APELBAUM A, AC BURGOON, JD ANDERSON, M LIEBERMAN, R BEN-ARIE, AK. MATTOO 1981 Polyamines inhibit biosynthesis of ethylene in higher plant.
Plant Physiol. (1988) 86, 0338-0340 0032-0889/88/86/0338/03/$0 1.00/0
Communication
Polyamine Content of Long-Keeping Alcobaca Tomato Fruit' Received for publication July 13, 1987 and in revised form October 13, 1987
ANDREW R. G. DIBBLE2, PETER J. DAVIES*, AND MARTHA A. MUTSCHLER Section of Plant Biology (A.R.G.D., P.J.D.) and Department of Plant Breeding (M.A.M.), Cornell University, Ithaca, New York, 14853 ABSTRACT Fruit of tomato landrace Alcobaca, containing the recessive allele alc, ripen more slowly, with a reduced level of ethylene production, and have prolonged keeping qualities. The levels of polyamines in pericarp tissues of alc and 'wild type' Alc (cv Rutgers and Alcobaca-red) fruit were measured by HPLC in relation to ripening. Putrescine was the predominant polyamine with a lower content of spermidine, while spermine was just detectable. The level of putrescine was high at the immature green stage and declined in the mature green stage. In Alc fruit the decline persisted but in alc fruit the putrescine level increased during ripening to a level smilar to that present at the immature green stage. There was no pronounced change or difference in spermidine levels. The enhanced polyamine level in ale fruit may account for their ripening and storage characteristics.
member of the polyamine sequence, the diamine PTC,3 is synthesized in most plant systems from arginine via ADC, though in tomato fruit the principal pathway appears to be similar to that in animals and microorganisms, namely from ornithine via ODC (4, 5, 8, 9). The level of ODC is highest shortly after pollination and declines to a low level of maturity (9), while inhibitors of ODC activity inhibit fruit development (4). Actual levels of polyamines have not been recorded through development and ripening, nor have ODC or ADC been followed through ripening in any tomato line. As the conversion of PTC to the other polyamines, SPD and SPM, involves SAM, which is also the precursor of ethylene (7), it follows that the synthesis of polyamines and ethylene are competitive. In view of the reduced ethylene production and delayed senescence of alc fruit, the polyamine content of these fruit was analyzed to determine whether it might vary from normal tomato fruit.
MATERIALS AND METHODS Plant Material. Fruit at various stages of ripening was harvested from field grown plants of tomato (Lycopersicon esculenAlcobaca is a landrace of tomato in which the fruit ripen more tum Mill.) landrace Alcobaca, Alcobaca-red and cv Rutgers VR slowly than standard horticultural varieties (60 versus 50 d from (a standard variety control of genotype Alc). Fruit stages used anthesis) and have prolonged keeping qualities. If picked ripe, were immature green, mature green, breaker (streaks of orange fruit can be kept at 20C for four times longer than standard at the distal end), and ripe. varieties (33 versus 9 d) (13, 15). This characteristic is determined Polyamine Analysis. Polyamine analysis was basically as deby a single recessive gene, designated alc, whose inheritance and scribed in Smith and Davies (17, 18). Only free polyamines were linkage have been described (13, 14). The storability of alc fruit measured. All glassware was silanized with Aquasil (Pierce, Rockis not due to extreme firmness of the harvested fruit, but to an ford, IL). attenuation of the overripening process, including a slower rate Pericarp tissue was homogenized in chilled tissue grinders with of softening possibly because of lower polygalacturonase levels matching pestles in ice-cold 0.2 N HC104 (0.1 g tissue/ml acid). (12; MA Mutschler, in preparation). alc Fruit lack a climacteric HDA at 0.1 Iumol/g fresh weight of tissue was added to the pattern of CO2 and ethylene production and produce less than extracts as an internal standard. After 1 h on ice the homogenates 25% of the ethylene produced by cv Rutgers (15). were centrifuged at 4° C in a clinical centrifuge. The supernatants A spontaneous mutant in Alcobaca has given rise to a plant were derivatized immediately for polyamine analysis; 0.1 ml with normal ripening fruit but with all the other characteristics aliquots of the supernatant were added to 0.2 ml of saturated of the original Alcobaca landrace. The fruit of this line, which Na2CO3 and 0.4 ml of dansyl chloride in acetone (7.5 mg/ml) in has been named Alcobaca-red, ripen to a crimson red as com- a 5-ml tapered reaction vial. The mixture was incubated in a pared to the medium orange color of the original Alcobaca fruits thermal reaction block at 60°C for 1 h in the dark. Excess dansyl and do not possess prolonged keeping qualities. Genetic analysis chloride was then removed by adding 0.1 ml proline in H20 (0.1 has shown that the alc allele has indeed mutated back to Akc g/ml) to the mixture. The mixture was incubated at 250 C for (MA Mutschler, in preparation). This spontaneous mutation 0.5 h in the dark. The polyamines were then extracted with 0.5 provides a line isogenic with the original Alcobaca landrace ml of toluene, vortexing vigorously for 30 s. The aqueous phase differing only in the Alc locus. was removed and discarded. The organic phase was dried comPolyamines have recently been implicated in several facets of pletely under N2. The polyamine residue was dissolved in 0.15 plant development and are particularly associated with continued to 0.7 ml of methanol, depending on the sample, centrifugally cell division and the prevention of senescence (7). The first 3Abbreviations: PITC, putrescine; SPD, spermidine; SPM, spermine; 'Supported by the Cornell University Biotechnology Program.
