Synthesis of DNA and the Development of Amylase and Phosphatase Activities in Cotyledons of Germinating Seeds of Vaccaria pyramidata

As in cotyledons of Agrostemma githago, synthesis of DNA takesplace after germination in cotyledons of Vaccaria pyramidataand is followed by the formation of hydrolases, in particular,-amylase and acid phosphatase. If DNA synthesis is inhibitedby hydroxyurea, no, or only slight, enzyme activity develops.The possible role of this DNA synthesis is discussed. Key words: DNA synthesis, amylase activity, phosphatase activity, seed germination, cotyledons, Vaccaria pyramidata     

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms ofacid phosphatase and ribonuclease from potato tubers were studiedby seasonal assays and Sephadex gel filtration. Ribonucleaseand p-nitrophenyl phosphatase activity fluctuated during storageof tubers, while ß-glycerophosphatase declined toa low level coincident with initiation of sprout growth. Inrecently-lifted tubers acid phosphatase activity occurred ina single high molecular weight peak. Two new forms of lowermolecular weight appeared during ageing of stored tubers. Theinfluence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica,on these seasonal changes was variable. No consistent effectson total hydrolase activities were observed, while post-infectionalchanges in molecular forms included a pronounced shift in themajor acid phosphatase peak. The possible significance of thismolecular weight change in infected samples is discussed inthe light of recent evidence concerning the sub-unit structureof acid phosphatase from potato tubers.     

Effects of Gibberellic Acid on the Activation, Synthesis, and Release of Acid Phosphatase in Barley Seed

Acid phosphatase activity was present in unimbibed barley seed,but rose during incubation of embryoless half-seeds and isolatedaleurone layers, and was further increased by 10^–6 M gibberellicacid (GA₃). Release of total acid phosphatase activity fromhalf-seeds and aleurone layers was markedly enhanced by GA₃.Inhibitor studies with cycloheximide and actinomycin D suggestedthat de novo synthesis of acid phosphatase occurred followingimbibition. Gel nitration, electrophoresis, and [¹⁴C]leucineincorporation studies revealed that a single molecular formof acid phosphatase was present in dry seed, whereas on incubationtwo further forms arose. A proportion of the three molecularforms of the enzyme was synthesized de novo. Gibberellic acidstimulated activation, but not de novo synthesis, of all threemolecular forms of acid phosphatase. Although a small amountof one of the molecular forms was secreted in the absence ofGA₃, the presence of gibberellin greatly increased secretionof the same form of acid phosphatase.     

Acid Phosphatase in Cotyledons of Germinating Vigna mungo Seeds

A marked increase in acid phosphatase activity took place incotyledons of germinating Vigna mungo seeds. The attachmentof axis organs was not required for this development of enzymeactivity in cotyledons. DEAE-cellulose column chromatographyrevealed that the phosphatase is composed of at least threeforms. (Received August 19, 1981; Accepted October 30, 1981)     

Multiple Forms of Acid Phosphatase in Cotyledons of Vigna mungo Seedlings: Immunological Detection and Quantitation

Using a combination of column chromatography and gel electrophoresis,we have found that acid phosphatase in cotyledons of Vigna mungoseedlings is composed of at least six forms (Ia₁, Ia₂, Ib₁,Ib₂, IIa and IIb). We purified one of the major forms, Ia₁,as a polypeptide of 53 kDa. Using an antiserum raised againstthe enzyme Ia₁, we examined the immunological relationshipsbetween the multiple forms from cotyledons and the distributionof the enzyme in organs of maturing and germinating seeds. (Received December 25, 1989; Accepted July 11, 1990)     

Structure and Expression of a-Amylase Gene from Vigna mungo

A single copy of the a-amylase gene, composed of three intronsand four exons, was found in Vigna mungo. Examination of levelsof a-amylase and its mRNA in detached cotyledons indicated thatattachment of the embryonic axis is not required for expressionof the gene in cotyledons of germinating seeds. (Received December 21, 1993; Accepted March 14, 1994)     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination     

A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)     

A Scanning Electron Microscope Study of Theobroma cacao Somatic Embryogenesis

This study describes the somatic embryogenesis of Theobromacacao L. with a scanning electron microscope. It revealed earlydevelopmental stages as globular and incipient heart-shaped.Morphological abnormalities, such as the occurrence of threeand four cotyledons and round or long forms of embryoids witha long and thin or short and thick stalk-like structure whichseems to equate to a suspensor, were also observed. A suspensorwas found in some embryoids. cacao, Theobroma cacao, embryoids, somatic embryogenesis, SEM     

Amylase Activities in Attached and Excised Cotyledons and in Embryonic Axes of Pisum sativum L.

