Cyclic AMP Promotion and Abscisic Acid Inhibition of α-Amylase Activity in the Seeds of Rice (Oryza sativa L.)

Cyclic AMP and GA3 stimulated both a-amylase activity in rice endosperm and the germination of the seed. In combination they also induced germination of ABA-treated seeds but cyclic AMP alone failed to neutralize the inhibitory effect of ABA; with added kinetin, however, it promoted the «-amylase activity of the dormant seeds. The enzyme activity decreased as the storage period of seeds increased. Cyclic AMP and GA3 did not produce any morphological expression of germination of the seeds in storage but induced the enzyme activity. INTRODUCTION It has been reported by Galsky and Lippincott (1969), Nickells, Schaefer, and Galsky (1971), Earle and Galsky (1971) that cyclic AMP can replace GA3 in promot ing the induction of «-amylase, protease, acid phosphatase, and ATPase activities in barley endosperm. Auxin-induced cell growth is also enhanced by cyclic AMP (Kamisaka and Masuda, 1970) produced in barley aleurone layers (Pollard, 1970). The function of cyclic AMP as a replacement for GA3 is, however, not supported by Kamisaka and Masuda (1970). The failure of rice seeds to germinate is due to the presence of supra-optimal concentrations of indole auxins and abscisic acid in the embryo (Dey and Sircar, 1968a, b). It has also been shown that gibberellins induce germination and <x amylase synthesis in rice endosperm excised from the embryo (unpublished). Results are presented in this paper to show that cyclic AMP can replace GA3 in stimulating the germination of seeds in storage. MATERIALS AND METHODS Rice seeds ev. Rupsail of different years of harvest from 1967 to 1971, supplied from the State Riee Research Station, Chinsurah, West Bengal, were used in this investigation. After steriliza tion with 0-1 per cent HgCl2, 100 seeds were allowed to germinate on filter-paper with 4 ml of test solution in total darkness at 32 °C. The germination percentage was counted after 48 h of incubation. The dehusked seeds after sterilization were soaked in sterile water for 20 h at 4 °C. The embryo was dissected out from the endosperm and 10 such endosperms were placed in a 25-ml Erlenmeyer flask containing 1 ml incubation medium of 2 /xM acetate buffer, 20 /xM calcium This content downloaded from 207.46.13.114 on Thu, 26 May 2016 06:28:04 UTC All use subject to http://about.jstor.org/terms Boy, Ghose, and Sircar—Cyclic AMP, ABA, and u-Amylase Activity 1065 chloride, 12-5 yg Chloromycetin in addition to GA3, kinetin, ABA, and cyclic AMP. Sterile solutions were used throughout the experiment and sometimes solutions were sterilized by filtration through bacterial filters with a pore diameter of 1 -0 ym. At the end of 48 h incubation, the homogenates of seeds were centrifuged at 3000 g for 15 min at 0 °C with 2 yM acetate buffer (pH 5-4). «-Amylase was measured by incubating 1 ml of the enzyme preparation with 1 ml of 1 per cent potato starch (pH 4-6) at 30 °C for 3 min. The reducing power of the control was measured by the addition of dinitrosalicylic acid according to the method of Bernfeld (1955). The colorimetric reading was converted to a-amalyase units by a standard curve determined from known amounts of maltose. Abscisic acid (ABA) was generously given by the F. Hoffman La Roche C. Ltd., Basle, Switzerland. The gibberellic acid (GA3), kinetin (kn) and cyclic AMP were obtained from Fluka A.G. Buchs and Sigma Chemicals Co., St. Louis, Mo., U.S.A. RESULTS AND DISCUSSION Both GA3 and cyclic AMP stimulated germination significantly at a concentration of 50 yM, but beyond this there was inhibition (Table 1). Table 1. Effect of GA3 and cyclic AMP on germination percentage of rice seeds (48-h incubation) Concentrations Total (differentiation Partial (sprouting pM into root and shoot) of embryo) GA3 10 64 32 50 72* 28 100 48 32 Cyclic AMP 10 64 32 50 72* 28 100 46 30 Control (sterile water) 56 40 Each value is the mean of triplicate experiments L.s.d. at 5 per cent level 120 2-71 L.s.d. at 1 per cent level 28-4 3-65 Significantly different from control at 5 per cent level. oncentrations Total (differentia ion Partial (sprout fiM into root and shoot) f embryo) * Si i i control at 5 per cent level. Table 2 shows that gibberellic acid can overcome the inhibition by ABA and partially induce germination, but cyclic AMP failed to act on the ABA-treated seeds. Cyclic AMP with GA3, however, promoted germination while without GA3 it showed 26 per cent sprouting of embryos (Table 2) and root or shoot elongation did not take place even after 96 h. Of the different concentrations of GA3 and cyclic AMP used, 50 pM in the medium containing excised endosperm resulted in a significant promotion in the activity of a-amylase after incubation to 48 h (Table 3). From the results it is evident that cyclic AMP induces a-amylase activity and thus the formation of cyclic AMP from adenine in the aleurone layers of barley (Pollard, 1970) may have some significance. ABA inhibited germination and the activity of hydrolyzing enzymes reversed partially by GA3 alone can be reversed completely by GA3 in combination with appropriate concentration of cytokinin (Table 4). Similar findings were also reported by Khan (1969) and Galston and Davies (1969). This content downloaded from 207.46.13.114 on Thu, 26 May 2016 06:28:04 UTC All use subject to http://about.jstor.org/terms 1066 Boy, Ghose, and Sircar—Cyclic AMP, ABA, and oc-Amylase Activity Table 2. Effects of cyclic AMP and GA3 on germination percentage of ABA treated rice seeds (48-h incubation) Treatment Concentrations of ABA (pg/ml)