Biosynthesis of ethylene in sweet potato root tissue

The biosynthetic pathway of ethylene in freshly cut and blackrot-diseased tissues of sweet potato roots was investigated.Glucose-U-¹⁴C administration gave labeled ethylene in both freshand diseased tissues, but at the early stage of infection, therewas ethylene production which was not derived from the fed ¹⁴C-glucose.Acetate-1-¹⁴C and acetate-2-¹⁴C were equally incorporated intoethylene produced from fresh tissue, but acetate-2-¹⁴C was preferentiallyincorporated into ethylene from diseased tissue. Pyruvate-3-¹⁴Cwas more efficient as a precursor than was acetate or glucosein fresh tissue, while its efficiency was the same as that ofacetate in diseased tissue. Monofluoroacetate promoted pyruvate-3-¹⁴Cincorporation in fresh tissue but inhibited incorporation indiseased tissue. We concluded that the TCA cycle is involvedin the case of diseased tissue but not in fresh tissue; thus,showing different pathways for ethylene production in each tissue.In addition, in diseased tissue, ethylene is assumed to be producedfrom some cellular component(s), not easily synthesized fromglucose through fungus infection, but is degraded as soon asinfection commences. ¹This paper constitutes Part 85 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury ²Present address: The Institute for Biochemical Regulation,Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464,Japan (Received April 20, 1970; )     

SOME PROPERTIES OF PEROXIDASE PRODUCED IN SWEET POTATO INFECTED BY THE BLACK ROT FUNGUS

Chemical and physicochemical properties of peroxidases producedback rotted sweet potato roots were investigated in comparisonwith those produced in cut one. Peroxidases in either diseased or cut tissue were composed offour major (D-A, D-B, D-C and D-D in diseased tissue and C-A,C-B, C-C and C-D in cut tissue) and several minor components.These peroxidases were separated from each other by DEAE-cellulosecolumn chromatography and other procedures. Several propertiesof the peroxidases were investigated.

1. Optimum pH's of peroxidase were in the range of 5.5 to 6.0.
2. The activity of each peroxidase was inhibited by acid, alkaliand some inhibitors such as cyanide, fluoride and azide. Azideinhibited more strongly D-A and C-A than D-B and C-B. On theother hand, cyanide and fluoride inhibited more strongly D-Band C-B than D-A and C-A.
3. Substrate specificity as determimedby using pyrogallol, guaiacol,chlorogenic acid, caffeic acidand umbelliferone differed betweenthe main peroxidases. Thedegree of indoleacetic acid oxidaseactivity of these peroxidaseswas also different from each other.
4. Light absorption spectraof the peroxidases showed that theybelonged to a-type peroxidaseexcept C-D. More precise investigationsof the spectra showedthat the spectra of D-A and C-A were differentfrom those ofD-B and C-B.

Peroxidase A (D-A), the main component in diseased tissue, waspurified by methods such as DEAE-cellulose chromatography andstarchgel electrophoresis to a grade higher than previouslyshown. It was homogeneous, according to investigations withultracentrifugation, immunochemical reaction and starch-gelelectrophoresis. Pyridine hemochrome of the peroxidase showedthat the heme in it was protoheme. Amino acid composition ofthe enzyme was determined. Peroxidase A oxidized various phenolicsubstances in the presence of H₂O₂. Indoleacetic acid oxidaseactivity of peroxidase A was inhibited by both chlorogenic acidand guaiacol. ¹Part 45 of Phytopathological Chemistry of Sweet Potato withBlack Rot. ²Present address: Central Research Institute, Japan MonopolyCorporation, Yutakamachi, Tokyo.     

