Mechanism of the Increase in Catalase Activity through Microbody Development in Wounded Sweet Potato Root Tissue

An increase in catalase activity accompanied by microbody developmentin wounded sweet potato root tissue was investigated with aspecific antibody against sweet potato catalase. The increasewas completely inhibited by cycloheximide. Analysis with singleradial immunodiffusion method showed that protein immunoprecipitatedby the antibody increased in wounded tissue, indicating theinvolvement of de novo synthesis of catalase protein in theactivity-increase. The activity-increase was, however, moreremarkable than the increase in immunoreactive protein and thisresults in an increase in specific catalase activity in woundedtissue, indicating the presence in intact tissue of an inertor less active protein, immunologically analogous to catalase.Actually, immunological analysis showed the presence in intacttissue of an immunoreactive protein which differed from activecatalase protein in the mobility on a polyacrylamide gel andprobably also in the molecular weight of subunit. The immunoreactiveprotein seemed to exist in a significant amount outside themicrobodies in intact tissue cells. Thus, there is a possibilitythat the increase in catalase activity in wounded tissue ispartly due to activation of the immunoreactive protein. (Received October 16, 1982; Accepted February 24, 1983)     

Effect of Ethylene on the Increase in Catalase Activity through Microbody Development in Wounded Sweet Potato Root Tissue

Catalase activity increases when slices of sweet potato roottissue are incubated in air. The increase is due to de novosynthesis of the enzyme protein and probably also to activationof a precursor protein [Esaka et al. (1983) Plant & CellPhysiol. 24: 615]. The activity-increase was partly depressedwhen tissue slices were incubated in ethylenecontaining air,while the immunologically determined amount of catalase proteindid not increase, rather it decreased, under the same conditions.We propose that ethylene inhibits the de novo synthesis of catalaseprotein but not the activation of precursor protein. Catalasefrom tissue slices incubated in ethylene-containing air migratedfaster on a polyacrylamide gel than that from intact tissueor tissue slices incubated in air. When either polyacrylamideor an SDS-polyacrylamide gel applied with crude extract fromtissue slices incubated in ethylene-containing air underwentimmunological blotting, the blots were much fainter than thosefor intact tissue. In addition, microbody membrane fractionfrom incubated tissue slices contained a significant amountof catalase which was sedimented at the bottom of a sucrosedensity gradient (20–70%) and was not solubilized by highconcentrations of lubrol PX. The fraction showed an exceptionallyhigh catalase activity per unit amount of immunoreactive proteinto anti-catalase antibody. We propose that ethylene causes somemodification of catalase protein which facilitates the formationof aggregates or cores. ¹Present address: Laboratory of Food Technology, Faculty ofApplied Biological Science, Hiroshima University, Fukuyama,Hiroshima 720, Japan. ²Present address: Terumo Co. Ltd., Omiya, Fujinomiya, Shizuoka418, Japan. (Received October 16, 1982; Accepted February 24, 1983)     

Mechanism of the Increase in Succinate Dehydrogenase Activity in Wounded Sweet Potato Root Tissue

The large subunit (mol wt: 65,000) of sweet potato succinatedehydrogenase was isolated by SDS-polyacrylamide gel electrophoresisof a succinate dehydrogenase preparation, which had been partiallypurified from root mitochondria by solubilizing the enzyme withEmulgen 810, DEAE-cellulose column chromatography, and polyacrylamidegel electrophoresis. Antibody to the purified large subunitwas produced in a rabbit, and the antiserum obtained was judgedto be specific to the large subunit based on the results ofdouble immunodiffusion tests and immunoelectrophoresis. Rocketimmunoelectrophoresis with the antiserum showed that the increasein succinate dehydrogenase activity during the ageing of sliced,sweet potato root tissue was due to an increase in the amountof enzyme protein. Both the increases in the activity of succinatedehydrogenase and in the amount of the large subunit proteinwere inhibited by cycloheximide or chloramphenicol. We proposethat synthesis of the large subunit of succinate dehydrogenaseon cytoplasmic ribosomes is controlled by a mitochondrial translationproduct(s). ¹ This work was supported in part by a research fund from TheIshida Foundation, Nagoya, Japan. (Received November 28, 1981; Accepted February 17, 1982)     

Stimulation by Ethylene of Mitochondrial Development in Wounded Sweet Potato Root Tissue

