Demonstratioin of de novo synthesis of RNase in Rhoeo leaf sections by deuterium oxide labeling

The increase in RNase activity that occurs in fresh cut Rhoeodiscolor leaf sections was followed in sections aged in wateror deuterium oxide for 24 hr. The increase in RNase activityis probably due to de novo synthesis, since the buoyant densityof the RNase from the deuterium labelled tissue was significantlyhigher (1.10 to 1.88%) than that of the controls. (Received June 29, 1973; )     

RNase in Lasallia hispanica and Cornicularia normoerica: multiplicity of electromorphs and activity changes during a hydration-dehydration cycle

The presence of RNase activity has been detected in the twosaxicolous lichen species, Lasallia hispanica (Frey) Sancho& Crespo and Cornicularia normoerica (Gunn.) DR. Activitywas localized in the soluble fraction and had an acid optimumpH in both species. When proteins from the soluble fractionof the two lichens were separated by isoelectric focusing, multipleelectromorphs with RNase activity were detected. L. hispanicaRNase was separated into seven bands, characterized by pls 7,6.28, 4.58, 4.45, 4.25, 3.95, and 3.47. In C. normoerica fourbands were detected, with pls of 6.28, 3.98, 3.57, and 3.39.The molecular mass of the main RNase of L. hispanica estimatedby SDS-PAGE was 31.86 kDa, which corresponds to the 33 kDa estimatedfor the undenatured RNase by gel chromatography. Proteins fromC. normoerica were resolved by SDS-PAGE in three bands, withestimated molecular mass of 36.07 kDa, 31.86 kDa and 17.13 kDa.In order to improve the detection of RNase activity, gels wereincubated after the run (electrophoresis or isoelectric focusing)in a RNA solution, instead of including the substrate in thegel. In both species, RNase activity increased during hydrationand decreased during desiccation. This pattern of activity resemblesthat of other enzyme activities in lichens, which decrease inresponse to water deficits, and is different from the responseof other poikilohydrous organisms such as bryophytes. Theseresults are discussed in relation to the mechanisms that lichenshave to withstand dehydration. Key words: Comicularia, Lasallia, lichens, ribonuclease, water stress     

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)     

Potato Lectin: A Cell-Wall Glycoprotein

The activity and the amount of potato lectin were measured inpotato tuber slices (Solanum tuberosum cv. Huinkul) aeratedfor 48 h. Lectin was found in a soluble form, liberated to themedium and associated with insoluble structures. Polyacrylamidegel electrophoresis in denaturating conditions and immunologicaltechniques indicated that the lectins associated to cell wall,soluble or liberated to the medium, were identical. The cell-wallfraction was found to contain 65% of total lectin in the tuber.The possible role of potato lectin in tubers was discussed. (Received June 5, 1985; Accepted September 3, 1985)     

Effects of Dazomet on Phenolase Activity in Sclerotia of Sclerotinia sclerotiorum

Developing sclerotia of the fungus Sclerotinia sclerotiorumexude a clear liquid which contains the enzyme o-diphenol oxidase.The activity of this enzyme, which is also present in the sclerotialtissue, is inhibited by Dazomet (a soil fumigant), sodium azide,and DIECA. These inhibitions can be prevented in the presenceof sufficient quantities of Cu²⁺. The activity of mushroom o-diphenoloxidase is affected by Dazomet and Cu²⁺ in a similar manner. Unpigmented, exposed surfaces of cut sclerotia darken withina few days due to the synthesis of a melanin-like pigment. Theformation of this pigment is prevented by Dazomet. This effectof Dazomet is compared with its action on the darkening of cutsurfaces of potato tubers which also possess appreciable amountsof o-diphenol oxidase.     

