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.     

Polar transport and content of indole-3-acetic acid in wounded sweet potato root tissues

In roots of sweet potato (Ipomoea batatas Lam. cv. Kokei 14),the metabolic response to wounding was remarkable in the proximalside and developed in the acropetal direction. We assumed thatthe polarity resulted from the increase in polar movement ofindoleacetic acid (IAA) (1977, Plant Physiol. 60: 563–566).Transport of IAA and change of the IAA level in the woundedtissue of sweet potato roots were investigated. Transport ofthe label from ¹⁴C-IAA was obviously polarized in the acropetaldirection. ¹⁴C-IAA administered to the wounded tissue was mainlymetabolized into two conjugates of IAA. The amount of IAA inthe wounded tissue, determined by the spectrofluorometric method,increased about 3-fold after 18 hr of incubation prior to thedevelopment of activities of some enzymes. The increase in IAAcontent was not affected with aseptic incubation, therefore,the possibility of IAA production by microorganisms on the woundedtissue was excluded. The results obtained strongly support ourhypothesis that IAA plays an important role in the metabolicresponse to wounding. (Received May 2, 1979; )     

Synthesis of RNA in tissue discs of sweet potato roots after cutting or mercuric chloride treatment

Time course analysis of RNA contents of tissue discs after cuttingdisclosed a remarkable increase in total RNA during the first12 hr after cutting and this elevated level remained unchangedfor 48 hr. The elevated RNA level at 24 hr of incubation wasnot changed by subsequent HgCl₂ treatment. The incorporationrate of the label from ³H-uridine into RNA rapidly increasedimmediately after cutting and reached a maximum at about 9 hrof incubation, then decreased sharply until 24 hr and continuedto decrease gradually thereafter. The incorporation rate at24 hr of incubation was not changed by subsequent HgCl₂ treatment.The results of polyacrylamide gel electrophoresis indicatedthat bulk RNA was synthesized most actively at 9 hr of incubationthen the rate of RNA synthesis decreased gradually. (Received August 26, 1977; )     

Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.     

Synthesis and apparent turnover of Acid invertase in relation to invertase inhibitor in wounded sweet potato root tissue

Previously we showed that acid invertase activity increased and then decreased rapidly in wounded sweet potato (Ipomoea batatas Liam.) root tissue, and that the tissue contained a heat-stable, proteinaceous inhibitor with a molecular weight of about 19,500 daltons.     

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; )     

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; )     

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; )     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

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; )     

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; )     

Enhancement of polyribosome formation and RNA synthesis of gibberellic Acid in wounded potato tuber tissue

As part of a more detailed study on plant tumorigenesis, the action of gibberellic acid (GA₃) in wounded potato tuber tissues as a model system has been evaluated. GA₃ stimulates total RNA synthesis in wounded tissues, the optimal concentration being 0.1 micromolar. The responsiveness of the tissue toward the hormone develops with time after wounding. Whereas freshly wounded tissue does not respond at all to the hormone, it becomes competent after about 6 hours, the competence being maximal after 1 day of wound healing.     

A new method for the preparation of beta-amylase from sweet potato.

A simple method for the preparation of sweet potato beta-amylase by thymol amylose adsorption is described. The method is far more efficient and gives higher recovery of the enzyme. The crystalline enzyme thus obtained is found to be homogeneous by gel chromatography, polyacrylamide gel electrophoresis.     

Regulated expression of ADPglucose pyrophosphorylase and chalcone synthase during root development in sweet potato

The phase transition in sweet potato root during tuber differentiationis a complex developmental process that involves changes in gene expression andmorphogenesis. Among the three kinds of root in sweet potato (white fibrousroot, thick-pigmented root and lateral root), ADP-glucose pyrophosphorylase(AGPase) and chalcone synthases (CHS) are expressed only in thick-pigmentedroots after 3 weeks, and this also depends on the developmental stage. Sinceexposing roots to the light or culturing under hydroponic conditions inhibitstuber formation in sweet potato, the expression of AGPase and CHS was studiedunder light and dark conditions. AGPase and CHS expression in sweet potatorootswas suppressed very sensitively by light or water stress, similar to rootdevelopmental patterns. Based on an analysis of AGPase and CHS expression indifferent kinds of root tissues and in different developmental stages, thesegenes were shown to be closely associated with the differentiation ofthickeningpigmented roots.     

Some investigations on inactivation of phenylalanine ammonia-lyase in cut-injured sweet potato root tissue

In sweet potato roots, activity of the phenylalanine ammonia-lyase(PAL)-inactivating system in crude enzyme solution increasedmarkedly in response to cut injury after a lag period of about10 hr and reached a maximum after 24 hr of incubation. The resultscoincided with previous results from experiments using a proteinsynthetic inhibitor. The inactivating system could be precipitatedby centrifugation and was distributed in a different patternfrom mitochondrial and microsomal marker enzymes, accordingto data from cellular fractionation by differential and sucrosedensity gradient centrifugation. The optimum pH of the inactivationwas 6.0. Previous studies showed that PAL content changed inparallel with PAL activity in vivo. However, immunochemicalstudies indicated that the inactivation was not due to proteolysis.Furthermore, proteinase activity in sweet potato tissue didnot change in response to cut injury. These results suggestedthat PAL was first inactivated by the inactivating system, thenthe inactivated PAL was rapidly decomposed by the proteinase. ¹ This paper constitutes Part 130 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. This work was supportedin part by a grant from the Ministry of Education. ² Present address: Faculty of Agriculture, Yamaguchi University,Yamaguchi 753, Japan. (Received May 14, 1977; )     

Establishment and characteristics of an anthocyanin-producing cell line from sweet potato storage root

 Anthocyanin pigments accumulated in a cell line derived from storage-root explants of sweet potato (Ipomoea batatas L.) cv 'Ayamurasaki'. Somatic pro-embryos were induced on the explants cultured on Murashige and Skoog medium supplemented with 1 mg/l 2,4-D. The pro-embryo structures produced callus when transferred to MS medium with 0.5 mg/l 2,4-D. A cell line was isolated from this callus which accumulated anthocyanin pigment. The color value of the pigment extracted after 27 days of culture in MS medium with 2 mg/l 2,4-D was 8.2, which was very close to that of a pigment extracted from roots, which was 8.9. Most of the pigments from the cell extract were hydrophilic and appeared on the ODS-column HPLC with a lower retention time than the main anthocyanins of the root tissues. The majority of the pigments were identical with the root anthocyanins. Cell line-specific anthocyanins were detected. Received: 8 January 1999 / Revision received: 2 March 1999 / Accepted: 30 June 1999     

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; )