Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

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1 Plant Growth Regulation 7: (1988) Kluwer Academic Publishers Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC JAN KFPCZYNSKI and EWA KFPCZYNSKA Department of Plant Physiology, University of Szczecin, Felczaka 3a, Szczecin, Poland (Submitted 27 April 1987 ; revised 28 September 1987 ; accepted 17 October 1987) Key words : Seed germination, Amaranthus paniculatus, paclobutrazol, gibberellin, ethylene Abstract. The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A 3 (GA 3 ), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA3 can be substituted by ethylene. Abbreviations : ACC, 1-aminocyclopropane-l-carboxylic acid ; AMO-1618, (2-isopropyl-5- methyl-4-trimethylammoniumchloride)-phenyl-1-piperidinium-carboxylate ; ancymidol, a- cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidine methanol ; chloromequat chloride, (2-chloroethyl)trimethylammoniumchloride ; ethephon, 2-chloroethylphosphonic acid ; GA 3, gibberellin A3 ; paclobutrazol, (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol ; Phosphon D, 2,4,dichlorobenzyl-tributhylphosphoniumchloride ; tetcyclacis, 5,(4-chlorophenyl)-3,4,5,9,1-pentaaza-tetracyclo)5,4,1,, 2.6,8.11 dodeca-3,9-diene 1. Introduction Several chemicals which retard plant growth processes have been identified as inhibitors of gibberellin biosynthesis. These include earlier compounds such as chlormequat chloride, AMO-1618 and Phosphon D and the more recent highly-active tetcyclacis and paclobutrazol [6]. The earlier inhibitors have low specificity, inhibiting the formation of ent-kaurene but the later ones are more specific since they block the biosynthetic step from entkaurene to ent-kaurenoic acid [2, 3, 5].

2 48 Seed germination was shown to be inhibited by AMO-1618 or chlormequat chloride [1, 9] and more recently inhibition of seed germination of Arabidopsis thaliana [7] and Amaranthus caudatus by tetcyclacis has been observed [8]. In this paper, the effect of paclobutrazol on Amaranthus paniculatus seed germination and its antagonism by GA 3, ethephon or a precursor of ethylene biosynthesis, ACC, is described. 2. Material and methods Seeds of Amaranthus paniculatus L. harvested in 1983 and then stored dry in a refrigerator were germinated in Petri dishes (5 cm) on filter paper moistened with distilled water or appropriate aqueous solution (1.5 cm3 ). In all experiments, conducted in the dark at 24 C, 5 seeds in 5 replications were used. The germination was recorded after periods specified for each experiment. Seeds with radicle about 2 mm long were considered as germinated. ACC was obtained from Sigma Chemical Company, ethephon from Union Carbide Agricultural Products Company, GA 3 from Polfa (Poland) and paclobutrazol (Cultar) from ICI. Plant Protection Ltd. 3. Results As expected paclobutrazol markedly inhibited the germination of Amaranthus paniculatus seeds ; the inhibition decreased with time (Fig. 1). About 6% inhibition after 3 days of incubation was observed when seeds were incubated at a concentration of 3 x 1-4 M. Inhibition due to the presence of paclobutrazol was overcome by addition of gibberellin A 3 (Fig. 2). At the lowest concentration used (1 - SM),GA 3 was only slightly less effective in reversing the inhibition caused by 1-4 M paclobutrazol solution than at higher concentrations. Gibberellin A 3 at the higher concentrations almost completely reversed the inhibition of seed germination. However, GA3 was also very effective in reversing inhibition caused by paclobutrazol at 3 x 1-4 M, at the highest concentration used. This reversal effect of GA 3 increased with time. Paclobutrazol application at 3 x 1-3 M completely inhibited seed germination. Addition of GA3 to the incubation medium partially antagonized this inhibition ; about 5% of seeds were able to germinate at 3 x 1-4 M of GA 3.

3 "5 1'' PACLOBUTRAZOL, M Fig. 1. The effect of paclobutrazol on germination of A. paniculatus seeds in the dark at 24 C. Key : Id, 2d, 3d - days of g ermination. S D does not exceed 1%. GA3 M Fig. 2. The effect of GA 3 on the inhibition of germination of A. paniculatus seeds by paclobutrazol. Key: A - 1-4, B - 3 x 1-4, C - 3 x 1 -'M of paclobutrazol. GA3, GA 3 + paclobutrazol off. 2d, 3d - days of germination. S D does not exceed 7, 8 and 11 % for A, B and C respectively. The inhibition of Amaranthus paniculatus seed germination caused by paclobutrazol was also reversed by ethephon (Fig. 3), in a manner similar to GA3 the effect being more pronounced with increasing concentration. The inhibition caused by paclobutrazol at the lowest concentration (1-4 M) was completely reversed by 7 x 1-5 M of ethephon, which was the highest

