The effect of Co2+ on ethylene production by mung bean (Phaseolus aureus Roxb.) and by apple tissues was studied. Co2+, depending on concentrations applied, effectively inhibited ethylene production by both tissues. It also strongly inhibited the ethylene production induced by IAA, kinetin, IAA plus kinetin, Ca2+, kinetin plus Ca22, or Cu2+ treatments in mung bean hypocotyl segments. While Co2+ greatly inhibited ethylene production, it had little effect on the respiration of apple tissue, indicating that Co2+ does not exert its inhibitory effect as a general metabolic inhibitor. Ni2+, which belongs to the same group as Co2+ in the periodic table, also markedly curtailed both the basal and the induced ethylene production by apple and mung bean hypocotyl tissues.In a system in which kinetin and Ca2+ were applied together, kinetin greatly enhanced Ca2+ uptake, thus enhancing ethylene production. Co2+, however, slightly inhibited the uptake of Ca2+ but appreciably inhibited ethylene production, either in the presence or in the absence of kinetin. Tracer experiments using apple tissue indicated that Co2+ strongly inhibited the in vivo conversion of L-IU_'4Clmethionine to 14C_ ethylene. These data suggest that Co2+ inhibited ethylene production by inhibiting the conversion of methionine to ethylene, a common step which is required for ethylene formation by higher plants.Co2+ is known to promote elongation, leaf expansion, and hook opening in excised plant parts in response to applied auxins or cytokinins. Since ethylene is known to inhibit these growth phenomena, it is suggested that Co2+ exerts its promotive effect, at least in part, by inhibiting ethylene formation.We have previously reported that Ca2+ or Sr2+ synergistically stimulated ethylene production in the presence of kinetin, whereas Co2+ or Ni2+, in contrast, greatly inhibited ethylene production in the presence or in the absence of kinetin (16, 18). As ethylene is endogenously produced by ripening fruit tissues (1, 3, 4, 5) and as its production in vegetative tissues can be regulated by the application of auxin (1,7,11,14,19,20), kinetin (1,15,16,19), auxin plus kinetin (1,15,19), Ca2+ (16,17), kinetin plus Ca2+ (16, 17), or Cu2+ (1,2,18), it is important to determine whether Co2+ also inhibits these ethyleneproducing systems. Evidence from tracer studies has established methionine as the in vivo precursor of ethylene in fruit and vegetative tissues (1,3,4,22,24,29). This paper describes the inhibitory effect of Co2+ on ethylene production by treated ness at 24 C. Segments 2 cm long were cut from hypocotyls at a point 1 cm below the hook, as previously described (16). Lots of 20 segments were incubated in 5 ml of a medium consisting of 50 mm potassium phosphate buffer (pH 6), 2% sucrose, various concentrations of Co2+, Ni2+, Ca2+, Cu2+, IAA, or kinetin as indicated, in a 50-ml Erlenmeyer flask. A plastic center well containing 0.2 ml of 40% KOH was hung in the flask to absorb CO2 evolved. The flasks were sealed with rubber serum-caps, and incubated...

In hypocotyl segments of mung bean (Phaseolus mungo L.) seedlings, exogenously supplied indoleacetic acid was rapidly conjugated mainly into indoleacetylaspartic acid, which was inactive in inducing ethylene production. Kinetin is known to stimulate indoleacetic acid-induced ethylene production. The mechanism of kinetin action on indoleacetic acid-induced ethylene production by hypocotyl segments of mung bean seedlings was studied in relation to indoleacetic acid uptake and indoleacetic acid metabolism. Kinetin enhanced indoleacetic acid uptake during the initial 2-hour incubation and markedly suppressed the conversion of indoleacetic acid to indoleacetic acid conjugates throughout the whole 7-hour incubation. As a result, there was more free indoleacetic acid and less conjugated indoleacetic acid in the segments treated with kinetin than in those receiving no kinetin. A close relationship was demonstrated between the rate of ethylene production and the level of free indoleacetic acid, which was regulated by kinetin.It is well known that auxins stimulate ethylene production in a wide variety of plant tissues (1-5, 8, 9, 11, 14, 20), and many of the effects of auxin are now attributed to its effects on ethylene production (13,20). Induction of new enzymes is suggested to be involved in this process, based on the observation that inhibitors of protein and RNA synthesis retard auxininduced ethylene production and that the stimulation of ethylene production by auxin has a substantial lag period (1,3,14). Apparently, the ethylene-producing system induced by auxin is labile and is characterized by a rapid turnover rate (9,14).In excised segments of etiolated pea shoots, Kang et al. (9) and others (2,4,5) have shown that IAA-induced ethylene production parallels the free IAA level, which in turn inversely depends upon the rate of IAA conjugation and decarboxylation.Kinetin alone slightly stimulates ethylene production by etiolated seedlings of several species, but a remarkable synergistic effect of kinetin on IAA-induced ethylene production has been observed (4,8,10 This study was undertaken to determine whether kinetin plays a causal role in the uptake and metabolism of IAA. The present paper presents evidence showing that in mung bean hypocotyl segments kinetin suppresses the conversion of IAA into IAA conjugates, the main pathway of IAA metabolism. As a consequence, a higher free IAA level is sustained and is, in turn, responsible for higher ethylene production. MATERIALS AND METHODSPlant Materials and Chemicals. Seeds of mung bean (Phaseolus mungo L.) purchased from a local market were sorted and surface-sterilized with 0.02% sodium hypochlorite solution for 10 min. After thorough washing, the seeds were imbibed and aerated for 12 hr and were then grown in vermiculite for 4.5 days in darkness at 24 C. Under dim green light, 2 cm long segments were cut from hypocotyls at a point 1 cm below the hook. Twenty segments were incubated in 5 ml of incubation medium containing 50 mM KH,PO4 (pH 4.5) and 2% sucrose in...

