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; )     

The Utilization of Cotyledonary Reserves in Phaseolus vulgaris L. cv. Carioca: II. THE EFFECTS OF 6-BENZY LADENINE AND GIBBERELLIC ACID UPON EMBRYONATED AND DETACHED COTYLEDONS

The presence of the embryo axis promotes starch hydrolysis incotyledons of Phaseolus vulgaris and, although reduced ratesof enzyme activity proceed in its absence, weight loss fromthe cotyledon is only significant in its presence. Applicationof gibberellic acid at various concentrations had no effectupon amylase or protease activity in either detached or embryonatedcotyledons: this was not the case for 6-benzyladenine whichpromoted both. In detached cotyledons amylolytic activity wasdirectly proportional to the concentration of applied 6-BA;concentrations below 10^–6 M were unable to completelysubstitute for the presence of the axis, while, above 10^–6M, hydrolytic activity higher than that of treated embryonatedcotyledons was observed. Increasing the concentration of 6-BAhad no effect, however, upon the amylolytic activity of embryonatedcotyledons. Detached cotyledons showed an increased chlorophyllcontent and 6-BA treatment further increased this; the proportionsof chlorophyll a and chlorophyll b remained unaltered. An inhibitor of amylase activity, destroyed by heating, is presentin embryonated cotyledons and can depress amylolysis in detachedcotyledons which appear to be inhibitor-free. The results intotal suggest that the developing axis may regulate reservehydrolysis in three distinct manners: firstly, by stimulatingthe synthesis of amylase via hormonal control, most probablya cytokinin; secondly, by checking the rate of amylolysis bymeans of an amylase inhibitor; and, thirdly, by being a sinkfor the products of reserve degradation.     

胚轴对萌发豌豆子叶中淀粉酶活性表达的影响

萌发豌豆的上、下肢轴均能诱导子叶中淀粉酶活性,外源GA和6—BA具有类似胚轴的作用。离体子叶的淀粉酶凝胶电泳只有一条活性极低的酶带,连生子叶中有两条酶带,其中由胚轴诱导新出现了一条活性很高的同工酶带,它的活性受亚胺环己酮的强烈抑制,而受放线菌素D影响不大。推测豌豆子叶中存在淀粉酶的长寿命mRN—A,胚轴和外源激素的作用在于促进mRNA的翻译。     

Subcellular Localization of Sorbitol-6-phosphate Dehydrogenase in Protoplast from Apple Cotyledons

The subcellular localization of sorbitol-6-phosphate (S6P) dehydrogenasein protoplasts of apple cotyledons was examined by differentialcentrifugation and linear sucrose density gradient centrifugation(30–60%, w/w). The distribution of S6P dehydrogenase activitywas 55% in the 500 x g pellet of the homogenate and 35% in thesupernatant of 105,000 x g. When the x g pellet was recentrifugedin a linear sucrose density gradient, one major peak of activitywas found at a density of 1.23. This peak coincided with themajor peak of chlorophyll and NADP⁺-triose-P dehydrogenase activity.When the 500 x g pellet was sonicated, the major peak of S6Pdehydrogenase activity shifted to a lighter density (d=1.18).The shifted peak also coincided with the peak of chlorophyll.The enzyme detected in the major peak of chlorophyll (d=1.23)was partially solubilized by sonic or detergent treatment, butnot by hypotonic solution. The results supported the localizationof S6P dehydrogenase in chloroplasts, and presumably their associationwith thylakoid membranes. Part of the enzyme was assumed tobe naturally present in the cytosol, too. (Received November 4, 1980; Accepted January 21, 1981)     

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)     

The influence of the embryonic axis and cytokinins on reserve mobilization in germinating lupin seeds