Influence of Ethylene and Kinetin on Tuberization and Enzyme Activity in Solanum tuberosum L.: Stolons Cultured in vitro

The response of potato stolons, cultured in vitro, to ethylenewas investigated utilizing 2-chloroethyl-phosphonic acid asthe source of ethylene. Concentrations of 0.067, 0.67, 6.7,and 67 µm did not promote tuber formation which occurredin the presence of 16 µm kinetin. In the presence of 2-chloroethyl-phosphonicacid stolon branching was promoted and they maintained a diageotropicgrowth habit in contrast to the negatively geotropic growthhabit of kinetin-treated cultures. The amount of soluble sugars accumulating at the stolon tipwas similar in the presence of kinetin and 2-chloroethylphosphonicacid. However, the level decreased in kinetin-treated stolonsas starch synthesis increased whereas a high specific activitywas maintained in the presence of 2-chloroethylphosphonic acidwhere no starch synthesis occurred. Higher levels of sucroseoccurred in kinetin-treated cultures. Both 2-chloroethylphosphonic acid and kinetin decreased invertaseactivity at the stolon tip. Peroxidase activity increased withtime in response to 2-chloroethylphosphonic acid whereas activityincreased appreciably in kinetin-treated stolons only afterday 5. Generally higher levels of 3'nucleotidase activity existedin 2-chloroethylphosphonic acid-treated stolons. RNase activitydecreased in kinetin treated stolons while activity increasedin the presence of 2-chloroethylphosphonic acid. It is suggestedthat ethylene may influence stolon growth but may not be directlyinvolved in tuber initiation.     

A Cytosolic Phospholipase A2 from Potato Tissues Appears to Be Patatin

Phospholipase (PL) A₂ is involved in signal transduction inthe resistance reaction that is induced in potato by inoculationof an incompatible race of Phytophthora infestans, the lateblight fungus, or by treatment with fungal elicitor hyphal wallcomponents (Kawakita et al. 1993). In this study, PLA₂ in thesoluble fraction from potato tuber was purified. The followingresults suggested that the enzyme was, in fact, patatin: (1)the molecular mass of the purified enzyme was 40 kDa, the sameas that of patatin; (2) the pI of the purified enzyme was approximately4.75, which corresponds to that of patatin; and (3) the amino-terminalamino acid sequence of the purified enzyme showed a high degreeof homology to that of patatin. Patatin is known as a storageprotein of the potato tuber and it has been shown to have esteraseactivity. However, other enzymatic activities and the function(s)of patatin are unknown. We investigated the PLA activities ofthe purified patatin. The PLA₂ activity of the patatin was muchhigher than the PLA₁ activity, even though the protein exhibitedboth activities. The PLA₂ activity of the enzyme was particularlyapparent when phosphatidylcholine with linoleic acid at thesn-2 position was used as substrate. Lower activity was observedwith phosphatidylcholine with palmitic acid, oleic acid andarachidonic acid at the sn-2 position. (Received October 5, 1995; Accepted February 9, 1996)     

The Measurement of Ribulose 1, 5-bisphosphate Carboxylase/Oxygenase Concentration in the Leaves of Potato Plants by Enzyme Linked Immunosorbtion Assays

Catt, J. W. and Millard, P. 1988. The measurement of ribulose1, 5-bisyphosphate carboxylase/ oxygenase concentration in theleaves of potato plants by enzyme linked immunosorbtion assays.—J.exp. Bot. 39: 157-164. An enzyme linked immunosorbtion assay (ELISA) has been developedfor the measurement of ribulose l, 5-bistfphosphate carboxylase/oxygenase(RUBISCO) concentrations in potato leaves. Extracts of leaveswere made using a buffer containing detergent and used to coatELISA plates. The amount of RUBISCO binding was estimated usinga double antibody technique. Various methods of extraction wereused and assessed and the efficiency of the assay was testedusing purified RUBISCO. The method is applicable to the determinationof RUBISCO from diverse sources. Key words: Ribulose 1, 5-bisphosphate carboxylase/oxygenase, immunosorbtion, Solanum tuberosum     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.     

Indoleacetic Acid-Induced Decrease of the Ribomiclease Activity in vivo

A study has been made on the influence of indole-3-acetic acid (IAA) on the ribonuclease (RNase) activity in wheat coleoptile sections and green pea stem sections. The hormonal effects on the enzyme activity, ribonncleic acid (RNA) metabolism and growth have been compared. Addition of 10^?5M IAA to the plant sections causes their RNase activity to decrease and their elongation to increase. Removal of the added IAA results in increasing enzyme activity and decreasing growth. The altered enzyme activities are paralleled by opposite changes in the RNA net synthesis. Administration of crystalline RNase to the plant tissue depresses growth. There is thus evidence that the in vivo effect of IAA on the RNase activity is of importance for the hormonal regulation of RNA metabolism and growth. The IAA-induced reduction in the enzyme activity involves cellular metabolism. The effect can be suspended by means of p-chloromercuribenzoate. A possible mechanism for the reduction is discussed.     

