349

Apple Scald Development and Regulation

P. Trivedi D. Caridhas and T. Solomos

Plant Gene Expression Laboratory Dept. of Natural Resource Science
National Botanical Research Institute and Landscape Architecture
Rana Pratap Marg University of Maryland
Lucknow 226001, College Park, MD 20742
India USA

Keywords: -farnesene, conjugated trienol, hypoxia, 1-MCP, HMGR, FPPS

Abstract
We have compared changes in -farnesene and conjugated trienols, as well as
gene expression, in scald-resistant cultivars, e.g., Gala and Braeburn, and in
scald-sensitive cultivars, e.g., Granny Smith, Delicious and Law Rome. We also
carried out similar comparisons in Granny Smith between controls and treatments
that diminish scald symptoms, e.g., low O
2
and 1-methylcyclopropene (1-MCP). The
data show that the initiation of the C
2
H
4
climacteric plays a crucial role in scald
development, since treatments that retard the onset of the climacteric, e.g., low O
2

and 1-MCP, also inhibit symptoms of scald development. Furthermore, if either low
O
2
or 1-MCP is applied after the initiation of the C
2
H
4
climacteric, their inhibitory
effects on scald development decrease. The treatments that retard the C
2
H
4

climacteric onset also strongly inhibit the rise in -farnesene and conjugated
trienols. In Granny Smith apples, we studied the effect of temperature on scald
development, as well as changes in the content of -farnesene and conjugated
trienols. The data show that, at temperatures above 7C, scald failed to develop but
the rise in -farnesene and conjugated trienols was not affected. It should be noted
that in scald-resistant cultivars stored at 1C there was an increase in both -
farnesene and conjugated trienols, though not as high as in sensitive cultivars.
However, if the auto-oxidation of -farnesene and trienols is the cause of scald
disorder, their levels in the resistant cultivars ought to have been sufficient for the
induction of the disorder. It is thus obvious that low temperature must induce
enzymes that create the scald-causing compounds. We observed that the
concentration of malondialdehyde (MDA) increased only in the areas showing scald
symptoms, which in turn indicates that oxidative processes were involved in scald
development. Alternatively, in the resistant cultivars the anti-oxidant capacity may
be higher than in the sensitive ones. In short, the data show that in addition to
cultivar type, low temperature and the induction of the C
2
H
4
climacteric play a
crucial role in scald development.

INTRODUCTION
Scald is a serious postharvest disorder, affecting the hypodermic cell layers and
producing brown patches on the skin of apples and pears (Bain and Mercer, 1963). The
mechanics of scald development are not well understood. The development of scald on
the skin of apples is influenced by physiological, environmental, developmental and
storage factors (Huelin and Coggiola, 1968; Ingle and DSouza, 1989). Forty years ago -
farnesene was found to be the major isoprenoid in the natural coating of the apple skin,
and correlative evidence has been presented that conjugated trienes, its auto-oxidation
products, are involved in the development of scald (see recent data by Rowan et al.,
2001). The role of -farnesene in scald development was further substantiated by the fact
that lovastatin, a competitive inhibitor of hydroxymethyl coenzyme-A reductase
(HMGR), inhibits both -farnesene biosynthesis and scald development (Ju and Curry,
2000). Experimental data comparing cultivars with varying susceptibility to scald do not
always correlate with the levels of conjugated triene hydroperoxides (CTH), however
(Whitaker et al., 2000). Furthermore, the auto-oxidation may not be the main source of
Proc. IX
th
Intl. Contr. Atmos. Res. Conf.
Ed.: R.M. Beaudry
Acta Hort. 857, ISHS 2010
350
CTH because scald-resistant cultivars contain sufficient quantities of -farnesene to
induce scald formation. Are there (a) special enzyme(s) induced in the sensitive
cultivars? Fernandez-Trujillo et al. (2003) have provided evidence that the antioxidant
ability of fruit contributes to scald control. In short, the resistance to scald may be due to
the ability of fruit to eliminate the free radicals generated by the oxidation of -farnesene.
Based on the fact that the sensitivity to scald decreases with maturity, and that
ethephon treatment that advance fruit maturity also enhance in certain cultivars resistance
to scald, it was suggested that C
2
H
4
may alleviate the disorder (Lurie et al., 1989). The
role of C
2
H
4
in scald development has been questioned (Soria et al., 1999). Nevertheless,
treatments with inhibitors of C
2
H
4
action greatly decrease the development of the
disorder. This indicates that induction of the C
2
H
4
climacteric is involved in scald
development (Fan et al., 1999). In this report we offer data supporting the view that the
initiation of the C
2
H
4
climacteric, hence fruit ripening, plays a critical role in scald
development.

