (presumed) importance of oxygen reductionin photoprotection is discussed. Key words: Water stress, gas exchange, fluorescence quenching, Lycopersicon esculentum, mutant (tomato, aurea), energy dissipation     

Phosphate Compounds of Scenedesmus C-2A' in Darkness or Blue Light as Measured by 31P NMR

Phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectra ofintact cells of Scenedesmus mutant C-2A' and of their perchloricacid extracts are presented. Sugarphosphates, including glucose-6-phosphateand fructose-1,6-bisphosphate, orthophosphate, nucleotide di-and triphosphates, NAD(P), UDPG and—in the case of intactcells—also polyphosphates were identified. Blue light,which is known to stimulate the carbohydrate breakdown of greenalgae, leads to a transient drop in P_i, a pronounced decreasein the ATP/ADP ratio, and an increase in sugarphosphates, givingrise to the idea that the enhancement of phosphorolytic starchbreakdown is a primary response to blue light. Addition of glucoseto Scenedesmus mutant cells leads to comparable changes (besidesan additionally enhanced glucose-6-phosphate level), which thussupport the view that blue light stimulates dark-type respiration.Altogether the results demonstrate the applicability of ³¹PNMR spectroscopy to the study of the metabolism of green algae. (Received December 7, 1984; Accepted February 12, 1985)