ABSTRACT. Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of ...
Plant Physiol. (1990) 92, 547-550
Received for publication September 22, 1989 and in revised form November 16, 1989
0032-0889/90/92/0547/04/$01 .00/0
Communication
Polyamine Levels and Tomato Fruit Development: Possible Interaction with Ethylene Robert A. Saftner* and Bruce G. Baldi' U.S. Department of Agriculture, Agricultural Research Service, Plant Hormone Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland 20705 opposite effects in relation to fruit ripening and senescence (3, 5, 9, 22, 26). Because of this, polyamine and ethylene physiologies may be linked during fruit development. This paper describes the changes in free polyamine levels and ethylene production during fruit development in pericarp from normal and slow ripening tomato cultivars.
ABSTRACT Fruits of tomato, Lycopersicon esculentum Mill. cv Liberty, ripen slowly and have a prolonged keeping quality. Ethylene production and the levels of polyamines in pericarp of cv Liberty, Pik Red, and Rutgers were measured in relation to fruit development. Depending on the stage of fruit development, Liberty produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. The polyamines putrescine, spermidine, and spermine were present in all cultivars. Cadaverine was detected only in Rutgers. Levels of putrescine and spermidine declined between the immature and mature green stages of development and prior to the onset of climacteric ethylene production. In Pik Red and Rutgers, the decline persisted, whereas in Liberty, the putrescine level increased during ripening. Ripe pericarp of Liberty contained about three and six times more free (unconjugated) polyamines than Pik Red and Rutgers, respectively. No pronounced changes in spermidine or cadaverine occurred during ripening. The increase in the free polyamine level in ripe pericarp of Liberty may account for the reduction of climacteric ethylene production, and prolonged storage life.
MATERIALS AND METHODS Plant Material
Fruits at various stages of development were harvested from greenhouse-grown plants of tomato (Lycopersicon esculentum Mill.) cv Liberty, Pik Red, and Rutgers. Fruits were graded for maturity and ripening stages (12, 17). The six fruit stages used were immature green, mature green, breaker, pink, light red, and red. Polyamine Analysis
A regulatory role for polyamines in plants is suggested by their ubiquity, their abundance in actively growing tissues and their decline in senescing tissues, the regulation of their production by factors that affect plant growth and development, and their effects on plant growth and development when applied to plants (9, 10, 22). Little is known about the role of polyamines in fruit development. In avocado (13, 26), apple (6), pear (25), and tomato cv Rutgers (4) fruits, free polyamine levels decline during fruit development. An increase is observed in the fruits of mandarin (16), Shamouti orange (11), and tomato landrace Alcobaca containing the recessive allele alc (7) during fruit maturation and ripening. Infusing polyamines into pear fruits delayed fruit ripening (25). These findings suggest that free polyamines serve as endogenous antisenescence agents. Ethylene is a senescence-promoting hormone and accelerates fruit ripening (1, 27). Free polyamines inhibited ethylene production in a variety of tissues (2, 5, 8,24) including tomato pericarp (18). Ethylene and polyamines are known to have
Polyamine analyses were performed as described elsewhere (23). Because free, but not conjugated, polyamines have been implicated as endogenous antisenescence agents (10, 15, 21), only free polyamines were analyzed. After tissue homogenization in chilled mortars with pestles in 0.2 N HC104 (100 mg tissue/mL acid) and dansylation of the extracts, dansylated polyamines were separated by HPLC on a 4.6 mm x 25 cm reverse phase C18 column packed with 5,um Whatman ODS3 resin. The polyamines were eluted with a 23 min 60% to 95% methanol in water gradient. Peaks were detected with an in-line fluorescence spectrophotometer (Perkin Elmer, model 650-10S, excitation = 365 nm, emission = 510 nm) and quantification was done using a relative calibration procedure as described in Smith and Davies (23). The identity of the dansyl polyamines was determined by retention times relative to an internal standard of 1,6-diaminohexane, by coinjection of known standards, and by mass spectrometric verification of HPLC-eluting peaks. A Kratos MS25 RFA mass spectrometer with a DS90 data system was used. Didensyl Put2 and Cad were analyzed by direct injection probe as dried residues using electron ionization (70 eV). Samples were volatilized using a temperature gradient of 100 to 400°C over 6 min with peak ion levels detected from 200 to 250°C. Samples from
' Current address: Frederich Miescher Institut, Postfach 2543, CH4002, Basel, Switzerland.
