Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons.

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 microM benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.

Uptake and effects of N-benzyladenine in excised watermelon cotyledons: influence of cotyledon age.

Some characteristics of uptake of [8-(14)C]N(6)-benzyladenine (BA) by watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either excised immediately after 24 hours inhibition (day 0) or cultured in the dark for 48 hours on moist filter paper (day 2) have been compared.The uptake of BA seems to be passive in cotyledons of both kinds. The initial rate of uptake is, however, much slower in day 2 cotyledons. This is probably due to a higher resistance of cell membranes to BA influx. When the day 2 cotyledons are frozen and thawed, so that the membrane barrier is abolished, the amount of BA taken up is the same as in day 0 cotyledons.In spite of the lower rate of uptake, the physiological effects of BA in day 2 cotyledons are as strong as in day 0 cotyledons and occur with a shorter lag time. Sensitivity to BA seems indeed to start 24 to 48 hours after excision.

Uptake of benzyladenine by excised watermelon cotyledons.

The uptake of 8-[(14)C]N(6)-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.There seems to be a correlation between the level of radioactivity (i.e. of BA + derivatives) present in the cotyledons and the magnitude of hormonal responses that are observed four days after uptake. This relationship holds regardless of whether a given level of radioactivity has been reached after a short period of uptake or after a long period of uptake followed by washing.

Effect of benzyladenine on the development of plastids and microbodies in excised watermelon cotyledons.

Excised watermelon (Citrullus vulgaris Schrad.) cotyledons were grown in the dark in the presence of 0.1 mM benzyladenine (BA). Under these conditions reserve breakdown and organelle differentiation progress very slowly. Treatment with BA accelerates, breakdown of reserves and stimulates development of organelles. Electron micrographs of cells from treated cotyledons show a larger number of plastids with a more developed inner membrane system. The levels of plastid pigments and enzymes are increased while starch content is reduced. Glyoxysomal enzyme levels are increased by BA during the first three days of development and their decline is accelerated thereafter. Also the activity of hydroxypyruvate reductase (EC 1.1.1.81.), a peroxisomal enzyme, is increased, but this increase is not followed by a decay phase. In water controls, hydroxypyruvate reductase bands together with glyoxysomal enzymes after equilibrium centrifugation in a sucrose gradient. In treated cotyledons the equilibrium position of glyoxysomal enzymes is uchanged while that of hydroxypyruvate reductase is shifted to a lower density.

Effect of benzyladenine on some enzymes of mitochondria and microbodies in excised sunflower cotyledons.

Benzyladenine (BA) increases the rate of expansion of dark-grown sunflower (Helianthus annuus L.) cotyledons. The hormone slightly enhances the development of the two glyoxysomal enzymes, isocitrate lyase and malate synthetase, during the first 3 days of germination and greatly accelerates their decay in the 2 following days. The levels of the peroxisomal enzymes, glycolate oxidase and glyoxylate reductase, are enhanced by BA more than those of the two glyoxysomal enzymes. These effects of BA on microbody enzymes are very similar to those of white light. Mitochondrial enzyme activities are increased to a varying extent by BA: the increase is minimal for fumarase, and maximal for cytochrome oxidase. The level of cytochrome oxidase is enhanced 346% at the 5th day of germination. Also, the rate of O(2) consumption is increased by BA, but the time course of this increased O(2) consumption does not match with that of cytochrome oxidase. Fusicoccin, a fungal toxin, mimics the effect of BA on cotyledon expansion, but fails to duplicate its action on microbody enzymes. This suggests that the effect of BA on microbody enzymes is not closely linked with the mechanism of growth promotion.

Solubilization of enzymes from glyoxysomes of maize scutellum.

Glyoxysomes isolated from maize scutella (Zea mays L.) were subjected to several disruptive treatments (osmotic shock, resuspension in an alkaline medium, addition of detergent). The damaged glyoxysomes were centrifuged at 89,500g for 40 minutes and several enzymic activities (isocitratase, malate synthetase, catalase, citrate synthetase, malate dehydrogenase) were measured in the supernatant fraction and in the pellet. Isocitratase is the most easily released of all glyoxysomal enzymes closely followed by malate synthetase. Citrate synthetase is in all instances the most insoluble enzyme. All of the enzymes had higher specific activities in the supernatant than in the pellet. These findings suggest that in corn scutellum glyoxysomes none of these enzymes is truly membrane-bound.

