Biochemical Properties of the Plasmalemma ATPase of Jerusalem Artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Enzymological properties of the ATPase of plasmalemma-enrichedfractions obtained from the parenchyma of Jerusalem artichoke(Helianthus tuberosus L.) tubers were studied in relation tothe morphogenetic properties of the tubers. Preparations fromdormant tubers showed higher V_max and K_m values than those fromnon-dormant tubers. These differences persisted after solubilizationof the enzyme with octylglucoside, but were slightly modifiedafter addition of K$ to the reaction medium. The implicationsof these results are discussed in relation to the morphogeneticproperties of the tubers (Received August 24, 1987; Accepted March 19, 1988)     

Transport and Fixation of Inorganic Carbon during Photosynthesis in Cells of Anabaena Grown under Ordinary Air: III. Some Characteristics of the HCO3--Transport System in Cells Grown under Ordinary Air

In cells of cyanobacterium Anabaena variabilis grown under ordinaryair (low-CO₂ cells), the transport of both CO₂ and HCO₃^–was significantly enhanced by Na⁺. This effect was pronouncedas the external pH increased. When low-CO₂ cells were treatedwith an inhibitor of carbonic anhydrase (CA), only CO₂ transportbut not HCO₃^– transport, was inhibited. The initial rateof photosynthetic carbon fixation as a function of the concentrationof internal inorganic carbon (IC) was practically the same irrespectiveof whether CO₂ or HCO₃^– was externally supplied. Theseresults suggest that IC is actively transported through theplasma membrane in a form of HCO₃^– probably by some transporterand that the transmembrane Na⁺ gradient is involved in thisIC transport system. Free CO₂ may be hydrated by CA to HCO₃^–and then transported to the cells by this transporter. On the other hand, CO₂ is actively taken up by cells grown withair containing 5% CO₂ (high-CO₂ cells) though the enhancingeffect of Na⁺ was much smaller in high- CO₂ cells than in low-CO₂cells. The initial rate of fixation as a function of internal IC concentrationindicated that the rate of the carboxylation reaction of accumulatedIC is higher in I0W-CO₂ cells than in high-CO₂ cells. The studieswith ethoxyzolamide indicated that even in low-CO₂ cells, CAdoes not function inside Anabaena cells. These results suggestthat inside the low-CO₂ cells of Anabaena, some mediator(s)facilitates the transport of IC to RuBPCase. (Received January 23, 1987; Accepted April 24, 1987)     

S-Locus-Specific Glycoproteins Associated with Self-Incompatibility in Brassica campestris

Specific S-glycoproteins were isolated from three Brassica campestriscultivars homozygous with respect to the S-alleles S₈, S₉ andS₁₂. Amino acid sequences of various peptide fragments of theS-proteins were determined using a gas-phase protein sequencer,and a nearly complete amino acid sequence of the S₈-glycoproteinwas determined on the basis of the revised cDNA sequence ofthe B. oleracea S-specific glycoprotein. The lysyl endopeptidasefragments of S₉ and S₁₂-glycoproteins were aligned in comparisonwith the sequence of the S₈-glycoprotein. Although extensivesequence homology was evident among the three S-glycoproteins,the sequences of the middle part were relatively different fromeach other. The numbers and positions of N-glycosylation alsodiffered among the S-glycoproteins of Brassica species. (Received April 20, 1987; Accepted July 29, 1987)     

Ion Efflux during Excitation of Characeae: Comparison of Voltage-Clamp and Conductometry Method for Efflux Measurement

Excitation of characean cells was studied in experiments usinga voltage-clamp. The efflux of monovalent anion was estimatedfrom the inward current (J₁) and was compared with that measuredby the conductometry method (J₈). It was found that, on theaverage, J₁ was larger than J₈ in Nitella. The Ca₂₊ influx isdiscussed with a view to explaining the discrepancy betweenJ₁ and J₈. (Received April 27, 1987; Accepted August 26, 1987)     

Regulation of Electron Transport Composition in Cyanobacterial Photosynthetic System: Stoichiometry among Photosystem I and II Complexes and Their Light-Harvesting Antennae and Cytochrome b6/fComplex

