Additive and Synergistic Effects of Kinetin and Ethrel on Germination, Thermodormany, and Polyribosome Formation in Lettuce Seeds

Abstract

The inhibition of germination of Grand Rapids lettuce (Lactuca sativa L.) seeds at 35 C was removed to a marked extent by kinetin and 2-chloroethylphosphonic acid (eth-rel). When both compounds were used together, an additive effect was observed. A synergistic effect was, however, noted when ethrel promoted the kinetin reversal of abscisic acid inhibition of seed germination (light-as well as gibberellic acid-, induced). Both kinetin and ethrel increased the total ribosomal material and the percentage of polyribosomes in lettuce seeds imbibed in the light for 24 hours. A combination of the two compounds showed a synergism in polyribo-some formation only at high ethrel concentration. The inability of ethrel to reverse abscisic acid inhibition indicates that kinetin action cannot always be substituted by ethrel. The possible mechanisms involved in the enhanced response by a combination of kinetin and ethrel are discussed. Plant hormones appear to have designated functions in seed germination (9). For example, the GA3-induced dark germination of Grand Rapids lettuce seeds is inhibited by ABA and this inhibition is reversed by cytokinins (8). Cytokinins release the dormancy of seeds presumably by counteracting the effects of inhibitors by an as yet unknown mechanism (9). Another feature of cytokinin is its ability to overcome thermodormancy in lettuce (5, 18, 20). There is evidence that this effect of cytokinin may be related to a higher level of ABA in lettuce seeds imbibed at 35 C compared to those imbibed at 25 C (Braun and Khan, unpublished data). Ethylene and ethrel, an ethylene-releasing compound, also release dormancy in various seeds (3, 6, 7, 21). It has been suggested that cytokinins act by stimulating ethylene production in seeds and other systems (7, 11). Synergistic and additive effects were noted when a combination of kinetin and ethrel was used to release dormancy in cocklebur and Indian rice grass seeds (21). An attempt is made here to determine whether the syner-gism or the promotion of kinetin effect by ethrel extends to processes in lettuce seeds affected by cytokinins. MATERIALS AND METHODS Seed Germination and Incubation. Fifty seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) were germinated in the light or dark, in 9-cm Petri dishes on two layers of Whatman No. 1 filter paper moistened with S ml of water or appropriate hormone solution. In the dark experiments, Petri dishes were wrapped in aluminum foil, covered with a dark cloth, and transferred to the incubator. Each treatment had at least three replications and was repeated twice. Polyribosome Isolation. Following 24 hr incubation of seeds in various test solutions, 150 germinating seedlings were frozen in dry ice. The tissue was powdered along with a small piece of dry ice and homogenized in 8 ml of 0.4 M sucrose, 50 mm tris-HCl, pH 8.5, 50 mm KCI, 10 mM MgCl2, and 5 ms mercaptoethanol plus 0.5 ml of 10% Triton X-100. The homogenate was centrifuged at 20,000g for 15 min. The supernatant was layered over a 2.5-ml pad of 1.5 M sucrose solution in a buffer containing 50 mM tris-HCl, pH 7.8, 20 mm KCI, 10 mm magnesium acetate, and 5 mm mercaptoethanol and centrifuged at 139,000g (average) for 2 hr in a Spinco Model 65 fixed rotor. The ribosomal pellet was resuspended in a 1-ml resuspension solution containing the above buffer. The ribosomal resuspension was layered over a 10 to 35% sucrose density gradient containing the buffer and centrifuged for 2 hr at 148,000g in a Spinco Model 41 Ti swinging bucket rotor. The distribution of polyribosomes was determined by recording the absorbance at 254 nm in an ISCO Model 640 density gradient fractionator. RESULTS Additive Effect of Kinetin-Ethrel in Release of Thermodormancy. The effect of various temperatures on germination is shown in Table I. A 35 C temperature completely inhibits germination, and the optimum temperature for germination is 25 C. This thermodormancy in lettuce seeds is released to a marked extent in the presence of kinetin or ethrel (Table II). An additive effect in the release of thermodormancy was observed when the two compounds were used simultaneously. No such effect was noted when a combination of GA3 and kinetin or GA3 and ethrel was used, suggesting that kinetin-ethrel effect on germination is rather specific. Additive Effect of Kinetin and Ethrel on Germination. Although kinetin and ethrel separately had very little effect on lettuce seed 263