2 Abbreviations: Put, putrescine; Cad, cadaverine; Spd, spermidine; Spn, spermine.
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Plant Physiol. Vol. 92, 1990
SAFTNER AND BALDI
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both HPLC fractions and standards had volatility and fragmentation patterns identical to each other and to those reported by Seiler et al. (20) for dansylated polyamines. Tridansyl Spd was analyzed as a dried residue by desorption chemical ionization (70 eV) using isobutane as reagent gas (-0.9 bar). Both HPLC fraction samples and standard samples of tridansyl Spd were detected within 30 s of a temperature gradient program of 100 to 600C over 1 min. The 'softer' ionization allowed detection of higher mass fragments as well as molecular ions with mass additions due to the reagent gas that were in agreement for all samples analyzed. Fragmentation was characteristic of tridansyl Spd as reported by Seiler et al. (20). Tetradansyl Spm was also analyzed using desorption chemical ionization; however, only detection of molecule fragments characteristic of dansylated polyamines was achieved.
Ethylene Analysis Pericarp slices were prepared as previously described (19). One g lots of pericarp slices were incubated at 230C in sealed 25-ml Erlenmeyer flasks containing 2 mL of 10 mM MesKOH (pH 6.0) and 200 mM sorbitol. Ethylene in air samples collected during incubation was analyzed with a gas chromatograph (14). The data presented in the "Results" are for single experiments with triplicate samples and are representative of a group of three or more experiments. RESULTS Ethylene Production and Fruit Ripening All cultivars exhibited a climacteric rise in ethylene pro-
duction (Fig. 1). Throughout fruit development, pericarp of Liberty fruits produced between 16 and 38% of the ethylene produced by Pik Red and Rutgers. Except for the red stages of development, pericarp of Rutgers fruits consistently produced more ethylene than that for Pik Red. Liberty fruits ripened more slowly than Pik Red and Rutgers fruits. Vine ripening from mature green to red took 17 to 21 d for Liberty fruits, 10 to 13 d for Pik Red fruits, and 8 to 10 d for Rutgers fruits. Detached red fruits of Liberty were noticed to have about twice the storage life (time until skin shriveling, 19 to 27 d versus 8 to 11 d) of Pik Red and Rutgers fruits. Polyamine Levels
Put, Spd, and Spm were present in the pericarp of all three cultivars with Spm being just detectable. In Rutgers pericarp, Cad was also present. Between the immature and mature green stages of development, Put and Spd levels declined in all three cultivars, whereas Cad, detected only in Rutgers, increased. The free polyamine level declined in the pericarp of all three cultivars in the initial stages of development (Fig. 2). Thereafter, the Put levels remained similar through the ripening stages in Rutgers and Pik Red while it increased in Liberty. The Spd level remained similar in all cultivars during ripening. Cad levels also remained similar in Rutgers pericarp
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Stage of fruit development Figure 1. Ethylene production in tomato pericarp slices of cv Rutgers, Pik Red, and Liberty fruits at various stages of fruit development. One g lots of pericarp slices from Rutgers, Pik Red, and Liberty fruits at various stages of fruit development were incubated at 230C in sealed 25-mL Erlenmeyer flasks containing 2 mL of 10 mM MesKOH (pH 6.0) and 200 mm sorbitol. Ethylene in air samples collected during incubation was analyzed with a gas chromatograph. SD did not exceed 8% of the mean values. IG = immature green, MG = mature green, B = breaker, P = pink, LR = light red, R = red.