This study was done to confirm our previous observation withthe pattern of changes in electron transport composition inducedby an imbalance of the electron transport state. Contents ofphotosystem (PS) I and II complexes and their antennae and Cytb₆/f complex were determined for systems of cyanobacterium SynechocystisPCC 6714 of different PS I/PS II ratios. The results indicatedthat (1) the observed changes in the PS I/PS II ratio are not-dueto regulation of the activities of the respective PS's but tochanges in their contents, (2) the molar ratio between PS IIand Cyt b₆/f complexes was fairly constant when marked changesoccurred in the PS I content, and (3) the PS II and Cyt b₆/fcontents per cell remained fairly constant while the PS I contentchanged markedly. These findings agree with our previous observationwith autotrophic cells of Anacystis nidulans Tx 20 and supportour argument that in cyanobacterial and red algal electron transportsystems, the content of the terminalcomponent(s), such as PSI complex, is regulated in order to maintain a balance betweenthe electron influx by PS II action to the system and the effluxby PS I action from it. (Received June 3, 1987; Accepted September 20, 1987)     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. II. Distribution and Role in Photosynthesis

Antibody was raised against Porphyridium carbonic anhydrase(CA) which was electrophoretically recovered from the gel afterSDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of thepartially purified enzyme. The antiserum reacted with CA ofPorphyridium, but not with that of Chlamydomonas reinhardtii.Even though the antiserum did not react with CA from P. cruentumR-l in Ouchterlony's double immunodiffusion, it blocked theenzyme activity in the presence of 1% Nonidet P-40 and 1% TritonX-100. After Western blotting and enzyme-linked immunostaining(ELIS), only one band which reacted with the antiserum was detectedin the extract of low-CO₂ cells (grown under ordinary air) ofP cruentum, while no significant band was detected in that ofhigh-CO₂ cells (grown under air enriched with 1–5% CO₂).Immunogold electron microscopy of low-CO₂ cells of P. cruentumR-l using this antibody revealed that most of the CA was localizedin the chloroplast, with some in the cytoplasm. No specificbinding of gold particles was observed in the high-CO₂ cells. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan (Received May 18, 1987; Accepted September 7, 1987)     

Radioimmunoassay of Gibberellins A5 and A20

Polyclonal anti-GA₅-antiseruin and anti-GA₂₀-antiserum wereprepared by immunizing rabbits with conjugates of N-GA₅-ß-alanylBSA and N-GA₂₀-ß-alanyl BSA. By radioimmunoassay usingthese antisera, GA₅ and GA₂₀ in developing fruits of Pharbitisnil Chois were analyzed after several purification steps andthe results were compared with those obtained by GC-MS to examinethe reliability of the analysis by radioimmunoassay. The analyticalresults by radioimmunoassay did not agree with those by GC-MSuntil two-step purifications by HPLC, indicating that samplesmust be suitably purified prior to radioimmunoassay to obtainreliable results. (Received November 13, 1986; Accepted April 17, 1987)     

Comparison between Electron Transfers through Plastocyanin in Spinach Chloroplasts and Cytocbrome C2 in Rhodopseudomonas sphaeroides

Photosynthetic electron transfers through the water-solubleperipheral membrane proteins of plastocyanin and cytochromec₂, were studied in spinach chloroplasts and the photosyntheticbacterium Rhodopseudomonas sphaeroides. In spinach chloroplasts,the rate of flash-induced oxidation of cytochrome f was highlydependent on the salt concentration in the suspending medium.The maximum rate with a half time of 200 µs was observedin the presence of 50 mas KCl or 5 mM MgCl₂. The salt effectwas similar to that on the reaction rate between P700 in thylakoidfragments and externally added plastocyanin. On the other hand,in intact cells of R. sphaeroides, in which cytochrome c₂ islocated in the periplasmic space exposed to the outer ionicenvironment, the rate of cytochrome c₁ oxidation via cytochromec₂ was almost independent of salt concentration. This independencewas a contrast to the strong dependence on salt concentrationof reactions between isolated reaction centers and cytochromec₂ These results suggest that plastocyanin reacts collisionallywith the photosystem I reaction center and cytochrome b₆f complexin a manner that is controlled by the surface electrostaticpotential. Cytochrome c₂, on the other hand, reacts with thebacterial reaction center and cytochrome bc₁ complex probablyby forming a complex prior to activation of the reaction center. ¹ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Fukazawa 2-1-1, Setagaya, Tokyo158, Japan.     

