Mechanism of increase in cytochrome c oxidase activity in sweet potato root tissue during aging of slices

The mechanism of an increase in cytochrome c oxidase [EC 1.9.3.1] activity during aging of sliced sweet potato root tissue was investigated with antibiotics and antibody to the purified enzyme. 1. The increase in cytochrome c oxidase activity was inhibited by chloramphenicol but not by cycloheximide. 2. Cytochrome c oxidase purified from wounded tissue was identical with that from intact tissue as judged by the subunit composition, sedimentation velocity, absorption spectrum, antigenicity, and activity per heme a. 3. An increase in the amount of cytochrome c oxidase protein took place during aging of slices. 4. Sweet potato cytochrome c oxidase consists of five subunits. When slices were aged in the presence of [3H]leucine, the three larger subunits (I, II, and III) of cytochrome c oxidase were labeled, while no radioactivity was incorporated into the other two subunits, IV and V. The results indicate that the increase in cytochrome c oxidase activity is due to an increase in the amount of the enzyme protein. We propose that excess amounts of subunits derived from the cytoplasm of the enzyme are present in intact tissue and are assembled with subunits of mitochondrial origin to form the holoenzyme after wounding of tissue.     

Increase in Catalase mRNA in Wounded Sweet Potato Tuberous Root Tissue

Catalase protein, as well as its activity, increases in woundedsweet potato tuberous root tissue [Esaka et al. (1983) PlantCell Physiol. 24: 615]. Whether catalase mRNA increases in woundedtissue was examined with a hybridization probe of a cDNA forsweet potato catalase mRNA. The content of catalase mRNA inthe tissue increased after a lag phase of 10 h to reach a maximumat 30 h after wounding, whereas total RNA content increasedwithout a lag phase. The increase in the mRNA content afterthe lag phase preceded that in catalase activity. In the earlystages after wounding, catalase mRNA in polysomes increasedin spite of no increase in the total content of mRNA in thetissue. We propose that an increase in catalase mRNA is responsiblefor the increase in catalase protein in wounded sweet potatotuberous root tissue. In the early stage after wounding, however,the translation of catalase mRNA seems to be activated throughincreased availability of ribosomes. (Received January 28, 1987; Accepted May 6, 1987)     

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)     

Presence of an inactive protein immunologically analogous to cytochrome c oxidase in the inner membrane of sweet potato root mitochondria

A protein, which was immunoreactive to antibody against cytochrome c oxidase, was found in the mitochondrial membrane fraction of sweet potato root tissue. The protein was associated relatively weakly with the mitochondrial inner membrane as compared with cytochrome c oxidase. It exerted no cytochrome c oxidase activity and contained no heme a. The protein was purified by phenyl-Sepharose column chromatography and polyacrylamide gel electrophoresis. The molecular weight of its polypeptide chain was 57,000. In addition, the protein decreased during aging of tissue slices. It is therefore not improbable that the protein is a precursor of cytochrome c oxidase composed of only the subunits of cytoplasmic origin, since aging of tissue slices has been shown to result in an increase in the enzyme activity which is inhibited by chloramphenicol but not by cycloheximide.     

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.     

Involvement of hydrogen peroxide and nitric oxide in expression of the ipomoelin gene from sweet potato

The IPO (ipomoelin) gene was isolated from sweet potato (Ipomoea batatas cv Tainung 57) and used as a molecular probe to investigate its regulation by hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) after sweet potato was wounded. The expression of the IPO gene was stimulated by H(2)O(2) whether or not the plant was wounded, but its expression after wounding was totally suppressed by the presence of diphenylene iodonium, an inhibitor of NADPH oxidase, both in the local and systemic leaves of sweet potato. These results imply that a signal transduction resulting from the mechanical wounding of sweet potato may involve NADPH oxidase, which produces endogenous H(2)O(2) to stimulate the expression of the IPO gene. The production of H(2)O(2) was also required for methyl jasmonate to stimulate the IPO gene expression. On the contrary, NO delayed the expression of the IPO gene, whereas N(G)-monomethyl-L-arginine monoacetate, an inhibitor of NO synthase, enhanced the expression of the IPO gene after the plant was wounded. This study also demonstrates that the production of H(2)O(2) stained with 3,3'-diaminobenzidine hydrochloride could be stimulated by wounding but was suppressed in the presence of NO. Meanwhile, the generation of NO was visualized by confocal scanning microscope in the presence of 4,5-diaminofluorescein diacetate after sweet potato was wounded. In conclusion, when sweet potato was wounded, both H(2)O(2) and NO were produced to modulate the plant's defense system. Together, H(2)O(2) and NO regulate the expression of the IPO gene, and their interaction might further stimulate plants to protect themselves from invasions by pathogens and herbivores.     