Ethylene (about 100 µl per liter) markedly stimulatedincreases in respiratory, Cyt c oxidase and succinate dehydrogenaseactivities of the crude mitochondrial fraction as well as mitochondrialmembrane protein during aging of sliced sweet potato root tissue,indicating that it stimulated mitochondrial development in woundedtissue. It had such an effect even when slices were pre-agedin its absence for 1 day and thereafter aged in its presence.The mitochondrial inner membrane from slices aged in ethylene-containingair was denser than that from fresh slices, while the membranefrom slices aged in ethylene-free air was lighter. Chloramphenicolcompletely inhibited the increase in Cyt c oxidase activitywhether slices were aged in the presence or absence of ethylene.Cycloheximide did not inhibit the increase in slices aged inethylene-free air, but did by 50% in those aged in ethylene-containingair. ¹ This work was supported in part by a Grant-in-Aid (No. 411308)for Scientific Research from the Ministry of Education, Scienceand Culture, Japan. (Received April 4, 1981; Accepted July 7, 1981)     

Studies on Microbody Development in Wounded Sweet Potato Root Tissue: Proposal for the Post-Translational Transport of Catalase into Preexisting Microbodies

Pure microbody fractions could be prepared in considerable yieldsfrom sweet potato root tissue slices incubated for 16 hr and3 days. The ratio of catalase activity to phospholipid contentin the fraction from slices incubated for 3 days was about 3times that from slices incubated for 16 hr. Total catalase activityin the former slices was about twice that in the latter. Thissuggests that catalase synthesized during incubation of theslices is transported into microbodies preexisting in intacttissue. ¹ Present address: Laboratory of Food Technology, Faculty ofApplied Biological Science, Hiroshima University, Fukuyama,Hiroshima 720, Japan. ² Present address: Terumo Co., Ltd., Omiya, Fujinomiya, Shizuoka418, Japan. (Received July 1, 1982; Accepted September 24, 1982)     

Purification and Properties of Catalase from Sweet Potato Root Microbodies

Catalase was isolated in a pure form from sweet potato rootmicrobodies by simple procedures including ammonium sulfatefractionation and Sepharose 6B column chromatography. A singleprotein band was detected after polyacrylamide gel electrophoresisof the purified preparation. The catalase consisted of polypeptideswith a molecular weight of 60,000 when analyzed by sodium dodecylsulfate-polyacrylamidegel electrophoresis, while the molecular weight of the enzymewas about 240,000 when estimated from sucrose density gradientcentrifugation. The enzyme's ratio of absorbance at 280 nm tothat at 405 nm was about twice that of mammalian catalase. Thecatalase showed a maximal activity at pH 6.5–8.5 but wasstable only at alkaline pHs. In double immunodiffusion tests,antiserum against the purified preparation formed a single precipitinline with the crude soluble fraction from sweet potato roottissue as well as with the purified preparation. The antiserumhad no ability to inhibit the activity, but catalase in boththe crude fraction and the purified preparation was completelyprecipitated by the antiserum. (Received August 20, 1981; Accepted January 5, 1982)     

Furano-sesquiterpene Reductase from Fungal-inoculated Sweet Potato Root Tissue

Cigarette flavor ingredients adsorbed on activated carbon were recovered by an organic solvent extraction method and a stem desorption method. Recovery by dichloromethane extraction showed the maximum effect and many volatile components, especially the thermal degradation products from sugar analogues, were recovered in a good yield. Their recovered yield and desorption rate depended on the physical properties of the carbons. Most of the volatile components were also recovered by the steam desorption method, but the recovery of components with high boiling points was very low by steam desorption. The difference in recovered yield from the carbons is explained by the specific surface area of the carbons measured before and after the desorption procedure.     

Increase of Mitochondrial Fraction in Sweet Potato Root Tissue after Wounding or Infection with Ceratocystis fimbriata

The acid-insoluble nitrogen content, lipid content, and cytochrome oxidase activity in the mitochondrial fraction are found to increase during incubation of slices of sweet potato (Ipomoea batatas) root tissue. These increases appear to be related to an increase in the number of the mitochondrial particles. The increase in the mitochondrial fraction is not accompanied by an increase in cell number. The nitrogen content in the mitochondrial fraction increases prior to the changes in the activity of cytochrome oxidase and lipid content. The increase in the numbers of the mitochondrial particles lags behind the increase in the cytochrome oxidase activity. Such findings are also found in the tissue infected by Ceratocystis fimbriata.     