2Current address: Department of Biochemistry, University of California, Riverside, CA 92521.
HDA, hexanediamine; ADC, arginine decarboxylase; ODC, ornithine decarboxylase; SAM, S-adenosylmethionine; ACC, I-aminocyclopropane- -carboxylic acid. 338
POLYAMINES IN RIPENING TOMATO FRUIT (A)
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PTC HDA
339
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FIG. 1. Fluorescence trace of polyamines as dansyl derivatives following separation by HPLC. A, Standards; B, from tomato fruit (cv Rutgers at immature green stage).
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ALCOB. RED
FIG. 3. Levels of spermidine in fruit pericarp of different lines of IG, Immature green; MG, mature green; BR, breaker; RP, ripe; ALCOB.RED, Alcobaca-red. tomato during ripening.
grator (model 3390A, Hewlett Packard, Allentown, PA). A relative calibration procedure was used to determine the amounts of
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I-.
(n%J
polyamines in samples using HDA as the internal standard. Chemicals. PTC dihydrochloride, SPD trihydrochloride, SPM tetrahydrochloride, HDA dihydrochloride and dansyl chloride were purchased from Sigma (St. Louis, MO). Acetone and methanol (HPLC grade) were from J. T. Baker (Phillipsburgh, NJ). Toluene (HPLC grade) was purchased from Burdick and Jackson (Muskegeon, MI).
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RESULTS Very clear, reproducible patterns of polyamines in the fruit tissue were produced (Fig. 1). Identity was determined by retention time and coinjection of a known standard. PTC is consist17: ently the major polyamine in these tomato fruit. The polyamine breakdown product diaminopropane has a retention time very close to that of PTC. However, by using a slower gradient the two can be separated. The endogenous soluble material was Iz shown to be PTC and not diaminopropane. SPD was present at only about one-seventh the level of PTC in immature green fruit w and about one-third the level of PTC in later stages of Alc fruit. The level of SPM was very low (less than one-twentieth the level of PTC) but was detectable as the sensitivity of the detector was ... 1-41 increased. IG MG BR RP IG MG BR RP IG MG BR RP IThe levels of PTC were about 0.3 gmol/g fresh weight in the F~ RUTGERS VR ALC OBACA ALCOB. RED of immature green fruit of all lines and this declined to pericarp FIG. 2. Levels of putrescine in fruit pericarp of different lines of 0.12 to 0.15 gmol/g fresh eight at the mature green stage (Fig. tomato during ripening. IG, Immature green; MG, mature green; BR, 2). Thereafter, the level remained relatively similar through the breaker; RP, ripe; ALCOB. RED, Alcobaca-red. ripening process in Alc lines (cv Rutgers and Alcobaca-red) while filtered through nylon membranes (0.2 um pore; Rainin, Wob- in alc fruit (Alcobaca) it increased during the ripening process to urn, MA), and assayed immediately, or stored for no longer than return to a level similar to that present in immature green fruit by the time the fruits were ripe. This change was not reflected in 1 week at -20° C in the dark. HPLC. The derivatives were separated on a C18 reverse phase the level of SPD which remained approximately similar in all column (0.4 x 25 cm, 5 u spherical particle, Alltech, Deerfield, lines during ripening (Fig. 3). IL) using a water to methanol solvent gradient going from 60 to 95% methanol in 23 min with detection by an in-line fluoresDISCUSSION cence spectrophotometer (excitation wavelength, 365 + 5 nm; emission wavelength, 510 ± 5 nm) (model 650-10 LC, Perkin The alc fruit show a clear increase in PTC in ripe fruit as Elmer, Norwalk, CT) with the peak areas recorded on an inte- compared to Ak lines. As polyamines have been shown to retard 0
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340
DIBBLE ET AL.
senescence in some tissues they may well also act in this way in tomato fruit, especially since alc conveys long-keeping by the attenuation of overripening, a senescent process. The elevated level of polyamines may also be responsible for the reduced production of ethylene by the alc fruit. Polyamines have been found to inhibit ethylene biosynthesis, in apple fruit and tobacco leaves, both from methionine and ACC (1, 2).