Citrate synthetase in mitochondria and glyoxysomes of maize scutellum.

Mitochondria and glyoxysomes were isolated from scutella of maize (Zea mays L.) by density gradient centrifugation. Citrate synthetase was partly solubilized from the organelles by sonication. The sonicated organelle suspensions were centrifuged at high speed, and the supernatants were used as enzyme preparations without further purification. The enzymes of the two organelles differ in all properties examined (pH activity curve, Km for substrates, elution volume on Sephadex G-100, mobility on starch gel at pH 7). Both enzymes are inhibited by ATP, but the inhibition is stronger for the mitochondrial enzyme. The inhibition is competitive for the mitochondrial enzyme and noncompetitive for the glyoxysomal enzyme. The glyoxysomal, but not the mitochondrial enzyme, is inhibited 40% with 1 mm ADP and cytidine triphosphate.

Cytochemical localization of catalase in glyoxysomes isolated from maize scutella.

The localization of catalase in isolated maize scutellum glyoxysomes was investigated by means of the diaminobenzidine histochemical reaction. Only the membranes of the glyoxysomes become heavily stained after incubation with diaminobenzidine and H(2)O(2). If the glyoxysomes are lysed with Tricine buffer at pH 9, 70% of the catalase is solubilized, while the remaining 30% is tightly bound to an insoluble fraction formed mostly by glyoxysomal membranes. This suggests that catalase may be present also in the matrix of the glyoxysomes. The lack of staining of the matrix with diaminobenzidine is probably due to the high concentration of catalase in the membranes of the organelles.

The development of glyoxysomes in maize scutellum: changes in morphology and enzyme compartmentation.

Changes in the ultrastructural aspect of the glyoxysome fraction obtained from maize scutella by density gradient centrifugation were followed during the first 6 days of germination. During the first 2 days the fraction consists of very electron-dense bodies about 0.3 to 0.5 micron in size while at the 4th day it is formed by larger and less dense membrane-bound particles. Some intermediate form between the two types of organelles can be seen at the 3rd day. Between the 4th and the 6th days of germination the glyoxysomes are destroyed, and their enzymes are released into the cytosol. At the peak of their development (4th day) the glyoxysomes contain 75 to 80% of the total isocitratase and 65% of the total malate synthetase of the scutellum. These values drop to very low levels during the next 2 days. Catalase bound to glyoxysomes amounts to 30 to 35% of the total activity present in the scutellum at the 1st day of germination: this value decreases steadily during the following days.

The development of glyoxysomes in peanut cotyledons and maize scutella.

The development of glyoxysomes during germination has been studied in isolated peanut (Arachis hypogaea L.) cotyledons and in maize (Zea mays L.) scutella. In peanut cotyledons isocitratase, malate synthetase, and protein associated with the glyoxysomal fraction increase simultaneously from the 3rd to the 8th day of incubation. In scutella of germinating maize seeds the specific activities of isocitratase, malate synthetase, and catalase associated with the glyoxysomes rise until the 4th day of germination and then decline while the total amount of protein present in the fraction stays constant during the first 5 days. If the peanut cotyledons are cultured in 2% glucose, the development of isocitratase and malate synthetase is severely inhibited, but the level of the glyoxysomal protein is not measurably affected.

Evidence for de novo synthesis of isocitratase and malate synthesis in germinating peanut cotyledons.

Evidence for de novo synthesis of isocitratase and malate synthetase in cotyledons of germinating peanut (Arachis hypogaea L.) was obtained by the density labeling method. When dry peanut cotyledons were cultured in H(2) (18)O, a 2.4% increase in the buoyant density of malate synthetase in a cesium chloride gradient was observed. In 100% D(2)O the buoyant density shift was 5.5% for isocitratase and 3.5% for malate synthetase in comparison to the water controls. These data suggest that isocitratase and malate synthetase do not pre-exist in some inactive form in the cotyledons, but are completely synthesized after onset of germination from a pool of amino acids which do not derive directly from hydrolysis of storage proteins.