Polyamine-induced prolongation of storage in tomato fruits'

Transcription

1 Plant Growth Regulation 10: , Kluwer Academic Publishers. Printed in the Netherlands. Polyamine-induced prolongation of storage in tomato fruits' DAVID M. LAW,* PETER J. DAVIES and MARTHA A. MUTSCHLER Section of Plant Biology (D.M.L. ; P.J.D.) and Department of Plant Breeding (M.A.M.), Cornell University, Ithaca, NY 14853, USA Received 18 December, 1990 ; accepted 4 April, 1991 Key words : Lycopersicon esculentum, tomato, fruit, polyamines, postharvest storage, shelf life, fruit softening Abstract. Mature green tomato fruit (Lycopersicon esculentum Mill) of cv. 'Rutgers' and the line 'Alcobaca-red' were vacuum infiltrated with solutions of polyamines, their precursors and metabolites, and other compounds which might affect ripening and/or storage duration. Putrescine (1,4diaminobutane), spermidine, spermine, diaminopropane, y-aminobutyric acid and methionine were found to increase the storage life of these fruit after vacuum infiltration of the test compounds and storage of fruit in darkness. Polyamines probably play a role in the normal ripening/overripening process and may prove commercially valuable in the extension of fruit shelf life. 1. Introduction Tomato fruits are often harvested while green because the firmness of these fruits facilitates shipping with reduced loss. Fruits are then exposed to ethylene to induce ripening. However, many fruits are immature and fail to respond to the hormone with full ripening, e.g., synthesis of flavor components, development of normal carotenoid levels, and softening. Detached fruit are more likely to ripen normally when more mature tomatoes are picked, but the rapid softening which accompanies ripening increases loss during transportation and reduces the shelf life of the marketed fruits. It would be desirable to produce fruits which can be harvested at the breaker stage (the first sign of color) or beyond and yet which retain firmness for a longer period after ripening. Edited by T. J. Gianfagna. ' The use of polyamines and related compounds in prolongation of the storage or shelf life of fruit is the subject of U.S. patent No. 4,957,757 (1990) awarded to the Cornell University Research Foundation. * Author for correspondence.

2 284 'Alcobaca' is a landrace of tomato which possesses the recessive gene ale [16]. The presence of this allele is associated with altered ripening characteristics, including prolonged storage capacity and the failure to produce normal levels of lycopene [11, 14, 16]. Analyses of polyamine levels in the pericarp of fruit of cv. Rutgers, 'Alcobaca' and 'Alcobaca-red' (an isogenic revertant possessing the Ale allele and exhibiting "normal" ripening) showed that free putrescine levels declined from the immature green to the mature green stage. However, while putrescine content remained low during the subsequent ripening of the "normal" (Alc) fruits, the level in 'Alcobaca' (alc) fruit rose through the breaker and ripe stages [6]. No differences in spermidine content were noted among lines, and spermine levels were Always extremely low. In an effort to understand the role of polyamines in the ripening processes of tomato fruits, and to determine if treatment with polyamines could maintain the firmness (and thus prolong the storage life) of fruits not possessing the a1c allele, detached mature-green fruits were treated with polyamines and related compounds which might affect ripening. 2. Materials and methods 'Alcobaca-red' is a bright-red-fruited line of tomato (Lycopersicon esculentum Mill) which arose from 'Alcobaca' as the result of a spontaneous reversion at the a1c locus [17] while cv. 'Rutgers' is a standard tomato cultivar. Field or greenhouse-grown mature-green fruits were vacuum infiltrated with aqueous solutions of the test compounds. Greenhouse-grown plants received supplemental light supplied by mercury halide lamps to extend the daylength to 16 h, under temperatures of 26 ± 3 C. Immediately prior to treatment calyces were removed, fruits were weighed, the stem scar was wiped with 95% ethyl alcohol, and the tomatoes were placed stem-side-up in a vacuum desiccator connected to a water aspirator. Field grown fruits were handled similarly except that sequential rinses with 0.5% sodium hypochlorite and water were added before calyx removal. After air drying to remove the alcohol, test solutions were applied to the stem scar depression, and the vacuum was applied for 30 ± 15 sec. This displaced air from the fruit, and quick vacuum release allowed uptake of the solution throughout the columella. Control fruits were treated with ultra purified water. Stem scars were blotted dry and the fruits were reweighed for more accurate determination of uptake volumes. Fifty ul to 1.5 ml of 1-50 mm solutions of each compound were used, giving internal concentrations of 5 to 500 µm of

