Postharvest Biology and Technology 20 (2000) 231 – 241
Peel tissue
a
-farnesene and conjugated trienol concentrations
during storage of ‘White Angel’
×
‘Rome Beauty’ hybrid
apple selections susceptible and resistant to superficial scald
Bruce D. Whitaker
a,*, Jacqueline F. Nock
b, Christopher B. Watkins
baHorticultural Crops Quality Laboratory,Plant Sciences Institute,Agricultural Research Ser
6ice,USDA,Building002,
10300Baltimore A6enue,Belts6ille,MD20705-2350, USA
bDepartment of Fruit and Vegetable Science,Cornell Uni
6ersity,Ithaca,NY14853,USA
Received 28 February 2000; accepted 20 June 2000
Abstract
In a 2-year study, fruit from eight red- and eight yellow-skinned ‘White Angel’בRome Beauty’ hybrid selections were stored for 21 weeks at 0.5°C plus 1 week at 20°C and evaluated for the incidence and severity of superficial scald. Five red-skinned (R-03, R-20, R-22, R-48 and R-85) and three yellow-skinned (Y-26, Y-55 and Y-65) selections were examined in both seasons. Peel-tissue samples taken at 0, 7, 14 and 21 weeks of storage were analyzed for concentrations of a-farnesene and its conjugated trienol (CTol) oxidation products by HPLC with UV detection.
Three red-fruited (R-44, R-48 and R-85) and five yellow-fruited (Y-38, Y-40, Y-55, Y-65 and Y-67) lines exhibited scald symptoms. The remaining lines (R-01, R-03, R-16, R-20, R-22, Y-07, Y-26 and Y-28) were free of scald. Overall, production ofa-farnesene and accumulation of CTols were not closely correlated with scald susceptibility.
Data for the selections most prone to scald, Y-65, Y-40 and R-44, were consistent with the proposed role of
a-farnesene oxidation products in scald induction, but for Y-55 and R-48, which developed mild to moderate scald
and accumulated very little CTols, the data conflicted with the a-farnesene oxidation – scald induction hypothesis.
Also, scald-resistant lines Y-07 and R-22 produced high levels of a-farnesene and reached CTol concentrations
comparable to those in several scald-susceptible lines. We conclude that if CTol do play a role in scald induction, there must be other mitigating factors of at least equal importance. Moreover, our findings support the proposal that oxidation products of a-farnesene are not essential for scald development in fruit with severely compromised
antioxidative defenses, but free radicals and/or toxic volatiles generated bya-farnesene oxidation can exacerbate scald
symptoms. © 2000 Published by Elsevier Science B.V.
Keywords:Peel-tissue samples;a-Farnesene; Conjugated trienol
www.elsevier.com/locate/postharvbio
1. Introduction
Superficial scald is a physiological disorder that arises in certain cultivars of apples and pears after
* Corresponding author. Tel.: +1-301-5046984; fax: + 1-301-5045107.
E-mail address: whitakerb@ba.ars.usda.gov (B.D. Whitaker).
long-term cold storage (Ingle and D’Souza, 1989). Largely on the basis of correlative data, the sesquiterpene a-farnesene, and more directly its
conjugated triene (CT) oxidation products, have long been thought to play a central role in scald induction (Huelin and Coggiola, 1968, 1970a; Anet and Coggiola, 1974; Whitaker et al., 1998). How-ever, the biochemical mechanism of the disorder has not been elucidated. Recent findings are consistent with the proposal that a volatile end-product of
a-farnesene oxidation, 6-methyl-5-hepten-2-one
(MHO), causes the discoloration and death of hypodermal cells which lead to development of scald symptoms (Mir and Beaudry, 1999; Mir et al., 1999). Exogenous MHO induced scald-like brown-ing in peel tissue from fruit of scald-susceptible apple cultivars (Mir and Beaudry, 1999), and a poststorage burst of MHO evolution was associated with intensification of scald symptoms in ‘Cortland’ apples (Mir et al., 1999). Moreover, it was recently shown that the CTs which accumulate in the skin and epicuticular wax of apple fruit during storage, mainly 7E,9E and 7E,9Z isomers of 2,6,10-trimethydodeca-2,7,9,11-tetraen-6-ol (conjugated trienols) in a ratio of 9:1 (Rowan et al., 1995; Whitaker et al., 1997), autoxidize at 20°C yielding MHO as a major product (Whitaker and Saftner, 2000).
