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; )     

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; )     

Heterogeneity of mitochondrial particles in fresh and wounded tissues of sweet potato roots

A mitochondrial fraction prepared from fresh tissue of sweetpotato root was subjected to sucrose density gradient centrifugation.The distribution of cytochrome oxidase activity, after the centrifugation,showed the presence of at least three kinds of mitochondrialparticles which differed in their sedimentation velocity. Byrepeating the sucrose density gradient centrifugation, it wasdemonstrated that they are not interconvertible. There seemedto be no difference in the distribution between cytochrome andsuccinate oxidase activities. In the case of malate or succinatedehydrogenase activity, however, the greater the sedimentationvelocity of the particle, the greater was the dehydrogenaseactivity per unit of cytochrome oxidase activity. Some changesin the distribution of cytochrome oxidase activity in responseto aging of the tissue slices were observed. ¹This paper constitutes Part 62 of the Phytopathological Chemistryof Sweet Potato with Black Rot.     

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; )     

Physiological behavior of peroxidase isozymes in sweet potato root tissue injured by cutting or with black rot

We found that the increase in total activity of peroxidase isozymesin diseased tissue of sweet potato roots was mainly caused bythe ethylene released from the tissue in response to infection. Component H, a peroxidase isozyme which moved toward the cathode,was formed close to the cut surface in cut-injured tissue, butnot in diseased tissue. We assumed that its absence in the latterwas due to physical and chemical damage caused by fungal invasion,of the component H formed and of the biosynthetic system. Ethylene seemed to be partly involved in inducing componentH formation in cut-injured tissue. Also, ethylene treatmentrather effectively maintained the increased activity, whichotherwise would have decreased at a later stage. Induction of component H formation was accompanied by the formationof a lignin-like substance on the cut surface. We suggest thatcomponent H contributes to the formation of this substance. ¹ This paper constitutes Part 103 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ² Present address: University of Tokyo Press, 7-3-1 Hongo, Bunkyo-ku,Tokyo 113, Japan. (Received July 11, 1972; )     

Comparison of Photosynthetic and Photorespiratory Enzyme Activities between Green Leaves and Colorless Parts of Variegated Leaves of a C4 Plant, Stenotaphrum secundatum (Walt.) Kuntze

The activities of enzymes involved in C⁴ photosynthesis andphotorespiration in colorless parts of variegated leaves ofStenotaphrum secundatum (Walt.) Kuntze were compared with thosein green leaves. Chlorophyll content of the colorless part wasonly about 0.3–3% of that of the green leaves. The activities of chloroplastic enzymes, pyruvate, P_i dikinase,NADP⁺-malic enzyme and NADP⁺-glyceraldehyde 3-phosphate dehydrogenasewere considerably lower in colorless tissue on a fresh weightor protein basis (the ratios of the activities in the green/colorlesstissues ranging from 5 to 20). A cytoplasmic enzyme, UDP-glucosepyrophosphorylase as well as aspartate and alanine aminotransferasesshowed comparable activities in the two types of tissue, whereasPEP carboxylase in the colorless tissue had only the one-thirdactivity of that in green tissue. Differences in activitieswere also observed for the glycolate pathway enzymes (the ratiosranging from 2 to 7 for glycolate oxidase, hydroxypyruvate reductaseand serine hydroxymethyltransferase, and 7 to 15 for catalase),while cytochrome c oxidase showed comparable activity in thetwo types of tissue. The results suggest that the deficiency of thylakoid developmentin the colorless tissue influences enzyme activities not onlyin plastids but also in other cellular compartments. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 26, 1986; Accepted June 17, 1986)     

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.     

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; )     

Flowering haploid plants obtained from protoplasts of tobacco leaves

Protoplasts were isolated enzymatically from haploid tobaccoleaves, then cultured in synthetic media. Cultured protoplastsstarted their first division within a few days after inoculationand subsequently developed into small colonies. After severalreinoculations, these colonies differentiated shoots and roots.Finally, haploid flowering plants were obtained. ¹ Present address: Sericultural Experiment Station, Suginami-ku,Tokyo, Japan. ² Passed away on July 29, 1971. (Received October 9, 1971; )     

Behaviors on gel-filtration and electrophoresis of 6-phosphogluconate dehydrogenase isozymes isolated from sweet potato

Sephadex G-200 gel-filtration of an enzymatic preparation fromsweet potato root tissue showed three activity peaks (I, IIand III) of 6-phosphogluconate dehydrogenase. Re-chromatographyof either Peak II or III produced two peaks corresponding toPeak II and III. Disc electrophoreses of the original preparation,Peak II, Peak III and the Peak II-like peak after re-chromatographygave idetical zymograms of the three isozymes which were notinterconvertible. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received May 30, 1972; )     

