Pawpaw [Asimina Triloba (L.) Dunal] Fruit Ripening. I. Ethylene Biosynthesis and Production

J. AMER. SOC. HORT. SCI. 130(4):638–642. 2005. Pawpaw [Asimina triloba (L.) Dunal] Fruit Ripening. I. Ethylene Biosynthesis and Production Rumphan Koslanund1 Post-Harvest Products and Processing Research and Development Offi ce, Department of Agriculture, Bangkok, Thailand Douglas D. Archbold2 Department of Horticulture, University of Kentucky, Lexington, KY 40546-0091 Kirk W. Pomper3 USDA National Clonal Germplasm Repository for Asimina spp., Atwood Research Facility, Kentucky State University, Frankfort, KY 40601-2355 ADDITIONAL INDEX WORDS. 1-aminocyclopropane-1-carboxylic acid, ACC, ACC synthase, ACC oxidase, malonyl-ACC ABSTRACT. Pawpaw fruit ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) activities, and tissue content of the ethylene precursor ACC and conjugate malonyl-ACC (MACC) were measured during postharvest ripening. Fruit were harvested near the advent of the ripening process and were ripened at room temperature. The fruit displayed increases in ethylene production and respiration rate during ripening with maxima for both 3 days after harvest. Mean ethylene maxima on a fresh weight basis were 4.7 and 7.6 μg·kg–1·h–1 and –1 –1 mean respiratory (CO2 production) maxima on a fresh weight basis were 220 and 239 mg·kg ·h in 1999 and 2001, respectively. The increase in ethylene evolution coincided with an increase in respiration and a rapid decline in fruit fi rmness. Internal and external fruit fi rmness declined in a parallel manner. The ethylene climacteric peak occurred after the greatest decline in fruit fi rmness, indicating that low levels of ethylene may be suffi cient to initiate the ripening process. The ethylene climacteric peak also coincided with the highest activities of both ACS and ACO as well as the maximum tissue ACC content. As ACC content increased, MACC content declined, suggesting a regulation of ethylene production via free ACC levels by malonylation of ACC. Thus, the climacteric development of ethylene production may be regulated by an increase of ACS activity and a decrease in ACC malonyltransferase activity, making more free ACC available for the production of ethylene by increased activity of ACO. The pawpaw belongs to the Annonaceae family, which in- Wills et al., 1984). For climacteric fruit, ethylene is generally cludes cherimoya (Annona cherimola Mill.), sweetsop or sugar thought to regulate fruit ripening by coordinating the expres- apple (Annona squamosa L.), custard apple (Annona reticulata sion of genes responsible for 1) enhancing a rise in the rate of L.), atemoya (Annona squamosa x A. cherimola), and soursop respiration, 2) autocatalytic ethylene production, 3) chlorophyll (Annona muricata L.). The genus Asimina Dunal is the only degradation, 4) pigment synthesis, 5) conversion of starch to temperate zone member of this tropical family (Darrow, 1975; sugars, 6) production of aroma volatiles, and 7) increased activ- Kral, 1960). The few earlier studies of pawpaw ripening have ity of cell-wall degrading enzymes (Gray et al., 1992). In the described qualitative changes including an increase in soluble ethylene-biosynthetic pathway, S-adenosylmethionine (SAM) is solids content, a rise in volatile production, and rapid loss of converted to ACC by ACS, while ACO catalyzes the conversion fi rmness (McGrath and Karahadian, 1994a, 1994b; Shiota, 1991). of ACC to ethylene (Yang and Hoffman, 1984). The levels of The shelf life of pawpaw fruit at ambient temperature does not free ACC may be regulated via malonylation of ACC to MACC, exceed 5 d, although cold storage may delay ripening for at least which could affect ethylene production (Lelievre et al., 1997). In 30 d (Archbold and Pomper, 2003). The rapid rate of pawpaw mature climacteric fruit, ethylene is autostimulatory and inhibi- ripening and deterioration may have led to its lack of horticultural tors of ethylene action block ethylene production and ripening attention to date. (Lelievre et al., 1997; Oetiker and Yang, 1995). Positive feedback Although pawpaw was initially confused in literature reviews regulation of ethylene biosynthesis is a characteristic feature of with papaya and mistakenly characterized as a climacteric fruit ripening climacteric fruit and senescing fl owers (Nakatsuka et many years ago, it does in fact exhibit respiratory and ethylene al., 1998). climacterics ≈3 d after harvest when held at ambient temperature Due to a need to slow the rapid rate of ripening and consequent (Archbold et al., 2003; Archbold and Pomper, 2003). The other deterioration of pawpaw fruit and the potential role of ethylene Annonaceae fruit noted above are climacteric as well (Brown et in mediating the process, the objective of this research was to al., 1988; Martinez et al., 1993; Paull, 1982; Tsay and Wu, 1989; study the pattern of ethylene production and respiration during pawpaw fruit ripening after harvest in relation to the activities of ACS and ACO and the levels of ACC and MACC. Materials and Methods Received for publication 25 May 2004. Accepted for publication 14 Jan. 2005. 