Biochemical studies on biogenesis of mitochondria in wounded sweet potato root tissue I. Time course analysis of increase in mitochondrial enzymes

Cytochrome oxidase and succinate dehydrogenase activities increasedduring aging of sliced tissue for sweet potato root after respectivelag phases of about 8 and 10 hr. The increase in the formerwas stepwise. On the other hand, malate dehydrogenase activityincreased slowly without a lag phase. Spectrophotometric determinationof heme contents in mitochondria indicated that the hemes increasedafter a lag phase for at least 8 hr during aging Treatment of tissue slices with cycloheximide at a concentrationof 10^–6 M prior to aging resulted in an extension of thelag phase in the increase of cytochrome oxidaseactivity andin complete inhibition of the increase of malate dehydrogenaseactivity. The antibiotic, at a concentration of 10^–5 M,completely suppressed the increases. Chloramphenicol (6 ? 10^–3M) also blocked the increases, except for that in malate dehydrogenaseactivity at an early stage of aging (Received December 22, 1970; )     

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; )     

Biochemical studies on biogenesis of mitochondria in wounded sweet potato root tissue II. Active synthesis of membrane-bound protein of mitochondria

Mitochondrial protein from sweet potato root tissue was fractionatedinto soluble and membrane-bound protein fractions. Membrane-boundprotein was further divided into structural, heme and bile salt-insolubleproteins. The increase in each protein component was investigatedduringaging of sliced tissue Proteins other than bile salt-insoluble protein were synthesizedduring an early stage of aging. A large amount of bile salt-insolubleprotein was synthesized after a lag phase lasting about 8 hrafter slicing Synthesis of soluble, structural and heme proteins was suppressedby bodi chloramphenicol and cycloheximide. However, synthesisof bile salt-insoluble protein was inhibited by cycloheximidebut not by chloramphenicol (Received December 22, 1970; )     

Modification of OGUR-ROSEN method for estimation of RNA in fresh and wounded sweet potato root tissues and change in RNA contents after the cutting

The suitability of the OGUR-ROSEN method for RNA extractionfrom sweet potato root tissue was investigated. The PCA extractfrom the freshly sliced tissue was colorless and showed a typicalspectrum of RNA. However, as the slices were incubated for 1to 3 days (wounded tissues), extract from them was brownishand revealed no typical spectrum of RNA. When wounded tissuewas treated with acetone containing isoascorbic acid and theresultant acetone powder was treated with PCA, extract indicatedthe spectrum very close to that of RNA. Interfering substancesstill remaining were removed satisfactorily with charcoal whichadsorbed RNA selectively. Thus, RNA could be assayed as theOD₂₆₀-difference between PCA extract from the acetone powderand a fraction of the extract non-adsorbable on charcoal. Timecourse analysis of RNA contents during incubation of the tissueslices disclosed a remarkable increase in the total RNA in theinitial stage of incubation. ¹This paper constitutes part 81 of Phytopathological Chemistryof Sweet Potato with Black Rot and Injury (Received September 5, 1969; )     

Biochemical studies on mitochondria formed during aging of sliced potato tuber tissue

Respiratory activities in mitochondria of potato tuber tissueincreased about 3-fold after aging sliced tissue for 1 day.Increased activity was insensitive to cyanide. The number ofmitochondrial particles also increased during aging. It seemslikely that newly formed mitochondria are heavier than pre-existingones and that respiration in the former is insensitive to cyanide. (Received January 26, 1970; )     

The Localization of Precursor Cells for Larval and Adult Hemopoietic Cells of Xenopus Laevis in two Regions of Embryos

For determination of the localization of lymphoid and erythroid precursor cells in embryos of Xenopus laevis , diploid-triploid chimeras were produced either by joining embryos antero-posteriorly or by orthotopic grafting of various tissues into N ieuwkoop -F aber st. 22–23 tailbud embryos. The sources of the hemopoietic cells were determined in the chimeric animals at various stages by microspectrophotometry of F eulgen -stained cells. Analyses of chimeras produced by joining embryos antero-posteriorly at different levels showed that the precursor cells that contribute to the hemopoietic cells are localized in the posterior half to three quarters. Orthotopic grafting of ventral or dorsal tissues revealed that the precursor cells that contribute to hemopoietic cells in early larvae are mostly localized in the ventral blood island (VBI) mesoderm, whereas those for late larvae and adults are localized both in the dorso-lateral plate (DLP) mesoderm comprising the prospective mesonephros and in the VBI mesoderm. Reciprocal heterotopic grafting of VBI- and DLP mesoderms showed that the two compartments differ in their capacities to differentiate into hemopoietic cells. It is proposed that the VBI-derived cells migrating towards the primary lymphoid organs constitute the transient hemopoietic population of early larvae, and the importance of the mesonephric region for definitive hemopoiesis is pointed out.     

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.     

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.