Activation of Electron Donation from Hydrogen Peroxide by Manganese in Non-oxygen evolving Photosystem II Particles

Analysis of the activation of H₂O₂-supported 2,6-dichloroindophenol(DCIP) photoreduction by MnCl₂ showed two Mn²⁺-binding sitesin non-oxygen evolving PS II particles, with large (0.4IM) andsmall (0.04 µm) K_m values for Mn²⁺. Photoreduction throughthe high-affinity Mn²⁺.-binding site was inhibited by treatmentwith H₂O₂. (Received April 20, 1987; Accepted July 13, 1987)     

The Role of Membrane Potential for the Control of Elongation Growth of Vigna Hypocotyl: Response of a Hollow Cylinder to Osmotic and Ionic Stress

We have devised an experimental system of perfusion througha hollow cylinder of a Vigna hypocotyl to examine the controlmechanism of plant stem elongation. When the cylinder was subjectedto osmotic stress, it began to shrink and then spontaneouslyresumed elongation. Not only the membrane potential differencebetween the parenchyma symplast and the central bore (V_px),but also that between the parenchyma symplast and the organsurface (V_ps), showed hyperpolarization a few minutes afterthe cylinder began to shrink. Removal of the stress caused animmediate increase in elongation rate followed by depolarizationof both membrane potentials a few minutes later. When the cylinderwas subjected to KCl stress, V_px showed transient depolarizationand recovery, while V_ps showed only immediate hyperpolarization.Increasing the KCl concentration caused V_px to depolarize, andthe cylinder simultaneously to cease to elongate for about 5min,even when the osmotic concentration of the perfusion solutionwas kept almost constant. An inverse reaction was observed whenthe KCl concentration was decreased. These two reversible responses suggest that control of V_px mayregulate the elongation of hollow cylinders, and that the xylempump plays an important role in the regulation of intact stemelongation. (Received January 7, 1987; Accepted April 30, 1987)     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. I. Purification and Properties of the Enzyme

Cells of Porphyridium cruentum R-l, a unicellular red alga,grown under ordinary air (0.04% CO₂) showed much higher activityof carbonic anhydrase (CA) than those grown under CCvenrichedair (2% CO₂). CA activity was not detected in a suspension ofintact cells, and was detectable only after the cells had beenhomogenized, indicating that this enzyme was localized onlywithin the algal cells. After partial purification of Porphyridium CA, its mol wt wasestimated as 59 kDa by SDS-PAGE and 55 kDa by gelfiltration.This suggests that the native enzyme is a monomer. Its activitywas not affected by benzensulfonamides, potent inhibitors ofCAs isolated from Chlamydomonas and other organisms. Chloride(or bromide) ions was essential for CA activity. CA activitymarkedly decreased when the cell extract had been incubatedat pH lower than 7 before assay. Upon readjusting the pH ofthe preincubation medium to 9 or higher, the enzyme activitywas restored, indicating that the inactivation is reversible. (Received April 17, 1987; Accepted July 21, 1987)     

Organic Acid Metabolism in Cellular Organelles of Vigna cylindrica (Mitorisasage) Cotyledons

In starchy cotyledons of Vigna cylindrica (L.) Skeels (Mitorisasage)during seed germination, the enzymes of the glyoxylate cyclewere located in the matrix of mitochondria. Glyoxysomes wereabsent. The glyoxylate cycle in the mitochondria supplies organicacids to the tricarboxylic acid cycle. In mitochondria, isocitratelyase activity was much higher than malate synthase activity.Part of the glyoxylate thus produced in mitochondria may benonenzymatically converted to formate by H₂O₂ and the formatethen converted to CO₂ by peroxidase or by formic dehydrogenase.The activity of superoxide dismutase, which supplies H₂O₂, washigher in mitochondria than in peroxisomes. The remaining glyoxylatein mitochondria is possibly converted to glycine by alanine-glyoxylateaminotransferase or transported to peroxisomes which lackedisocitrate lyase activity but had high malate synthase activity.In peroxisomes, glyoxylate may be also produced from urate,as is suggested by the fairly high activities of uricase, allantoinaseand allantoicase. Judging from the enzyme distribution, Vignaperoxisomes should be capable of producing malate, oxalacetate,citrate, isocitrate and a-ketoglutarate. ¹Present address: Department of Horticulture, College of Agricultureand Animal Science, Yeugnam University, Gyeongsan 632, Korea. (Received May 27, 1987; Accepted October 7, 1987)     

Inhibitory Effect of N,N'-Dicyclohexylcarbodiimide on Reduction of Cytochrome b560 and Stoichiometry of the Reduction in Chromatium Chromatophores

