Light-induced chemiluminescence of luminol in spinach chloroplast fragments: Reaction of O2- with electron transfer components

Chemiluminescence of luminol (CLL) was induced by illuminatedspinach chloroplast fragments. CLL was diminished by superoxidedismutase or under anaerobic conditions and increased by anautoxidizable electron acceptor, methyl viologen. The optimumpH for CLL was 10.0-10.5. Ferredoxin and cytochrome c reducing substance (CRS) did notaffect the intensity of CLL, but accelerated the dark decayin the absence of methyl viologen. In the presence of methylviologen, ferredoxin and CRS lowered the intensity and acceleratedthe dark decay. 3-(4-Chlorophenyl)-1,1-dimethylurea diminishedCLL. Carbonylcyanide m-chlorophenylhydrazone accelerated theinitial rate of CLL increase at low concentration and inhibitedit at high concentration. Half-decay time of CLL after the cessationof light was shortened by inhibiting electron transfer on theoxidizing side of photosystem II. We conclude that most of the CLL observed in illuminated chloroplastsis dependent on O₂^–. The results also suggest that O₂^–is reduced by reduced ferredoxin or CRS and oxidized on theoxidizing side of photosystem II. The half life of O₂^–in illuminated chloroplasts was estimated from the half-decaytime of CLL to be a few sec. ¹ Present address: Kyushu Dental College, Department of Biology,Kitakyushu 803, Japan. (Received May 30, 1977; )     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Effects of Light on Chlorophyll Formation in Cultured Tobacco Cells II. Blue Light Effect on 5-Aminolevulinic Acid Formation

5-Aminolevulinic acid (ALA) accumulation in dark-grown tobaccocallus cells in the presence of levulinic acid (LA) was followedunder blue or red light or in continuous darkness. Significantformation of ALA continued in the dark. The protochlorophyll-(ide) (Pchl) content of dark-incubated cells remained low becauseof its turnover. We inferred that the feedback inhibition ofALA synthesis by Pchl would not occur in darkincubated calluscells. ALA formation was enhanced by blue light, and this effectreached saturation at an intensity of about 800 mW.m^–2.Neither weak nor strong red light affected ALA formation. Fullenhancement of ALA formation by blue light was attained afterfairly long continuous illumination of the callus cells. Thisblue lightenhanced activity of ALA synthesis declined very slowlyduring the subsequent dark incubation. The blue light enhancement of ALA formation was observed incallus cells supplied with sucrose over a wide range of concentrations.Pchl regeneration in carbon-starved callus cells, supplied withglutamate at various concentrations, was also markedly enhancedby blue light. Respiration of the callus cells was not enhancedby blue light. A possible role of blue light in regulating ALAformation in callus cells is discussed. ¹Dedicated to the late Professor Joji Ashida. (Received September 3, 1982; Accepted April 5, 1983)     

Correlation of cytokinins and abscisic acid with monocarpic senescence in soybeans

The involvement of cytokinins and abscisic acid (ABA) in themonocarpic senescence (foliar yellowing following fruit development)of soybeans was examined. Foliar sprays of cytokinin (10^–4M zeatin or 10^–5 M benzyladenine), begun when the plantsfirst set fruit and repeated every other day, significantlydelayed, but did not prevent, monocarpic senescence. Foliarsprays of 10^–4 M ABA, applied in the same manner, significantlyhastened senescence of fruiting soybeans but apparently hadno effect on depodded plants. Leaf and stem material from pre-senescentand senescent plants was extracted, chromatographed, and bioassayedfor cytokinin-lilce activity (Amaranthus betacyanin productionassay) and ABA-like activity (oat coleoptile straight growthassay for inhibitors). ABA-like activity increased, and cytokinin-likeactivity decreased in shoot tissue before the plants began tosenesce. Cytokinin-like activity in the fruit also declinedduring this period. These results implicate a decrease in cytokininsand an increase in ABA-like inhibitors in the control of monocarpicsenescence of soybeans, but neither alone is causal. ¹ Supported in part by Research Grant 416-15-79 from the USDACooperative State Research Service under PL 89–106. ² Present address: Biology Dept., College of St. Benedict'sSt. Joseph, Minn. 56374, U.S.A. (Received February 4, 1978; )     

