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 in-cell growth and development, especially those involving nucleic acids (3,7,11,27 (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...
Polyamine biosynthesis in senescing leaves of A vena sativa L. was measured by determining the activities of arginine decarboxylase (EC 4.1.1.19), ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50). Polyamine content was also estimated by thin layer chromatography and high performance liquid chromatography. Arginine decarboxylase activity decreases progressively in aging attached first leaves and in senescing excised leaves in the dark. Conversely, it increases during Ught exposure ofexcised leaves, which retards senescence. Ornithine decarboxylase activity is high and constant in the attached leaf, irrespective of age; it decreases in excised leaves kept in the dark and in the Ught, irrespective of senescence. S-Adenosyl-L-methionine decarboxylase shows no correlation with age or senescence. Levels of putrescine, diaminopropane, agmatine, and spermidine are high in young leaves and declne with age. The best single indicator of senescence is usually spermidine, which decreases in excised leaves incubated in the dark, but increases in such leaves with time of lght exposure. Spermidine generally has a reciprocal relationship with putrescine, indicating that spermidine synthase, which converts putrescine to spennidine, may exert important physiological control. These data support the view that polyamines play an important role in the regulation of plant development.
The effects of the polyamines spermidine and 1,3-diaminopropane on ethylene biosynthesis and chlorophyll (Chl) loss were studied in peeled leaves of oat (Avena sativa L., var. Victory) incubated in the dark. Peeling off the epidermal cells induces an increase in 1-aminocyclopropane-lcarboxylate (ACC) synthase activity, resulting in an enhanced ACC and ethylene formation. Both polyamines inhibit ethylene biosynthesis from methioine by inhibiting ACC synthase activity and, more effectively, the conversion of ACC to ethylene. They also inhibit Chi loss occurring between 24 and 48 h of dark incubation; but, as shown by inhibitor experiments, inhibition of Chl loss does not result from inhibition of ethylene formation. Ethylene production and Chl loss, both associated with senescence, require membrane integrity, thus, treatments which promote deterioration of membranes inhibit both processes. Ca" in the incubation medium competitively reduces the polyamine-mediated inhibition of ACC conversion and Chl loss. The data suggest that polyamines initially attach to membranes, thereby inducing changes which, in turn, lead to inhibition of ethylene biosynthesis and retardation of senescence.In the preceding study, we investigated the effects of exogenous Dap4 and Spd on Chl loss, protease activity, and ethylene production in senescing oat leaves, in relation to the endogenous levels of PAs (26). We found an accumulation of Dap after application of exogenous Spd, presumably due to the activity of PA oxidase. We described the inhibitory effect of Dap on protease activitJr, Chl breakdown, and ethylene production. The observed Ca + antagonism of these senescence processes suggested that Dap exerts its influence through an initial interaction with specific negatively charged sites on membranes. We have here extended our study to investigate the possible mode of action of Dap and Spd, including effects on the biosynthesis of ethylene. Formation of ethylene is generally associated with leaf senescence in the dark, although an obligatory connection between the two seems not to exist (1 1).
Abstract. During the 2002During the -2003 Exotic Newcastle Disease (END) outbreak in Southern California, a highthroughput real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) system was developed to respond to the large diagnostic and surveillance sample workload. A 96-well RNA extraction method, using magnetic bead technology, combined with a 96-well RRT-PCR assay, allowed 1 technician to process and test more than 400 samples per day. A 3-technician team could complete testing on approximately 1,900 samples per day. The diagnostic sensitivity of the high-throughput RRT-PCR assay was 0.9967 (95% CI 0.9937-0.9997) based on 926 virus isolation confirmed positive samples. Diagnostic specificity using an initial 434 virus isolation confirmed negative samples was 100%. A diagnostic specificity of 0.9999 (95% CI 0.9999, Ͼ0.9999) was subsequently calculated on the basis of 2 false-positive results among 65,343 surveillance samples collected after the final END-positive case was confirmed in May 2003. Assay performance over 500 replicates, including reproducibility of the combined extraction and RRT-PCR amplification steps yielded a standard deviation of 0.70 RRT-PCR cycle thresholds (Ct) and a standard deviation of 0.59 Ct for the RRT-PCR steps alone. The high-throughput RRT-PCR developed for END contributed significantly to the 2002-2003 END control effort, reducing the predicted timeline for eradication from 3 years to just 11 months, primarily because of the large number of samples that could be rapidly tested. The 96-well approach described for high-throughput END RRT-PCR could be adapted to other rapid, high-volume testing needs, as required for potential foreign animal disease responses or intensive surveillance efforts.
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