MATERIALS AND METHODS
Apples were harvested from the University orchard in Western Maryland. We
used Gala and Braeburn as scald-resistant cultivars, and Delicious, Rome and
Granny Smith as scald-sensitive cultivars. In this report we present only the data for
Granny Smith and Gala as representatives of the sensitive and resistant cultivars,
respectively. The rate of C
2
H
4
evolution under different gas treatments was determined in
a flow-through system (Kanellis et al., 1991). -farnesene and conjugated trienol were
measured using a previously published method (Whitaker et al., 1997). The fruit were
treated with 1-MCP by following the instructions of Rohm and Haas, the supplier of
SmartFresh powder that generates the required concentration of 1-MCP. For the
cloning and labeling of HMGR1 and FPPS, we used the Invitrogen TOPO TA cloning
and Invitrogen Radprime labeling kits, following the instructions provided in the kits.
Scald was scored visually, and texture was assessed using a penetrometer.
Malondialdehyde was monitored by liquid chromatography using the OD at 529 nm.

RESULTS AND DISCUSSION
The development of scald in Gala apples was evaluated after storage at 1C for
285 d and 8 additional d at 20C. Three sets of conditions were used: controls, 2 ppm 1-
MCP, and 1.5% O
2
. Under none of these conditions did the fruit show any scald
symptoms (Table 1). Granny Smith that were stored at 1C for 247 d showed severe
scald symptoms, however, which were further enhanced when fruit were transferred from
1C to 20C (Table 2). Conversely, the Granny Smith treated with 1-MCP and 1.5% O
2

was free of scald symptoms (Table 2). Granny Smith contains higher levels of -
farnesene and conjugated trienol than Gala (Figs. 1A, B, 2A, B). Furthermore, in both
cultivars the levels of these compounds in fruit that were treated with 1-MCP and 1.5%
O
2
were negligible (Figs. 1A, B, 2A, B). In the fruit treated with 1-MCP and low O
2
, the
rate of C
2
H
4
evolution was also suppressed at 1C (data not shown). In order to
investigate the correlation between -farnesene accumulation and C
2
H
4
evolution, we
stored Granny Smith apples at 1C under various treatments. We present here only the
data on the controls, and on the fruit treated with 1-MCP and with 1.5% + 1-MCP. In the
controls, C
2
H
4
increased after about 10 d, whereas the 1-MCP-treated fruit took about 45
d (Fig. 3B). On the other hand, the fruit treated with 1-MCP + low O
2
showed no increase
in C
2
H
4
evolution for 100 d (data not presented). The concentration of -farnesene was in
close temporal relationship with that of C
2
H
4
evolution. After 96 d, in the 1-MCP-treated
fruit the concentration of -farnesene approached the peak value of the controls, which
paralleled an increase in C
2
H
4
evolution (Fig. 3B). In fruit that were treated with 1-MCP
+ 1.5% O
2
, -farnesene did not increase for 145 d, and the lack of increase was reflected
in a suppression of the C
2
H
4
rise (data not shown). Moreover, fruits treated with 1-MCP +
low O
2
and kept at 1C for 260 d showed no climacteric rise in C
2
H
4
and no scald
symptoms (data not shown).
351
All this demonstrates that the induction of the C
2
H
4
climacteric rise controls the
rise in -farnesene biosynthesis. Furthermore it bolsters the evidence for the crucial role
of -farnesene in scald development.
We also monitored the effect of storage at different temperatures (1, 5, 10, and
20C) on scald development. We found that fruit stored at 10 and 20C did not develop
scald symptoms (Table 3). This supports previous evidence that scald development is a
chilling injury (Watkins et al., 1995), although recently it was reported that scald
symptoms were more severe at 10C than at 5C (Fan et al., 1999).
As pointed out in the Introduction, the development of scald is a response to
oxidative stress. On measuring the changes in MDA content in the peel of Granny Smith
and Gala fruit, we found that only scalded peel areas contained MDA, since we detected
no MDA either in Gala or 1-MCP-treated Granny Smith (Table 3). Both sets of these
fruit were scald-free.
We treated Granny Smith with compactin, an inhibitor of 3-hydroxy-3-
methylglutaryl-CoA reductase (HMGR), and found that it appreciably inhibited the
synthesis of -farnesene and trienol for 63 d postharvest (Table 4). After investigating the
expression of HMGR1 and farnesene pyrophosphate synthase (FPPS) we found that it is
unrelated to the -farnesene content, since 1-MCP, while inhibiting -farnesene
biosynthesis, did not affect the level of its expression (Fig. 4). In addition, 1.5% O
2
,
which strongly inhibited -farnesene biosynthesis, greatly enhanced the accumulation of
the HMGR1 transcripts (Fig. 4). Analysis of the HMGR promoter revealed that it contains
hypoxic responsive elements (Fig. 5). Farnesene synthase seems to be the controlling step
in the biosynthesis of -farnesene (Pechous et al., 2005).
In short, the data show that: (a) the induction of the C
2
H
4
climacteric regulates
scald development together with fruit ripening, since fruit kept for long storage periods
with no rise in System II C
2
H
4
show no symptoms of either ripening or scald
development; and (b) -farnesene appears to be a crucial contributor to scald
development.