4 5 1 A 2,3 id.2g3d C 3d 3 Z 4 2d o[ 2 I I I I ~~ I I 1 I 7.1' ' 7. 1-' 7. 1-' 716 ETHEPHON, M Fig. 3. The effect of ethephon on the inhibition of germination of A. paniculatus seeds by paclobutrazol. Key: A- 1-4, B- 3 x 1-4, C- 3 x 1 - 'M of paclobutrazol. Ethephon, Ethephon + paclobutrazol. 2d, 3d - days of germination. SD does not exceed 14, 4 and 9% for A, B and C respectively. concentration used. Complete inhibition at 3 x 1-3 M of paclobutrazol could be reduced to about 25% following ethephon application. The inhibitory effect of paclobutrazol at 3 x 1-3 M was also partially counteracted by ACC, a precursor of ethylene biosynthesis (Fig. 4) ; At the highest concentration (3 x 1-4 M) ACC allowed germination of ca 5% seeds. 1 2dpd 8 8 i 6 z Z 4 I ne W 2 I 1' ACC, M I 319 Fig. 4. The effect of ACC on the inhibition of germination of A. paniculatus seeds by paclobutrazol. ACC, ACC + paclobutrazol (3 x 1 - '), 2d, 5d - days of germination. SD does not exceed 1%.

5 51 4. Discussion Since paclobutrazol inhibited gibberellin biosynthesis in seed tissue [4], it seems likely that the inhibition of seeds germination of Amaranthus paniculatus is associated with the inhibition of gibberellin biosynthesis and thus endogenous gibberellins are required for germination. The addition of GA 3 to the incubation medium counteracted the inhibition caused by paclobutrazol. Similarly, GA4+, was effective in reversing of the inhibition of Am-. aranthus caudatus seed germination due to presence of tetcyclacis, another inhibitor of gibberellin biosynthesis [8]. The antagonism of the inhibition of seed germination by paclobutrazol in the presence of GA 3 supports the concept that endogenous gibberellins are required for the germination of these seeds. Interestingly, inhibition of germination due to the presence of paclobutrazol is reversed also by ethylene liberated from ethephon or synthesised from exogenous ACC, a precursor of ethylene biosynthesis. This is in accordance with results of experiments in which seeds of Amaranthus caudatus were incubated in the presence of tetcyclacis and ethephon or ACC [8]. Such simulation of action of endogenous gibberellins by ethylene may indicate that ethylene and gibberellins affect the same or similar processes leading to germination. The comparison of molecular concentration of GA 3, ethephon and ACC applied to the incubation medium lead to the conclusion that ethephon is much more efficient than gibberellin or ACC, the precursor of ethylene biosynthesis, in reversing paclobutrazol inhibition. However, the different efficiencies of these compounds might be related to difference in their penetration. It is interesting to speculate whether the action of GA3 on A. paniculatus seeds requires the presence of endogenous ethylene since it was suggested that endogenous ethylene is involved in the response of A. caudatus seeds to exogenous GA4+7 [8]. In conclusion, endogenous gibberellins seem to be involved in germination of Amaranthus paniculatus seeds. The effect of exogenous GA 3 on the inhibition of germination by paclobutrazol can be replaced by ethylene released from ethephon or synthesised from ACC. Acknowledgment We wish to thank ICI Plant Protection Ltd, for the gift of paclobutrazol.

6 52 References 1. Black M (1969) Light-controlled germination of seeds. Symp Soc Exp Biol 23 : Coolbaugh R C and Hamilton R (1976) Inhibition of ent-kaurene oxidation and growth by a-cyclopropyl-a(p-methoxyphenyl)-5-pyrimidyne methyl alcohol. Plant Physiol 57 : Graebe J E and Ropers H J (1978) Gibberellins. In : D S Letham, P B Goodwin and T J V Higgins, eds. Phytohormones and Related Compounds : A Comprhensive Treatise, Vol. 1 : 17. Amsterdam : Elsevier North Holland 4. Hedden P and Graebe J E (1985) Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates of Cucurbita maxima endosperm and Malus pumila embryos. J Plant Growth Regul 4 : Hildbrandt E, Graebe J E, Rademacher W and Jung J (1982) Mode of action and biological activity of new potent plant growth retardants : BAS 16..W and triazole compounds. Abstract 11th Int. Conf. on Plant Growth Substances, p.65. Aberystwyth, UK 6. Jung J and Rademacher W (1983) Plant growth regulating chemcials cereal grains. In : Nickell, ed. Plant Growth Regulating Chemicals, Vol Boca Raton, CRC Press 7. Karssen C M, L4cka E, Kgpczynski J and Koorneef M (1985) Gibberellin biosynthesisdependent and-independent light effect on seed germination of Arabidopsis thaliana. European Symp. Photomorhogenesis in Plants p. 59. Wageningen 8. Kgpczynski J (1986) Ethylene-dependent action of gibberellin in seed germination of Amaranthus caudatus. Physiol Plant 67 : Knypl J S (1967) Growth retardants in relation to the germination of seeds. III. The synergistic inhibitory effect of (2-chloroethyl)-trimethylammonium chloride and coumarin on germination of kale seeds, and its reversal by kinetin and gibberellic acid. Acta Soc Bot Pol 36 :