Production of ethylene by injured sweet potato root tissue

Ethylene production by sweet potato root tissue was examinedwith special emphasis on tissue injury. The root tissue producedethylene in response to cut injury. Increasing the cut surfacearea increased ethylene production, and the amount was proportionalto logarithm of the surface area. Tissue discs washed with waterbefore incubation produced less ethylene than unwashed discs. When the tissue was treated with chemicals that might destroythe cells, ethylene production remarkably increased. Monoiodoacetamide,trichloroacetic acid and sodium ethylmercurithiosalicylate wereparticularly effective in inducing ethylene production. Here,again, ethylene production was related to the degree of injury.Treatment of the tissue with increasing concentrations of thesechemicals resulted in increasing ethylene production, but concentrationsover a certain limit rather decreased the ethylene production.This may be due to the rapid destruction of the whole tissueused before ethylene production commenced. For thylene production,the presence of injured but still living cells was necessary. Relationship of the injury-induced ethylene production to metabolicactivation is discussed. ¹Part 67 of the Phytopathological Chemistry of Sweet Potatowith Black Rot and Injury. ²Fulbright grantee of 1967. Present address: Department of Biochemistry,University of Wisconsin, Madison, Wisconsin, U.S.A.     

Fungal extracts that induce phytoalexins in sweet potato roots

Soluble extracts from mycelia and conidia of two strains ofCeratocyslis fimbriata induced formation of terpenes in sweetpotato root tissue. Factors inducing terpene formation are water-or 0.02 M KCl-soluble, heat stable, organic solvent-insoluble,and dialyzable, and have neither cationic nor anionic properties.They caused cellular injury of root tissue, accompanied by productionof ethylene. ¹This paper constitutes Part 115 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury, and Contributionof Research Branch, Agriculture Canada, Winnipeg, Canada. Thiswork was supported in part by a grant from the Ministry of Education,Japan. ²Present address: Research Branch, Research Station, AgricultureCanada, Winnipeg, Manitoba, Canada. (Received July 27, 1974; )     

IPOMEAMARONE ACCUMULATION AND LIPID METABOLISM IN SWEET POTATO INFECTED BY THE BLACK ROT FUNGUS I. IDENTIFICATION OF STEROL AND CHANGES IN LIPID METABOLISM DURING INFECTION PROCESS

Changes in lipid contents during infection process in the non-infectedtissue adjacent to the infected region of diseased sweet potatoroots with black rot were examined in comparison with cut controltissue. Incorporation of 2 ¹⁴C-acetate and ³²P-phosphate intolipid fraction was also investigated. Although there was nosignificant change in lipid ester groups in both tissues, increasein phospholipids was found in diseased tissue. Sterol isolatedfrom fresh material was identified with ß-sitosterol.Chromatographic patterns of non-phospholipid fraction of diseasedtissue suggested that some metabolic alteration of this fractionmight occur in response to infection. ¹This paper constitutes Part 40 of Phytopathological Chemistryof Sweet Potato with Black Rot.     

Polarity of Production of Polyphenols and Development of Various Enzyme Activities in Cut-injured Sweet Potato Root Tissue

Investigation of polyphenol production in cut-injured sweet potato (Ipomoea batatas Lam. cv. Kokei 14) roots by histochemical and quantitative methods showed that polyphenols were produced in striking amounts in the proximal side of the tissue pieces (2 cm thick), but only in small amounts in cells of the distal side. In response to cut injury, formation of the enzymes related to polyphenol biosynthesis, phenylalanine ammonia-lyase and trans-cinnamic acid 4-hydroxylase, was also pronounced in the proximal side of the tissue pieces and slight in the distal side. The similar polarity was observed in the development of activities of various enzymes, such as NADPH-cytochrome c oxidoreductase, acid invertase, peroxidase, o-diphenol oxidase, and cytochrome c-O₂ oxidoreductase. Acropetal development of polyphenol contents and of various enzyme activities may be related to the acropetal movement of indoleacetic acid (IAA) in roots of various plants. Treatment of the distal surface of tissue pieces with IAA or 2,4-dichlorophenoxyacetic acid caused polyphenol production but treatment with gibberellic acid, abscisic acid, kinetin, or ethylene had little effect. The results suggest that IAA may play a role in the metabolic response to cut injury.     