Regardless of the role of a-farnesene oxidation
products, scald development very likely involves the adverse effects of oxidative stress that occurs with prolonged storage at chilling temperatures (Du and Bramlage, 1995; Shewfelt and Purvis, 1995; Watkins et al., 1995; Rao et al., 1998). Huelin and Coggiola (1970b) and Anet (1972) first proposed that scald susceptibility or resistance in different apple cultivars is, at least in part, determined by the efficiency of their natural antioxidant defenses. Anet (1974) subsequently examined the levels of endogenous lipophilic antioxidants in the cuticle of 16 apple cultivars and found a correlation between scald resistance and the presence of antioxidant levels sufficient to curtail a-farnesene oxidation
during storage. Meir and Bramlage (1988) also showed a negative correlation between scald suscep-tibility and high levels of unidentified lipophilic antioxidants with an absorbance maximum at 200 nm in the cuticle of ‘Cortland’ apples. More
recent investigations have compared the accumula-tion of peroxides and lipid peroxidaaccumula-tion products, and the activities of enzymes that detoxify active oxygen species (AOS), in peel tissue of scald-suscep-tible and -resistant apple fruit stored at 0 – 1°C in air (Du and Bramlage, 1994a, 1995; Rao et al., 1998). Du and Bramlage (1994a, 1995), compared apple cultivars with wide variation in scald susceptibility (‘Cortland’, ‘Delicious’ and ‘Empire’) and found no marked changes in peroxidation or activities of antioxidative defense enzymes related to scald de-velopment. In contrast, Rao et al. (1998) found a close correlation between increasing levels of H2O2
and lipid peroxidation products, declining perox-idase and catalase activities, and the occurrence and severity of scald symptoms in fruit of susceptible and resistant ‘White Angel’בRome Beauty’ hybrid selections grown at the same location under identi-cal conditions.
In their study of the ‘White Angel’בRome Beauty’ selections, Rao et al. (1998) assessed changes ina-farnesene and CT levels
spectrophoto-metrically using hexane- dip extracts of individual fruit. Althougha-farnesene and CT concentrations
appeared somewhat higher in scald-susceptible than in scald-resistant lines early in storage,a-farnesene
synthesis and oxidation were not elevated in apples that eventually developed scald symptoms. In addi-tion, Rupasinghe et al. (1998) determined that
a-farnesene synthase activity was about threefold
lower in scald-developing compared with scald-free ‘Delicious’ apple peel tissue. These findings chal-lenge the hypothesis that oxidation ofa-farnesene is
directly linked with scald induction. Thus, the present study was undertaken to rigorously examine
a-farnesene synthesis and oxidation in individual
red- and yellow-pigmented ‘White Angel’בRome Beauty’ selections during storage using HPLC – UV analysis (Whitaker et al., 1997), and to assess the relationship ofa-farnesene metabolism to the
inci-dence and severity of scald after storage.
2. Materials and methods
2.1.Plant material and fruit source
B.D.Whitaker et al./Posthar6est Biology and Technology20 (2000) 231 – 241 233
apple trees derived from a ‘White Angel’ and ‘Rome Beauty’ cross growing at the New York State Agricultural Experiment Station in Geneva, NY (Hemmat et al., 1994; Lawson et al., 1995). Trees derived from this cross segregate for red-and yellow-skinned fruit (Cheng et al., 1996) with wide variation in scald susceptibility (Weeden, 1993).
2.2. Tissue sampling, storage conditions, and scald assessment
Over two harvest/storage seasons (1997/98 and 1998/99), fruit from 16 selections, eight red- and eight yellow-skinned, were sampled and exam-ined. Among these, three red-fruited (R-44, R-48 and R-85) and five yellow-fruited (Y-38, Y-40, Y-55, Y-65 and Y-67) lines exhibited at least mild scald symptoms after storage, whereas the remaining lines were completely free of scald (R-01, R-03, R-16, R-20, R-22, 07, 26 and Y-28). Five red-skinned (R-03, R-20, R-22, R-48 and R-85) and three yellow-skinned (Y-26, Y-55 and Y-65) selections were harvested, stored, and analyzed in both years.