CONCLUSIONS
The induction of the C
2
H
4
climacteric controls both synthesis of -farnesene and
scald development.

ACKNOWLEDGEMENTS
I wish to thank the Washington Tree Fruit Research Commission for financial
support.

Literature Cited
Bain, J.M. and Mercer, F.J. 1963. The submicroscopic cytology of superficial scald, a
physiological disease of apples. Aust. J. Biol. Sci. 16:442449.
Fan, X., Mattheis, J.P. and Blankenship, S. 1999. Development of apple superficial scald,
soft scald, core flush and greasiness is reduced by MCP. J. Agric. Food Chem.
47:30633068.
Fernndez-Trujillo, J.P., Nock, J.F., Kupferman, E.M., Brown, S.K. and Watkins, C.B.
2003. Peroxidase activity and superficial scald development in apple fruit. J. Agric.
Food Chem. 51:71827186.
Huelin, F.E. and Coggiola, I.M. 1968. Superficial scald, a functional disorder of stored
apples. IV. Effects of variety, maturity, oiled wraps, and diphenylamine on
concentration of -farnesene in fruit. J. Sci. Food. Agric. 19:297301.
Ingle, M. and DSouza, M.C. 1989. Physiology and control of superficial scald of apples.
A review. HortScience 24:2831.
Ju, Z. and Curry, E.A. 2000. Lovastatin inhibits -farnesene biosynthesis and scald
development in Delicious and Granny Smith apples and dAnjou pears. J. Amer.
Soc. Hort. Sci. 125:626629.
Kanellis, A.K., Solomos, T. and Roubelakis-Angelakis, K.A. 1991. Suppression of
352
cellulose and polygalacturonase and induction of alcohol dehydrogenase isoenzymes
in avocado fruit mesocarp subjected to low oxygen stress. Plant Physiol. 96:269274.
Lurie, S., Klein, J.D. and Ben-Arie, R. 1989. Pre-harvest ethephon sprays reduce
superficial scald in Granny Smith apples. HortScience 24:104106.
Pechous, S.W., Watkins, C.B. and Whitaker, B.D. 2005. Expression of -farnesene
synthase gene AFS1 in relation to levels of -farnesene and conjugated trienols in peel
tissue of scald-susceptible Law Rome and scald-resistant Idared apple fruit.
Postharvest Biol. and Technol. 35:125132.
Rowan, D.D., Hunt, M.B., Fielder, S., Norris, J. and Sherburn, M.S. 2001. Conjugated
triene oxidation products of -farnesene induce symptoms of superficial scald on
stored apples. J. Agric. Food. Chem. 49:27802787.
Soria, Y., Recasens, I., Gatius, F. and Puy, J. 1999. Multi-variable analysis of superficial
scald susceptibility on Granny Smith apples dipped with different postharvest
treatments. J. Agric. Food Chem. 47:48544858.
Watkins. C.B., Bramlage, W.J. and Cregoe, B.A. 1995. Superficial scald of Granny
Smith apples is expressed as typical chilling injury. J. Amer. Hort. Sci. 120:8894.
Whitaker, B.D., Solomos, T. and Harrison, D.J. 1997. Quantification of -farnesene and
its conjugated trienol oxidation products from apple peel by C18-HPLC with UV
detection. J. Agric. Food Chem. 45:760171.
Whitaker, B.D., Nock, J.F. and Watkins, C.B. 2000. Peel tissue -farnesene and
conjugated trienol concentrations during storage of White Angel Rome Beauty
apple selections susceptible and resistant to superficial scald. Postharvest Biol.
Technol. 20:231241.