Time sequence of the effect of ethylene on transport, uptake and decarboxylation of auxin

The effect of ethylene on the uptake, decarboxylation and basipetaltransport of IAA-1-¹⁴C, IAA-2-¹⁴C and NAA-1-¹⁴C in cotton stemsections (Gossypium hirsutum L., var. Stoneville 213) was studied.A reduction in the capacity of cotton stem sections to transportauxin basipetally appears in sections excised from plants exposedto ethylene for only 3 hr and increases with fumigation time. In addition to reducing transport, increasing ethylene pretreatmentperiods from 3 to 15 hr also progressively reduced the uptakeof ¹⁴C and increased the release of ¹⁴C as ¹⁴CO₂ from IAA-1-¹⁴C.The effect of ethylene on the decarboxylation of IAA-1-¹⁴C wassignificant when expressed as either the cpm of ¹⁴C releasedper hr per mg dry weight or the cpm released per hr per mm²in contact with the IAA donor. Comparative experiments usingIAA-1-¹⁴C and IAA-2-¹⁴C demonstrated that the effect of ethyleneon the decarboxylation of IAA was primarily a cut surface effectwhich apparently contributes to the reduction of IAA uptakeby ethylene. Although ethylene significantly reduced the transport of NAA-1-¹⁴C,uptake was significantly increased rather than decreased aswith IAA-1-¹⁴C while decarboxylation was unaffected. Ethylene pretreatment caused no significant changes in the dryweight or the cross-sectional area of the absorbing surfaceof the transport tissue. ¹A contribution of the Texas Agricultural Experiment Station.Supported in part by Grant GB-5640, National Science Foundationand grants from the Cotton Producers Institute and the NationalCotton Council of America. ²Present address: Central Research Department, E. I. Du PontDe Nemours and Company, Wilmington, Delaware 19898, U. S. A. (Received May 29, 1969; )     

STUDIES ON PHOTOPHOSPHORYLATION: I. TWO-STEP EXCITATION KINETICS OF PHOTOPHOSPHORYLATION

Based on the observations on the time lag and the low efficiencyof photophosphorylation at low light intensities, a ‘two-stepexcitation kinetics’ of photophosphorylation of chloroplastswas developed, which could explain some of the hitherto unexplainedfacts concerning photophosphorylation. By analyses of the experimental results along the line of thetheory, it was inferred that the assumed substance (X) responsiblefor ATP formation occurs in a relatively large quantity of about1 mole/5–20 moles of chlorophyll. It was concluded thatthe high energy intermediate (X^**) of ATP formation which wasassumed to accumulate on pre-illuminating the chloroplasts (inthe absence of ADP and Pi) does not represent a side pool ofextra energy, but a substantial intermediate in the normal courseof ATP formation. Alternative possibilities for the explanation of the experimentalfacts were also discussed. ¹This paper is submitted to the University of Tokyo to fulfilla part of the requirements for the doctorate of H. SAKURAI.     

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.     

Occurrence of Peroxidases in Sweet Potato Infected by the Black Rot Fungus

Changes in the peroxidase activity and the patterns in sweet potato tissue infected by Ceratocystis fimbriata were investigated, by the method of starch-gel electrophoresis, DEAE-cellulose column chromatography and immunochemical analysis, compared with that in cut and healthy tissue. Time-course analysis of the increment of the total peroxidase activity also showed noticeable differences between diseased and cut tissues. The peroxidases in diseased and cut tissues were composed of four major and several minor components. Though electrophoretic analysis did not show so striking differences in the patterns of peroxidases between diseased and cut tissues, there were distinct differences in the ratio of activities of major peroxidase components between both tissues. Immunochemical works indicated that peroxidase A which showed the most prominent increase in diseased tissue was found to be formed in cut tissue though the amounts were appreciably small. The activity of peroxidase C in diseased tissue was not so high as seen in cut tissue.     

