SOME PROPERTIES OF THE CYTOCHROME C REDUCING SUBSTANCE, A FACTOR FOR LIGHT-INDUCED REDOX REACTION OF CYTOCHROME C IN PHOTOSYNTHETIC LAMELLAE

Cytochrome c reducing substance (CRS), a redox substance discoveredin photoreactive lamellar fragments, was purified by Sephadexcolumn chromatography. Chromatographic behaviours of CRS ofAnabaena and spinach were essentially the same. Purified CRSof Anabaena showed an absorption spectrum having one absorptionmaximum around 260 mµ. The absorption peak disappearedon addition of excess amount of borohydride. Similar absorptionchange on borohydride addition was observed with spinach CRSpreparation. Purified preparations of Anabaena and spinach CRS supportedphotophosphorylation in spinach broken chloroplasts. The phosphorylationwas found to couple the electron flow from water to molecularoxygen. ¹This work was supported by grant GM-11300 from the NationalInstitute of Health, U. S. A. ²Present address: Institute of Applied Microbiology, The Universityof Tokyo, Tokyo, Japan.     

STUDIES ON THE MECHANISM OF GROWTH INHIBITING EFFECT OF LIGHT

1. A substance which inhibits indoleacetic acid (IAA)-and naphthaleneaceticacid (NAA)-induced elongation of Avena coleoptile section andIAA-induced Avena coleoptile curvature was found in an ethersoluble neutral fraction of water extract of sunflower leavesand in agar blocks containing the diffusate from young sunflowerleaves.
2. This substance also inhibits the growth of isolatedsunflowerepicotyl.
3. The R_f value (0.9) of the substance ona paper chromatogramdeveloped with ammoniacal iso-propanolindicates that it isidentical with the inhibitor reported byAUDUS et al. (1956),but not with inhibitor-ß.
4. Theinhibitor can be transported from leaf to stem, and thetransportseems to be accelerated by illuminating the leaf.
5. The auxindiffused from sunflower leaf into agar block may beidenticalwith IAA.
6. A substance, which has the same properties as theinhibitorfrom sunflower leaf, was obtained in crystalline formfrom theleaf of Jerusalem artichoke.
7. The mechanism of growthinhibition caused by this crystallinesubstance seems to involveinactivation of a sulfhydryl group.
8. The reason why the stemgrowth of sunflower seedlings is reducedby strong light isdiscussed: the amount of the inhibitor transportedfrom leafto stem is increased under strong light, and in thestem, growthinhibition is caused by a direct effect of thisinhibitor ongrowth and by its inhibiting effect on the transportof IAAfrom leaf to stem.

¹ Present address: Botanical Garden, Faculty of Science, Universityof Tokyo, Tokyo (Received February 15, 1961; )     

EVIDENCE ON THE SITE OF ACTION OF GROWTH RETARDANTS

1. The ability of five growth retardants to inhibit the GA-inducedand endogenous growth of Avena leaf sections has been investigated.The retardants vary in effectiveness. The order, from most effectiveto least, is Phosfon D, Amo-1618, C011, CCC and B995. 2. The inhibition of growth caused by Phosfon D and Amo-1618is not reversed by GA. It is apparent that the retardants donot compete with GA at the site of GA-action. 3. Addition of IAA will partially reverse the inhibition inducedby Phosfon D or Amo-1618. It is concluded that the retardantsact in part in Avena leaf sections by interfering with the auxinmetabolism of the tisssue. ¹ Supported in part by grants G-14578 and GB-1950 from the NationalScience Foundation ² Present address: Department of Botany, University of Washington,Seattle, Washington     

THE ROLE OF AUXIN IN THE PHOTOPERIODIC TUBERIZATION IN BEGONIA EVANSIANA ANDR.

