Giovanna P. Longo

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.

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 μM 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.

Variations in carbohydrate and lipid content and in osmotic potential of watermelon cotyledons treated with benzyladenine

Abstract Benzyladenine (BA)-stimulated growth of excised watermelon cotyledons is accompanied by a stimulation of the conversion of reserve lipids to soluble sugars. After the 3rd day of development most of the carbohydrate is found as starch in the controls and as soluble sugars in treated cotyledons. As a result of these two effects the amount of soluble sugars per cotyledon is always higher in BA-treated cotyledons. Osmotic potential (measured as variation in fresh weight in presence of mannitol solutions or as osmolarity of cell sap) is, however, always lower in BA-treated cotyledons. These data suggest that BA increases the capacity of water uptake not only by build-up of solutes, but also by a direct influence on cell walls.

Evidence for two classes of responses of watermelon cotyledons to benzyladenine

Abstract Watermelon ( Citrullus vulgaris Schrad.) cotyledons were excised after 24 h of imbibition and cultured for 5 days on filter paper saturated with benzyladenine (BA) solution at concentrations ranging from 10 −8 to 10 −4 M. Alternatively the cotyledons were exposed to 10 −4 M BA for 5 min or 4 h and then cultured in water. We examined the effects of these treatments on several parameters: growth, carotenoid level, lipid disappearance and representative enzymes for plastids, mitochondria and microbodies. Two groups of responses to BA could be distinguished. Growth and mitochondrial enzyme levels showed a dose-response curve with maximal stimulation at the highest concentration tested (10 −4 M). A 5-min treatment with 10 −4 M BA yielded a very small response. Other responses (increase in carotenoids and in marker enzymes for plastids and microbodies) showed a sharp optimum at 10 −5 M concentration in the dose-response curve. A 5-min treatment with 10 −4 M BA was sufficient to yield a large response. Irradiation of the cotyledons with white light mimicked several responses to BA belonging to the second group, but none belonging to the first group.

Benzyladenine stimulates the development of mitochondria in watermelon cotyledons

Abstract The development of mitochondria was followed for 5 days in watermelon cotyledons excised from the seedling 24 h after soaking and cultured in the dark on filter paper saturated with water or with a 10−4 M benzyladenine (BA) solution. Treatment with BA increases the total mass of mitochondria as shown by electron microscopy and by measurements of mitochondrial protein after density gradient centrifugation. Enzyme levels are increased to the same extent as total mitochondrial protein so that specific activities in mitochondria from treated cotyledons are the same as those in mitochondria from water controls. Only the specific activity of cytochrome oxidase and the specific content of cytochrome a 3 are higher in mitochondria of BA-treated cotyledons. Mitochondria isolated from cotyledons treated with BA seem to be in a better state of preservation than those isolated from water controls.

Expansion growth is not the main driving force for cytokinin-promoted development of cotyledons

Abstract Excised watermelon cotyledons were treated with benzyladenine (BA) or fusicoccin (FC), both at 10 −5 M concentration. The effects of the two growth regulators were compared by monitoring the time course of the following parameters: growth (increase in fresh wt.), carotenoid and lipid content and the activities of cytochrome oxidase and hydroxypyruvate reductase (HPR). The treatment with FC induces a large growth response, of the same order of magnitude as that observed with BA. All other developmental responses that are strongly stimulated by BA are little or not-at-all affected by FC. These results indicate that growth by itself is not sufficient to entrain the other developmental responses induced by BA. On the other hand BA can partially enhance carotenoid content and HPR activity even when expansion growth is completely suppressed by osmotic stress.

Effect of benzyladenine on proteolytic activity of excised watermelon cotyledons

Abstract Excised watermelon cotyledons were grown in presence or absence of 10−4 M benzyladenine (BA). The time course of proteolytic activity was assayed with hemoglobin and azocasein. Total protease activity shows a large peak at pH 4.4 (with hemoglobin) or 5.4 (with azocasein) and a smaller one at pH 8.5. BA does not enhance proteolytic activity. On the other hand electron microscopical data show an accelerated breakdown of protein bodies in BA-treated cotyledons. BA increases autolytic activity (release of free aminoacids without exogenous substrate) in a 17 500 × g pellet and in a band that sediments to equilibrium at 1.28–1.30 g/cm3 on a sucrose gradient. This band is most probably formed by protein body fragments. It is likely that BA enhances the action of proteases that are sequestered inside protein bodies and are inaccessible to exogenous substrates.

Malate dehydrogenase from maizes cutellum glyoxysomes I. localization within the organelle

Abstract Lysis of maize scutellum glyoxysomes by sonication or dilution with buffer at pH 9 releases 60–70% of the total malate dehydrogenase (MDH) activity. The fraction of MDH that remains bound to the glyoxysome ghosts after lysis is strongly activated by Triton X-100 and salts. The solubilized enzyme is not significantly activated. MDH that remains associated with the glyoxysome ghosts is partly bound to the membrane and partly present as a high molecular weight aggregate. Gel filtration on Sephadex inactivates glyoxysomal MDH, but not the mitochondrial and the cytoplasmic enzyme. This inactivation can be partially reversed by addition of salts.

Development of mitochondrial enzyme activities in germinating maize scutellum

Abstract We have checked the time course of specific activities of several mitochondrial enzymes in maize scutellum during the first 6 days of germination. The activities were measured in organelles purified on sucrose gradients. All of the enzymes studied show a peak of activity on the 5th day followed by a very rapid decline during the subsequent 12 h, the only exception being citrate synthase (CS) which peaks on the 4th day. Particulate β-oxidation activity is present in mitochondria as well as in glyoxysomes of maize scutellum. Glyoxysomes have higher specific activities, but total activities (per organ or per g fresh weigth) are of the same order of magnitude as in mitochondria. The β-oxidation activity of mitochondria is not likely to result from glyoxysome contamination. β-Oxidation reaches a maximum value on the 3rd day of germination in mitochondria and on the 4th day in glyoxysomes, 1 day before the peak of acetyl-CoA-utilizing enzymes in the respective organelles (CS in mitochondria and citrate synthase + malate synthase in glyoxysomes).

Interaction between benzyladenine and fusicoccin on the development of excised watermelon cotyledons. II. Correlation between growth and proton extrusion

Abstract Treatment of excised watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons with 10−5 M fusicoccin (FC) induces stimulation of growth and a marked decrease in pH of the external medium. Treatment of the same cotyledons with 10−5 M benzyladenine (BA) also stimulates growth, but does not induce a decrease in pH. When BA and FC are administered together the growth rate is higher than with either of the two regulators alone, but the decrease in pH is slightly smaller than with FC alone. The accompanying paper, Fantelli et al., Plant Sci. Lett., 33 (1984) 267, reports that a 2-day preincubation in 10-5 M BA prevents the inhibitory effect of FC on BA-induced organelle development. We have measured pH changes of the external medium under these experimental conditions. After transfer to FC the pH decreases for 8 h as observed in absence of preincubation with BA, but then increases again, while without preincubation the pH of the medium continues to decrease for an indefinite time. The results suggest that cytokinin-stimulated cotyledon expansion does not involve the continued operation of a proton pump, thus confirming the data obtained by Ross and Rayle, Plant Physiol., 70 (1982) 1470, with cucumber cotyledons. The activation of the pump by FC probably causes an imbalance in ion transport that blocks BA-stimulated organelle development. The preincubation in BA may help to keep the proton pump under control.