Studies on chlorophyll formation in Chlorella protothecoides III. Effects of chloramphenicol, cycloheximide, and ethionine on chlorophyll formation

Effects of chloramphenicol, cycloheximide, puromycin and ethionineon the light-independent and subsequent light-dependent processesof chlorophyll formation in "glucose-bleached" cells of Chlorellaprotothecoides were studied. These substances, except puromycin,strongly suppressed different phases of chlorophyll formation.Ethionine most strongly suppressed the light-independent phaseand chloramphenicol an early, relatively short process in thelight-dependent phase of chlorophyll formation. Cycloheximideseverely suppressed all phases of chlorophyll formation. Possibleimplications of these results for the biosynthesis of chlorophyllin algal cells are discussed. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. ² Laboratory of Entomology, Faculty of Agriculture, TamagawaUniversity, Machida-shi, Tokyo, Japan (Received October 5, 1972; )     

Effect of wounding or infection by Phytophthora infestans on the contents of terpenoids in potato tubers

Inoculation of the potato cut surfaces with an incompatiblerace of Phytophthora infestans induced an accumulation of rishitin,but only a trace occurred when infected by a compatible race.When the tuber was cut, a large amount of steroid-glycoalkaloids(solanine) accumulated in the cut surface tissue, although onlya trace was found in intact tissue. Only a small amount of solanine,if any, was contained in the wound periderm tissue. Most ofthe solanine seemed to be distributed in tissue neighbouringthe newly formed meristematic tissue zone. Distribution of solanineas a function of distance from the cut surface was exponential.Infection of the cut surface by an incompatible race of Phytophthorainfestans reduced the accumulation of solanine. The higher theconcentration of zoospore used, the less the solanine content.It has been reported that the higher the concentration of zoosporesused for inoculation of the cut surface, the less the numberof renewed meristematic cells in the wound tissue. In experimentsusing fresh and aged tubers, a good correlation between thenumber of renewed meristematic cells and solanine content wasfound. The accumulation of solanine in the wound tissue andits reduction due to infection by an incompatible race may berelated to renewed meristematic cells formation and its reductioncaused by the infection. No drastic change in carotenoids or sterol contents was found2 days after cutting or inoculation, when the tubers were cut,or cut and then infected by the incompatible race. ¹ Studies on the phytoalexin (No. 10). (6) in References constitutes"Studies on the phytoalexin No. 9". ² Present adress: Plant Pathology Laboratory, Faculty of Agriculture,Nagoya University, Chikusa-ku, Nagoya, 464, Japan. (Received April 1, 1972; )     

Changes in glutamate dehydrogenase activity in cut-injured sweet potato root tissue

Changes in glutamate dehydrogenase activity in sweet potatoroot tissue in response to slicing were investigated with mitochondrialand supernatant fractions. Results indicated that activity inmitochondrial fraction had decreased, whereas activity in supernatantfraction had increased, 12 hr after slicing. The increase inactivity in the supernatant fraction may be related to the regenerationof phenylalanine, a precursor of polyphenols. ¹This paper constitutes Part 104 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ²Present address: Laboratory of Biochemistry, Faculty of Agriculture,University of Tokyo, Bunkyoku, Tokyo 113, Japan. (Received August 15, 1972; )     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

The effect of ethylene on auxin-induced growth of excised rice coleoptile segments

Elongation growth induced by exogenous auxin of apical coleoptilesegments of etiolated rice seedlings was promoted by ethylene.In the absence of exogenous auxin, growth promotion was notobserved. The highest promotion by ethylene was obtained at10^–6 M of indole-3-acetic acid, a suboptimal concentrationfor auxin-induced elongation. Level of ethylene which achievedthe effect was less than 1 µl per liter of an incubationatmosphere. ¹Present address: The Ocean Research Institute, University ofTokyo, Nakano, Tokyo, Japan (Received May 27, 1970; )     

BIOGENESIS OF ETHYLENE IN APPLE TISSUE: I. FORMATION OF ETHYLENE FROM GLUCOSE, ACETATE, PYRUVATE, AND ACETALDEHYDE IN APPLE TISSUE