The formation of aerial tubers in Begonia plants, an SD response,was inhibited by IAA, NAA and IBA applied to their leaves duringthe dark periods. The effectiveness of IAA differed accordingto the time of application during the dark periods, and themost sensitive time varied with daylengths employed. In order to inhibit the tuberization under optimal photoperiods(8-hr SDs), IAA had to be applied during the first 2 days orso of the SDs. Under non-optimal photoperiods, however, IAAwas effective even when applied somewhat later. The auxin activity of leaf extracts from the plants subjectedto 8-hr SDs decreased during the first 2 or 3 days to a minimum,and then increased until finally began to decrease, again; undernon-optimal photoperiods, the minimum of auxin activity wasattained more slowly. The paper-chromatographic study suggestedthat the change in auxin activity was mainly due to the changein IAA content. The number of SDs making the auxin content minimal agreed withthe minimum number of SDs required for tuberization. On the basis of the above results, the part played by endogenousauxin in photoperiodic induction is discussed. ¹Present address: Institute for Agricultural Research, TôhokuUniversity, Sendai. ²Present address: Biological Institute, Yamaguchi University,Yamaguchi.     

Changes in Endogenous Ribosyl-trans-zeatin and IAA Levels in Relation to the Proliferation of Tobacco Crown Gall Cells

Changes in the contents of major endogenous plant hormones intobacco crown gall cells, namely IAA and ribosyl-trans-zeatin,during cell growth were examined using HPLC and ¹⁴C-labeledplant hormones. The content of IAA was high at the early logarithmicstage, while that of ribosyl-trans-zeatin was high at the middlelogarithmic stage. This suggests that cell growth is affectedfirst by IAA, then by ribosyl-trans-zeatin. ³ Present address: Department of Agricultural Chemistry, TottoriUniversity, Koyama, Tottori 680, Japan (Received July 13, 1981; Accepted September 11, 1981)     

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.     

PENETRATION OF SUGARS FROM THE CHEMOSENSORY HAIR TIP IN THE FLESHFLY

(1) The penetrations of ¹⁴C-D-glucose and 3-O-methyl-¹⁴C-D-glucoseinto the isolated head of the fleshfly, Boettcherisca peregrina,from the tip of the longest chemosensory hair on the labrumwere investigated. (2) Both ¹⁴C-D-glucose and 3-O-methyl-¹⁴C-D-glucosepenetrated into the hair almost linearly with time and movedrapidly into the labrum. The isotopic activity was finally detectedin the head. (3) The isotopic activity of the hair dipped into¹⁴C-D-glucose solution increased in the preparation which hadbeen pretreated with 7.5 x 10^–2M colchicine for 30 min,whereas in the case of 3-O-methyl-¹⁴C-D-glucose no effect by7.5 x 10^–2M colchicine was found. (4) Both ¹⁴C-D-glucoseand 3-O-methyl-l4C-D-glucose which penetrated from the poreof the hair tip were detected in the dendritefree lumen andin the dendrite-containing lumen of the chemosensory hair. (5)These results suggest that D-glucose not only moves in the dendrite-freelumen and the dendrite-containing lumen but also in the dendrite(s).The suggestive results that 3-O-methyl-D-glucose moves in thedendrite(s) could not be obtained. ^*Present address: Department of Physiology, Medical Collegeof Miyazaki, Miyazaki, Japan.     

Effects of Exogenous Auxin on the Regulation of Elongation Growth of Excised Segments of Vigna Hypocotyls under Osmotic Stress

IAA applied simultaneously with osmotica greatly enhanced theadaptive recovery of the elongation growth of segments of Vignahypocotyls during osmotic stress irrespective of whether ornot absorbable solutes were present. IAA stimulated both thesurface pump and the xylem pump, which have been shown to bestimulated by osmotic stress and to control the yielding ofthe cell wall and the absorption of solutes. Thus, wall extensibilityand the effective turgor were further enhanced under osmoticstress in the presence of IAA. These results indicate that thesimultaneous presence of IAA can reduce the inhibition of growthby osmotic stress, and they support numerical predictions basedon the apoplast canal model. The mechanism involved in the rapidrecovery of growth is discussed. ¹ Present address: Research Centre, Guangxi Agricultural University,Xiu Ling Rd., Nanning, Guangxi 530005 China. ² Present address: Biology Institute, Department of GeneralEducation, Nagoya University, 1 Furo-cho, Chikusa-ku, Nagoya,464 Japan. ³ Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2, Seto, Kanazawa-ku, Yokohama, 236 Japan.     