1Former: Graduate Student, Dept. of Horticulture, Univ. of Kentucky, Lexington, KY 40546-0091. PLANT MATERIAL. Pawpaw fruit were harvested at the fi rst sign 2Professor of Horticulture. To whom reprint requests should be addressed. of softening as determined by touch from 22 and 28 genotypes 3Principal Investigator of Horticulture and Curator. at the Kentucky State Univ. Research Farm, Frankfort, on several 638 J. AMER. SOC. HORT. SCI. 130(4):638–642. 2005. BBookook 11.indb.indb 663838 66/13/05/13/05 33:35:49:35:49 PMPM dates in 1999 and 2001. Immediately following harvest, fruit were Lizada and Yang (1979). The reaction mixture consisted of a transported to the laboratory at the Univ. of Kentucky. Because 500-μL aliquot of the above ACC extract and 4 μM HgCl2, and preliminary work in 1998 indicated no appreciable variation in the volume was brought up to 1800 μL with water. These reaction ripening pattern among genotypes and the intent was to character- tubes were kept on ice. After the addition of 200 μL of a cold ize general trends of pawpaw fruit ripening, not possible clonal mixture of 5% NaOCl and saturated NaOH (2:1; v/v), the test differences, the fruit from the different genotypes were pooled for tube was immediately sealed with a serum stopper. The test tube use, and data was pooled across harvest dates within each year. was then vortexed for 10 s and returned to ice for 2 min. Prior MEASUREMENT OF ETHYLENE PRODUCTION AND RESPIRATION. to gas sampling, the reaction mixture was vortexed for another Ethylene production and respiration rate were determined at 10 s, and a 1-mL gas sample was then withdrawn for ethylene ambient temperature (22 ± 2 °C) during ripening after harvest analysis by gas chromatography (GC). in 1999 at 0, 1, 2, 3, 5, and 7 d with replication of 72, 53, 43, MACC was quantifi ed by the method of Hoffman et al. (1983). 43, 30, and 30 fruit, respectively, and in 2001 at 0, 1, 2, 3, 4, 5, A 500-μL aliquot of the above ACC extract was treated by add- and 6 d with replication of 59, 55, 40, 34, 22, 17, and 8 fruit, ing 1 mL 2 N HCl and placing it in a 100 °C water bath for 3 h to respectively. Replicate numbers declined as fruit were sampled hydrolyze MACC to ACC. The solution was cooled on ice for 15 for destructive analyses in this and other studies. min, and was then neutralized with 0.5 mL 4 N NaOH. The solu- Individual fruit were weighed and placed in a 0.98-L bottle at tion was frozen, lyophilized, and re-dissolved in 0.5 mL water. room temperature that was capped for 2 h. For C2H4 quantifi cation, The ACC content was determined as above. The concentration 1-mL gas samples were withdrawn from the bottle headspace by of MACC was calculated as the difference in ACC before and inserting a syringe through a fi tted septum, and injected into a after hydrolysis. The ACC and MACC results are expressed as Varian 2100 gas chromatograph (Varian, Palo Alto, Calif.) fi tted nanomoles per 100 g dry weight (DW). with a 1-m alumina column, run at 100/70/100 °C for the injector/ IN VITRO ACS ACTIVITY. ACS activity was measured by the column/fl ame ionization detector temperatures, respectively. The N2 methods of Yip and Yang (1993) with some modifi cations. Upon carrier gas fl ow rate was 30 mL·min–1. Ten-milliliter gas samples removal of fruit from –80 °C storage, fl esh tissue (10 g) was were withdrawn from the bottles to determine CO2 production ground in liquid N2 with a mortar and pestle. The macerate was using an Oxygen/Carbon Dioxide Headspace Analyzer ZR 892 HS homogenized (Omni 5000; Omni Intl., Gainesville, Va.) in 30 (Illinois Instruments, McHenry, Ill.). Fruit volume was subtracted mL ice-cold buffer containing 400 mM potassium phosphate (pH from bottle volume using a fruit weight vs. volume regression 8.5), 10 μM pyridoxal 5-phosphate (PLP), 4 mM dithiothreitol (volume = 1.79 + 0.81 × fresh weight; r = 0.8, P < 0.01) to calculate (DTT), and 20% glycerol. The homogenate was fi ltered through C2H4 and CO2 production per unit fruit weight per hour. four layers of cheesecloth and 1 layer of Miracloth (Calbiochem, FRESH WEIGHT AND FIRMNESS MEASUREMENT. Fruit external EMD Biosciences, La Jolla, Calif.) and then centrifuged at 48,400 fi rmness was determined using a Chatillon Digital Force Gauge gn and 4 °C for 20 min. The resulting supernatant (0.5 mL) was (model DFM 10; John Chatillon and Sons, Greensboro, N.C.) fi ltered through a G-50 Sephadex (Sigma Chemical Co., St. mounted on a model LTC test stand and equipped with a 2-mm Louis) column (10 mL bed volume) that had been equilibrated fl at probe.

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