The influence of the embryonic axis and cytokinins (CKs) onreserve mobilization has been examined in yellow lupin (Lupinusluteus L. cv. JSG 6167) seed during germination and during earlygrowth for up to 9 d in the dark. The study included determinationof starch, soluble sugars, proteins, and amino acid content.Amylolytic and proteolytic enzyme activity was also measuredin untreated cotyledons with intact embryo (attached) or detachedcotyledons (embryo removed), and in detached cotyledons followingtreatment with CKs namely, dihydrozeatin, (diH)Z, and 6-benzylaminopurine,BAP. Generally, the detached cotyledons showed reduced mobilizationand decreased enzymatic activity in comparison to attached cotyledons,indicating the importance of the embryonic axis in this process.However, a rise in protease activity and free amino acid contentwas detected in 9-d-old detached cotyledons suggesting thatthe end products do not necessarily inhibit enzyme activity.While (diH)Z was partially effective in inducing reserve mobilizationand enzymatic activity in detached cotyledons, the effect ofBAP was more pronounced and appeared to replace the embryonicaxis. The embryonic axis of this species has recently been shownto synthesize CKs which are transported to the cotyledons, arehighly stabe and induce cotyledon expansion and chlorophyllsynthesis. The results of the present investigation and previousstudies from this laboratory collectively indicate that theregulation of reserve mobilization in yellow lupin seeds appearsto be mediated, at least in part, by a stimulus, probably aCK, emanating from the embryonic axis. Key words: Lupinus luteus, cytokinins, benzylaminopurine, dihydrozeatin, embryonic axis, lupin seeds, reserve mobilization     

Gibberellin A3 reverses the effect of salt stress in chickpea (Cicer arietinum L.) seedlings by enhancing amylase activity and mobilization of starch in cotyledons

The percentage germination of chickpea seeds (Cicer arietinum L.cv. PBG-1) gradually decreased with increasing concentration of NaCl in the growth medium and was completely inhibited with 200 mM NaCl. In the presence of 75 mM NaCl, only 51% of the seeds germinated. Gibberellic acid (GA3) and kinetin at 6 µM concentration induced the maximum increase in % germination and seedling growth under salt stress. However, IAA further inhibited both the germination and growth of stressed seedlings. The reduction in amylase activity in cotyledons of stressed seedlings was partially reversed with GA3 and kinetin whereas IAA did not show any positive effect. GA3 was more effective than kinetin in enhancing the reduced germination and seedling growth of chickpea seeds along with amylase activity in cotyledons under NaCl induced saline conditions. The reduced uptake of radiolabelled 14C sucrose by cotyledons and its reduced distribution in the shoots and roots of stressed seedlings was increased with addition of GA3 in the medium. Cotyledonary amylase was separated into amylase 1 and amylase 2 by sephadex G 150 column chromatography. The reduced activities of both amylase 1 and amylase 2 in cotyledons under salt stress was returned to near normal levels with GA3 and there was also an increase in starch utilization, resulting in its lower concentration in cotyledons of GA3-supplemented stressed cotyledons.     

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     

Peroxidase and Glucose-6-phosphate Dehydrogenase Levels in Cotyledons of Cucumis sativus (L.)

Peroxidase and glucose-6-phosphate dehydrogenase become increasinglyactive in cucumber cotyledons excised from the plant althoughthere is a fall in total protein content. The increases areinhibited by dinitrophenol, D, and L-threo-chloramphenicol,cycloheximide, puromycinj and actinomycin D, and it is concludedthat the enzymes are synthesized de novo. Disc electrophoresisof cotyledon extracts revealed three bands of peroxidase, oneof which becomes much enhanced in excised cotyledons. Citrullineaccumulates markedly in excised cotyledons. If the shoot ofcucumber plants is cut away above the cotyledons, very largeincreases in peroxidase and glucose-6-phosphate dehydrogenaseare observed although yellowing and protein loss are delayed.     

Metabolic alterations in cotyledons of Cucurbita pepo infected by cucumber mosaic virus