THE RELATIONSHIP OF GIBBERELLIN AND AUXIN IN PLANT GROWTH

No synergism was found between IAA and gibberellin in the Avenucurvature test and this bioassay thus measures changes in diffusibleauxin resulting from gibberellin treatment and not a synergisticaction of the gibberellin on the curvature response to auxin.Gibberellin treatment causes an increase in diffusible auxinfrom the stem apex of dwarf pea (Pisum sativum L. var. LittleMarvel) 24 to 48 hours before the elongation response in thestem. The increase in diffusible auxin in the stem apex of Centaureacyanus L. var. Blue Boy occurs four to six days before the boltingresponse to gibberellin treatment under short days. The stemtissues of both the dwarf pea and Centaurea show an elongationresponse to IAA when the IAA is applied in a manner simulatingthe stem apex. Thus the growth of the dwarf pea and the boltingof Centaurea brought about by treatment with gibberellin aredependent on an increase in diffusible auxin. ¹Present address: Biological Institute, College of General Education,University of Tokyo, Komaba, Meguro, Tokyo.     

Effects of Osmotic Stress, Ionic Stress and IAA on the Cell-Membrane Resistance of Vigna Hypocotyls

The cell-membrane resistance (R_m) of Vigna hypocotyls was examined,and the effects of osmotic stress, ionic stress and IAA on R_mwere investigated. R_m decreased by 64 to 77% under osmotic stressin the presence of absorbable solutes (40 mM sorbitol, 15 mMKC1, 30 mM sucrose; or 40 mM sorbitol, 15 mM KC1, 30 mM sucroseplus 10^–4 M IAA) or under ionic stress (50 mM NaCl or50 mM KC1). R_m was not changed by perfusion with 10^–4M IAA. Therefore, the hyper-polarizations of the membrane potentialobserved in both cases should be ascribed totally to the activationof the electrogenic proton pump. Although R_m showed an increaseof 1.6 fold when the hypocotyls were subjected to osmotic stress(100 mM sorbitol or 100 mM sorbitol plus 10^–4 M IAA),83.6% or 92.4% of the hyperpolarization of the membrane potential(V_px was also the result of the activation of the pump. Theresults, calculated on the basis of the current source model,support the viewpoint that the hyperpolarization of the cellmembrane potential of Vigna hypocotyls under osmotic stress,ionic stress or in the presence of IAA is an expression of theactivation of the proton pump, and is not caused by an increasein R_m. ¹ Present address: Researchers and Planners of Natural Environment, Yotsugi Bldg. (2F), 1-5-4 Horinouchi, Suginami-Ku, Tokyo,166 Japan ² Present address: Graduate School of Integrated Science, YokohamaCity University, 22-2 Seto, Kanazawa-Ku, Yokohama, 236 Japan (Received February 14, 1991; Accepted July 24, 1991)     

Studies on anaerobic biphasic growth of a denitrifying bacterium, Pseudomonas stutzeri

Growth of Pseudomonas stutzeri(VAN NIEL strain) in the presenceof a limiting amount of nitrate under anaerobic conditions ischaracterized by 2 logarithmic phases separated distinctly byan intermediate phase where the growth rate is very low. Inthe first logarithmic phase nitrate is reduced stoichiometricallyto nitrite stage, and in the second phase nitrite is reducedto nitrogen gas. The nitrite reducing activity of cells in the second growthphase is 3–4 times higher than that of cells in the firstphase. The rise in nitrite reducing activity is correlated witha remarkable increase in the content of cytochromes a₂ and c-552. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan. ²Present address: Institute of Molecular Biology, Faculty ofScience, Nagoya University, Nagoya, Japan. (Received June 16, 1969; )     

Effect of indoleacetic acid on the penetration of leucine and glucose through epidermal tissue of onion bulb