1. With the aim of elucidating the path of carbon in the formationof ethylene in plants, studies were made on the incorporationof ¹⁴C into ethylene evolved from apple slices, using several¹⁴C- labeled compounds as substrates. The effects of inhibitorswere also investigated. 2. The formation of ethylene-¹⁴C from glucose-¹⁴C was inhibitedby fluoride, but unaffected by arsenite, thus suggesting thatglucose is converted to ethylene via pyruvate. 3. Acetate is converted to ethylene after cleavage of C-l andC-2. Only a small portion of the latter (C-2) enters the moleculeof ethylene, the former (C-l) is detected in carbon dioxide.On the other hand, 2, and 3-carbons of pyruvate are converted,without splitting, to ethylene. 4. On removal of air, the incorporation of ¹⁴C into ethylenefrom acetate-2-¹⁴C was depressed, while that from pyruvate-¹⁴Cwas unaffected. 5. Acetaldehyde-l,2-¹⁴C is converted to ethylene without conversioninto ethanol. 6. These results are interpreted to suggest the occurrence ofthe pathway in which pyruvate and acetaldehyde may serve asprecursors of ethylene. ¹ A part of this paper was read at the regular Meeting of KansaiBranch of the Agricultural Chemical Society of Japan in Kyoto,October, 1964, and at the Annual Meeting of the Japanese Societyof Plant Physiologists in Tokyo, April, 1965 and presented ina preliminary form elsewhere (10).     

Involvement of cellular membrane in regulation of ethylene production

Effects of various substances which would change membrane structureson auxin-induced ethylene production in etiolated mung beanhypocotyl segments were investigated. Auxin-induced ethyleneproduction was not affected by treatment of tissue segmentswith pronase, trypsin, Con A and amphotericin B. PhospholipaseD inhibited ethylene production and its action was reversible,suggesting that the inhibitory action may not be due to enzymaticaction. Lecithin, Tween 20, Triton X 100 and SDS also significantlyinhibited ethylene production. Inhibition by the former twosubstances was completely, and that of the latter two was partiallyreversed by removing them from the tissue segments. All thesubstances which inhibited ethylene production also suppressedIAAsp formation by the tissue. It was concluded that inhibitionresulted from structural changes of cell membranes caused byreversible interaction with the lipophilic substances and thatethylene production and IAAsp formation were under the controlof membrane function. ¹ Supported in part by grants from the Ministry of Educationand the Ministry of Agriculture and Forestry, Japan. (Received December 24, 1976; )     

THE MECHANISM OF GIEBERELLIN ACTION IN THE DWARF PEA

The increase in diffusible auxin in the dwarf pea followingtreatment with gibherellin has been shown not to involve theinhibition of IAA oxidase, the rate of basipetal auxin transport,decrease in growth inhibitor, the formation of a complex betweengibberellin and auxin, or the enzymatic conversion of tryptophanto ether-soluble auxin. The tryptophan conversion system fromplants treated with gibberellin formed four times more water-solubleauxin than did the enzyme preparation from control plants. Thusthe increase in water-soluble auxin appears to be the majorcause of the higher auxin level in plants treated with gibberellin. ¹Present address: Biological Institute, College of General Education,University of Tokyo, Komaba, Meguro, Tokyo.     

COMPARATIVE STUDIES ON GROWTH, RESPIRATION, PHOTOSYNTHESIS AND PIGMENT CONTENT IN RHODOPSEUDOMONAS PALUSTRIS

1. Comparative studies were performed on growth, photosyntheticand respiratory activities, and pigment content in Rhodopseudomonaspalustris.
2. The growth of the organism, as influenced by variousculturalconditions such as light, aerobiosis, anaerobiosisand nutritionalfactors was investigated.
3. The respiratoryactivity of the bacterium was found to be higherin dark-growncells than in cells grown in the light. The photosyntheticactivitydid not significantly depend on the growth conditionsof theculture. Cells of younger cultures were found to be moreactivethan those of older cultures, with respect both to respirationand photosynthesis.
4. The pigment content was found to be higherin the light-growncells than in the dark-grown ones. The ratiophotosyntheticactivity/bacteriochlorophyll was significantlyhigher in thelatter than in the former.
5. Light, as well asvarious nutritional factors, was found toexert a marked accelerationon pigment formation, although ithas not yet been possibleto culture cells completely lackingin photosynthetic pigmentsand accordingly in photosyntheticactivity.

¹ Present address: Division of Dermatology and Urology, TokyoMetropolitan Hiroo Hospital, Tokyo. ² Present address: Department of Biology, Saitama University,Urawa. ³ Present address: Department of Biochemistry, School of Medicine,Yokohama University, Yokohama. ⁴ Present address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Tokyo. (Received July 23, 1961; )     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )