Relation of Polyamine Biosynthesis to the Initiation of Sprouting in Potato

The polyamines putrescine, spermidine, and spermine and their biosynthetic enzymes arginine decarboxylase, ornithine decarboxylase and Sadenosyl-L-methionine decarboxylase are present in all parts of dormant potato (Solamm tuberosum L.) tubers. They are equally distributed among the buds of apical and lateral regions and in nonbud tissues. However, the breaking of dormancy and initiation of sprouting in the apical bud region are accompanied by a rapid increase in ornithine decarboxylase and Sadenosyl-L-methionine decarboxylase activities, as well as by higher levels of putrescine, spermidine, and spermine in the apical buds. In contrast, the polyamine biosynthetic enzyme activities and titer remain practically unchanged in the dormant lateral buds and in the nonbud tissues. The rapid rise in ornithine decarboxylase, but not arginine decarboxylase activity, with initiation of sprouting suggests that ornithine decarboxylase is the rate-limiting enzyme in polyamine biosynthesis. The low level of polyamine synthesis during dormancy and its dramatic increase in buds in the apical region at break of dormancy suggest that polyamine synthesis is linked to sprouting, perhaps causally. The PA,2 Put, Spd, and Spm are present throughout the microbial, animal, and plant worlds (3, 7). In microbial and plant cells, Put is derived either from arginine via ADC and the intermediate Agm, or from ornithine by ODC. In mammalian cells, Put synthesis occurs only by the latter pathway. The Put thus formed is converted successively to Spd and Spm through propylamino group transfer from SAM mediated by SAMDC in all the above types of cells (3, 11). These amines have been implicated in several important processes involved incell growth and development, especially those involving nucleic acids (3, 7, 11, 27). Both the activity of PA biosynthetic enzymes and PA titer have been reported to increase dramatically during rapid growth in many plant systems, such as germinating seeds of Zea, Pisum, Triticum, and Tragopogon (30), developing seedlings of Phaseolus (4) and Lathyrus (21, 22), ovaries of tomato, and rapidly dividing tobacco cells in suspension culture (12), during crown gall-tumor development (6) and embryogenesis of carrot suspension cells (18, 19). Although little is known about the activities of PA biosynthetic enzymes in dormant tissues such as tubers and senescencing plant parts, contents of PA in these systems are extremely low (1, 26). We have observed that activities of PA biosynthetic enzymes 'Supported by grants from the National Science Foundation and Binational Agricultural Research and Development Fund to A. W. G. 2Abbreviations: PA, polyamine; Put, putrescine; Spd, spermidine; Spm, spermine; ADC, arginine decarboxylase; Agm, agmatine; ODC, ornithine decarboxylase; SAM, S-adenosyl-L-methionine; SAMDC, S-adenosyl-Lmethionine decarboxylase. and PA titer decrease during aging and senescence of oat leaves (17). Furthermore, exogenous application of the PA or their precursor amino acids retards senescence of leaves in several monocotyledonous and dicotyledonous plants (16) and of protoplasts from oat leaves (2, 10, 14, 15). PA appear to inhibit senescence by preventing Chl, protein, and RNA breakdown in leaves (16) and by increasing macromolecular synthesis and mitotic activity in protoplasts (15). The antisenescence properties of PA and their correlation with cell proliferation and differentiation lend support to the contention that they act as growth factors (3, 5, 11). Despite these observations, the relative roles of individual PA biosynthetic enzymes in dormant and actively dividing plant tissues is not well understood. We have, therefore, examined the activities of ADC, ODC, and SAMDC and the endogenous levels of Put, Spd, and Spm in dormant and actively growing tissues of potato tubers. MATERIALS AND METHODS Plant Materials. Idaho Russet baking potatoes (Solanum tuberosum L.) (U.S. No. 1) were purchased from the local supermarket. The tubers were firm, with no sign of sprouting, and were therefore regarded as dormant. Medium-size tubers were selected and allowed to sprout by storing them in the dark at room temperature for about 2 months. Samples of tissue were taken from dormant, initial, and advanced (profuse) sprouted tubers. Three areas from the same tuber showing different sprouting activity were selected for sampling (Fig. 1): (A) bud tissue in the apical region (hereafter referred to as apical buds) which developed sprouts; (B) bud tissue from the lateral region (hereafter referred to as lateral buds) which remained dormant throughout the storage period; and (C) nonbud tissue which does not develop sprouts. Tissue sections (7 mm in diameter, 3 mm in length) containing the skin and outer cortex were removed. During the periods when sprouting occurred, the sprouts were excised with the apical bud tissue and assayed together. Six to eight tubers were used for each experiment and slices of each selected area were randomized. Suitable amounts were selected for determining ADC, ODC, and SAMDC activities and PA titer. Extraction and Measurements Polyamine Biosynthetic Enzymes. For each extraction, approximately 5 g (9 discs) of tissue was homogenized in chilled mortars with 1.5 ml of 100 mm phosphate buffer at pH 7.6. The homogenates were centrifuged at 26,000g for 15 min at 4°C and the resulting clear supernatant fractions were assayed for enzyme activities. Enzyme assays were done by modification of conventional methods (8, 18, 24) in 12 x 75 mm polystyrene culture tubes sealed with polyethylene caps. A filter paper disc (6 mm diameter) impregnated with 50 1d2 N KOH was supported on a 22-gauge syringe needle through the cap and used to trap the '4CO2 liberated. ADC activity was determined by measuring the release of "CO2 from the substrate, L-[U-'4Clarginine. The reaction mixture