Changes in Acid-soluble Nucleotides in Cut-injured Sweet Potato Root Tissue

ATP and ADP increased in cut-injured sweet potato root tissue during the 3 to 6-hr incubation period, and showed the maximum for the 9 to 18-hr, and 6 to 9-hr incubation periods, respectively, then decreased. ATP was present in the highest amount among ATP, ADP and AMP throughout the 72-hr incubation period, while AMP was in the lowest. Total acid-soluble nucleotides increased gradually, and showed the peak content at the 12-hr incubation period, and decreased thereafter. Adenine mononucleotides such as ATP, ADP and AMP occupied about 40 to 65% of total acid-soluble nucleotides.     

甘薯块根生长及其淀粉体发育过程的解剖结构特征

为了探明甘薯块根的生长及其淀粉体的发育规律,该试验以甘薯品种‘徐薯22’为材料,采用树脂半薄切片等方法对甘薯块根的生长及其淀粉体的发育进行观察研究。结果表明:(1)甘薯块根完成初生生长的时间短,块根初生结构由表皮、皮层和中柱构成,块根横截面上皮层所占比例比中柱大。(2)甘薯移栽后10 d块根开始次生生长,次生生长形成维管形成层和木栓形成层;随着块根次生生长,位于次生木质部分散导管周围的薄壁细胞脱分化,通过平周分裂产生副形成层;维管形成层、木栓形成层和副形成层的共同作用使块根快速膨大。(3)淀粉体在块根进入次生生长时首先在皮层细胞产生,随后大量出现在次生生长产生的薄壁细胞中,块根中淀粉体的发生及发育总体上表现出由外向内的顺序。(4)块根薄壁细胞中的淀粉粒有单粒和复粒两种类型;块根生长早期,薄壁细胞中主要以复粒淀粉为主,生长后期主要以单粒淀粉为主;块根生长过程中,包含复粒淀粉的淀粉体可通过分裂形成包含单粒淀粉的淀粉体。(5)淀粉可在块根生长的整个时期积累,其中以块根生长中期积累速度最快。     

Ethylene Production in Sweet Potato Root Tissue Infected by Ceratocystis fimbriata

The rate of ethylene production by sweet potato (Ipomoea batatasLam. cv. Norin No. 1) root tissue infected with Ceratocystisfimbriata Ell. & Halst. increased markedly during incubationat 29?C under high relative humidity. During incubation thefungus progressively invaded root tissue. The rate of ethyleneproduction reached a peak two days after inoculation when thebrowning region that contained the penetrating mycelia had expandedinward about 0.3 mm from the surface, followed by a declinein ethylene production. Apparently, the 1-aminocyclopropane-1-carboxylicacid (ACC) synthase activity was not high enough, and the amountof ACC in the infected tissue was too low to account for thehigh rate of ethylene production throughout the incubation period.Ethylene production by the infected tissue showed scarcely anyinhibition by amino-ethoxyvinylglycine, a specific inhibitorof ACC synthase. These findings suggest that the pathway ofethylene biosynthesis that operates in infected sweet potatoroot tissue may differ from the methionine pathway in whichACC serves as an intermediate. (Received March 24, 1984; Accepted June 27, 1984)     

Induction of Polysome Formation in Sweet Potato Root Tissue in Response to Wounding

We determined the effects of yolk water-soluble protein (YSP) on bone resorption. YSP potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven by tumor necrosis factor-α (TNF-α). YSP (200 μg/ml) abolished the formation of tartarate-resistant acid phosphatase (TRAP)-positive osteoclasts. Furthermore, TNF-α induced TRAP activity was greatly inhibited by YSP (100 μg/ml) treatment. Our results suggest that YSP has therapeutic potential for bone-erosive diseases.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Structure of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Amino acid sequence and peptide-carbohydrate linkage of GP–I–a and GP–I–b, which are two glycopeptides out of three obtained from the saccharogenic amylase of Rhizopus javanicus sp. 3–46, were investigated. By the dinitrophenylation and the subtractive Edman degradation the sequence of GP–I–a and GP–I–b was determined to be respectively.

A glycosylamine type linkage between asparagine and N-acetylglucosamine residue was proved for GP–I–a by the use of the enzyme, β-aspartylglycosylamine amide hydrolase. It seems likely that the peptide-carbohydrate linkage of GP–I–b is also a glycosylamine type linkage.     