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibited the flash-inducedreduction of cytochrome b₅₆₀, by blocking the electron flowbetween the secondary electron acceptor and cytochrome b₅₆₀presumably in the vicinity of the ubiquinone pool. The stoichiometryof the reduced cytochrome b₅₆₀ per reaction center bacteriochlorophylldimer was 0.77?0.12 throughout the redox potential range of150 to 390 mV at pH 7.0. The high stoichiometry suggested thatmost of the electrons ejected from the reaction center reducedcytochrome b₅₆₀. (Received January 19, 1982; Accepted March 15, 1982)     

Specific Inhibition of Electron Transfer for Nitrate Reduction by a Low Concentration of Cyanide in Rhodobacter sphaeroides f. s. denitrificans

The effects of cyanide on the electron flow in NO₃^– andNO₂^– reductions and photosynthetic electron transfer wereinvestigated with intact cells of a photodenitrifier, Rhodobactersphaeroides f. s. denitrificans. In the presence of 30 µMKCN, electron transfer for NO₃^– reduction was inhibitedby about 70% and the concomitant H translocation was completelyinhibited. However, neither NO₂^– reduction nor photosyntheticcyclic electron transfer was affected at 30 µM. Theseresults suggested that the electron transfer pathway to NO₃^–has, in addition to a b-type cytochrome and the nitratereductase,a component sensitive to a low concenration of cyanide whichis not involvedin the cytochrome bc₁ complex. (Received April 13, 1987; Accepted July 23, 1987)     

A Comparative Study on the Effects of H2S and SO2 Fumigation on the Growth and Accumulation of Sulphate and Sulphydryl Compounds in Trifolium pratense L., Glycine max Merr. and Phaseolus vulgaris L.

Maas, F. M., De Kok, L. J., Peters, J. L. and Kuiper, P. J.C. 1987. A comparative study on the effects of H₂S and SO₂ fumigationon the growth and accumulation of sulphate and sulphydryl compoundsin Trifolium pratense L., Glycine max Merr. and Phaseolus vulgarisL.—J. exp. Bot. 38: 1459-1469. The effects of 0—25 mm³ dm³ H₂S and SO₂ on growth andsulphur content of shoots of Trifolium pratense, Glycine maxand Phaseolus vulgaris were studied. After 2 weeks of fumigationthe yield of T. pralense was reduced by 32% by H₂S, but notaffected by SO₂. Yield of G. max was not affected by H₂S, butreduced by 20% by SO₂, whereas that of P. vulgaris was increasedby 11% by H₂S and not affected by SO₂. Increases in sulphydrylcontent were already observed after 24 h of exposure to H₂Sand SO₂ in all plants. The increase was greatest in T. pratenseand smallest in P. vulgaris and, except for T. pratense, alwaysgreater in the H₂S-exposed than the SO₂-exposed plants. Oneday of exposure resulted in an increase in sulphate contentonly in the SO₂-fumigated plants, with the highest accumulationin T. pratense and the lowest in P. vulgaris. After 2 weeksan increase in sulphate content was also observed in the H₂S-exposedplants. This increase was also highest in T. pratense and lowestin P. vulgaris. Transpiration rate was not affected by a 24 h exposure to H₂Sor SO₂ and was highest in T. pratense, intermediate in G. maxand lowest in P. vulgaris. The order of theoretical rates of deposition of H₂S and SO₂correlated with the observed increases in sulphydryl contentduring the first 24 h of exposure in both H₂S and SO₂-fumigatedplants and with the increase in sulphate content in the SO₂-exposedplants. The increases in sulphydryl content were only 8% ofthe theoretical H₂S and SO₂-deposition fluxes, whereas sulphateaccumulation accounted for at least 57% of the theoretical SO₂-depositionflux. Key words: Air pollution, clover, French bean, Glutathione, Soybean, sulphur metabolism.     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Formation of Isodityrosine by Peroxidase Isozymes

Fry, S. C. 1987. Formation of isodityrosine by peroxidase isozymes.—J.exp. Bot. 38: 853–862. Tyrosine residues of extensin are oxidatively coupled in vivoto form isodityrosine bridges, whereas treatment of purifiedextensin with H₂O₂+ peroxidase in vitro yields only dityrosine.Two explanations for the correct mode of coupling in vivo weretested. The first, that the pH of the cell wall is lower thanthat (pH 9-0) at which in vitro experiments have been conducted,provided part of the answer since treatment of L-tyrosine withH₂O₂+peroxidase in vitro at pH 37–5 yielded some isodityrosine.The second, that the wall contains other isozymes of peroxidasethan the basic isozyme usually studied in vitro, appeared unlikelybecause several sharply contrasting isozymes yielded similarisodityrosine: dityrosine ratios from L-tyrosine+ H₂O₂ at anygiven pH. The isozymes were also similar in their ability tooxidize tyrosine-dimers further to higher polymers. It is concludedthat the formation of isodityrosine in vivo is dictated by neighbouringwall molecules, possibly ionically-bound pectins, which modifythe local environment of the tyrosine residues of extensin. Key words: Isodityrosine, peroxidase isozymes, extensin     