Effects of Some Growth Regulators on Polyamine Biosynthetic Enzymes in Etiolated Pea Seedlings

This study was designed to examine possible links between polyaminebiosynthesis and effects of growth regulatory compounds. Anauxin (IAA), a cytokinin [benzyladenine; benzylaminopurine (BAP)],an ethylene source (ethephon) and abscisic acid (ABA) were individuallyapplied to terminal buds of excised etiolated or red light (R)-exposedpea epicotyls. Effects were noted on bud fresh weight and onthe two main enzymes of putrescine biosynthesis, arginine decarboxylase(ADC; EC 4.1.1.19 [EC] ) and ornithine decarboxylase (ODC; EC 4.1.1.17 [EC] ).As previously reported [Dai and Galston (1981) Plant Physiol.67: 266], both bud growth and ADC activity are increased byR light. In such buds, ADC is raised further by 1–10 µMBAP or ABA and inhibited by 1–10 µM IAA or ethylene(50 mg/liter or more of ethephon). In all cases, effects ofR-irradiation plus 1 mM growth regulators on ODC activity wasthe inverse of their effects on ADC, indicating independentcontrol of these pathways. These results do not support theview that putrescine biosynthetic activity is correlated withgrowth in etiolated pea seedlings. ¹Supported by a grant from NSF to A.W.G. ²Supported by a grant from the Turkish Government. Permanentaddress: Department of General Botany, University of Istanbul,S?leymaniye, Istanbul, Turkey. ³On sabbatical leave from the Department of Horticulture, HebrewUniversity of Jerusalem, Rehovot, Israel. (Received September 22, 1983; Accepted February 28, 1984)     

Promotion by gibberellin of lettuce seed germiation as a function of presoaking period

Germination of lettuce seeds (Lactuca sativa L. cv. Grand Rapids)was examined in the presence of various doses (10^–5.0–10^–3.0M) of gibberellic acid applied at various times (hour 0–8)of soaking. Germination promotion by gibberellic acid was greateras the dose of gibberellic acid was increased and attenuatedwith the length of the presoaking period. As an exception, ca.95% germination was always evoked by the largest dose (10^–3M) of gibberellic acid given at any time of soaking. The dose-responsecurve obtained for each presoaking period had a distinct sigmoidalprofile. Synergistic and photoreversible promotion by red light of thegibberellin-induced germination was also investigated. Far-redlight pulse given 6 hr after the red pulse was still effectivein removing the red light action. Application of enzyme kineticsto the gibberellin action and also to the synergism betweengibberellin and red light was suggested. ¹National Institute for Basic Biology, Myodaiji, Okazaki 444,Japan. (Received December 18, 1979; )     

The occurrence and properties of methenyltetrahydrofolate cyclohydrolase in plants

Methenyltetrahydrofolate cyclohydrolase (E.C. 3.5.4.9 [EC] ), whichis responsible for the enzymatic conversion of 5,10-methenyl-H₄FAto 10-formyl-H₄FA, has been found in various plant tissues.The enzyme was partially purified from pea seedlings and someof its properties were investigated. It was unstable, but wasstabilized by the addition of 25% glycerol. The enzyme was purifiedabout 60-fold by fractionation with ammonium sulfate and columnchromatography on DEAE-cellulose in the presence of 25% glycerol.Optimum pH for the reaction was 7.7. Michaelis constants for5,10-methenyl-H₄FA in the forward reaction, and for 10-formyl-H₄FAin the reverse reaction were 4x10^–5M and 2x10^–4M,respectively. The apparent equilibrium constant for the reactionwas calculated as 50. Enzyme activity was greatly inhibitedby the reduced forms of folate derivatives. The probable participationof this enzyme in the regulation of folate coenzyme levels inplant tissues has been suggested. ¹ Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants, VI. (For Part V, see Reference (5) ). Partof this paper was presented at the 22nd annual meeting of theJapan Vitamin Society held at Hiroshima on October 14, 1970. ² Present address: Sizuoka Eiwa Junior College, Ikeda, Shizuoka. (Received September 9, 1972; )     