Tables




Table 1. Percent of fruit with scald scores from 1 to 5 for Gala fruit stored 285 d in air
(control and 1-MCP) or in CA (1.5% O
2
) at 1C plus an additional 8 d at 20C, where
1 = no scald; 2 # of surface scalded; 3 # of surface scalded; 4 # of surface
scalded; 5 = extensive scald.

285 d at 1C 285 d at 1C + 8 d at 20C
Scald score
Control 1-MCP 1.5% O
2
Control 1-MCP 1.5% O
2

1 100 100 100 100 100 100
2 0 0 0 0 0 0
3 0 0 0 0 0 0
4 0 0 0 0 0 0
5 0 0 0 0 0 0


353
Table 2. Percent of fruit with scald scores from 1 to 5 for Granny Smith fruit stored 247
d in air (control and 1-MCP) or in CA (1.5% O
2
) at 1 C plus an additional 10 d at
20C, where 1 = no scald; 2 # of surface scalded; 3 # of surface scalded; 4 # of
surface scalded; 5 = extensive scald.

285 d at 1C 285 d at 1C + 10 d at 20C
Scald score
Control 1-MCP 1.5% O
2
Control 1-MCP
1 33.33 100 100 9.10 100
2 29.17 0 0 27.3 0
3 12.50 0 0 36.3 0
4 20.83 0 0 0 0
5 4.17 0 0 27.3 0








Table 3. Percent of fruit with scald scores from 1 to 5 for Granny Smith fruit stored 247
d in air (control and 1-MCP) or in Low O
2
(1.5% O
2
) at 1, 5, 10 and 20C for various
durations, where 1 = no scald; 2 # of surface scalded; 3 # of surface scalded; 4 #
of surface scalded; 5 = extensive scald.

Temp. (C) and duration Score Control MCP Low O
2
1 (190 d) 1 10 100 100
2 30 0 0
3 30 0 0
4 15 0 0
5 15 0 0

5 (176 d) 1 22.22 95.65
2 22.22 4.35
3 38.89 0
4 16.67 0
5 0 0

10

(153 d) 1 100 100
2 0 0
3 0 0
4 0 0
5 0 0

20 (150 d) 1 100 100
2 0 0
3 0 0
4 0 0
5 0 0

354
Table 4. Malondialdehyde (MDA) content and its standard deviation for tissue samples of
Granny Smith and Gala apple fruit taken from scalded and non-scalded tissues
expressed on a fresh weight basis.

Sample MDA content (g/g) STD
Scalded area in Granny peel 0.5061 0.0758
MCP-treated without scald 0 0
Gala without scald 0 0







Table 5. Effect of Compactin on the -farnesene and conjugated trienol content in
Granny Smith apple fruit stored in air at 1C expressed on a fresh weight basis.

