Claudio 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.

Interaction between benzyladenine and white light on excised watermelon cotyledons

Abstract We tested whether some typical responses of watermelon (Citrullus vulgaris Schrad.) cotyledons to benzyladenine (BA) that had been previously studied in the dark are modified by continuous exposure to white light. The responses tested were increase in fresh weight and in the levels of several enzymes related to the development of plastids, glyoxysomes and peroxisomes. Three different types of interactions between BA and light are described. (a) Increase in fresh weight: light does not have any effect and does not modify the response to BA; (b) isocitrate lyase (peak level): light and BA both increase enzyme activity, but light does not enhance further on the response if BA concentration is at a saturating level; (c) enzymes of plastids and peroxisomes: light and BA are both active and interact following a multiplicative pattern. The multiplicative interaction common to all plastid and peroxisome-linked responses suggests that the development of the two types of organelles is coordinated, but that the mechanisms of regulation by BA and light are independent from each other although some step may be shared.

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.

Purification and properties of malate synthetase from maize scutella

Abstract Malate synthetase was purified over 200-fold from maize scutella. Catalase remains associated with malate synthetase during most of the purification procedure. The two enzymes can be separated by gel filtration on Sepharose 6B. The molecular weight of malate synthetase as estimated by gel filtration and density gradient sedimentation is approximately 500 000. 5.6 m M ATP causes a 57.5% inhibition of the enzymatic activity. The inhibition is competitive with respect to acetylcoenzyme A. ADP and AMP have a smaller inhibitory effect.

Sexual differentiation in Asparagus officinalis L. III. Hormonal content and peroxidase isoenzymes in female and male plants.

SummaryUsing an immunological method we assayed the levels of auxin, abscisic acid and three cytokinins (transzeatin riboside, dihydrozeatin riboside, isopentenyladenosine) in flowers of female and male plants of Asparagus officinalis L. at different stages of development. The largest differences between the sexes were found for auxin: auxin content was found to be about three times higher in young male flowers than in female flowers at a corresponding developmental stage. In order to identify some of the biochemical markers linked to sex differentiation, we also examined peroxidase isoenzyme patterns during flower development. We found five flower-specific peroxidase bands, three of which appear to be localized in the anthers. In young flowers still sexually undifferentiated in their morphology these bands are present in both sexes. They subsequently rapidly disappear in the female flower (approximately at the same time as when anther development is blocked), while they persist for a much longer time in the male. The temporary presence of these peroxidase isoenzymes in female young flowers together with the large difference in auxin content indicate that the stage of the young flower is a crucial moment in the process of sex determination.

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.

The effect of benzyladenine on anthocyanin accumulation in excised sunflower cotyledons

Abstract We have studied the effect of some growth factors on the increase in fresh weight and anthocyanin content of isolated sunflower cotyledons. In the dark, benzyladenine (BA), kinetin (K) and fusicoccin (FC) stimulate the increase in fresh weight with respect to the water control. BA and K also stimulate the accumulation of anthocyanins in the dark while FC completely inhibits it. Indole-3-acetic acid (IAA) has no effect on either response while abscisic acid (ABA) inhibits growth and is without effect on anthocyanin synthesis. Results of studies on the interaction between different growth regulators are consistent with the hypothesis that the effects of BA on growth and on anthocyanin accumulation are not linked by a common step.