HORTSCIENCE 41(6):1462–1465. 2006. systems also catalyze similar conversions during O2 deprivation when an abundance – and Nitrite Treatments of NO2 can provide an adequate electron acceptor in lieu of O2 (Neill et al., 2003). Under sufficiently acidic conditions, HNO2 Reduce Ethylene Evolution from can be reduced to produce _NO by common metabolites including ascorbate (Weitzberg Fruit Disks and Lundberg, 1998) and various phenyl- propanoids (Bethke et al., 2004; Peri et al., David R. Rudell1 and James P. Mattheis2,3 2005). U.S. Department of Agriculture, Agricultural Research Service, Tree Fruit Interest in amending horticultural crop Research Laboratory, 1104 N. Western Avenue, Wenatchee, WA 98801-1230 storage practices with _NO exposure follows reports of decreased _NO evolution coinci- Additional index words. Malus sylvestris var. domestica (Borkh.), fresh-cut, dioxide, dent with increased ethylene production dur- N-nitrosoglutathione, sodium nitroprusside ing fruit maturation (Leshem and Pinchasov, 2000; Leshem et al., 1998). Furthermore, Abstract Ô Õ Malus sylvestris domestica . Golden Delicious apple [ var. (Borkh.)] cortex disks exogenous _NO (g)or_NO donor treatment _ S suspended in solutions containing a nitric oxide ( NO) donor [ -nitrosoglutathione of a variety of agricultural commodities in- _ (GSNO) or sodium nitroprusside (SNP)], NO gas, or nitrite (KNO2)wereusedto cluding carnation (Bowyer et al., 2003), _ – _ identify impacts of NO on ethylene production and NO2 on NO and ethylene broccoli, strawberry, kiwi (Leshem et al., production. Treatment with GSNO or SNP reduced ethylene biosynthesis compared 1998), and pear (Sozzi et al., 2003), purport- with control treatments containing equimolar concentrations of oxidized glutathione edly increases storage . _ (GSSG) or Na4(CN)6 respectively. Apple disk exposure to NO gas did not impact The objective of the current study was to – _ ethylene production. Treatment with NO2 resulted in increased NO production and determine whether _NO evolved from do- _ decreased ethylene biosynthesis. Generation of NO increased linearly whereas nors or supplied as a gas has a role in reg- – ethylene generation decreased exponentially with increasing NO2 treatment concen- ulation of ethylene evolution from apple _ – tration. NO was enhanced in autoclaved tissue disks treated with NO2 , suggesting that tissue disks. its production is produced at least in part by nonenzymatic means. Although this _ – evidence shows NOisreadilygeneratedinapplefruitdisksbyNO2 treatment, and _ – Materials and Methods ethylene synthesis is reduced by NO/NO2 generated in solution, the exact nature of _ – NO generation from NO2 and ethylene synthesis modulation in apple fruit disks ÔGolden DeliciousÕ apple fruit were har- remains to be elucidated. vested from a commercial orchard and stored at 0 C and 95% RH in air for 2 to 4 months. Upon removal from storage, were held _ The uncharged, gaseous radical nitric with O2, the stability of NO increases under at 20 C for 2 to 3 d. The top and bottom of oxide (_NO) has an established role in the hypoxic and anoxic conditions (Bonner and each apple was removed and a 7-mm brass physiological processes of many organisms. Stedman, 1996). In aqueous media, dissolved cork borer was used to excise tubes of cortex _ _ In plants, NO is implicated in cell damage, NO2 typically forms and nitrites tissue. Each tube was sliced into disks ~2 to signal transduction, and wound response (Bonner and Stedman, 1996). _NO can be 3 mm thick. About 5 g of disks were weighed (Neill et al., 2003). _NO is involved in the oxidized or reduced, producing NO+ or NO– into 50-mL Erlenmeyer flasks, to which regulation of a wide variety of specific pro- (Bonner and Stedman, 1996), species that a buffered solution containing 5 mL 0.1 M cesses including regulation of seed germina- also exhibit biological activity (Stamler and 4-morpholineethanesulfonic acid (MES; (Ph, _ tion (Beligni and Lamattina, 2000), Feelisch, 1996). Reactions between NO and 6.0), 0.01 M CaCl2, and a specific treatment _– hypocotyl elongation (Beligni and Lamatti- the reactive species O2 can produce compound were added. Treatments included na, 2000), stomatal aperture (Lum et al., the highly reactive and potentially damaging 0.01 to 10 mM KNO2, 0.5 mM N-nitrosoglu- – 2002), programmed cell death (Pedroso species peroxynitrite (OONO ) (Bonner and tathione (GSNO), 0.5 mM oxidized glutathi- et al., 2000), xylem differentiation (Gabaldo´n Stedman, 1996). Also, unlike many other one (GSSG), 0.5 mM sodium