3 285 the respective added compound, if uniform dispersion occurred in each fruit. Acidic compounds were neutralized in the application solution. Treated fruits were placed in a dark chamber at 21 ± 1 C. At 2 d intervals they were examined quickly under dim, indirect fluorescent light to determine the stage of ripening and the condition of ripened fruit. Fruits were stored until subjectively judged to be unacceptably oversoft for retail sale to consumers [1, 21]. In a second trial volumes of test solutions (10mM) were adjusted so as to give internal concentrations of about 100,uM and fruits were stored in darkness in humidified chambers (80% R.H.) at 24 ± 2 C. A penetrometer was employed to determine softening by comparing deformations under a 500 g weight applied to a 5 cm 2 plastic disc placed on the fruit at three equatorial points over the locules 10 d after treatment. IAA and GA 3 were obtained from Calbiochem (San Diego, CA). All other compounds were purchased from Sigma (St. Louis, MO). 3. Results When vacuum infiltrated into normal (Ale) fruit, solutions of the polyamines putrescine (1,4-diaminobutane) (PTC), spermidine (SPD) and spermine (SPM), the polyamine metabolite 1,3-diaminopropane (DAP), and precursors including agmatine (AGM) and methionine (MET) all increased storage life (Table 1). These compounds did not affect the time between infiltration and ripening (12.5 ± 2.8d and 12.0 ± 2.4d for Alcobaca-red and Rutgers, respectively) but shelf-life was prolonged after ripening occurred, often doubling the storage life. Fruits were usually rejected for overripening (oversoftening), though fruits were also discarded in response to desiccation, and occasionally upon pathogenic infection. A number of tested compounds failed to increase the storage life of greenhouse-grown 'Alcobaca-red' and 'Rutgers' tomato fruits when internal concentrations of the added compounds were in the range of 5-500,uM (if uniformly distributed). These included abscisic acid, a-aminoisobutyric acid (a-aib), aminooxyacetic acid (AOA), aminoethoxyvinylglycine (AVG), cobalt chloride, galactose, GA 3, IAA, silver nitrate, zeatin, and zeatin riboside. Silver nitrate, a-aib, and AOA delayed ripening of these fruit to some extent (data not shown). Since amines were the only substances implicated in storage prolongation, a second experiment was performed with field-grown mature-green fruit of 'Alcobaca-red' to further examine amine effects. These fruits were stored at a slightly higher temperature (26'C) under 80% relative humidity to reduce desiccation. Ripening was complete within 7 d under these conditions ("red

4 Table 1. Effect of amines on storage life of Alcobaca-red and cv. Rutgers tomatoes. Mature green fruit were vacuum infiltrated with test compounds (1-50 mm) so as to give internal concentrations ranging from pm (mean internal concentration = 102 pm) and were then stored in darkness at 21 C. Fruits were scored for the number of days of storage after attaining ripeness before rejection for oversoftening, desiccation, or pathogenic infection. Compound Number of Days of storage % of Compound Number of Days of storage % of samples after ripening (± S.E.) control samples after ripening (± S.E.) control Controls Control' AGM` ± AGM * 204 ARG ** 181 ARG ** 241 CIT ** 203 CAD ** 185 DAP ± 3.0 * 172 DAP ** 228 MET ** 217 ORN ** 196 PTC * 165 PTC * 194 SPD ** 225 SPD ** 202 SPM ** 219 SPM * 174 SAM ** 297 a = 18µl water g' fresh weight; b = 7,ul water g' fresh weight ; `Abbreviations : AGM = agmatine; ARG = arginine ; CAD = cadaverine ; CIT = citrulline; DAP = 1,3-diaminopropane; MET = methionine ; ORN = ornithine; PTC = putrescine ; SPD = spermidine ; SPM = spermine; SAM = S-adenosylmethionine. * = significant at P = 0.05 by `t' test ; ** = significant at P = 0.01 by `t' test. Cultivar Alcobaca-red cv. Rutgers