Changes in the capacities of enzymes in various metabolic pathwayshave been measured during infection of cotyledons of Cucurbitapepo L. with cucumber mosaic virus (CMV). Starch accumulationand low sucrose content, which are characteristic of the earlystages of infection, are reversed in the later stages of infection.The decline in starch correlated with a reduced capacity forstarch synthesis (ADP-glucose pyrophosphorylase) and a risein the capacity for starch degradation (total starch hydrolase,starch phosphorylase). ¹⁴CO₂ feeding experiments, conductedat saturating CO₂ concentration, show that the newly-assimilatedcarbon was lost at a lower rate from infected cotyledons andless was incorporated into structural carbohydrates, phosphorylatedintermediates plus organic acids, more into soluble sugars,amino acids and proteins. At a later stage of infection therewere dramatic increases in respiratory capacity and a substantialalteration of carbohydrate metabolism. The infection had a largestimulatory effect on the capacity for oxidative pentose-phosphatepathway (glucose-6-phosphate dehydrogenase, 6-phospho-gluconatedehydrogenase), glycolysis (ATP- and pyrophosphate-dependentphosphofructokinases), tri-carboxylic acid cycle (isocitratedehydrogenase, fumarate hydratase), anaplerotic reactions (NAD-dependentmalic enzyme, phosphoenolpyruvate car-boxylase) and oxidativeelectron transport (cytochrome c oxidase). While there wereno overall changes in photosynthetic rate (measured in saturatingCO₂), infection either reduced (Rubisco and glycerate kinase)or did not affect (chloroplastic fructose bis-phosphatase andhydroxypyruvate kinase) the capacities of the photosyntheticcarbon reduction pathway or the photosynthetic carbon oxidationpathway. Key words: Plant-virus interaction, sucrose, starch, enzymes, ¹⁴CO₂ incorporation, O₂ flux     

Gibberellic acid and kinetin partially reverse the effect of water stress on germination and seedling growth in chickpea

The percent germination and seedling growth of chickpea (Cicer arietinum L. cv. PBG-1) decreased with increasing concentrations of exogenous polyethylene glycol 6000 (PEG). With 15% PEG in the growth medium germination was only 33% while with 10% PEG it was 58% as compared to 93% in control. Addition of gibberellic acid (GA₃) and kinetin to medium containing 10% PEG increased the germination and seedling growth and the effect was maximum with 6 µM GA₃ which was a better inducer of growth and germination under reduced water potential than kinetin. However, indole acetic acid (IAA) inhibited germination and growth of stressed seedlings. The activity of amylase in cotyledons under stress was significantly increased with GA₃ while kinetin and IAA were less effective. Gibberellic acid also enhanced the mobilization of starch from cotyledons of stressed seedlings resulting in low starch levels in cotyledons compared with stressed seedlings.     

Water stress enhances beta-amylase activity in cucumber cotyledons

Cotyledons detached from 4-d-old cucumber (Cucumis sativus L.) seedlings were subjected to water stress (air-drying or PEG-treatment) to examine the effects of the stress on carbohydrate metabolism. Amylolytic activity in the cotyledon was increased about 6-fold by water stress within 1 d. The substrate specificity and the action pattern indicated that beta-amylase is responsible for the activity. Activities of azocaseinase, malate dehydrogenase and triose-phosphate isomerase were not affected by water stress, indicating that the effect of the stress on beta-amylase is rather specific. Cycloheximide-treatment strongly reduced the enhancement of beta-amylase activity. The hypocotyl of cucumber seedlings also exhibited an increase in the enzyme activity when subjected to water stress. The major free sugars in cucumber cotyledons were glucose, fructose, maltose, and sucrose; sucrose being the most abundant. Sucrose content in excised, unstressed cotyledons increased markedly during the incubation. Changes in other free sugars were small compared with that of sucrose. Starch also accumulated in unstressed cotyledons. In stressed cotyledons more sucrose and less starch accumulated than in unstressed ones. Such results were discussed in relation to the enhancement of beta-amylase activity.     

Rhythmic Oscillations in Starch Concentration and Activities of Amylolytic Enzymes and Invertase in Medicago sativa Nodules

Alfalfa (Medicago sativa L.) nodule amylase, starch phosphorylaseand invertase activities and concentrations of starch and proteinwere determined every 4 h for 44 h to determine if daily fluctuationsoccur. Plants experienced 12 h light: 12 h dark (LD) duringentrainment and the first 24 h of the experiment. The last 20h were under continuous darkness (DD). Temperature (21°C)and relative humidity (72%) were always constant. Data wereanalyzed by the cosinor method to determine probabilities ofsinusoidal rhythms with periods between 12.0 and 30.0 h. UnderLD conditions, significant 12.1 h rhythms were found for activitiesof amylase and starch phosphorylase and for starch content.Rhythms in amylase activities and starch content were inverselycorrelated whereas rhythms of starch content and starch phosphorylaseactivity were positively correlated. These data indicate thatnodule starch can be rapidly turned over and that amylases maybe responsible for the degradation. None of these rhythms persistedunder DD conditions. In contrast, invertase activity was rhythmicunder LD (24 h period) and DD conditions (30 h period). No significantrhythmic variations were detected in protein levels throughoutthe entire experiment. (Received August 12, 1985; Accepted November 22, 1985)     