The penetration of leucine-(U)-¹⁴C and glucose-(U)-¹⁴C throughthe bulb epidermal tissue of Allium cepa was examined in thepresence of indoleacetic acid (IAA). Not only the uptake ofleucine-¹⁴C and glucose-¹⁴C in epidermal tissue but also theirtranscellular penetration were accelerated by IAA treatment.N-Ethylmaleimide (NEM) inhibited their uptake and transcellularpenetration, and the inhibitory effect was relieved by additionalIAA. In the presence of IAA, leucine-¹⁴C and glucose-¹⁴C weremore penetrable by adaxial than abaxial application, but inthe absence almost no difference due to application side wasobserved. IAA appears to promote permeability of the epidermaltissue only to substances applied adaxially. N,N'-Dicyclohexylcarbodiimide(DCCD) showed a little inhibitory effect on the IAA-inducedpromotion of the uptake and penetration of leucine-¹⁴C appliedadaxially. Leucine-¹⁴C and glucose-¹⁴C penetrated more easilythrough killed than fresh tissue, with little difference betweenabaxial and adaxial applications. ¹ Present address: Department of Biology, Faculty of Science,Kochi University, Kochi 780, Japan. ² Present address: Department of Medical Zoology, Medical School,Mie University, Tsu 514, Japan. (Received October 13, 1977; )     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

IONIC COMPOSITION OF THE CYTOPLASM OF NITELLA FLEXILIS

The K, Na and Cl concentrations of the chloroplast layer andthe flowing cytoplasm of Nitella flexilis have been determinedby applying an internal perfusion technique, which enabled usto avoid contamination of ions from the cell sap. K, Na andCl concentrations of the chloroplast layer are 110, 26 and 136mM and those of the flowing cytoplasm are 125, 5 and 36 mM respectively.The cell sap contains 80 mM K, 28 mM Na and 136 mM Cl. Althoughthere are some variations in these values among samples, theflowing cytoplasm is rich in K and poor in Cl and especiallyin Na. The exchange of K and Na across the tonoplasl occursfairly easily (half-time, a few hours), while that of Cl occursextremely slowly (half-time, a few days). ¹This work was supported by Research Grants from the Ministryof Education of Japan     

Auxin- and Acid-Induced Changes in the Mechanical Properties of the Cell Wall

Auxin- and acid-induced changes in the mechanical propertiesof the cell wall were analyzed by measuring the creep of thecell wall using pumpkin (Cucurbita moschata Duch cv. Shirakikuza)hypocotyl segments. Hypocotyl segments were treated with orwithout IAA and stored in 50% glycerol at –15°C formore than 2 weeks before measurements. Creep rate increasedwith the increase in the load. The increase was first very slowup to the phase shift point (yield threshold, y), and afterthat, it was steep. The rate of the creep rate increase (creepcoefficient, Cm) was larger and y was smaller at pH 4.5 thanpH 6.8. This indicates cell wall loosening was facilitated underacidic conditions. IAA-pretreatment of the segments resultedin the lowering of y at pH 6.8 only. Around pH 5 and 45°C,Cm was highest and y was lowest. Boiling in distilled wateralmost lost the differential effect of Cm and y on pH and IAA.The differential effect of pH on Cm and y were recovered bythe addition of a crude extract of cell wall-bound proteins.It is implied that some enzymatic processes are involved inthe control of acid-induced cell wall extension. ¹Present address: Nihon Shokuhin Kako Co., Ltd. 30 Tajima, Fuji-City,Shizuoka, 417-8530 Japan. ²Present address: Graduate School of Environmental Earth Science,Hokkaido University, Sapporo, 060 Japan.     

Studies on the metabolism of a sulfur-oxidizing bacterium VII. Oxidation of sulfite by a cell-free extract of Thiobacillus thiooxidans

Properties of the cell-free extract, prepared from a strainof Thiobacillus thiooxidans by sonic disruption followed byfractionation with centrifugatiori, were investigated with referenceto its sulfite-oxidizing activity. Without the addition of cofactors the particulate fraction(F-P)catalyzed oxidation of sulfite with oxygen or bacterial cytochromec-552 obtained from Pseudomonas stutzeri as electron acceptor.TMPD reduced by ascorbic acid was also oxidized by F-P. Thesoluble fraction(F-S) showed no activity in oxidizing sulfiteand TMPD, but stimulated TMPD oxidation by F-P. Oxygen uptake with either sulfite or TMPD as substrate was inhibitedby KCN, NaN₃, CO and c-phenanthroline. CO-Inhibition was reversedby light. Reduction of cytochrome c-552 by sulfite was insensitiveto these agents. Antimycin A markedly inhibited sulfite oxidation with eitheroxygen or cytochrome c-552 as electron acceptor, but was withouteffect on TMPD oxidation. DDC and SAO, both strong inhibitors of sulfur oxidation, didnot affect sulfite and TMPD oxidations. Cytochromes of the a, b and c types were contained in F-P. Thesecytochromes were rapidly reduced when F-P was incubated withsulfite. Cytochrome(s) of the c type was present in F-S, too. ¹VI.=References (3) ²Partly supported by a grant from the Ministry of Education ³Present address: Sanyo Women's College, Hatsukaichi, Hiroshima738, Japan ⁴Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima 734, Japan (Received May 15, 1970; )     