Percent PFR-Dependent Germination of Spores in Pteris vittata

The phytochrome-dependent germination of spores was studiedin the fern Pteris vittata. Brief irradiations with red lightgiven at 0 and 25?C resulted in very similar germination rates.Irradiation with far-red light cancelled this promotive effect,irrespective of the temperature at which tested. The maximumrate of germination was induced by red light of ca. 70Jm^–2and half of the rate was induced by ca. 15Jm^–2 When sporesimbibed in the dark were kept for 1 h at 0 or 25?C under irradiationswith monochromatic lights from 660 to 730 nm at 10 nm intervals,spore germination was induced depending upon the establishedphotostationary states of phytochrome at both temperatures tested.The percent of P_FR estimated in spores that had been irradiatedbriefly with red light was consistent with that resulted fromphotostationary states under different monochromatic lightsin terms of the percent of germination of a spore population.The threshold of the % P_FR required for the germination of eachspore ranged widely from a few percent to 80% of the P_FR. Thisdiversity may vary the timing of germination in nature. ¹ Partial preliminary results of this research were introducedin a review by M.F. (1978). ³ Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya, Tokyo 158, Japan. (Received May 15, 1982; Accepted August 5, 1982)     

Polysome formation in Pinus resinosa at initiation of seed germination

Ribonucleic acid systems present in dormant embryos of red pine(Pinus resinosa Ait.) were studied. Sucrose gradient centrifugationwas used to isolate ribosomes of dormant embryos and embryosimbibed for various times in the light. In dormant embryos,ribosomes existed as monomers. After imbibition, a gradual decreasein the monomers was observed, with subunits and polymers ofribosomes detected within 4 hr. When poly U was added to homogenatesof dormant embryos, formation of polysomes was observed aftera 15-min incubation at 25°C. However, artificial polysomeformation required some factors from heavy particles in thehomogenates. ¹ Contribution from the Missouri Agricultural Experiment Station,Journal Series No. 7079. ² Present address: Government Forest Experiment Station, Meguro,Tokyo, Japan. (Received April 20, 1971; )     

Elicitor Activity of a Fungal Product Assessed at the Single-Cell Level by a Novel Gel-Bead Method

Elicitor from Erysiphe pisi was incorporated into gel beads.Individual beads were placed on single cells from barley coleoptiles.The elicitor induced unusual cytoplasmic responses and temporaryresistance to infection in coleoptile cells. The technique isapplicable to assessment of elicitor activity at the single-celllevel. ¹Contribution no. 118 from the Laboratory of Plant Pathology,Mie University. ²Present address: Laboratory of Plant Pathology & GeneticEngineering, College of Agriculture, Okayama University, Okayama,700 Japan     

A Ca2+- and Voltage-Dependent Cl- -Sensitive Anion Channel in the Chara Plasmalemma: A Patch-Clamp Study