Kennzeichnung der Bildung und Alterung von Wurzeln in Callus-und Organ-Kulturen von Daucus carota durch die Aktivität der Glutamatdehydrogenase

Summary In callus tissues of Daucus carota rhizogenesis was influenced by addition to the cultures of solutions with different kinetin concentrations (0–0.1–0.5–2 mg/l) and a constant auxin content (1 mg/l). In these cultures the specific activity of glutamate-dehydrogenase (E.C. 1.4.1.2.), aspartate-aminotransferase (E.C. 2.6.1.1.), isocitrate-dehydrogenase (E.C. 1.1.1.42) and of acid phosphatase (E.C. 3.1.3.2.) was determined. Before root initiation can be seen morphologically in tissues grown on low kinetin concentrations, the specific activity of glutamate-dehydrogenase increases to more than three times the level found in cultures with no subsequent root initiation, whereas the specific activity of the other three enzymes changes far less. Total soluble protein measured as percentage of fresh weight remains nearly the same in all callus cultures. The activity of the same enzymes was measured in liquid grown roots of Daucus carota during a period of ageing of 130 days. During this time, the specific activity of glutamate-dehydrogenase is reduced to 1/10 of the value found in growing roots, whereas the activity of the other three enzymes is reduced only to 1/2 or to 1/3 of this value. Therefore, the state of senescence and the capacity for further growth can be characterized by the specific activity of the glutamate-dehydrogenase in the roots. The changes in the specific activity of glutamate-dehydrogenase in callus tissue and in the roots do not depend on inhibitory substances in the cell-free extracts.     

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.     

The nature of the dual effect of auxin on root formation in Azukia cuttings

In disbudded Azukia stem cuttings, auxin exerted a dual effecton root formation. The first phase of auxin action is identifiedwith the acceleration of cell division, especially longitudinaldivision. In cuttings treated with auxin during the first 24hr, longitudinally divided cells were observed in all 12 rootprimordia, while in water-treated cuttings, such cells wereobserved only in 8 root primordia. The second phase is the promotionof the reaction in which root primordia unable to develop furtherwithout auxin supply develop into roots. Irrespective of thetreatment during the first 24 hr, the auxin-treatment duringthe second 24 hr increased the number of roots protruding fromthe cuttings. Portulal applied during the first 24 hr increased the numberof root primordia which contained longitudinally divided cells.Gibberellin applied during the first 24 hr inhibited both transverseand longitudinal divisions in root primordia. ¹ Supported in part by Grant No. 139011 from the Ministry ofEducation, Japan. ² Present address: Junior College of Toyo University, Hakusan,Bunkyo-ku, Tokyo 112, Japan. (Received June 13, 1978; )     

Effect of antibiotics on biogenesis of mitochondria during aging of sliced sweet potato root tissue

The number of mitochondrial particles, and the activities ofperoxidase and cytochrome oxidase increased during aging ofsliced sweet potato root tissue. Blasticidin S inhibited allthese processes, indicating the occurrence of de now synthesisof protein. Chloramphenicol suppressed the synthesis of cytochromeoxidase and the reproduction of mitochondria but did not suppressperoxidase synthesis. Cylcoheximide suppressed peroxidase synthesisleaving the activities of cytochrome oxidase synthesis and mitochondrialreproduction unimpaired. A differentiation of ribosomal andmitochondrial protein syntheses is suggested. Mitomycin G and5-iododeoxyuridine inhibited the reproduction of mitochondriabut not the synthesis of enzymes. ¹This paper constitutes Part 76 of the Phytopathological Chemistryof Sweet Potato with Black Rot. (Received January 4, 1969; )     

Inhibition of sweet potato glucose 6-phosphate dehydrogenase by NADPH2 and ATP

NADPH₂ and ATP competitively inhibit sweet potato glucose 6-phosphatedehydrogenase with NADP and glucose 6-phosphate (G6P), respectively.At pH 8.0, a Lineweaver-Burk plot of the reciprocal rate againstreciprocal G6P concentration was concave downwards in the presenceand absence of ATP, whereas a double reciprocal plot followedthe Michaelis-Menten relationship at pH 7.0, irrespective ofthe presence of ATP. Many of the other metabolic intermediatestested had no effects on the enzyme reaction. ¹ This paper constitutes Part 96 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. ² Present address: Institute of Applied Microbiology, Universityof Tokyo Bunkyo-ku, Tokyo 113. (Received October 20, 1971; )     

