Interaction of Kinetin and Calcium in Relation to Their Effect on Stimulation of Ethylene Production

Abstract

Application of kinetin and Ca2+ caused a striking synergistic increase in ethylene production by mung bean (Phaseolus aureus Roxb) hypocotyl segments. The effect of kinetin on Ca2+ uptake and of Ca'+ on the uptake and metabolism of kinetin in relation to their effect on ethylene production was studied. Tracer experiments showed that kinetin greatly increased the uptake of 4"Ca2' after 6 hours of incubation. Reciprocallv, Ca2+ stimulated the uptake of kinetin-8-14C and remarkably enhanced the metabolism of kinetin-8-14C into several polar metabolites. Consequently, the quantity of free kinetin-8-'4C remaining in Ca2+-treated segments was much less than in control segments. A possible mechanism accounting for the synergism between kinetin and calcium on ethylene production is discussed.We have previously reported a large synergistic increase in ethylene production by mung bean (Phaseoluts aureus Roxb) hypocotyl segments when kinetin and Ca2+ were applied (12). The amount of ethylene produced was dependent on the concentrations of kinetin and Ca2+ ion applied. Analysis of the synergistic effect of kinetin and Ca2+ on ethylene production required examination of the effect of kinetin on the uptake of Ca>2 and the reciprocal effect of Ca+ on the uptake and metabolism of kinetin. This paper describes the stimulatory effect of kinetin on Ca2+ uptake and of Ca>2 on kinetin uptake and metabolism. A possible mechanism accounting for the synergism between kinetin and Ca2+ ion on ethylene production is discussed. MATERIALS AND METHODSSeeds of mung bean (Phaseolus aureus Roxb) were grown in vermiculite for 3.5 days in darkness at 24 C. Segments 2 cm long were cut from hypocotyls at a point 1 cm below the hook, as previously described (11). Lots of 20 segments were incubated in 5 ml of a medium consisting of 50 mM potassium phosphate buffer (pH 6), 2% sucrose, and a given concentration of labeled and unlabeled kinetin or Ca2+, as indicated, in a 50-ml Erlenmeyer flask. A plastic centerwell containing 0.2 ml of 40% KOH was hung in the flask to absorb the CO2 evolved. The flasks were sealed with rubber serum caps and incubated in a shaker at 27 C in darkness.At the time intervals indicated, 1 -ml gas samples were 1 This work was supported by National Science Foundation Grant GB-33907X.withdrawn by hypodermic syringe, and ethylene was assayed with a gas chromatograph equipped with an alumina column and a flame ionization detector. The flask was then flushed with air and recapped for the next determination.For uptake studies, the hypocotyls incubated with 4"Ca'+ (104 /Ci, 41.5 ,umoles) were washed with five changes of distilled H20. The tissue was then ground in a glass homogenizer in 4 ml of 95% ethanol. The debris was pelleted by centrifugation and the supernatant was collected. The pellet was serially extracted with 2 ml of H20; 2 ml, 3 ml, and 5 ml of 10 mM HCl; and lastly with 5 ml of 0.5 mM EDTA. The radioactivity of the incubation medium, of each extract, and of the debris was determined with a liquid scintillation ...

Stimulation of Ethylene Production in the Mung Bean Hypocotyls by Cupric Ion, Calcium Ion, and Kinetin

“…During the course of the study of the synergistic stimulation of ethylene by kinetin and Ca2 , we found that kinetin greatly enhanced the uptake of Ca2+ into the tissue, with a lag period of about 5 hr, which corresponds to the time required for induction of ethylene production (15). Our results are compatible with the view that kinetin plays a role by releasing and transporting Ca2+ from cell wall to an intracellular site where Ca2+ is required for ethylene biosynthesis.…”

“…We have found that kinetin greatly enhances the uptake of Ca2+ with a lag period of 6 hr which corresponds to the time required for induction of ethylene production. We suggested that the synergism between kinetin and Ca2+ on ethylene production is due to the enhanced intake of Ca2+ by kinetin into a specific site for ethylene production (15 Figure 1, the pattern of ethylene production in the Sr2+ plus kinetin system was identical to the Ca2+ plus kinetin system, indicating that Sr2+ can substitute for Ca2+ in the present system. The magnitude of ethylene production in response to Sr2+ was dependent on Sr2+ concentration (Fig.…”

“…We have shown that kinetin enhanced the uptake of 45Ca2+, and that the increase in uptake was paralleled by an increase in ethylene production (15). We have further compared kinetinenhanced ethylene production by mung bean seedlings which had been germinated in the presence of different levels of Ca2 , and found greater ethylene production by high Ca2+ seedlings than by low Ca2+ seedlings (unpublished results).…”

“…It is therefore pertinent to ask whether the synergistic stimulation of ethylene production by Ca2+ and Cu2+ may be due to the enhanced uptake of Ca2+ by Cu2+. To examine this possibility, we studied the effect of Cu2+ on the uptake of 45Ca2+, using the same technique as we previously used for the kinetin plus Ca2+ system (15). As shown in Figure 5A, Cu2+ (14).…”

Calcium: A Central Regulator of Plant Growth and Development

“…Yet other studies identified a Ca 21 / cytokinin/ethylene connection, although there was disagreement between published reports on the nature of the interaction. Lau and Yang (1975), in studies on mung bean hypocotyl segments, reported that kinetin greatly stimulated uptake of 45 Ca 21 , and Ca 21 stimulated the uptake and metabolism of kinetin 14 C. Of particular note, both Ca 21 and kinetin caused a striking increase in ethylene (Lau and Yang, 1975). By contrast, Poovaiah and Leopold (1973a), in studies of leaf abscission in bean petiole explants, found that Ca 21 inhibited ethylene production.…”