5 287 Table 2. Effects of amines on softening of 'Alcobaca-red' fruit. Mature green fruit were infiltrated with a variable volume of 10 mm solutions of test compounds to give final internal concentrations of 100 µm (if evenly distributed). Penetrometer deformations were measured 10 d later. Compound Number of samples Penetrometer deformation (mm) Significant difference vs controls (x ± S.E.) Water a-aba' NS fl-aba ± 0.20 NS GABA _ a-aib NS fl-ala ± 0.30 NS DAP ORN NS PTC _ SPD a Confidence level according to `t'-test; NS = not significantly different. n Abbreviations : a-aba = a-aminobuytric acid ; #-ABA = Q-aminobutyric acid ; GABA = y-aminobutyric acid ; a-aib = a-aminoisobutyric acid ; #-ALA = f-alanine; DAP = 1,3- diaminopropane; ORN = L-ornithine; PTC = putrescine ; SPD = spermidine. ripe" according to the USDA color chart for tomato ripening), though the incidence of pathogenic infection was greater (infected fruit were discarded). PTC, SPD, amines metabolically related to them, and unrelated amines (the a and /3 isomers of aminobutyric acid) were compared. Results of penetrometer readings (Table 2) confirmed that effective compounds increased the keeping capacity of tomato fruit by slowing the rate of softening. Furthermore, the different amines varied greatly in relative effectiveness of maintaining firmness. PTC, SPD, and their metabolites DAP and y-aminobutyric acid (GABA), maintained fruit firmness, while ornithine (ORN), /3-alanine (fl-ala), and other isomers of aminobutyric acid had no effect (Table 2). 4. Discussion The addition of amines in amounts similar to the endogenous soluble PTC content [6, 211 increased shelf life about two-fold. While polyamines have been implicated as important regulators in early stages of tomato fruit growth [4, 5, 9], the role they play in the ripening process is unknown. Polyamines are often regarded as anti-senescence agents and have been proposed to act through binding to nucleic acids or membranes [25]. AGM and ORN are potential precursors of PTC; MET and PTC are precursors of SPD and SPM ; and DAP, GABA, and /3-ALA can arise as products of

