Uptake of Benzyladenine by Excised Watermelon Cotyledons

Abstract

The uptake of 8-14C1N'-benzyladenine (BA) was studied in excised watermelon (Citrulls 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 (ie. 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.Although the action of exogenous cytokinins on excised plant organs is well documented (6,8,9) much less is known about their absorption. Studies on cytokinin uptake have been done with cultured cells (3) and with moss protonemata (5), but comparatively little work has been done with whole organs from higher plants (2, 10). Some interesting data about uptake of BA in Amaranthus half-seedlings have appeared in a recent report (4), but they were not discussed because cytokinin absorption was not the main issue of the paper.Earlier work done in our laboratory showed that treatments with lO-' M BA for as short as 1 min are sufficient to evoke longlasting responses in watermelon cotyledons (8). These results suggested that cotyledons are able to take up BA quite efficiently from the external medium. In this paper we present some characteristics of BA uptake and release in isolated cotyledons and some data on the relationship between total amount of BA taken up and magnitude of the hormonal response.MATERIALS AND METHODS Plant Material. Watermelon (Citrullus vulgaris Schrad.) seeds were bought from SRS seeds, Modesto, CA. The cotyledons were excised from the embryonic axis after 24 h of imbibition at 27 C in the dark (day zero) and immediately used for the uptake tests.Uptake of Labeled BA. Batches of 20 cotyledons were placed in a small Petri dish (50 mm diameter) containing one filter-paper disc imbibed with 1 ml 8-[4C]BA solution (1.34,Ci/,tmol) at l-4 M concentration. After an appropriate time of incubation in darkness at 27 C or 2 C the cotyledons were blotted dry, thoroughly rinsed with distilled H20 as described (8) and extracted in boiling 80%1o ethanol (v/v) for 1 h. For each uptake experiment we used 4 batches of 5 cotyledons that were extracted separately with 5 ml ethanol. One ml of the ethanol used for extraction was added to 10 ml Luma Gel in a scintillation vial. The radioactivity was determined in a Packard Tri-Carb scintillation counter with 78% efficiency. SD between replicate samples did not...

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