ACTIVATION OF PROTEIN SYNTHESIS IN THE IMBIBITION PHASE OF SEED GERMINATION

Initiation of germination in many seeds requires only contact with water at the appropriate temperature. The nature of the metabolic phenomena occurring during water imbibition is an intriguing question. Recognizing that the ability to make new protein is certainly an early requirement during germination, we have undertaken a study of the protein synthesis system in dry and imbibed seeds. While germination manifests itself visibly only in the growth of the embryo, nevertheless studiesl' 2 have indicated that the supporting tissue (cotyledon or endosperm) also develops new enzymatic activities with germination. Thus, concern for the activation of the protein synthesis system would apply similarly to this supporting tissue. The studies to be described indicate that the entire apparatus necessary for protein synthesis is functional in the cotyledon of the ungerminated peanut and that messenger RNA is limiting. Imbibition corrects this inadequacy. Studies with peanut and wheat embryos suggest that the formation or activation of messenger RNA during imbibition may be a general phenomenon in seed germination. Materials and Methods.-The medium for preparation of microsomes was 0.5 M sucrose, 0.01 M MgCl2, 0.05 M tris buffer pH 7.9, 0.025 M KC1, and 0.005 M 2-mercaptoethanol. With cotyledons from peanuts germinated 1-4 days,3 2 ml of medium were added per seed. With cotyledons from dry seed, an additional 0.2 ml of water was added per seed. The cotyledons were blended twice for 15 sec; the suspension was filtered through cheesecloth and centrifuged for 20 min at 22,000 X g. The supernatant was removed carefully (to keep out most of the upper lipid layer) and centrifuged in a Spinco ultracentrifuge for 60 min at 105,000 X g. Supernatant was removed from the upper third of the tube (105,000 X g supernatant), and the remaining supernatant was discarded. The pellet was suspended in a solution containing 0.01 M tris pH 7.9, 0.004 M MgCl2, and 0.005 M mereaptoethanol, and centrifuged for 10 min at 23,500 X g. The opalescent supernatant was used as the "microsome" preparation. In some experiments the 105,000 X g pellets were resuspended in blending medium and recentrifuged. These preparations are referred to as "washed microsomes." In addition, "105,000 X g supernatant" was dialyzed for 4-5 hr against 60 vol of 0.01 M tris pH 7.9, 0.01 M KCI, 0.01 M MgCl2, 0.005 M mereaptoethanol, and recentrifuged for 2 hr at 105,000 X g. The top third of these supernatants were pooled and stored as "dialyzed supernatant." Peanut axes (attached to the cotyledon) were allowed to imbibe for 20 hr at 25° in the usual manner.3 Wheat embryos imbibed for 16 hr at 200 on paper towel moistened with a solution of streptomycin sulfate (100 -y ml). In some experiments the wheat embryos were germinated on sucrose-agar containing antibioties.4 Both embryonic materials were ground by hand with sand in a mortar. In the peanut experiment (Table 5, expt. 1) 100 dry axes and 71 one-day axes were ground with 15 ml of sucrose medium. In the wheat experiment, 2 gm of viable wheat embryos, prepared according to Johnston and Stern,4 were ground with 14 ml of sucrose medium. The RNA content of all microsome preparations was determined from the optical density at 260 mIA, assuming E = 20 for 0.1% solution of RNA. Protein content was determined according to Lowry et al.5 In general, the microsome preparations contained approximately two parts protein to one part RNA. The spectra were those expected for ribonucleoproteins, except for the preparations from peanut cotyledon. The latter absorbed more in the 230-250 my region, apparently due to occluded lipid.