Conversely, ethylene inhibits polyamine biosynthesi, acting by inhibiting the activity of both arginine decarboxylase (3, 1 1) and SAM decarboxylase (10, 11) in pea seedlings. When ethylene biosynthesis is inhibited by polyamines in orange peel discs, more radiolabel from labeled methionine was incorporated into SPD and less into ACC, though SPM was found to be unaffected (6). When inhibitors of the production of either ethylene (or ACC) (e.g. aminoethoxyvinylglycine) or polyamines (e.g. difluoromethyl arginine or methylglyoxalbisguanylhydrazone) were tried, they caused an increase in the production of the other product (more notably SPD in the former case) (6, 16). When polyamine synthesis was inhibited in carnation flowers there was a promotion of both ethylene production and senescence (16). Thus, the two pathways are closely linked and may be of importance in fruit ripening and senescence. Acknowledgment-We thank David Law for extensive discussion and constructive criticism. LITERATURE CITED 1. APELBAUM A, AC BURGOON, JD ANDERSON, M LIEBERMAN, R BEN-ARIE, AK MATTOO 1981 Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts. Plant Physiol 68: 453-456 2. APELBAUM A, I ICEKSON, AC BURGOON, M LIEBERMAN 1982 Inhibition by polyamines of macromolecular synthesis and its implication for ethylene production and senescence processes. Plant Physiol 70: 1221-1223 3. APELBAUM A, A GOLDLUST, I ICEKSON 1985 Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal
Plant Physiol. Vol. 86, 1988
regulation of plant growth. Plant Physiol 79: 635-640 4. COHEN E, SM ARAD, YM HEIMER, Y MIZRAHI 1982 Participation of ornithine decarboxylase in early stages of tomato fruit development. Plant Physiol 70: 540-543 5. COHEN E, YM HEIMER, Y MIZRAHI 1982 Ornithine decarboxylase and arginine decarboxylase activities in meristematic tissues of tomato and potato plants. Plant Physiol 70: 544-546 6. EVEN-CHEN Z, AK MATroo, R GOREN 1982 Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-['4C] methionine into spermidine in aged orange peel discs. Plant Physiol 69: 385388 7. GALSTON, AW, R KAUR-SAWHNEY 1987 Polyamines as endogenous growth regulators. In PJ Davies, ed, Plant Hormones and Their Role in Plant Growth and Development. Martinus Nijhoff, Dordrecht, pp 280-295 8. HEIMER YM, Y MIZRAHI 1982 Characterization of ornithine decarboxylase of tobacco cells and tomato ovaries. Biochem J 201: 373-376 9. HEIMER YM, Y MIZRAHI, U BACHRACH 1979 Ornithine decarboxylase activity in rapidly proliferating plant cells. FEBS Lett 104: 146-148 10. ICEKSON I, A GOLDLUST, A APELBAUM 1985 Influence of ethylene on Sadenosyl methionine decarboxylase activity in etiolated pea seedlings. J Plant Physiol 119: 335-345 11. ICEKSON I, M BAKHANASHVILI, A APELBAUM 1986 Inhibition by ethylene of polyamine biosynthetic enzymes, enhanced lysine decarboxylase activity and cadaverine accumulation in pea seedlings. Plant Physiol 82: 607-609 12. KOPELIOVITCH E, Y MIZRAHI, HD RABINOWITCH, N KEDAR 1980 Physiology of the tomato mutant Alcobaca. Physiol Plant 48: 307-31 1 13. MUTSCHLER MA 1981 Inheritance and characterization of "Alcobaca" storage mutant in tomato. Hortiscience 16: 399 14. MUTSCHLER MA 1984 Inheritance and linkage of the Alcobaca ripening mutant in tomato. Am J Hortic Sci 109: 500-503 15. MUTSCHLER MA 1984 Ripening and storage characteristics of the "Alcobaca" ripening mutant in tomato. Am J Hortic Sci 109: 504-507 16. ROBERTS DR, MA WALKER, JE THOMPSON, EB DUMBROFF 1984 The effects of inhibitors of polyamine and ethylene biosynthesis on senescence, ethylene production and polyamine levels in cut carnation flowers. Plant Cell Physiol 25: 3 15-322 17. SMITH MA, PJ DAVIES 1985 Separation and quantitation of polyamines in plant tissue by high performance liquid chromatography of their dansylated derivatives. Plant Physiol 78: 89-91 18. SMITH MA, PJ DAVIES 1987 Monitoring polyamines in plant tissues by high performance liquid chromatography. In HF Linskens, JF Jackson, eds, High Performance Liquid Chromatography in Plant Sciences (Modern Methods of Plant Analysis, New Ser. Vol 5). Springer-Verlag, New York, pp 209-227