Treatment Days of storage -Farnesene
(g/g)
Conjugated trienol
(l/g)
Control 57 477.90 104.97
1.5 M Compactin 63 75.05 1.3






Figures






Fig. 1. Alpha farnesene content of Granny Smith (A) and Gala apple fruit peels as a
function of storage duration and treatment with 1-MCP and storage in air (1-
MCP), CA storage (1.52 kPa O
2
), or storage in air (Control) at 1C.
355


Fig. 2. Conjugated trienol content of Granny Smith (A) and Gala apple fruit peels as a
function of storage duration and treatment with 1-MCP and storage in air (1-
MCP), CA storage (1.52 kPa O
2
), or storage in air (Control) at 1C.


















Fig. 3. Alpha farnesene content of Granny Smith (A) apple fruit peels and ethylene
biosynthetic rate (B) as a function of storage duration and treatment with 1-MCP
and storage in air (1-MCP), CA storage (1.52 kPa O
2
), or storage in air (Control)
at 20C.
356




Fig. 4. Accumulation of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1 (HMGR1)
and farnesene pyrophosphate synthase (FPPS) transcripts when the -farnesene
content was close to its peak value during storage in air (C), in air with 1-MCP
treatment (M), or in CA at 1.5, 2, 4 or 5% O
2
. Storage temperatures were 1, 5, 10,
and 20C as indicated.







357





Fig. 5. Analysis of the HMGR1 and HMGR2 promoter regions.
HMGR1 AATTAAAATAATTTGTAAAAAAA AAAATCTAAAATCT AAAAT-TAATGAGAGAGC AAAG CAA
HMGR2 ------------------------- AATG-AAAAA-TAAAATA------------ AAAG - AA
HMGR1 - G - AA AGAAA GT - GGT - AGGAAAGA-GA-CAAAAAG-A-G-G-TG-CATTCC TTTGACT GT
HMGR2 AGT - A AGAAA G - AGGTC - GGAAAGATGAAAC--AAAGCAAGAGGTGT-ACTCC TTTGACT AT
HMGR1 TTATT -ATATGCCAT - GTAA------------------------------------------
HMGR2 TTATTT -TATGCC - TTGTAACTCCGA CACGTGCCGATAGGACACTCTTTACTCCTTCCCTCT
HMGR1 -- GT ----- CC ----- T - ACACGTGGTC--GTCG-CTTCG-AACGCGTACGCCAGCCC -- AC
HMGR2 GCGTAGCTTCCCCACCCTCG CACGTGCCAC-TC-CCTTCGCA-CGCGTACGCCAGC TT - AC
HMGR1 CT - CC TATATAAA CCCCGCCTA--TGCACTCGCTCCACCATTCAAATCAA - C ---- C -- C -
HMGR2 - TTCC TATATAAA CCCC - CCTAAA--CACTCGCTCCACCATTCAAA -CA-GACTAAACGGCA
HMGR1 ---- ACCAACA A ATTA - AA---AA-CAAATGTTTATTCAATCATTTTTTGTGAGTTTCCGGT
HMGR2 ACATAACCAACAAATT - TAAGTTAAA-AA------ATTCAATCATTTTT -GTGAGTTTCCCGT
HMGR1 TTTGCCCACAGAAGCCTCAGTCC -C-CGCTGTCCTTT-CCTC-TTC-TCTCCTCCGCCGCTC
HMGR2 TTTGACA - AGCCTCAGT -ACTCGCTGTCCTTTTCCTCC -CCT-CGATCTCCTCCGCCGCTC
HMGR1 GATCGCCGCCGGCGCATTTTAGCATCACCA -ACCGCGTCAACACA TACG M DVRRRSTMDTP
HMGR2 GATCGCCGCCGGCGCATTTTAGCATCACCAG -CCGCGTCAACACACC CG MDVRRRSTMDTP
HMGR1 A TKARSGPMKVKVVDHEGDVGVVGAKASDALPLPLYLTNAVFFTLFFSVVYFLLTRWREKIRT
HMGR2 ATKARSGPIKVKVVDHENDVVVVGAKASDALPLPLYLTNAVFFTLFFSVVYFLLTRWREKIRT
HMGR1 S TPLHVVNLSEIVAIL
HMGR2 STPLHVVDLSEIVAIL
Transcription Start Site TATA Box Defence Responsive Element
Hypoxia Responsive Element Pollen Specific Element
Transcription Start Site TATA Box Defence Responsive Element
Hypoxia Responsive Element Pollen Specific Element
358