during ripening. Cad levels also remained similar in Rutgers pericarp during ripening. At the red stage, Liberty pericarp contained about three and six times the amount of free polyamines as Pik Red and Rutgers pericarp, respectively. DISCUSSION
Pericarp of Liberty shows increased free polyamine levels and decreased climacteric ethylene production in ripening fruits as compared to pericarp of Pik Red and Rutgers. Fruits of Liberty also ripen more slowly and have a longer keeping quality than fruits of Pik Red and Rutgers. Since applied polyamines have been shown to inhibit ethylene production in a variety of plant tissues (2, 5, 8, 24) including tomato pericarp (18), the elevated level of free polyamines may be responsible for the reduction in both ethylene production and ripening of Liberty fruits. Alternatively, but less likely, the increase in the free polyamine level may reflect an otherwise inhibited ripening process. Because free polyamines act as antisenescence agents in some tissues, the elevated level of free polyamines in ripe tissue may also be responsible for the longer keeping quality of Liberty fruits in that overripening is a process associated with senescence. Elevated levels of free polyamines have similarly been observed in the pericarp of tomato landrace Alcolaca having the recessive allele alc (7). Like Liberty fruits, alc fruits also ripen slowly and have prolonged keeping qualities (7). Furthermore, when inhibitors of polyamine biosynthesis, i.e., difluoromethyl ornithine, Darginine, L-canavanine, were applied to pericarp of Liberty, free polyamine levels decreased (R Saftner, unpublished data) and ethylene production increased (18). The ripening process also can be delayed in tomato fruits just beginning to turn red by infusing them with Put (P Davies, personal communica-
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ETHYLENE PRODUCTION AND POLYAMINE LEVELS IN TOMATO PERICARP
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Stage of fruit development Figure 2. Free polyamine levels in tomato pericarp of cv Uberty, Pik Red, and Rutgers fruits at various stages of fruit development. Freshly prepared tomato pericarp of Rutgers (A), Pik Red (B), and Liberty (C) fruits at various stages of development were extracted with 0.2 M perchloric acid, the extracts dansylated, and dansyl polyamines separated by an HPLC system with a 5 jm Whatman ODS-3 reverse phase column (4.6 mm x 25 cm). The mobile phase was 60% to 95% (vol/vol) methanol in water in 23 min at a flow rate of 1 mL min-'. Detection was by an in-line fluorescence spectrophotometer, excitation = 365 nm, emission = 510 nm. SD did not exceed 5% of the mean values. Abbreviations are the same as in Figure 1.
tion). Thus, free polyamine and ethylene physiologies are interrelated and may account, at least in part, for the ripening and storage characteristics of tomato fruits. LITERATURE CITED 1. Abeles FB (1973) Ethylene in Plant Biology. Academic Press, New York 2. Apelbaum A, Burgoon AC, Anderson JD, Lieberman M, BenAire R, Mattoo AK (1981) Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts. Plant Physiol 68: 453-456 3. Apelbaum A, Goldlust A, Icekson I (1985) Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth. Plant Physiol 79: 635-640 4. Bakanashvili M, Barkai-Golan R, Kopeliovitch E, Apelbaum A (1987) Polyamine biosynthesis in Rhizopus-infected tomato fruits: possible interactions with ethylene. Physiol Mol Plant Pathol 31: 41-50 5. Ben-Aire R, Lurie S, Mattoo AK (1982) Temperature-dependent inhibitory effects of calcium and spermine on ethylene biosynthesis in apple discs correlate with changes in microsomal membrane microviscosity. Plant Sci Lett 24: 239-247 6. Biasi R, Bagni N, Costa G (1988) Endogenous polyamines in apple and their relationship to fruit set and fruit growth. Physiol Plant 73: 201-205 7. Dibble ARG, Davies PJ, Mutschler MA (1988) Polyamine content of long-keeping Alcobaca tomato fruit. Plant Physiol 86: 338-340 8. Even-Chen Z, Mattoo AK, Goren R (1982) Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunt label from 3,4-['4C]methionine into spermidine in aged orange peel discs. Plant Physiol 69: 385-388