CHANGE OF B-TYPE HAEM CONTENT IN RELATION TO PEROXIDASE BIOSYNTHESIS IN INJURED SWEET POTATO ROOTS

The methods of quantitative analysis of b-type haem in plantswere investigated. With an improved method developed was determinedthe haem content in the supernatant, mitochondrial, and microsomalfractions of sweet potato tissue. The activities of peroxidase,catalase, and cytochrome oxidase, as well as the contents ofb-type haem and acid-insoluble nitrogen in the cellular fractionswere determined at different incubation times after cuttingof sweet potato tissue. Peroxidase and catalase increased withtime in each celluler fraction, following a short lag phase.In the mitochondrial fraction, b-type haem, cytochrome oxidase,and acid insoluble nitrogen increased linearly with time. Inthe microsomal and supernatant fraction, b-type haem increasedwith time following a short lag phase. The increase in haemcontent of the supernatant fraction appeared to be associatedwith peroxidase formation. Time course analysis showed that ⁵⁹Fe-incorporation into b-typehaem of the supernatant fraction increased with time and thatincorporation was markedly inhibited by blasticidin S. The incorporationof ⁵⁹Fe into mitochondrial haem did not increase with time andwas not inhibited by blasticidin S. Blasticidin S inhibited⁵⁹Fe-incorporation into microsomal haem. Time course analysisof b-type haem content, ⁵⁹Fe-incorporation into b-type haem,and peroxidase activity suggest that in the injured tissue haemis synthesized from low molecular weight compounds and is incorporatedinto peroxidase as the haem moiety. ¹ This paper constitutes Part 57 of the Phytopathological Chemistryof Sweet Potato with Black Rot. ² Present address: Institute for Plant Virus Research, Chiba.     

Effect of the age of tobacco leaves on photosynthesis and photorespiration

Relationships among the activities of enzymes related to photosynthesisand photorespiration, and ¹⁴CO₂ photosynthetic products wereinvestigated with individual tobacco leaves attached to thestalk from the bottom to the top. P-glycolate phosphatase ofthe chloroplasts and glycolate oxidase of the peroxisomes hadtheir maximum activities in the 25th leaf from the dicotyledons.Maximum photorespiration was similarly distributed. The highestratio of serine-¹⁴C to glycine-¹⁴C in the photosynthesates andmaximum glycolate formation were also observed in the 25th leaf.Glutamateglyoxylate aminotransferase, serine hydroxymethyltransferaseand glycine decarboxylase were more active in the upper leaves.RuDP carboxylase had nearly constant activity in all leaves,except for the youngest in which activity decreased. MaximumCO₂ photosynthesis and enzyme activity for the C₄ dicarboxylicacid cycle occurred in the upper, youngest leaf. Distributionof photosynthetic CO² fixation among the leaves did not coincidewith RuDP carboxylase activity. The photosynthetic capacityappeared to be better related to the distribution pattern forenzymes of the C₄ dicarboxylic acid pathway, i.e. PEP carboxylase,pyruvate Pi dikinase and 3-PGA phosphatase in the upper leaves.The results suggest that the C₄ dicarboxylic acid pathway participates,to some extent, in photosynthesis in young leaves of tobacco,a dicotyledonous plant. ¹This work was reported at the Annual Meeting (1970) of theJapanese Plant Physiologists in Kobe. ²The Central Research Institute, Japan Monopoly Corporation1-28-3, Nishishinagawa, Shinagawaku, Tokyo, 141 Japan. (Received November 2, 1972; )     

Effects of cycloheximide, actinomycin D and ethylene on the increase and subsequent decrease in acid invertase activity in wounded sweet potato

In wounded sweet potato root tissue, the mechanisms of the increaseand subsequent decrease of acid invertase activity were examinedin compariosn with phenylalanine ammonia lyase (PAL) and peroxidaseactivities. The increase in acid invertase and PAL activitiesin response to wounding was inhibited by both cycloheximideand actinomycin D. However, the increase in peroxidase activitywas inhibited by cycloheximide but was not inhibited by actinomycinD, which repressed RNA synthesis 60 to 70%. The increase inacid invertase activity was supressed by ethylene, while PALand peroxidase activities were stimulated. The results of cycloheximideco-treatment suggest that the fluctuation in enzyme activitiesby ethylene treatment involves de nove protein synthesis. Further,the effect of ethylene on enzyme activities was under competitionby carbon dioxide. The decrease in acid invertase activity atthe late stage of incubation was stimulated by cycloheximidetreatment, but that of PAL activty was prevented. The resultssuggest the existence of different inactivating systems betweenacid invertase and PAL. ¹ This paper constitutes Part 117 in the Series "The PhytopathologicalChemistry of Sweet Potato with Black Rot and Injury". (Received September 2, 1974; )     

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; )