All apples were picked on 15 October in 1997, and on 14 October in 1998. Ten fruit from each selection were used to determine the internal ethylene concentration (IEC) as described by Rao et al. (1998). Thirty fruit per selection were sampled at harvest and after 7, 14 and 21 weeks of air storage at 0.5°C in perforated plastic bags. The skin and outer 2 – 3 mm of cortical tissue were peeled manually, frozen immediately in liquid N2, and stored at −80°C. All fruit
were peeled within 30 min of removal from 0.5°C storage. Samples of the pooled peel tissue from each selection×duration of storage (40 – 50 g) were sealed in individual ziplock bags, packed in dry ice, and shipped overnight to Beltsville, MD for HPLC analysis of a-farnesene and
CTols. When received, the samples were stored at −80°C until analyzed.
After 21 weeks at 0.5°C plus 1 week at 20°C, at least 100 fruit of each selection were visually assessed for scald, except for R-20 (27 fruit), R-44 (62 fruit) and Y-28 (39 fruit)
in the 1997/98 season, which had a high inci-dence of decay. On fruit showing scal sym-ptoms, scald severity was rated on a scale of 0 – 4 based on the percentage of the sur-face area affected, where 0=no scald, 1=
1 – 10%, 2=11 – 33%, 3=34 – 66% and 4=67 – 100%.
2.3. Preparation of samples and HPLC analysis
Frozen peel samples (3 g) were pulverized in liquid N2 and transferred to 50-ml screw-cap
culture tubes containing 9 ml of HPLC-grade hexane. The tubes were flushed with N2, sealed,
and agitated at 5°C for 1.5 h. Extracts were vacuum filtered through glass fiber disks and re-stored to 9 ml total volume. Aliquots (1.5 ml) were transferred to 2-ml vials and the hexane evaporated under a gentle stream of N2 without
heating. The residue was dissolved in 400 ml of
HPLC-grade methanol and filtered through a 0.45 mm PTFE membrane prior to HPLC
analy-sis. Samples (80ml) were injected manually into a
Waters 600MS HPLC system fitted with a 4.6×
250 mm, Luna C18 column (Phenomenex,
Tor-rence, CA). The mobile phase was meth-anol/acetonitrile/water (90:7.5:2.5) pumped at 0.8 ml min−1. Absorbance at 232 nm (
a-farnesene)
and 269 nm (CTols) was monitored by a Waters 490 programmable wavelength detector and data were gathered and processed using the Waters Baseline 810 program in a 286 PC. a
-Farnesene gave a single peak that eluted at 11.9 min and CTols gave a prominent peak at 5.8 min with a small shoulder at 6.0 min. Cal-culations of a-farnesene and CTol concentrations
were based on their molar extinction coefficients as previously described (Whitaker et al., 1997). A second aliquot of the hexane extracts was used for spectrophotometric estimation of a-farnesene
and CTol concentrations according to the method of Rao et al. (1998). Absorbance at 232 nm (a-farnesene) and 281 – 290 nm (CTols) was
recorded using a Shimadzu UV-160 spectro-photometer. Samples were diluted as required to maintain A232 nm in the linear range (51.2
Fig. 1. a-Farnesene concentration in peel tissue of ‘White
Angel’בRome Beauty’ selections at harvest as a function of the log of the internal ethylene concentration (IEC). Data points represent both red- and yellow-skinned lines from the 1997 and 1998 harvests. The polynomial equation derived from regression analysis was Y=12.77+10.54X+2.25X2, with anR2value of 0.478.
3. Results
3.1. Relationship between a-farnesene and internal ethylene concentrations at har6est
In apples with IECs at or above log 0 ml l−1, a-farnesene concentrations increased with higher
IECs (Fig. 1). Although the R2 for the
relation-ship was low (0.478), few exceptions to the trend occurred, and in fruit with IECs below log 0 ml
l−1 the farnesene content was always B10 mg
kg−1 fresh weight.