9. Galston AW (1982) Polyamines as modulators of plant development. BioScience 33: 382-388 10. Galston AW, Kaur-Sawhney R (1987) Polyamines and senescence in plants. In WW Thomson, EA Nothnagel, RC Huffaker, eds, Plant Senescence: Its Biochemistry and Physiology. American Society of Plant Physiologists, Rockville, MD, pp 167-181 11. Hasdai D, Bar-Akiva A, Goren R (1986) Chemical and morphological characteristics of developing fruits from old clone v. nucellar Shamouti orange trees. J Hort Sci 61: 389-395. 12. Kader AA, Morris LL (1976) Correlating subjective and objective measurements of maturation and ripeness of tomatoes. In Proceedings of the 2nd Tomato Quality Workshop, University of California, Davis, pp 57-62 13. Kushad MM, Yelenosky G, Knight R (1988) Interrelationship of polyamine and ethylene biosynthesis during avocado fruit development and ripening. Plant Physiol 87: 463-467 14. Lieberman M, Kunishi A, Mapson LW, Wardale DA (1966) Stimulation of ethylene production in apple tissue slices by methionine. Plant Physiol 41: 376-382 15. Martin-Tanguy J (1985) The occurrence and possible function of hydroxycinnamoyl acid amides in plants. Plant Growth Regul 3: 381-399 16. Nathan R, Altman A, Monselise SP (1984) Changes in activity of polyamine biosynthetic enzymes and in polyamine contents in developing fruit tissues of 'Murcott' mandarin. Sci Hort 22: 359-364 17. Ryall AL, Lipton WJ (1972) Handling, Transportation and Storage of Fruits and Vegetables, Volume 1, Vegetables and Melons. AVI Publishing Co, Westport, CT, pp 155-156 18. Saftner RA (1989) Effects of organic amines of a-amino-isobutyric acid uptake into the vacuole and on ethylene production by tomato pericarp slices. Physiol Plant 75: 485-491 19. Saftner RA, Baker JE (1987) Transport and compartmentation of 1-aminocyclopropane-l-carboxylic acid and its structural
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SAFTNER AND BALDI analog, a-aminoisobutyric acid, in tomato pericarp slices. Plant Physiol 84: 311-317 Seiler N, Schneider H, Sonnenberg K (1970) Massenspektrometrische Identifizierung von biogenen Aminen in Form ihrer 1Dimethylamino-naphthalin-5-sulfonyl-Derivate. Z Anal Chem 252: 127-136 Slocum RD, Galston AW (1985) Changes in polyamine biosynthesis associated with postfertilization growth and development in tobacco ovary tissues. Plant Physiol 79: 336-343 Slocum RD, Kaur-Sawhney R, Galston AW (1984) The physiology and biochemistry of polyamines in plants. Arch Biochem Biophys 235: 283-303 Smith MA, Davies PJ (1985) Separation and quantification of polyamines in plant tissue by high performance liquid chro-
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matography of their dansyl derivatives. Plant Physiol 78: 8991 Suttle JC (1981) Effect of polyamines on ethylene production. Phytochemistry 20: 1477-1480 Toumadje A, Richardson DG (1988) Endogenous polyamine concentrations during development, storage and ripening of pear fruits. Phytochemistry 27: 335-338 Winer L, Apelbaum A (1986) Involvement of polyamines in the development and ripening of avocado fruits. J Plant Physiol 126: 223-233 Yang SF (1987) The role of ethylene and ethylene synthesis in fruit ripening. In WW Thomson, EA Nothnagel, RC Huffaker, eds., Plant Senescence: Its Biochemistry and Physiology. American Society of Plant Physiologists, Rockville, MD, pp 156166
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