Amylase activity increased in attached cotyledons of peas, Pisumsativum L. var. Bördi, only during imbibition and remainedalmost constant up to 96 h after germination, but in excisedcotyledons the activity increased slightly at first then markedly.In contrast, the content of the reducing sugars was higher inattached cotyledons than in excised ones. A similar inverserelationship has been found between the concentration of reducingsugars in axes (both attached and excised) and amylase activity. The leakage from intact seeds contained more reducing sugarsthan the leakage from excised cotyledons, whereas the amountof proteins released from the cotyledons was four times greaterduring imbibition. This increase in amylase activity in excisedcotyledons is not thought to be the result of axis excision,but to be the result of the leakage of sugars from the cotyledonsduring incubation. These results suggest that the concentration of reducing sugarsmay be a factor that regulates amylase activity in vivo in boththe cotyledons and axis during the germination of pea seeds. (Received August 4, 1982; Accepted December 14, 1982)     

A promotive action of kinetin on amylase activity in cotyledons of Phaseolus vulgaris

Kinetin and the embryo axis acted similarly in bringing abouta promotion of amylase activity in cotyledons of Phaseolus vulgaris.No promotive effect of gibberellic acid or indole-3-acetic acidon amylase activity could be detected. It is suggested thatthe regulatory action of the embryo axis on starch degradationin the cotyledons of P. vulgaris is mediated by cytokinins. (Received May 4, 1970; )     

Distribution of {beta}-Thioglucosidase Activity in Intact Plants, Cell and Tissue 6Brassica napus L.

Distribution of myrosinase activity in extracts from seeds,intact plants, cell cultures and regenerated callus and plantsof Brassica napus L. was determined by the rate of glucose formationfrom glucosinolate hydrolysis. Calli with shoots and regeneratedplants were obtained from protoplasts or from explants. Of the seedling organs from Brassica napus L. cv. Niklas, hypocotylsshowed the highest myrosinase activity. In cotyledons a nearlyconstant enzyme activity was determined over the first 6 d,followed by a gradual decline. Roots showed a fast decline inenzyme activity over the investigated period. Freshly-isolated protoplasts contained less myrosinase activitythan the original intact tissue. The enzyme activity in developingcalli generally decreased during the first culture periods.After the initial decline a low activity was found which wasstable for a period of more than 2 years. The enzyme activityshowed fluctuations when measured at different times after mediumchange. Protoplast calli with regenerated shoots showed a considerablyhigher myrosinase activity than calli without shoots. Myrosinaseactivity was also found in explant calli including explant callifrom cotyledons and hypocotyls after induction of shoots. Myrosinase activity in seeds from 21 cultivars of Brassica napus,Brassica campestris, Sinapis alba and Raphanus sativus was testedand the highest myrosinase activity was found in seeds fromthe Sinapis alba cultivar Trico while the lowest activity wasfound in the Brassica campestris cultivar Rapido III. Leaf, stem and inflorescence from flowering regenerated or seed-grownplants contained a low but significant myrosinase activity.In contrast, roots showed a high myrosinase activity. The resultsobtained from regenerated plants indicate that the myrosinasesystem is stable in vitro culture, and that the glucosinolate-myrosinasesystem is active in calli tissue. Key words: Myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1), in vitro cultures, intact plants     

Formation of multiple forms of acid and alkaline phosphatases in relation to the culture age of Aspergillus niger