3.2. Incidence and se6erity of scald in relation to
a-farnesene synthesis and oxidation in red-skinned selections
Three of the eight red-fruited lines harvested in 1997 (R-44, R-48 and R-85) developed scald after 21 weeks at 0.5°C plus 1 week at 20°C (Table 1). Two of the scald-resistant (SR) lines (R-01 and R-16) were absent from the 1998 harvest, but the poststorage outcome was the same for the remain-ing six lines; R-44, R-48, and R-85 exhibited scald and R-03, R-20 and R-22 did not. For both R-44 and R-48, the percentage of scalded fruit and scald severity were similar in the 2 years, whereas for R-85, the number of scalded fruit was much lower in 1998/99 than in 1997/98.
R-44 was clearly the most scald-prone red-skinned selection, with incidence \90% and about one-third of the fruit surface affected in both years.
In both SR and scald-susceptible (SS) red-skinned selections, peel a-farnesene levels
gener-ally peaked at 7 weeks in fruit harvested in 1998 and at 14 weeks in those harvested in 1997 (Fig. 2). Also, among the selections harvested in both years, total a-farnesene accumulation was greater
in 1998/99 in SR fruit, but not in SS fruit. Of the lines that produced the most a-farnesene, peak
concentrations were at least as great in peel of SR lines R-20 and R-22 as in peel of SS lines R-48 and R-85. In R-44, the red-skinned selection most prone to scald, peel concentrations of a-farnesene
were only determined in 1998/99 and were consis-tently lower than in R-48 or R-85 that year.
Fig. 2.a-Farnesene concentration in peel tissue of
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Table 1
Scald evaluation in red- and yellow-skinned ‘White Angel’בRome Beauty’ selections after 20 weeks at 0.5°C in air plus 1 week at 20°C, and relativea-farnesene and conjugated trienol (CT) levels in peel tissue over the course of storage
1997/98 Harvest and storage 1998/99 Harvest and storage
Hybrid Scald percent Scald severity a-Farnesene CTs Hybrid Scald percent Scald severity a-Farnesene CTs
selection selection
Red fruit
– –
c01 0 0.0 Very low Mod. High c01 – –
Very low c03 0 0.0 Moderate Very low
0
c03 0.0 Low
– – – –
c16 Very low
c16 0 0.0 Low
0 0.0 Very high Mod. low
c20 0 0.0 High (early) Mod. Low c20
0 0.0 Mod. high Mod. high
c22
c22 0 0.0 High (late) High (late)
92 2.5 Moderate
c44 98 2.5 Not analyzed Not analyzed c44 Mod. high
19 1.9 Mod. high Very low
c48 Very low
c48 27 1.9 Mod. high
c85 63 1.3 Very high Very high c85 4 1.8 Mod. high Very high
Yellow fruit
High c07 – – – –
0.0
c07 0 Very high
0 0.0 Moderate Very low
c26
c26 0 0.0 Very low Low
0 0.0 Mod. high c40 100 4.0 Very high Moderate
c28 Low
47 1.8 Mod. high Moderate
c67 Mod. low
Very high
c38 35 1.1
c55
70 1.4 Very low Very low 54 1.0 Very low Very low
c55
Very high c65 76 2.0 Mod. low Very high
Fig. 3. Conjugated trienol concentration in peel tissue of scald-resistant and -susceptible red-skinned selections over 21 weeks at 0.5°C in air. Closed and open symbols indicate fruit from the 1997 and 1998 harvests, respectively. Data points represent the average of two replicate analyses; variation be-tween replicates was generally less than 10% except for values 52 mg kg−1 fresh weight.
lated no more than 6 mg kg−1of CTols in either
storage season. Most red-skinned SR selections did not exceed 6 mg kg−1 CTols throughout
storage, but fruit of SR line R-22 reached 13 – 15 mg kg−1 CTols in both years, comparable to
levels in highly SS line R-44 fruit. The pattern of CTol accumulation in peel of R-22 fruit was quite different in the two seasons and reflected the different pattern of a-farnesene accumulation
(Fig. 2). Botha-farnesene and CTols began to rise
much sooner, and peaked 7 weeks earlier, in 1998/99 than in 1997/98.