Mechanism of the Increase in Ribonuclease Activity in Potato Tuber Slices

Upon wounding of potato tubers (Solanum tuberosum L. cv. Spunta)RNase activity increases, peaks in about 16 hours, then declines.To see if the increase of the activity is due to de novo synthesisof the enzyme protein, the extracts were compared for theirability to react with a rabbit antibody prepared against thewound activated RNase. The enzyme was purified by polyacrylamidegel electrophoresis of a RNase preparation, which had been partiallypurified from aged potato slices by ammonium sulfate precipitation,carboxymethyl-Sephadex column chromatography and gel filtrationthrough Sephadex G-100. Using rocket immunoelectrophoresis RNase-proteinimmunoprecipitated by the antibody increased in wounded tissue.This observation implies that the activity increase involvesenzyme synthesis. The increase was inhibited by actinomycinD and cordycepin, but not by 5-fluorouracil, suggesting a requirementfor mRNA synthesis. (Received April 9, 1985; Accepted December 16, 1985)     

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.     

Wound-induced phenylalanine ammonia-lyase in potato (Solanum tuberosum) tuber discs. Significance of glycosylation and immunolocalization of enzyme subunits.

1. Excised discs of potato (Solanum tuberosum) tuber were incubated with [3H]fucose and extracts were prepared and incubated with an antibody to phenylalanine ammonia-lyase. Analysis of the resulting immunoprecipitated proteins by SDS/PAGE showed [3H]mannose- and [3H]fucose-labelled bands with Mr values corresponding to those of phenylalanine ammonia-lyase subunits. 2. When potato discs were incubated with [3H]sugars in the presence of tunicamycin, an inhibitor of N-linked protein glycosylation, incorporation of radioactivity from [3H]mannose into the immunoprecipitated enzyme subunits was virtually eliminated, whereas that from [3H]fucose was only marginally inhibited. 3. Tunicamycin reduced the level of extractable phenylalanine ammonia-lyase activity induced in excised potato tuber discs. Kinetic analysis revealed that the Vmax value of the enzyme in crude extracts from tunicamycin-treated tissue was reduced, whereas the apparent Km values were unaffected. 4. Immunoprecipitation of the enzyme labelled in vivo with [35S]methionine showed that tunicamycin did not inhibit the synthesis of the enzyme protein per se, nor did it increase the degradation of the enzyme protein. 5. Immunoprecipitation of the enzyme labelled in vitro with [14C]nitromethane showed that tunicamycin did not affect the introduction of the dehydroalanine residue into the active site. 6. These results are consistent with the following hypothesis: tunicamycin inhibits the N-linked glycosylation of phenylalanine ammonia-lyase which, in turn, results in imperfect folding of the enzyme protein. The orientation of the active site is changed in such a way that the affinity of the enzyme for its substrate is unaffected, whereas the catalytic activity of the enzyme is reduced. 7. Both optical- and electron-microscopic immunolocalization studies with antibody to phenylalanine ammonia-lyase showed increased deposition of silver granules in cells in sections of potato discs in which induction of the enzyme was allowed to occur compared with cells from newly wounded tissue. The enzyme was located in the cytoplasm, and was possibly membrane-associated.     

Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato

Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H₂O₂ homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl₂, but not MgCl₂ or CoCl₂. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H₂O₂ level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl₂ and SPCAM, and plays an important role in H₂O₂ homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band.     

Detection of catalase in rat heart mitochondria.

The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.     

Wounding-induced Enhancement of the Activity of Chromatin-bound DNA-Dependent RNA Polymerases in Sweet Potato Root

Chromatin fractions were isolated from intact and wounded sweet potato root tissues. The synthesis of RNA by the chromatin fractions was dependent on four ribonucleoside triphosphates and a divalent cation such as Mg²⁺ and Mn²⁺, Mn²⁺ being most effective. Whereas phosphate did not interfere with the polymerase reaction, it was totally blocked by pyrophosphate. The reaction was inhibited by DNase and actinomycin D as well as RNase and trypsin. The RNA polymerases of sweet potato root needed SH-groups for catalysis. Activity of chromatin-bound RNA polymerases (EC 2.7.7.6) promptly increased in the 6 hr after wounding and then decreased gradually up to 24 hr. Under the present experimental conditions it was mostly due to the activity of RNA polymerase I. RNA polymerase II contributed only about 5 to 15% to the total activity. The increase in the activity after wounding was completely inhibited by cycloheximide. Plant hormones such as 2,4-dichlorophenoxyacetic acid, gibberellic acid and dibutyryl cyclic adenosine 3′,5′-monophosphate stimulated the increase in RNA polymerases three to four times after wounding. Ethylene partially suppressed the wound-induced increase of RNA polymerases.     