Untersuchungen über rnasen in kotyledonen von nachgereiften und dormantenagrostemma githago-samen

Activities of RNasea were studied in cotyledons of dormant and afterripenedAgrostemma githago seeds. Activity of RNase increases during imbibition and germination. This increase in activity cannot be observed in variants which are not able to germinate (dormant seeds and seeds blocked by higher temperature). The development of RNase activities during germination cannot be inhibited by concentrations of cycloheximide or actinomycine D completely preventing phosphatase synthesis. These results may be indicative for the assumption that the increase of RNase during germination is caused by enzyme activation and not by enzyme synthesis. Cytokinins and a combination of cycloheximide and gibberellic acid stimulate the activity of RNase in dormant cotyledons, whereas neither cycloheximide nor gibberellic acid, applicated by themselves, show any effect. Cytokinins and gibberellic acid do not influence the activity of RNase of afterripened cotyledons, abscisic acid inhibits the increase of enzyme activity. There are characteristic changes in the pattern of RNases during germination revealed by polyacrylamide gel electrophoresis. The increase in RNase activity of dormant cotyledons caused by cytokinins is accompanied by obvious changes in the RNase pattern on polyacrylamide gel. Treating dormant cotyledons with cytokinins dormancy is partially overcome. In consequence of the application of cytokinins the differences in the electrophoretic RNase pattern between dormant and afterripened cotyledons can be nearly balanced.     

Physiological Basis for a Cycloheximide-induced Soft Rot of Potatoes by Pseudomonas fluorescens

Cycloheximide renders discs of potato tissue (Solanum tuberosum,cultivar Kennebec) susceptible to soft rot by a non-pathogenicisolate of Pseudomonas fluorescens. Pectate lyases (E.C. 4.2.99.3 [EC] )are the dominant extracellular macerating agents produced bythe test organism. Potato discs aged 24 h become resistant tomaceration by purified lyase preparations. Cycloheximide blocksthe development of resistance by inhibiting suberization. Thesite of inhibition is thought to be the cycloheximide-sensitivesynthesis of phenylalanine ammonia-lyase (E.C. 4.3.1.5 [EC] ) in potatodiscs. This enzyme is necessary for production of phenolic precursorsof suberin. Comparison of tissue from a number of potato cultivarscorrelates the synthesis of phenylalanine ammonia-lyase withresistance of discs to attack by the Pseudomonad. Resistance of potato tissue to pectate lyase is also affectedby intrinsic reactions not involving suberization. Resistanceincreases in fresh unsuberized discs when tubers are transferredfrom cold storage to room temperature before use. Resistancedecreases rapidly when tubers are transferred back to the cold.The intrinsic resistance appears to increase in the surfacelayer of cells in ageing discs. It is estimated that intrinsicreactions and suberization contribute equally to resistanceof aged discs to pectate lyase maceration.     

Purification and Some Properties of Alcohol Acetyltransferase from Banana Fruit

Isoamyl acetate, one of the major characteristic aroma compoundsof banana fruit (Musa sapientum L.), was synthesized by thecondensation of acetyl-CoA and isoamyl alcohol under the actionof alcohol acetyltransferase, which was found to be localizedin the soluble fraction of pulp cells. The activity of thisenzyme increased with the ripening of banana fruit. The enzyme was purified about 62-fold. The purified enzyme wasvery labile at pHs lower than pH 7.0 but relatively stable atpH 7.5{small tilde}9. The enzyme was most active at 30C andpH 8.5. The molecular weight was estimated to be about 40,000by gel filtration. Its K_m values for acetyl-CoA and isoamylalcohol were 50 µM and 0.4 mM, respectively. (Received January 28, 1985; Accepted May 30, 1985)     

Effects of inhibitors on the enzyme system producing C6-aldehydes from C18-unsaturated fatty acids in chloroplasts of Japanese silver (Farfugium japonicum) leaves