3.3. Incidence and se6erity of scald in relation to
a-farnesene synthesis and oxidation in
yellow-skinned selections
Five of the 8 yellow-fruited lines tested (Y-38, Y-40, Y-55, Y-65 and Y-67) developed scald (Table 1). One SR line (Y-28) and 1 SS line (Y-38) included in the 1997/98 storage experiments were replaced by two other SS lines (Y-40 and Y-67) in 1998/99. The SR selection Y-26 exhibited no scald in both years. Of the 3 SS lines examined in only one season, the incidence and severity of scald were extreme in Y-40 (1998)/99), intermediate in Y-67 (1998/99), and relatively low in Y-38 (1997/
98). SS lines Y-55 and Y-65 were harvested in both years, and for each of them, the incidence and severity of scald were greater in 1997/98 than in 1998/99. Scald was more extensive in Y-65 than in Y-55 fruit, the incidence and severity index averaging 88% and 2.8, and 62% and 1.2, respectively.
As noted for red-skinned selections,a-farnesene
content tended to increase earlier in yellow-skinned fruit harvested in 1998 compared with those harvested in 1997 (Fig. 4). However, the two SS lines that were harvested in both years, Y-55 and Y-65, did not show this trend. In con-trast, the only SR selection harvested in both years produced much morea-farnesene in 1998/99
than in 1997/98. Thea-farnesene content of both
SR and SS yellow-skinned selections varied con-siderably. Fruit of the most acutely SS line, Y-40, showed the earliest rise in a-farnesene and
main-tained the highest levels throughout storage, whereas fruit of the moderately SS line Y-55 had Overall, peel tissue CTol levels were somewhat
higher in SS than in SR red-skinned selections (Fig. 3). However, both groups included lines with relatively high and relatively low concentrations of CTols. SS line R-85 accumulated the most CTols among red-skinned selections. In R-85 fruit harvested in 1998, the rate of CTol accumulation increased throughout storage and CTol concen-tration peaked at 27 mg kg−1
at 21 weeks, whereas in 1997, CTols accumulated most rapidly from 7 to 14 weeks and reached a maximum of 18 mg kg−1 at 21 weeks. Peel CTols in fruit of
the most highly SS line, R-44, increased linearly for 14 weeks to a peak level of 15 mg kg−1
accumu-B.D.Whitaker et al./Posthar6est Biology and Technology20 (2000) 231 – 241 237
Fig. 4.a-Farnesene concentration in peel tissue of
scald-resis-tant and -susceptible yellow-skinned selections over 21 weeks at 0.5°C in air. Closed and open symbols indicate fruit from the 1997 and 1998 harvests, respectively. Data points represent the average of two replicate analyses; variation between repli-cates was generally less than 10% except for values 510 mg kg−1fresh weight.
scald were lesser, in 1998/99 than in 1997/98 (Table 1). The most acutely SS line, Y-40, and the moderately SS line Y-67 had similar, moderate peel CTol levels throughout storage, with maxima at 21 weeks of 6 and 8 mg kg−1,
respec-tively. The pattern and final level of CTol accu-mulation in peel of SR line Y-07 were almost identical to those in peel of SS line Y-67, although the data were from different years. SR line Y-26 in both years, and SR line Y-28 in 1997/98, did not exceed a peel CTol content of 3 mg kg−1.
Finally, and most notably, in accord with their low production of a-farnesene (Fig. 4), fruit of
moderately SS Y-55 accumulated no more than 1 mg kg−1CTols throughout storage in both years.
Fig. 5. Conjugated trienol concentration in peel tissue of scald-resistant and -susceptible yellow-skinned selections over 21 weeks at 0.5°C in air. Closed and open symbols indicate fruit from the 1997 and 1998 harvests, respectively. Data points represent the average of two replicate analyses; varia-tion between replicates was generally less than 10% except for values 52 mg kg−1fresh weight.
the lowesta-farnesene levels after 21 weeks of any
of the ‘White Angel’בRome Beauty’ selections. In addition, among the selections harvested in 1997, the rate and extent of a-farnesene
accumu-lation in SR lines Y-07 and Y-28 were comparable to those in SS line Y-38.