Multiple effects of the inhibitory protein of ethylene production on cellular metabolisms

The effects of an inhibitory protein of ethylene productionisolated from etiolated mung bean hypocotyls (Planta 113: 115,1973) were investigated. Etiolated mung bean hypocotyl segmentsincubated with IAA for 3 hr (1st incubation) to induce ethylene-producingactivity were incubated for 1 hr with IAA in the presence ofthe inhibitory protein and a radioactive material to measuremetabolic activity. Under the conditions where ethylene productionwas inhibited 80% or more by the protein, RNA synthesis, proteinsynthesis and phosphate uptake were suppressed 55–60,65–80, and 60–75%, respectively. Conversion of 1-¹⁴C-acetateto CO₂, lipid, basic and neutral fractions was also inhibited,but the degrees of inhibition were much less than those forthe other processes. When the segments pretreated with the inhibitoryprotein during the 1st incubation period were washed free ofthe protein and assayed for their metabolic activities, theinhibition of RNA and protein syntheses and of phosphate uptakewas partially restored, while ethylene-producing activity wasfully restored to the control level. Similar reversible inhibitoryeffects were also observed for those metabolic activities inthe tissue segments not treated with IAA, thus not producinginduced ethylene. Oxygen uptake and conversion of U-¹⁴C-glucoseto CO₂ were not affected by the inhibitory protein. The possibilitythat the inhibitory protein acts on cell surface membranes andthe modified membranes affect the regulatory mechanism of cellularmetabolism is discussed. ¹ This investigation was supported in part by grants from theMinistries of Education (B-248009), and of Agriculture and Forestryof Japan. (Received November 4, 1977; )     

A factor in cell-free extracts inactivating sexual agglutinability of a mating type in Saccharomyces cerevisiae

Cell-free extracts of both a and a mating-type strains of Saccharomycescerevisiae contained a substance which irreversibly inactivatedsexual agglutinability of a cells, but not that of a cells. ¹ Present address: Department of Pharmacology, Osaka Collegeof Pharmacy, 2-10-65 Kawai-cho, Matsubara, Osaka 580, Japan. (Received January 9, 1976; )     

Natural Occurrence of Indoleacetamide and Amidohydrolase Activity in Etiolated Aseptically-Grown Squash Seedlings

Etiolated seedling tissues of aseptically grown squash (Cucurbitamaxima Duch) contain indole-3-acetamide (IAM) as a natural endogenouscompound, conclusively identified by gas chromatography-massspectrometry (GC-MS). Roots of aseptically raised seedlingsalso contain amide hydrolysing activity, which converts IAMto IAA, indoleacetonitrile (IAN) to IAM and IAA, and 1-naphthaleneacetamideto 1-naphthaleneacetic acid. This activity was enriched 48-foldby fractional precipitation with ammonium sulphate, Sephadexgel nitration and anion exchange chromatography. Being hydrolytic,it works equally well in air and in vacuo, without added cofactors.The partially purified enzyme works optimally between pH 7 and7.5, and a K_m value of 80 µM was calculated with IAM asthe substrate. The product of this reaction was definitivelyidentified as IAA by GC-MS. The temperature optimum of thisamidohydrolase lies around 45°C, and it is stable to freezing.A comparison of its properties with the amidohydrolase of Agrobacteriumor crown gall tissue, shows it to be different. In view of thenatural occurrence of both IAM and the amidohydrolase, it issuggested that the IAM pathway of IAA biogenesis is feasiblein etiolated squash seedlings. ⁴Deceased 2/2-1993.