nitroprusside _ et al., 2005), plant defense (Delledonne et al., radical species, NO can disperse from its (SNP), and 0.5 mM Na4Fe(CN)6. GSNO and 1998), development (Pagnussat et al., cellular origin across membranes to neigh- SNP are commonly used _NO donors, and 2002), regulation of photosynthesis (Takaha- boring cells (Neill et al., 2003). GSSG and Na4Fe(CN)6 the respective con- shi and Yamasaki, 2002), and fruit Elucidation of _NO synthesis mechanisms trols (decomposition products) for these do- and senescence (Leshem and Pinchasov, in plants is less advanced than in animal nors. All chemicals were purchased from 2000; Leshem et al., 1998; Sozzi et al., 2003). systems. In animal systems, a family of FAD/ Sigma-Aldrich (St. Louis, Mo.). Experiments _ The free radical nature of NO contributes NADH-containing proteins called nitric ox- using GSNO, SNP, GSSG, or Na4Fe(CN)6 to variations in its longevity that are de- ide synthases catalyze the conversion of were performed in the dark to prevent light- _ pendent in part on environmental O2, con- L-citrulline to L-arginine and NO (Wendeh- induced decomposition. Additional cortex centration. In aerobic environments, _NO is enne et al., 2001). Although evidence of disks were autoclaved at 113 C, 68 N, for unstable, reacting with O2 to produce another similar catalytic activity and proteins with 13 min before treatment to evaluate the _ – _ gaseous free radical, nitrogen dioxide ( NO2) analogous structures exists for plants (Guo conversion of NO2 to NO in nonviable (Bonner and Stedman, 1996), although the et al., 2003; Ninnemann and Maier, 1996; tissue. After introduction of the treatment _NO half-life is longer than many other bi- Zeidler et al., 2004), additional evidence solution, flasks were left open for at least ologically significant radical species (Kop- suggests that other mechanisms of _NO pro- 30 min, then the head space was evacuated from penol, 1998). Because _NO readily reacts duction are prevalent in plant systems (Neill the flasks using ethylene-free compressed air et al., 2003; Yamasaki, 2005). Under neutral and the flasks were sealed with a rubber and alkaline conditions, a reaction catalyzed serum stopper. Flask head space was sampled by reductase (NR) uses NAD(P)H to (0.5 mL) after 30 min using a 1.0-mL syringe. Received for publication 16 May 2006. Accepted reduce (NO –) and produce _NO (Dean and for publication 4 July 2006. 2 Ethylene concentration was determined by 1Postdoctoral Research Plant Physiologist. Harper, 1988; Yamasaki et al., 1999). This injecting head space samples into a Hewlett- 2Research Plant Physiologist. reaction has been demonstrated in plants both Packard 5880 gas chromatograph (Agilent, 3To whom reprint requests should be addressed; in vitro (Yamasaki et al., 1999) and in vivo Palo Alto, Calif.) equipped with a 46-cm e-mail [email protected]. (Rockel et al., 2002). Other plant enzyme (length) · 0.32-cm (diameter) glass column

1462 HORTSCIENCE VOL. 41(6) OCTOBER 2006 POSTHARVEST BIOLOGY AND TECHNOLOGY packed with Porapack PQ (Supelco, Belle- fonte, Pa.) and a flame ionization detector. Flow rates for N2 carrier, H2, and compressed air makeup gases were 30, 10, and 100 mLÁmin–1 respectively. Oven, injector, and detector temperatures were 60, 100, and 200 C respectively. When repeat measure- ments were made, the serum stopper was removed until the next collection period. To test the efficacy of _NO gas for re- ducing ethylene evolution, tissue disks were weighed, placed in flasks, and the flasks were sealed as outlined earlier. The flasks were pur- ged with N2 until the head space atmosphere reached 0.5 kPa O2. Flask oxygen concentra- tion was measured using a CheckPoint hand- held digital O2/CO2 meter (PBI Dansensor, Glen Rock, N.J.). _NO (g) concentration in the flasks was adjusted to 10 uLÁL–1 by injecting the gas into the flask head space. Flask _NO concentration was verified using a chemiluminescent-based analysis system (Sievers 280i; Sievers, Boulder, Colo.). Head space was drawn into the instrument at 75 mLÁmin–1 and the initial (maximum) value recorded. Head space ethylene measurements were made as described previously from samples collected 15, 30, 60, and 75 min after _NO addition. All experiments were performed using three or more replications per treatment. Experiments using _NO donors were repeated three times. SE was calculated for the repeated _ measurements of C2H4 and NO. Regression analyses using the general linear model was performed using the SAS v.9.0 software package (SAS Institute, Cary, N.C.).