Change in invertase activity of sweet potato in response to wounding and purification and properties of its invertases

When root tissue of sweet potato (Ipomoea batatas Lam.) was sliced, acid invertase activity, initially absent in freshly sliced tissue, appeared after a 3- to 6-hour lag phase, rapidly reached a maximum in 18 hours, and thereafter decreased. The increase in invertase activity was accompanied by a decrease in sucrose content of the root tissue. Alkaline invertase activity was present in fresh root tissue, but changed little after wounding. Acid invertase in wounded tissue and alkaline invertase in fresh tissue were purified and their properties were investigated. The acid invertase was a ß-fructofuranosidase and was unaffected by substrate or by any of the cations and several metabolites. The alkaline invertase was more specific for sucrose, was inhibited by glucose and glucose 6-phosphate, and displayed non-Michaelis-Menten kinetics.     

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; )     

Reduced susceptibility to waterlogging together with high-light stress is related to increases in superoxide dismutase and catalase activities in sweet potato

We investigated the changes in antioxidative enzyme activities of two sweet potato cultivars under waterlogging and high-light conditions in the growth chamber. The activities of antioxidative enzymes were measured from leaf crude extract of sweet potato during the first five days of the treatments. Activities of superoxide dismutase and catalase were consistently increased in Taoyuan 1 sweet potato over time under waterlogging and high-light conditions. However, decreases in both superoxide dismutase and catalase activities were observed for cultivar Yongtsai under waterlogging and high-light conditions. Waterlogging, together with high-light intensity, impairs superoxide dismutase and catalase activities in the cultivar Yongtsai indicating its greater susceptibility to waterlogging and high-light stress. In contrast, the increase in activities of superoxide dismutase and catalase in Taoyuan 1 indicated its greater ability to detoxify reactive oxygen species during the treatment and ensured its reduced susceptibility to waterlogging and high-light stress. The activities of peroxidase may be inactivated by high-light treatment and, therefore, may not be associated with tolerance of sweet potato plants to waterlogging and high-light stress. Differences in susceptibility to waterlogging and high-light conditions in the leafy vegetable Yongtsai and storage root Taoyuan 1 are discussed.     

The effect of injury and infection by Gibberella zeae on the polyacrylamide gel electrophoretic pattern of soluble proteins in potato tuber tissue

Polyacrylamide gel electrophoretic patterns of soluble proteinsfrom intact potato tubers and from wounded tuber tissue wereidentical. The patterns from diseased tissue showed some minordifferences not before 3–4 days after inoculation. Itis concluded that primarily proteins of minor fractions areinvolved in protein synthesis after wounding or infection. (Received March 22, 1969; )     

Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures

The effect of various stages of normal cell growth on human fibroblast collagenase found in the culture medium was studied, so that the regulatory mechanisms of synthesis, secretion and activity of the enzyme could be established. Specific activity of collagenase increased 6- to 10-fold shortly after confluence was reached when compared with low density levels and decreased in post-confluent cultures, suggesting that synthesis and/or release of the enzyme changes with culture density. To assess this possibility, culture medium was examined for immunoreactive collagenase protein by radioimmunoassay. After confluence was reached, immunoreactive collagenase had increased approx. 2-fold, indicating greater secretion, and probably synthesis, of the enzyme. However, the increase in specific activity of the enzyme observed shortly after confluence was greater than could be accounted for by an increase in immunoreactive enzyme protein. As a result of the disproportionate increase in collagenase activity, the collagenase activity per unit immunoreactive protein was also found to be greatest shortly after confluence and decreased in post-confluent cultures. This density-associated modulation of collagenase expression could be reproduced by initiating the cultures at high density after subculture. Expression of collagenase activity was dependent upon intact protein synthetic mechanisms, since cultures maintained in the presence of cycloheximide failed to secrete collagenase into the culture medium.     

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; )     

甘薯愈伤组织对干旱胁迫和盐胁迫的生理反应对比

研究干旱胁迫和盐胁迫对“芦选一号”。日‘薯愈伤组织可溶性蛋白、可溶性糖、脯氨酸含量、SOD活性等的影响,从而在细胞水平上探讨甘薯抵御渗透胁迫的生理机制。并分析甘薯细胞对干旱处理（PEG-6000）和盐处理（NaCl）的反应差异。结果表明,可溶性蛋白质含量在干旱胁迫下缓慢升高,在轻度和中度盐胁迫的生长前期和中期有较大幅度的上升。但后期下降,表明短时间盐胁迫下,Na^＋可能促进可溶性蛋白的合成;MDA在重度干旱胁迫下的含量显著低于重度盐胁迫,而SOD活性显著高于盐胁迫。表明在盐胁迫下细胞膜透性增加的主要原网是膜脂过氧化作用。干旱处理则是PEG-6000脱水的直接结果;重度干旱胁迫下,可溶性糖含量在短期内迅速升高,然后下降,而脯氨酸含量则在胁迫中后期迅速上升。脯氨酸可能有补偿可溶性糖含量降低的作用。