Peel tissue CTol levels were generally higher in SS than in SR yellow-skinned selections (Fig. 5). However, among the SS lines there was wide variation in the production and peak level of CTols. Highly SS line Y-65 accumulated the most CTols of any yellow-skinned selection in both years, with a maximum concentration of 15 – 18 mg kg−1 at 21 weeks. It is noteworthy, though,
3.4. Comparison of a-farnesene and CTol
concentrations determined by HPLC–UV analysis and spectrophotometric estimation
Using aliquots from the same peel tissue hexane extracts, a-farnesene and CTol values calculated
by the UV absorbance method (Anet, 1972) were compared with those determined by HPLC – UV analysis (Whitaker et al., 1997). Data for three representative scald-susceptible yellow-skinned se-lections (Y-40, Y-55 and Y-65), which include values in the low, moderate, and high ranges, are shown in Fig. 6.
a-Farnesene concentrations determined by the
two methods were generally quite similar. For Y-40, which had the highesta-farnesene levels of
any selection, spectrophotometric values were
typ-ically 5 – 10% higher than HPLC values. Con-centrations of CTols determined by the two methods did not differ substantially for Y-65, which had the highest CTol levels among the yellow-skinned selections, but were markedly dif-ferent for Y-40 and Y-55. Hexane extracts from these two lines, which accumulated very low (Y-55) to low-moderate (Y-40) levels of CTols, gave UV-absorbance-derived values that were from 1.4- to 6.2-fold higher than those obtained by HPLC – UV analysis.
4. Discussion
Beginning with Huelin and Coggiola (1970a), there have been numerous reports of a correlation between high concentrations of CTs and scald development, including recent of reports from our labs encompassing the effects of harvest maturity, air versus low O2storage, and inhibition of
ethyl-ene action by 1-methylcyclopropethyl-ene treatment on scald development and on a-farnesene synthesis
and oxidation in apple cultivars with a broad range of scald susceptibility/resistance (Whitaker and Solomos, 1997; Whitaker et al., 1997, 1998; Watkins et al., 2000). Contrary to this body of evidence, the results of this study generally sup-port the conclusion of Rao et al. (1998) that abundant synthesis of a-farnesene and
accumula-tion of its CTol oxidaaccumula-tion products in the epider-mis and cuticle are not closely correlated with scald susceptibility in fruit of ‘White Angel’×
‘Rome Beauty’ hybrid selections.
Ethylene appears to stimulate a-farnesene
syn-thesis shortly after apples are stored and to pro-mote scald development (Watkins et al., 1993, 1995; Du and Bramlage, 1994b; Whitaker and Solomos, 1997). Three recent studies showed that prestorage treatment of scald-susceptible apples with blockers of ethylene action reduced a
-far-nesene synthesis and oxidation, and controlled scald (Gong and Tian, 1998; Fan et al., 1999b; Watkins et al., 2000). However, because ethylene is a general promotor of ripening and senescence (Yang, 1987), and treatments that reduce ethylene production or responsiveness improve poststorage quality and alleviate storage disorders (Gong and
B.D.Whitaker et al./Posthar6est Biology and Technology20 (2000) 231 – 241 239
Tian, 1998; Fan et al., 1999a,b; Watkins et al., 2000), it remains uncertain whether a-farnesene
oxidation products are involved in scald induction or are merely byproducts of free radical reactions which cause metabolic dysfunction and cell death. In this investigation, the level of a-farnesene in
peel tissue at harvest was generally much greater in fruit with IECs above log 0ml l−1, suggesting a
link between the two factors. Meigh and Filmer (1969) showed that an increase in a-farnesene
concentration is associated with the climacteric. More recently, Watkins et al. (1993) found that onset of the increase ina-farnesene occurs sooner
in apples with higher IECs at harvest. Thus, the failure to demonstrate a relationship between a
-farnesene concentrations at harvest and scald sus-ceptibilty (Huelin and Coggiola, 1968) may have been partly due to differences in the climacteric state among the cultivars examined. A general increase in production of volatiles occurs with apple ripening, so ethylene may stimulate a
-far-nesene synthesis simply by promoting ripening. The results of this study reinforce the view that cultivar differences override any relationship be-tween sesquiterpenoid accumulation and scald su-ceptibility (Meigh and Filmer, 1969).