Vigna Radiata Seed Germination under Salinity

Salinity reduced mung bean (Vigna radiata Wilczek) radicle and root elongation, delayed and inhibited hypocotyl elongation and mobilization of reserves from the cotyledons to the embryo axis. Fresh and dry masses and water content of the embryo axes were reduced. Under salinity, a net leakage of K to the media increased with time and increasing NaCl concentrations. Sugars present in the cotyledons of seeds were of primary importance for growth of the embryo axis upto 18 h after sowing whereas breakdown of starch by amylase contributed later, the contribution being delayed and reduced with increasing NaCl concentration. Even when amylase activity in the cotyledons was progressively reduced with increasing NaCl concentration, the increasing contents of soluble sugars in the cotyledons indicated that sugars were not limiting for mung bean seedling growth under salinity.     

Histochemical studies on mobilization of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark at 27 C, and time-sequence changes of mobilization of protein and starch reserves in cotyledons were observed by histochemical techniques. The distributions of amylase and protease activities in cotyledon sections were also examined during germination by use of the starch-polyacrylamide gel film and India ink-gelatin film methods, respectively. Amylolytic and proteolytic processes occurred more or less simultaneously during the germination. At the day 2 stage, low levels of hydrolytic enzyme activities were observed throughout cotyledon sections. At day 4, both amylase and protease activities appeared to increase in tissue areas farthest from vascular bundles, and the mobilization of starch and protein reserves also proceeded in these areas. At day 6, the reserves were found to remain only in the cells around vascular bundles. When cotyledons were detached from axis organs, allowed to imbibe water and incubated for 4 days at 27 C, the breakdown of reserves was markedly retarded and the patterns of enzyme localization in cotyledon sections appeared not as conspicuous as those in the sections from intact cotyledons. These histochemical results are discussed with reference to the previous results ofin vitro experiments.     

Promotion of radish cotyledon enlargement and reducing sugar content by zeatin and red light

Effects of zeatin on amino acid and sugar contents of detached radish (Raphanus sativus L.) cotyledons were investigated to determine if accumulation of these solutes contributes to cytokinin-enhanced growth. Protein and amino acid levels were not significantly affected, but in cotyledons incubated in light the hormone caused greater accumulations of reducing sugars than occurred in light controls. Continuous fluorescent light or a few minutes of red light increased both the growth rate and the reducing sugar levels compared to dark controls. A far red treatment following red light overcame the promoting effect of the latter. Amounts of reducing sugars were closely associated with growth under the above conditions. Activity of an unidentified amylase was elevated by continuous light or a red light treatment (nullifiable by far red), suggesting that reducing sugars were derived from starch. Zeatin-treated cotyledons exhibited less amylase activity than did light controls, perhaps implicating cytokinin-stimulated conversion of fats to sugars in light. In darkness zeatin promoted cotyledon growth but did not increase sugar levels nor amylase activity, suggesting that enhanced ion accumulation also contributes to the more rapid water uptake leading to growth.     

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     

Effects of Sucrose on Petiolar Carbohydrate Accumulation and Photosynthesis in Excised Sinapis Cotyledons

Petioles of Sinapis cotyledons cultured in 6x10^–2 M sucrosein the light increased 28-fold in total carbohydrate contentover a 7-d period compared with an 8-fold increase in petiolesof cotyledons cultured on water. The starch and reducing sugarfractions were the major components of this accumulation. Labelledsucrose applied to the petiole base moved quickly up the petioleand into the main veins of the lamina. Some basipetal redistributionoccurred subsequently and after 24 h radioactivity accumulatedstrongly at the petiole base. Culture in sucrose reduced basalaccumulation and increased acropetal movement of the label. Fixation of ¹⁴CO₂ by petioles remained constant when cotyledonswere cultured in water, whereas in sucrose, fixation fell by50 per cent during the first 2 d. The pattern of inhibitionof fixation matched the pattern of sucrose distribution in thepetiole. Petiolar chlorophyll content remained constant in controlsbut fell rapidly after 4 d culture in sucrose. The results are discussed in relation to the role of petiolarcarbohydrate accumulation in the regulation of CO₂ fixation,primordium development, and senescence in this system.