Polyamine Metabolism in Ripening Tomato Fruit1

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele aic) and contain three times as much putrescine as the standard Rutgers variety (Aic) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-akc-a near isogenic line possessing the allele akc, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in ak pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and aic fruit showed a decrease in the metabolism of [1,4-14C]putrescine and [terminal labeled-3H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-akc fruit, and as a result it was significantly higher in aic fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in alc fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase. The fruit of Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) ripen more slowly than the standard commercial varieties and if picked ripe they can be kept four times longer (9). The delayed overripening feature is conferred by a single recessive gene a/c, whose inheritance, linkage, and effects on ripening-related mRNAs have been described (8, 10). In an earlier paper (3), we reported on the levels of free PAs3 in the fruit pericarp tissues of Alcobaca (alc), its spontaneous revertant Alcobaca-red, and the standard variety Rut' Supported by grants from the Herman Frasch Foundation and the Cornell University Biotechnology Program. 2Current address: Department of Molecular Biology and Genetics, University of Guelph, Guelph, Ontario, N 1G 2W 1, Canada. 3Abbreviations: PA(s), polyamine(s); Ptc, putrescine; Spd, spermidine; Spm, spermine; SAM, S-adenosylmethionine; PCA, perchloric acid; ODC, ornithine decarboxylase; ADC, arginine decarboxylase; GABA, -y-aminobutyric acid. gers (both Alc) during the process of ripening. In all the lines, the levels of Ptc were high at the immature green stage and then declined at the mature green stage. Thereafter, Ptc levels in the fruit of Alc lines remained low but in fruit of alc they rose to levels similar to that of immature green fruit (3). The level of Spd, however, was not significantly different either during ripening or in different genotypes. The Spm content was always very low. Similar changes in Ptc content were recently documented in the ripening fruit of another long keeping variety of tomato (14). The increase in Ptc level in the alc fruit may be due to either decreased Ptc metabolism or increased biosynthesis. We, therefore, examined PA metabolism, i.e. PA conjugate levels and Ptc and Spd breakdown, and Ptc biosynthesis in the fruit ofalc and A/c genotypes at different stages ofripening. In a companion paper (1 1), we characterized the pathways of Ptc and Spd metabolism in tomato pericarp tissues. MATERIALS AND METHODS Plant Material and Cultivation Methods The fruit of tomato (Lvcopersicon esculentum Mill.) isogenic lines Alcobaca (alc) versus Alcobaca-red (Alc-red: a revertant of alc) and near isogenic lines Rutgers versus Rutgers-alc (a/c backcrossed into cv Rutgers) were used for experiments. Seed was originally obtained from Dr. M. A. Mutschler (Plant Breeding Department, Cornell University, Ithaca, NY). Plant cultivation methods were similar to those described previously (1 1). Ripening stages used were immature green, mature green, breaker (streaks of orange at the distal end), and ripe. For experiments on storage in light and in dark, fruit of different lines were excised at mature green stage and stored at 22 ± 2°C in darkness or under fluorescent lights. The Alc fruit (Rutgers and A/c-red) were analyzed after 12 to 15 d of storage by which time the fruit ripened under both conditions. The alc fruit (a/c and Rutgers-a/c) were analyzed after 28 to 35 d of storage and at that time the fruit in the light were ripe but the dark-kept fruit were bleached. Analysis of Free PAs and PA Conjugates Polyamines were extracted in 0.2 N PCA (100 mg tissue/ mL acid) and after 1 h on ice the homogenates were centrifuged. For soluble PA conjugates, 0.2 mL of supernatant was mixed with 0.2 mL concentrated HCI in a glass ampule, the