Among the ‘White Angel’בRome Beauty’ se-lections most prone to scald (Y-65, Y-40 and R-44), results were consistent with thea-farnesene
oxidation – scald induction hypothesis; Y-65 had high CTol levels in both years, Y-40 accumulated only a moderate amount of CTols but produced the most a-farnesene, and R-44 showed a rise in
CTols early in storage and a moderately high peak concentration. However, the SR selections Y-07 and R-22 also produced high levels ofa
-far-nesene and reached CTol concentrations com-parable to those in several SS lines. From this we infer that if CTol do play a role in scald induc-tion, there must be other mitigating factors of at least equal importance. Huelin and Coggiola (1970b), Anet (1972) emphasized that detection of CTs early in storage was the best predictor of scald development and proposed that natural an-tioxidants delay a-farnesene oxidation and thus
are important in scald resistance. In accord with this, scald resistance was correlated with the level of lipophilic cuticular antioxidants present at
har-vest (Meir and Bramlage, 1988), or maintained during storage (Anet, 1974). More recently, Rao et al. (1998) found that maintenance of peroxidase and catalase activities sufficient to prevent a marked increase H2O2 late in storage is a key
factor in scald resistance.
In both years, SS selections Y-55 and R-48 accumulated only small amounts of CTols, yet they exhibited scald symptoms, albeit mild. Y-55 in particular produced relatively little a-farnesene
and consistently had among the lowest CTol lev-els of all the selections examined. Thus, the results for lines Y-55 and R-48 oppose the a-farnesene
oxidation – scald induction hypothesis, and appear to contradict the statement by Anet (1972) that ‘no instance of superficial scald has been ob-served… from apples which showed no a
-far-nesene autoxidation.’
Fruit of the ‘White Angel’בRome Beauty’ selections produced similar amounts of a
-far-nesene but accumulated 2- to 50-fold lower levels of CTols than fruit of SS ‘Granny Smith’ and ‘Delicious’ (Whitaker et al., 1997, 1998). It is possible, then, that those hybrid selections which do develop scald are hypersensitive to the oxida-tion products of a-farnesene. Also, it was recently
proposed that MHO, a volatile product of CTol autoxidation (Whitaker and Saftner, 2000), is the compound directly responsible for induction of scald symptoms (Mir and Beaudry, 1999; Mir et al., 1999), and production of MHO by SS and SR ‘White Angel’בRome Beauty’ selections has not yet been examined. Overall, our results and those of Rao et al. (1998) are consistent with the hy-pothesis that oxidation products of a-farnesene
are not essential for scald development in fruit with severely compromised antioxidative defenses, but free radicals and/or toxic volatiles (e.g. MHO) generated bya-farnesene oxidation can exacerbate
scald symptoms. Further experiments focused on SS selections that accumulate very little CTol, as well as SR lines that reach relatively high CTol concentrations, should help to elucidate the fac-tors involved in susceptibility and resistance to superficial scald.
A concern about the a-farnesene and CT data
Anet (1972), is the spectrophotometric method used to determine the concentrations of these sesquiterpenoids. Calculations were based on ab-sorbance of crude hexane dip extracts at 232 nm (a-farnesene) and at 281-290 nm (CTs), raising the
question of whether, at least in some instances, UV absorbance by interfering compounds might give spurious results (Whitaker et al., 1997). Here, comparison of data obtained by HPLC – UV anal-ysis and UV absorbance measurements (Fig. 6) indicated that a-farnesene concentrations
deter-mined by the two methods were generally in close agreement. However, CTol levels calculated from UV absorbance data tended to be higher than those measured by HPLC – UV, particularly when peel tissue concentrations were relatively low (e.g. in Y-55). Since the concentration ofa-farnesene is
typically much greater than that of its CTol oxi-dation products, absorbance by other compounds is less likely to introduce significant error. As determined by Huelin and Coggiola (1968), pas-sage of apple skin hexane extracts through a Florisil column gives good partial purification of
a-farnesene and thus further improves the
accu-racy of the spectrophotometric method (Whitaker et al., 1997).
Acknowledgements
We wish to thank Mr Michael Schifanelli for his excellent technical assistance in tissue extrac-tion and HPLC – UV analysis. This research was supported in part by funds from the USDA NE-103 Cooperative Regional Research Project and from the New York Apple Research and Devel-opment Program.
References
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Anet, E.F.L.J., 1974. Superficial scald, a functional disorder of stored apples. XI. Apple antioxidants. J. Sci. Food Agric. 25, 299 – 304.
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