Results and Discussion Treatment of ÔGolden DeliciousÕ apple cortex disks with _NO donors caused a re- duction in ethylene production (Fig. 1). Treatment with GSNO reduced ethylene pro- duction within the first 30 min. SNP treat- Ô Õ m ment initially stimulated ethylene production Fig. 1. Ethylene production by Golden Delicious cortex tissue disks during treatment with 500 M of either GSNO and GSSG (control) or SNP and Na (CN) in 0.1 M MES (pH, 6.0) buffer and 0.01 M at 45 to 75 min, but production thereafter was 4 6 CaCl . Error bars represent SE (n = 3). SE was smaller than the symbol size where bars are not present. lower than that of the control. A previous 2 report indicated the impact of _NO (g) fumi- gation treatment on ethylene production was Nitrite treatment enhanced _NO and de- centration appears to occur via an intermedi- concentration dependent in whole ÔBartlettÕ creased ethylene emissions from tissue disks ary process contained within the tissue. – pear fruit (Sozzi et al., 2003). Similarly, (Fig. 2). Treatment with 1 mM NO2 resulted The results indicate decreases in ethylene tomato, lettuce, and pea plant growth was in an initial 80-fold increase in _NO pro- synthesis may have resulted from the pres- inhibited at high and enhanced at low _NO duction that coincided with a 12-fold de- ence of increased tissue concentration of treatment concentrations (Neill et al., 2003). crease in ethylene emission by the end of the _NO; however, the nature of the conversion _ – _ SNP treatment may have induced a similar experiment. NO production increased with of NO2 to NO in this system is less clear. – _ concentration-dependent response from ap- NO2 treatment concentration (Fig. 3A). Nitrite can be reduced to NO in reactions ple tissue through higher initial release of Maximum inhibition of ethylene emission catalyzed by various enzymes (Neill et al., _ + – NO or possibly NO , a species also purport- occurred between 1 mM and 10 mM NO2 2003), including NR (Yamasaki et al., 1999). _ – _ edly generated by SNP (Stamler et al., 1992). (Fig. 3B). Regression of NO production with Conversely, NO2 can be reduced to NO in Exposure of apple disks to _NO (g) under ethylene production demonstrates a similar the presence of polyphenols found in apple _ hypoxic conditions did not alter ethylene inverse relationship between NO and C2H4 homogenate (Peri et al., 2005) under acidic – production (data not presented). The lack of concentrations (Fig. 3C). Balancing ethylene conditions where NO2 is converted to HNO2 _ _ a NO treatment effect may have been the and N2 (g) or air with increasing levels of NO (Takahama et al., 2003). Autoclaved apple _ _ – result of the unstable nature of NO gas in air (g) in a gas burret did not affect the ethylene disks produced 19-fold more NO from NO2 (Bonner and Stedman, 1996), the suppressive concentration, indicating there is no direct than control disks, suggesting _NO produced _ – effect of hypoxia on apple fruit ethylene interaction between ethylene and NO (data in apple tissue from NO2 may arise at least in production and metabolism, or insufficient not presented) that would contribute to the part from reactions not requiring metabolic _ accumulation of NO in tissue disks compared reduced C2H4 concentrations detected in activity. In autoclaved tissue, phenylpropanoids with treatments during which _NO was gener- these experiments. Therefore, reduction of were likely available for interaction with _ – ated from aqueous donor compounds. ethylene production with increased NO con- NO2 as a result of decompartmentalization

HORTSCIENCE VOL. 41(6) OCTOBER 2006 1463 1998), including a climacteric fruit, ÔBartlettÕ pear (Pyrus communis) (Sozzi et al., 2003), to _NO gas has been reported to promote de- creased rates of ripening or senescence. Results of numerous _NO fumigation trials using a variety of treatment concentrations and treatment durations, application timing during storage regimes, fruit maturities, and storage periods after treatment using both whole ÔBartlettÕ pear and other intact apple and pear cultivars in our laboratory have failed to demonstrate consistent efficacy of this treat- ment for reducing whole apple and pear senescence or for quality maintenance (data not presented). The disparity in ethylene re- duction of whole fruit and excised tissue after _NO treatment in our studies suggests apple fruit ethylene production systems may be differentially affected by these treatments. For example, our evidence suggests wound ethylene (system 1), stimulated by tissue dis- ruption, may be reduced in the presence of _ Fig. 2. Ethylene and _NO production by ÔGolden DeliciousÕ cortex tissue disks during treatment with 1 mM NO, whereas ethylene production resulting KNO2 in 0.1 M MES (pH, 6.0) buffer and 0.01 M CaCl2. Error bars represent SE error (n = 3). SE was from climacteric ripening (system 2) may be smaller than the symbol size where bars are not present. impacted less, if at all. The efficiency of _NO treatment for reducing senescence of noncli- macteric commodities (Leshem et al., 1998) may also be consistent with this interpretation. The current study illustrates reduction of ethylene production using _NO treatment of excised tissue of a climacteric fruit. This treatment may ultimately have utility for extending storage life of fresh-cut fruit. – Application of NO2 to tissue disks was highly effective for both enhancing _NO and reducing ethylene levels, indicating that in- – creasing NO2 levels in apple fruit tissue before processing may influence storage life of apple slices while avoiding direct applica- – tion of NO2 .

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