The inside-out patch-clamp technique was applied to the plasmolyzedplasmalemma of inter-nodes of Chara corallina without enzymatictreatment. We found two different types of channel activitythat were CP^–-sensitive. Both types of channel were Ca²⁺-dependent.However, the one that exhibited greater dependence on Ca²⁺ ionswas the focus of our studies, and we named it the Ca²⁺-dependentCP^–-sensitive anion channel. When the concentration ofCa²⁺ ions on the cyto-plasmic side was 1.0 µM, the Ca²⁺-dependentCP^–-sensitive channel opened most frequently between approximately–80 and –100 mV. At 10 µM Ca²⁺, it openedless frequently, and at 0.1 µM Ca²⁺ it scarcely openedat all. These observations indicate that the anion channel ofinterest is voltage-dependent over a restricted range of concentrationsof Ca²⁺ ions. The dependence on Ca²⁺ and voltage of the channelcan explain the behavior of the excitable Ca²⁺-activated Cl^–channel in the Chara plasmalemma. The channel activity was blockedby several antagonists of calmodulin. ⁴ Present Address: Department of Biology, College of GeneralEducation, Osaka University, Toyonaka, 560 Osaka, Japan (Received October 8, 1990; Accepted April 4, 1991)     

13C/12C Ratios of carbon dioxide from peanut and sunflower seedlings and tobacco leaves in light and in darkness

Shoots of intact peanut and sunflower seedlings evolved CO₂in the light which was enriched more than 10 per mille in ¹³Ccompared with simultaneous CO₂ evolution from the roots. Carbondioxide collected from tobacco leaves in the light was 10 permille enriched in ¹³C compared with that collected in the dark.Anaerobic conditions inhibited photorespiration but did notchange isotopic ratios of dark respiration. ¹ Department of Biology, Fresno State College, Fresno, California93710, U. S. A. ² Deceased. (Received February 29, 1972; )     

Photoreversible and photoirreversible absorbance changes in the red and far-red spectral regions in Zea primary roots

When 2-mm apical segments of the primary roots of Zea mays L.(cv. Golden Cross Bantam 70) were irradiated successively withred and far-red light, a photoirreversible absorbance decreasewas separated from the red far-red reversible absorbance changetypical of phytochrome. The difference spectrum of the reversiblechange showed maximum absorbance changes at 666 and 730 nm,while the photoirreversible change induced by red light showeda maximum decrease at 640 nm. The photoreversible absorbancechange was linearly proportional to the fluence of red lightbetween 1 and 6 J m^–2, while the photoirreversible absorbancechange was proportional to its logarithm. Red light of approximately6 J m^–2 induced 50% of the maximum photoirreversible absorbancechange at 640 nm but only about 25% of the maximum photoreversibleabsorbance change. Moreover, no effect of ascorbate on the twoabsorbance changes was observed. ¹Faculty of Education, University of Yamagata, Yamagata 990,Japan. (Received November 2, 1980; )     

Photoreversible and photoirreversible absorbance changes in the red and far-red spectral regions in Zea primary roots

When 2-mm apical segments of the primary roots of Zea mays L.(cv. Golden Cross Bantam 70) were irradiated successively withred and far-red light, a photoirreversible absorbance decreasewas separated from the red far-red reversible absorbance changetypical of phytochrome. The difference spectrum of the reversiblechange showed maximum absorbance changes at 666 and 730 nm,while the photoirreversible change induced by red light showeda maximum decrease at 640 nm. The photoreversible absorbancechange was linearly proportional to the fluence of red lightbetween 1 and 6 J m^–2, while the photoirreversible absorbancechange was proportional to its logarithm. Red light of approximately6 J m^–2 induced 50% of the maximum photoirreversible absorbancechange at 640 nm but only about 25% of the maximum photoreversibleabsorbance change. Moreover, no effect of ascorbate on the twoabsorbance changes was observed. ¹Faculty of Education, University of Yamagata, Yamagata 990,Japan. (Received November 2, 1980; )     

Isolation and characterization of the 337 m{micro} UV-absorbing substance in red alga, Porphyra yezoensis UEDA