Characterization of Two Proton Transport Systems in the Tonoplast of Plasmalemma-Permeabilized Nitella Cells

ATP-dependent and PP_i-dependent H⁺-transport systems of thetonoplast were characterized in plasmalemma-permeabilized Nitellacells, where direct access to the protoplasmic surface of thetonoplast was possible. Since H⁺ transport across the tonoplastcan be measured in situ, the identity of the membrane responsiblefor H⁺ pumping is unequivocal. H⁺ transport was evaluated bythe accumulation of neutral red. While both transport systemswere obligately dependent on Mg²⁺, the two transport systemsshowed completely different sensitivity to NO₃^– and K⁺,suggesting the presence of two types of H⁺-pumps in Nitellatonoplast. NO₃^– applied to the protoplasmic surface, completelyand reversibly inhibited ATP-dependent transport but had noeffect on PP_i-dependent transport. By contrast, NO₃^– appliedinto the vacuole by the vacuolar perfusion technique did notinhibit ATP-dependent or PP_i-dependent H⁺ transport. Replacementof K⁺ with the organic cation, BTP, inhibited PP_i-dependenttransport but not the ATP-dependent one, indicating that PP_i-dependenttransport is K⁺ dependent. The sensitivities of the H⁺ transportsystems found in the tonoplast of Nitella are quite similarto those of higher plant tonoplasts. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received February 21, 1987; Accepted May 27, 1987)     

LIGHT-INDUCED OXYGEN EVOLUTION CAUSED BY HYDROGEN PEROXIDE IN CATALASE-INHIBITED CHLORELLA CELLS

1. Chlorella cells and spinach chioroplasts, whose catalase activityhad been more than 90% inhibited by 10^–5 M azide, werefound to decompose H₂O₂ photochemically to liberate oxygen,indicating that H₂O₂ was used as an oxidant of the HILL reaction.
2. That, however, the observed phenomena cannot be fully accountedfor in terms of the HILL reaction with H₂O₂ was revealed bythe observation that an extract of Chiorella cells, which hadbeen completely freed from chlorophyll, also showed a light-acceleratedO2 evolution from H₂O₂ in the presence of 10^–5 M azide.This extract contained a large quantity of catalase, which seemedto have been, in some way, involved in the reaction in question.
3. The catalatic H₂O₂ decomposition caused by crystalline catalaseof mammalian liver (in the presence of 10^–5 M azide) wasnot accelerated by the effect of light.

¹ Present address: Department of Biology, Faculty of Science,Niigata University, Niigata. (Received June 4, 1961; )     

Characterisation of Non-Uniform Photosynthesis Induced by Abscisic Acid in Leaves Having Different Mesophyll Anatomies

The effects of abscisic acid (ABA) on photosynthesis in leavesof Helianthus annuus L. were compared with those in leaves ofVicia faba L. After the ABA treatment, the response of photosyntheticCO₂ assimilation rate, A, to calculated intercellular partialpressure of CO₂, P_i, (A(p_i) relationship) was markedly depressedin H. annuus. A less marked depression was also observed inV.faba. However, when the abaxial epidermes were removed fromthese leaves, neither the maximum rate nor the CO₂ responseof photosynthetic oxygen evolution was affected by the applicationof ABA. Starch-iodine tests revealed that photosynthesis was not uniformover the leaves of H. annuus treated with ABA. The starch contentwas diffferent in each bundle sheath extension compartment (thesmallest subdivision of mesophyll by veins with bundle sheathextensions, having an area of ca. 0.25 mm² and ca. 50 stomata).In some compartments, no starch was detected. The distributionof open stomata, examined using the silicone rubber impressiontechniques, was similar to the pattern of starch accumulation.In V.faba leaves, which lack bundle sheath extensions, distributionof starch was more homogeneous. These results indicate that the apparent non-stomatal inhibitionof photosynthesis by ABA deduced from the depression of A(pⁱ)relationship is an artifact which can be attributed to the non-uniformdistribution of transpiration and photosynthesis over the leaf.Intercellular gaseous environment in the ABA-treated leavesis discussed in relation to mesophyll anatomy. ¹ Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received September 30, 1987; Accepted January 13, 1988)