Acid phosphatase (EC 3.1.3.2 [EC] ) was extracted from mycelia ofAspergillus niger, then separated and purified into four fractions.These acid phosphatases, designated IA, IB, II and III, hadpH optima at 5.0, 4.5–5.0, 4.5 and 2.5, respectively.None required the presence of divalent cations, and all werestrongly inhibited by NaF. They were non-specific acid phosphatasesbut varied in their activities with various substrates. Thealkaline phosphatase (EG 3.1.3.1 [EC] ) of A. niger was also separatedinto two fractions, alkaline phosphatases I and II. Changes in the activity ratios of these acid and alkaline phosphataseswere studied during culture in a peptone medium. The activityof acid phosphatase II was higher than the others when the culturewas young. The activity of acid phosphatase III increased toa maximum in the actively growing phase, then decreased. Thatof acid phosphatase I became highest in the mature culture.In contrast, the activity of alkaline phosphatase I was higherthan the others in young cultures, while alkaline phosphataseII became dominant in the mature culture. Activities of the various acid and alkaline phosphatases indifferent regions of the growing colonies were also studied.The changing patterns of these enzymes in both liquid and surfacecultures were compared. When A. niger was cultured in a medium containing a low concentrationof phosphate, acid phosphatase activity greatly increased afterthe consumption of phosphate, but alkaline phosphatase activitydid not. ¹ The present experiments were carried out, for the most partat the Institute of Applied Microbiology of the University ofTokyo. (Received February 10, 1975; )     

The significance of the Presence of Stomata on Seedling Roots

Slightly squashed and transversely sectioned sprouts (seedlingroot + hypocotyl to cotyledons) of Pisum arvense, Ornithopussativus and Helianthus annuus were examined. The presence ofstomata is described on seedling roots, including the root hairzone, in Pisum and Ornithopus and in the root hair zone of thehypocotyl of Helianthus. The stomata found in the root hairzones are almost always open, usually without chloroplasts andare not sensitive to the action of abscisic acid (ABA). TheABA sensitivity of the stomata appears first above the roothair zone and increases gradually towards the cotyledons. Thepossible role of stomata in the root hair zone is discussed. Seedling root, hypocotyl, root hair zone, stomata     

Localization of Sorbitol Oxidase in Vacuoles and Other Subcellular Organelles in Apple Cotyledons

To investigate the function and subcellular localization ofsorbitol oxidase, free cells, protoplasts and isolated vacuolesof apple cotyledons (Malus pumila Mill. var. domestica Schneid.)were examined by differential and sucrose density gradient centrifugation.Twenty percent of the activity of sorbitol oxidase in the wholetissue was contained in the subcellular fraction (d=1.06) whichcorresponded closely to the main peaks of activity and proteinafter the recentrifugation of the 150,000?g pellet of rupturedvacuoles with a linear sucrose density gradient. The enzymethus appears to be derived from the tonoplast membrane. Thistonoplast membrane-bound sorbitol oxidase may play an importantrole in the transport of vacuolar sorbitol into the cytoplasm,rather than in the transport of sorbitol into the vacuole. About10% of the enzyme activity also occurred in the subcellularfraction having a density of 1.12–1.16, which coincidedwith the peaks of acid phosphatase and ATPase activities. Thereforesorbitol oxidase may also be associated with the plasma membrane.Furthermore, 30–40% of its activity was located in theinterspace between the cell wall and the plasma membrane, orperhaps attached weakly to them. These results suggest thatsorbitol is transported into the cytoplasm by being convertedto glucose by sorbitol oxidase. ¹ This paper is contribution A-138 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

ATPase and Proton-Translocating Activities in a Plasma Membrane-Enriched Fraction from Cotyledons of Ricinus communis

Sucrose gradient centrifugation was used to separate the microsomalmembranes and purify the plasma membrane ATPase from Ricinuscotyledons. The pellet from a three-step (30, 34, 38%) sucrosegradient was enriched in plasma membrane as determined by acombination of marker assays. The partially purified plasma membrane ATPase was magnesium-dependentand had a pH optimum of 6.5. It showed high sensitivity to vanadate,erythrosin B, SW 26, DCCD and PCMBS but low sensitivity to azide,nitrate and NEM. Substitution of calcium for magnesium resultedin low activity, and in the presence of magnesium, calcium wasinhibitory. KCl stimulation was low (less than 50%) and of thepotassium salts tested all were stimulatory except which was inhibitory. Specificity for nucleotide triphosphateswas high, greatest activity occurring with ATP. Proton-pumping activity measured using quinacrine fluorescencequenching was inhibited by vanadate and erythrosin B but notby nitrate and oligomycin indicating that activity was mainlydue to the plasma membrane ATPase. Key words: ATPase, cotyledons, plasma membrane, proton pumping, Ricinus communis     