Using a variety of Sephadex gel filtrations, starch block zoneelectrophoresis, Avicel SF preparative TLC and DEAE cellulosecolumn chromatography, the characteristic 337 mµ UV-absorbingsubstance from marine red alga, Porphyra yezoensis UEDA wasisolated. The molecular weight of this substance was about 1,000,and its chemical composition was 43.11% C, 5.85% H, 7.23% N,34.73% O and 9.08% Na. In vivo it is located in the chloroplast.On irradiation at 378 mµ, it has a fluorescence actionspectrum peak at 470 mµ. Structural studies on this substanceare still underway, but it could be a kind of aminosugar judgingfrom NMR, IR spectra and other chemical properties. ¹This work was partially supported by the grant of the Ministryof Education in 1969 ²Present address: Department of Biochemistry and Biophysics,University of Hawaii, Honolulu, Hawaii 96822, U.S.A. (Received December 21, 1969; )     

Effect of shade treatment on biosynthesis of catechins in tea plants

When tea plants were shaded with black lawn cloth for severaldays in the field, the accumulations of (—)-epicatechin,(—)-epicatechin-3-gallate, (—)-epigallocatechinand (—)-epigallocatechin-3-gallate decreased in newlydeveloping tea shoots. Radioactive tracer studies showed thatthe conversions of glucose-U-¹⁴C, shikimic acid-G-¹⁴C and phenylalanine-U-¹⁴Cinto (—)-epicatechin and (—)-epigallocatechin moietieswere depressed by the shade treatment for tea plants but theincorporation of trans-cinnamic acid-3-¹⁴C was not affected.The treatment was found to have no significant effect on theactivities of phospho-2-keto-3-deoxy-heptonate. aldolase (EC.4.1.2.15 [EC] ), 3-dehydroquinate synthase (EC. 4.6.1.3 [EC] ), 3-dehydroquinatedehydratase (EC. 4.2.1.10 [EC] ), shikimate dehydrogenase (EC. 1.1.1.25 [EC] )and trans-cinnamate 4-monooxygenase (EC. 1.14.13.11 [EC] ) in theshoots, whereas the activity of phenylalanine ammonia-lyase(EC. 4.3.1.5 [EC] ) clearly decreased. (Received March 17, 1980; )     

Effects of Light on Degradation of Chlorophyll and Proteins during Senescence of Detached Rice Leaves

Regulatory effects of light on senescence of rice leaves wereinvestigated by measuring degradation of chlorophyll and proteinsin leaf segments which had been kept in the dark or under illuminationwith light of different intensities and colors. When leaveshad been left in total darkness for three days at 30°C,there was an initial long lag that lasted for one whole dayand then chlorophyll was rapidly degraded in the second andthird days. Breakdown of chlorophyll was strongly retarded bycontinuous illumination with white light of intensity as lowas 0.5 µmol photons m^–2 s^–1 but the effectof light decreased at intensities above 10 µmol photonsm^–2 s^–2. The initial lag and subsequent degradationof chlorophyll in the dark were little affected by illuminationwith red or far red light at the beginning of dark treatment.However, a brief illumination with red light at the end of thefirst and/or second day significantly suppressed degradationof chlorophyll during subsequent dark periods and the effectof red light was nullified by a short irradiation with far redlight. Thus, degradation of chlorophyll is regulated by phytochrome.Thylakoid membrane proteins and soluble proteins were also largelydegraded during three days in the dark. Degradation of membraneproteins such as the apoproteins of light-harvesting chlorophylla/b proteins of photosystem II and chlorophyll a-binding proteinsof reaction center complexes showed a long lag and was stronglysuppressed by illumination with weak white light. Thus, theloss of chlorophyll can be correlated with degradation of chlorophyll-carryingmembrane proteins. By contrast, light had only a weak protectingeffect on soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenaserapidly disappeared under illumination with weak white light.Thus, breakdown of thylakoid membrane and soluble proteins aredifferently regulated by light. Artifacts which would be introducedby detachment of leaves were also discussed. ¹ Present address: Department of Applied Biology, Faculty ofScience and Technology, Science University of Tokyo, Yamazaki,Noda-shi, Chiba, 278 Japan. ² Present address: Department of Life Science, Faculty of Science,Himeji Institute of Technology, Harima Science Park City, Hyogo,678-12 Japan.     