6 288 further metabolism of PTC, polyamines, and ORN [7, 18, 19, 22, 25]. In tomato fruit PTC is metabolized to SPD, GABA, and glutamic acid, while SPD is metabolized to PTC and fl-alanine [18]. Compounds which prolonged the storage life of tomatoes included MET, AGM, PTC, SPD, SPM, GABA, and DAP (Tables 1 and 2). All are specifically related to endogenous polyamine metabolism. The metabolically unrelated isomers of aminobutyric acid and a-aib (which is an ethylene synthesis inhibitor and structural analogue of 1-aminocyclopropane-l-carboxylic acid (ACC) [20]) were ineffective. ORN and /3-ALA did not reduce softening ; perhaps the fruits were unable to significantly metabolize or incorporate these compounds as active forms. Thus, prolongation of storage seems not to be a generalized effect of amine infiltration, but to be related specifically to some compounds involved in the polyamine pathway. It is notable that effective amines did not delay the duration of ripening (i.e., the time between treatment at the mature green stage and full color development) but rather slowed the process of softening after ripening. Other substances have been reported to extend fruit shelf life, though often through delayed ripening ; these include auxins [1, 3, 15, 26], gibberellins [1, 3, 15], calcium [15], and succinic acid-2,2-dimethylhydrazide [15]. Inhibition of softening by exogenous amines may have mimicked a function of endogenous PTC in the long-keeping lines Alcobaca [6] and Liberty [21]. In these lines levels of free PTC decreased in pericarp tissue between the immature and mature green stages, as happened in the normal-keeping lines Rutgers, Alcobaca-red, and Pik-Red. However, in the long-keeping lines PTC levels subsequently increased throughout ripening, while they remained low in the normal lines. It has been proposed that elevated PTC inhibited ethylene synthesis [21]. Polyamines have been shown to be capable of inhibiting ethylene production [2, 10], and neither Alcobaca nor Liberty fruits exhibit a sharp climacteric during ripening [16, 21]. However, the enhancement of ACC synthesis at the start of ripening is not associated with any drop in the level of PTC or SPD in normal tomato fruit [4]. More than an inhibition of ethylene synthesis and/or action by polyamines must be involved in decreased softening rates, since inhibitors of ethylene formation (a-aib, AOA, AVG) and action (silver, cobalt) failed to prolong the storage life of 'Alcobaca-red' and 'Rutgers fruits'. The effective amines may have had some more direct action. Recent experiments on the addition of polyamines to apples have shown that these compounds delay softening and extend shelf life in these fruits [12]. In this case the mechanism of action has been shown not to be via a change in ethylene production, and a direct action on the cell wall was proposed. Certain amines and their conjugates are incorporated into cell

7 289 walls [8, 23]. Such incorporation might decrease the activity of wall-degrading enzymes by affecting enzyme production or enzyme ability to act on substrate. Kramer et al. [13] have found that polyamines inhibit the action of polygalacturonase (PG) in vitro. While PG may not be the major regulator of softening [24], this inhibition of PG indicates that polyamines may also inhibit the action of other, as yet uncharacterized, wall-degrading enzymes. In support of this proposal, the presence of polyamine conjugates has been associated with resistance to pathogenic attack [23]. Alternatively, the free amines may act within the cell or cell wall to alter the level or activity of the wall-degrading enzymes. The level of amines applied was not vastly greater than that naturally present, so that the reason for the action is not immediately clear. However, applied compounds may reach different sites compared to their endogenous counterparts, or exogenous amines may alter endogenous amine metabolism [23, 25]. By analogy, exogenous abscisic acid can alter ripening when applied in amounts that are a small fraction of endogenous levels, perhaps as a result of differences in compartmentation [15]. Acknowledgements We thank Ruth Calhoun for typing the manuscript. This research was supported by grants from The Cornell University Biotechnology Program and The Frasch Foundation. References 1. Abdel-Kader AS, Morris LM and Maxie EC (1966) Effect of growth-regulating substances on the ripening and shelf-life of tomatoes. HortSci 1 : Apelbaum A, Burgoon AC, Anderson JD, Lieberman M, Ben-Arie R and Mattoo AK (1981) Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts. Plant Physiol 68 : Ben-Arie R and Lurie S (1986) Prolongation of fruit life after harvest. In : SP Monselise, ed. CRC Handbook of Fruit Set and Development, Boca Raton, FL: CRC Press 4. Casas JL, Acosta M, del Rio JA and Sabater F (1990) Ethylene evolution during ripening of detached tomato fruit: Its relation with polyamine metabolism. Plant Growth Regulation 9: Cohen E, Arad S and Heimer YM (1982) Participation of ornithine decarboxylase in early stages of tomato fruit development. Plant Physiol 70 : Dibble ARG, Davies PJ and Mutschler MA (1988) Polyamine content of long-keeping Alcobaca tomato fruit. Plant Physiol 86: Flores HE and Filner P (1985) Polyamine catabolism in higher plants : Characterization of pyrroline dehydrogenase. Plant Growth Reg 3 : Goldberg R and Perdrizet E (1984) Ratio of free to bound polyamines during maturation in mung-bean hypocotyl cells. Planta 161 :

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