Chemiluminescence in Wounded Root Tissue : EVIDENCE FOR PEROXIDASE INVOLVEMENT

Root and stem segments from soybean (Merrill cv. `Bragg') showed an enhanced chemiluminescence upon mechanical injury. Roots emitted more light than did stems. Light emission was diminished by CN⁻ and N₃⁻ but was not affected by rotenone and antimycin A. Catalase quenched chemiluminescence in wounded root segments as did ascorbic acid and hydroquinone. Superoxide dismutase addition resulted in a small diminution in light emission, but mannitol, an OH· scavenger, was without effect. The addition of H₂O₂ to wounded root segments markedly elevated chemiluminescence in the presence of air as well as under N₂. It is concluded that peroxidases, found abundantly in roots, predominantly contribute to light emission in wounded plant tissue.     

Morphological Changes in Chilling Injured Sweet Potato Root

Hydrophobicity and amounts of polar groups on the cell surface were determined by the use of fluorescent probe, colloid titration, and acid titration. Surface hydrophobicity of hydrocarbon-grown cells was about 7 times greater than that of glucose-grown cells of the same strain, while amounts of polar groups did not differ so much. Adsorption of cells to air bubbles was maximum at pH 3. Langmuir’s adsorption isotherm held for the adsorption. Affinity to air bubbles of hydrocarbon-grown cells was 1.8 times greater than that of glucose-grown cells, while the theoretical maximum amounts of cells to adsorb per unit area of bubbles were equal for the both cells.     

Suppression by Exogenous Phospholipid of Cyanide-Insensitive Respiration of Submitochondrial Particles from Sweet Potato Root Tissue

Submitochondrial particles from sweet potato root tissue retainedthe respiratory characteristics of the intact mitochondria withrespect to the sensitivity to cyanide and salicylhydroxamicacid. The activities of total, cyanide-insensitive, and salicylhydroxamate-sensitiverespiration of the submitochondrial particles yielded from adefinite weight of tissue slices incubated under aerobic conditions,particularly in ethylenecontaining air, were higher than thosefrom the same weight of intact tissue. The less phospholipidthe submitochondrial particles contained relative to protein,the higher the activities of cyanide-insensitive and salicylhydroxamate-sensitiverespiration tended to be relative to total respiratory activity.When the submitochondrial particles were incubated with phospholipidliposomes, the activities of cyanide-insensitive and salicylhydroxamate-sensitive,but not cyanide-sensitive, respiration became extremely low.All phospholipids showed this effect. Such incubation of thesubmitochondrial particles with phospholipid liposomes yieldedlighter particles, indicating close association of exogenouslyadded phospholipid with the particles. Phospholipid moleculesseemed to enter the membrane of the particles. We propose thatphospholipid deficiency in the mitochondrial inner membranefacilitates operation of the cyanide-insensitive electron transportpath. (Received March 30, 1984; Accepted June 15, 1984)     

Sucrose-Induced Expression of Genes Coding for the Tuberous Root Storage Protein, Sporamin, of Sweet Potato in Leaves and Petioles

Sporamin, a major tuberous root protein of sweet potato, wasfound to accumulate in large quantities in excised leaves andpetioles when such explants were supplied with high concentrationsof sucrose. Although a small amount of sporamin could be detectedin leaves and petioles treated with 1% or lower concentrationsof sucrose, the maximum level of induction required sucroseat a concentration of 3% or higher. The appearance of sporaminpolypeptides in leaves and petioles treated with 3% sucrosefollowed a lag period of about one day, while a significantamount of sporamin mRNAs was already detectable in petiolesafter one day of treatment with sucrose. Addition of silvernitrate to the medium did not affect the accumulation of sporamin,suggesting that this induction is not due to the effect of ethyleneinduced by wounding of the tissue. The accumulation of sporamincould also be induced by glucose and by fructose, but not byman-nitol, suggesting that changes in carbohydrate and/or energymetabolism in the cell may be involved in the induction. Callustissues obtained by treatment of leaf segments with 1-naphthaleneaceticacid did not accumulate sporamin even though these cells werecultured on agar medium that contained 3% sucrose. However,when callus tissues were allowed to grow after transfer to amedium that contained 6-benzylaminopurine and sucrose, accumulationof large amounts of sporamin was induced. These results suggestthat, while expression of genes coding for sporamin can be inducedin organs other than the tuberous root by a process that doesnot accompany the differentiation of tissue, the induction ofexpression of sporamin genes by sucrose requires that cellsbe competent in some specific, but as yet unidentified, way. (Received August 27, 1990; Accepted November 5, 1990)