THE INCORPORATION OF 32P ORTHOPHOSPHATE INTO THE PHOSPHOLIPIDS OF ELONGATING AVENA COLEOPTILES

Four phospholipids of Avena coleoptile tissue were identifiedas phosphatidyl inositol, phosphatidyl glycerol, phosphatidylethanolamine and phosphatidyl choline. IAA caused an increase in total uptake of ³²P and incorporationof ³²P in phospholipids. IAA also caused a shift in the proportionsof identified ³²P phospholipids. Incorporation of ³²P into phosphatidylcholine was greater while incorporation into phosphatidyl glyceroland phosphatidyl ethanolamine was less in IAA-treated tissuecompared with untreated control tissue. ¹Contribution No. 338 from the Department of Botany, PennsylvaniaState University and 3001 from the Pennsylvania AgriculturalExperiment Station. ²Present address: Juniata College, Huntingdon, Pennsylvania,U.S.A.     

Demonstration of light-induced potential change in Chara cells lacking tonoplast

Chara cells without tonoplasts, prepared by replacing the cellsap with EGTA-containing media, showed essentially the samepattern of light-induced changes in membrane potential and membraneresistance as normal cells although the concentrations of ionsand ATP in the cytoplasm decreased considerably (1/3–1/10)after loss of the tonoplast. Removal of the tonoplast reducedthe rate of photosynthetic O₂ evolution to about 50% of thatof normal cells but did not affect the magnitude of light-inducedpotential change. Not a full but a certain level of electronflow seems necessary to activate the putative electrogenic H⁺-pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Japan. ² Present address: Niigata College of Pharmacy, Niigata 950-21,Japan. (Received September 4, 1978; )     

Collapse of Peroxide-Scavenging Systems in Apple Flower-Buds Associated with Freezing Injury

Changes in the metabolic activities of peroxide-producing systemsand peroxide-scavenging systems after freezing and thawing inflower buds of the apple, Malus pumila Mill., were studied withspecial reference to freezing injury. In flower buds of the‘McIntosh’ apple that were frozen below lethal temperatures,the activity of NADH-Cyt c reductase (EC 1.6.99.3 [EC] ), one of theenzymes in the electron-transport chains that are related tothe peroxide-producing systems, decreased slightly, while thatof Cyt c oxidase (EC 1.9.3.1 [EC] ) hardly changed. By contrast, theactivities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 [EC] ),dehydroascorbate reductase (EC 1.8.5.1 [EC] ) and ascorbate peroxidase(EC 1.11.1.11 [EC] ), which are involved in the peroxide-scavengingsystems, decreased to very low levels. The activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) also decreased markedly. However,little change was observed in the activities of hexokinase (EC2.7.1.1 [EC] ), glucosephosphate isomerase (EC 5.3.1.9 [EC] ), glutathionereductase (EC 1.6.4.2 [EC] ) and glutathione peroxidase (EC 1.11.1.9 [EC] ).Examination of substrates involved in the peroxide-scavengingsystems revealed that the levels of glucose-6-phosphate andfructoses-phosphate decreased to approximately 10^–4 to10^–5 M and 10^–5 M, respectively, and the levelsof GSH decreased to about 10^–5 M or became barely detectable.A decrease in the levels of GSSG also occurred while levelsof ascorbate rose slightly. Similar results were observed withflower buds from ‘Starking Delicious’ and ‘Jonathan’apple trees. These results suggest that the freezing injury to apple flower-budsis closely related to the collapse of the peroxide-scavengingsystems that are coupled with the pentose phosphate cycle. Theresults also suggest that the dysfunction of these peroxide-scavengingsystems is caused by H₂O₂, which may be produced during freezingand thawing. (Received March 14, 1992; Accepted June 5, 1992)