Increases in activities of membrane-bound hydrolases in pea cotyledons during germination

Membrane-bound proteinase and acid phosphatase activities, butnot cytosol proteinase activity, in pea cotyledons increasedafter lag phases during germination. The activity hydrolyzingN--benzoyl-D,L-arginine P-nitroanilide in the membrane fractionincreased rapidly in the imbibition stage. Whether the increasesare due to de novo synthesis of the enzyme proteins was studied. ¹ Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received May 28, 1973; )     

In vitro Culture of Immature Cotyledons of Soya Bean (Glycine max L. Merr.)

An in vitro procedure promoting the rapid growth and proteinincrease of soya bean cotyledons has been developed. The amountof protein synthesized varied greatly depending on the nitrogen(N) source provided. Glutamine was the most effective N source,while inorganic forms of N were ineffective. Growth and proteinsynthesis were both more rapid in vitro than in vivo. Underthe best conditions, soya bean cotyledons increased 8-fold bothin dry weight and in protein in 6 days. The formation of the7S and 11S storage proteins in vitro was similar to that invivo. Hence, this in vitro culture method is appropriate forstudying legume seed storage protein synthesis under controlledconditions.     

Genome Comparison In Silico in Neisseria Suggests Integration of Filamentous Bacteriophages by their Own Transposase

We have identified filamentous prophages, Nf (Neisserial filamentousphages), during an in silico genome comparison in Neisseria.Comparison of three genomes of Neisseria meningitidis and oneof Neisseria gonorrhoeae revealed four subtypes of Nf. Elevenintact copies are located at different loci in the four genomes.Each intact copy of Nf is flanked by duplication of 5'-CT and,at its right end, carries a transposase homologue (pivNM/irg)of RNaseH/Retroviral integrase superfamily. The phylogeny ofthese putative transposases and that of phage-related proteinson Nfs are congruent. Following circularization of Nfs, a promoter-likesequence forms. The sequence at the junction of these predictedcircular forms (5'-atCTtatat) was found in a related plasmid(pMU1) at a corresponding locus. Several structural variantsof Nfs—partially inverted, internally deleted and truncated—werealso identified. The partial inversion seems to be a productof site-specific recombination between two 5'-CTtat sequencesthat are in inverse orientation, one at its end and the otherupstream of pivNM/irg. Formation of internally deleted variantsprobably proceeded through replicative transposition that alsoinvolved two 5'-CTtat sequences. We concluded that the PivNM/Irgtransposase on Nfs integrated their circular forms into thechromosomal 5'-CT-containing sequences and probably mediatedthe above rearrangements.     

Functional characterization of the enzymes with 2,3-bisphosphoglycerate phosphatase activity from pig skeletal muscle

In pig skeletal muscle exist four enzymes with 2,3-bisphosphoglycerate phosphatase activity. Two of them (forms I-A and I-C) are multi-functional enzymes which, in addition to the phosphatase activity, possess 2,3-bisphosphoglycerate synthase and phosphoglycerate mutase activities. The other two enzyme forms (II-A and II-B) only show the phosphatase activity. The four enzymes differ in substrate specificity. Form I-C is highly specific for glycerate 2,3-P2; form I-A also hydrolyzes the monophosphoglycerates and forms II-A and II-B are specific for phosphoester bonds adjacent to a C-1 carboxylic group. The enzymes possess similar Km, Kcat and optimum pH value, but they are differently inhibited by the reaction products. They are also differently affected by glycolate-2-P (their main activator) and by other modifiers. Probably form I-A, which corresponds to M-type phosphoglycerate mutase, is the main enzyme implicated in the breakdown of glycerate 2,3-P2 in pig muscle.