A slight increase in ethylene production resulted from the application of either kinetin or Ca(2+) to mungbean (Phaseolus mungo L.) hypocotyl segments, but a remarkable synergistic increase in C2H4 production was observed when they were applied together. The induction time was about 6 h as compared to 1 h for auxin-induced C2H4 production. A slight stimulation of C2H4 production was also observed when Ca(2+) was applied with abscisic acid, but no synergistic effect of Ca(2+) was observed with indole-3-acetic acid or GA3.

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 ...

The synergistic stimulation of ethylene production by kinetin and Ca2l in hypocotyl segments of mung bean (Phaseolus aureus Roxb.) seedling was further studied. The requirement for Ca2l in this system was specific. Except for Sr", which mimicked the effect of Ca2", none of the following divalent cations, including Ba2+, Mg6", Cu2l, Hg2+, Co2+, Ni2+, Sn2+, and Zn2+, showed synergism with kinetin on ethylene production. Fe2+, however, showed a slight synergism with kinetin. Some of them (Hg2+, Co2l, and Ni2+) had a strong inhibitory effect, while others (Zn2+, Mg2+, Sn2+, and Ba2l) had a slight or no inhibitory effect on ethylene production in the absence or presence of kinetin.Cu2+ alone, depending on the concentration applied, stimulated ethylene production with a lag period of about 2 hours and had no synergism with kinetin on ethylene production. When Cu2+ was applied with Ca2l, a remarkable synergistic stimulation of ethylene production was observed.Tracer experiments indicated that Cu2l enhanced the uptake of 45Ca2+ into the tissues during the first few hours of incubation, and this increase of 45Ca2+ uptake paralleled the enhancement of ethylene production. When Ca2+ was applied together with kinetin plus Cu2+, both the ethylene production and the 45Ca2+ uptake were greatly increased over those from the segments treated with Cu2l or kinetin alone. The increase in ethylene production as a result of kinetin plus Ca2l plus Cu2+ treatment is equal to the combined increases caused by kinetin plus Ca2l and Cu2l plus Ca2l. A possible mechanism accounting for such cooperative effects of Cu2+, Ca2l, and kinetin 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 I cm below the hook, as previously described (14). Lots of 20 segments were incubated in 5 ml of a medium consisting of 50 mm potassium phosphate buffer, pH 6, 2% sucrose, various concentrations of different divalent cations, kinetin, or labeled 45Ca2+ (100 ,uCi, 50 ,umoles) as indicated, in a 50-mi Erlenmeyer flask. A plastic center well containing 0.2 ml of 40% KOH was hung in the flask to absorb CO2 evolved. The flasks were sealed with rubber serum caps and incubated in a shaker at 27 C in darkness.At time intervals indicated, 1-ml gas samples were withdrawn by hypodermic syringe, and ethylene was assayed with a gas chromatograph equipped with an alumina column and a flame ionization detector. The flasks were flushed with air and recapped for the next ethylene determination.For 45Ca2+ uptake studies, the hypocotyls, incubated for a given time interval, were washed with 10 changes of distilled H20, and then ground with a glass homogenizer in 9 ml of 80% ethyl alcohol. The debris was pelleted by centrifugation, and the supernatant was collected. The pellet was serially extracted three times with 5 ml of 20 mm HCI. The radioactivity in each extract and in the debris was determined with a ...