When chloroplasts isolated from Farfugium japonicum (Japanesesilver) leaves were used as an enzyme source, the activity ofthe enzyme system producing C₆-aldehydes (cis-3-hexenal andn-hexanal) from C₁₈-unsaturated fatty acids (linolenic and linoleicacids) decreased upon treatment with LAHase from potato. Thisenzyme system could not be separated from chlorophylls and lipidsby detergent treatment and was not affected by light illumination,CCCP or DCMU. The activity of the enzyme system was inhibitedby MB and NTB used as a redox reagent, SKF 525-A as an oxidaseinhibitor and DABCO as a quencher of singlet oxygen, but notby DCIP, PMS and SOD. These data suggest that; i) interactionof the enzyme system with lipids is required for maximal enzymeactivity, ii) this enzyme system may involve electron mediator(s),and iii) singlet oxygen takes part in the enzyme reaction. (Received October 28, 1977; )     

Function of Lysosomes and Lysosomal Enzymes in the Senescing Corolla of the Morning Glory (Ipomoea purpurea)

The rapid senescence of the Ipomoea corolla is characterizedby the breakdown of protein and nucleic acids. At the onsetof wilting the activities of deoxyribonuclease (DNase), ribonuclease(RNase), and ß-glucosidase are increased dramatically,while other hydrolytic activities such as the actions of protease,aminopeptidase, -glucosidase, phosphatase, esterase, and -amylaseare only slightly changed. Isolated corolla discs show a course of senescence similar tothat of the intact organ. When floating on solutions of cycloheximidethe activities of DNase, RNase, and ß-glucosidasedo not increase. Actinomycin D inhibits the increase in RNaseactivity. It is concluded that protein synthesis is a prerequisitefor the changes in these enzyme activities in the senescingcorolla. The function of the lysosomal compartment in the process ofsenescence is illustrated by electron micrographs showing theautophagic activity of vacuoles. The last phase of senescenceis characterized by the breakdown of the tonoplast and completedigestion of the cytoplasmic constituents in the autolysingcells.     

The Relationship of Abscisic Acid to Nitrate Reductase Activity in the Potato Plant

Abscisic acid (ABA) at 3.8 µM suppressed both in vivoand in vitro nitrate reductase activity in roots, stems andleaves of potato plants grown in solution culture. Suppressionwas maximal between 24 and 48 h, followed by recovery of activityat 72 h in roots and leaves and at 96 h in stems. Removal from ABA after 24 h resulted in complete recovery ofnitrate reductase activity in roots by 24 h and partial recoveryin leaves. ABA treatment enhanced nitrate accumulation in roots,decreased that of leaves, but had no effect on stem nitratecontent. ABA enhanced decay of the enzyme following nitrate removal;by 7 h activity in roots was 22.5% of the initial value comparedto 55% in the control. ABA showed a less drastic effect on lossof activity in leaves and stems. These results indicate thatABA suppression of nitrate reductase activity is not dependenton nitrate uptake, and although it reduced leaf nitrate contentthere was no clear relationship between tissue nitrate levelsand the ABA response. (Received September 13, 1984; Accepted July 1, 1985)     

Morphology, RNase and transaminase of root protoplasts

RNase and transaminase activities were analysed for mechanicallyand enzymatically prepared protoplasts from Allium Cepa roots.The comparative analyses at three root regions show that theenzymes were less active in the protoplasts than in the cellsfrom which they had been obtained. The enzyme gradients (fromapex to base of the root: RNase increase and transaminase decrease)noted previously in the intact roots were found to be similarin the protoplasts, however to a lesser degree. On the otherhand, the relative activity of both tested enzymes was lowerin the enzymatically prepared protoplasts than in those obtainedby the mechanical technique. In connection with their physiologicalproperties, the respective effects of the mode of preparingthe protoplasts were discussed. (Received November 22, 1971; )     

Inhibition of an aa3-Type Two-Subunit Cytochrome c Oxidase from Nitrobacter agilis by N,N'-Dicyclohexylcarbodiimide

The electron transfer activity of an aa₃-type two-subunit cytochromec oxidase of Nitrobacter agilis was inhibited by DCCD. Althoughthe activity of the purified cytochrome c oxidase dissolvedin 1% Triton X- 100 was not affected by DCCD even at a ratioof 1,000 mol DCCD per mol cytochrome aa₃, the activity of theenzyme dissolved in 0.02% Tween 20 or 0.02% Triton X-100 wasinhibited by 60% or more at a ratio of 1,000 mol DCCD per molcytochrome aa₃. The results of SDS polyacrylamide gel electrophoresisof the enzyme incubated with DCCD suggested that subunit IImight be a binding site for DCCD. (Received February 23, 1985; Accepted April 23, 1985)