The mechanisms by which plants perceive and transduce the ethylene signal are topics that have
been researched intensively. Following the isola- tion of Arabidopsis ethylene insensitive mutants
Bleecker et al., 1988 and the cloning of two genes in particular involved in the pathway ETR
1 gene, Chang et al., 1993; CTR 1 gene, Kieber et al., 1993, models of the chain of events that
proceed ethylene binding have been proposed Bleecker and Schaller, 1996; Pallin et al., 1996;
Kieber, 1997; McGrath and Ecker, 1998. Subse- quent to ethylene binding with the ethylene bind-
ing protein EBP, a suspected heterotrimetric G-protein Harpham et al., 1996, they suggest a
negative regulation of the receptors His kinase activity which inactivates the activity of the CTR
1 protein kinase. It is predicted that this in turn influences through a MAP kinase cascade the
downstream products, EIN 2, EIN 3, EIN 5 and ultimately ethylene sensitivity.
Several inhibitors of ethylene synthesis have assisted in the study of the pathway Yu and
Yang, 1980; Yoshii and Imaseki, 1982; Kende, 1989; Bouquin et al., 1997; Nakatsuka et al.,
1997; Mullins et al., 1999. 1-MCP is a non-toxic compound that inhibits ethylene perception by
irreversibly binding to and hence inactivating the EBP Sisler et al., 1996. Shown to be an effective
inhibitor of ethylene action in banana Golding et al., 1998, Petunia hybrida Serek, 1995 and
Gypsophila paniculata Newman, 1998, this an- tagonist has been suggested as a possible alterna-
tive for the commercially used silver thiosulfate STS in improving product longevity Porat et
al., 1995.
Wound-induced production of ethylene in cit- rus flavedo is strongly inhibited by treatment with
ethylene Riov and Yang 1982, a phenomenon referred to as ‘autoinhibition’. Investigations into
the mechanisms by which ethylene modifies its own synthesis have focused on the action of ACS
Yoshii and Imaseki 1982; Yang and Hoffman 1984; Hyodo et al., 1985 and how autoinhibition,
by acting at the level of ACS gene expression Peck and Kende, 1998; Mullins et al., 1999,
suppresses induction of the protein Yang and Hoffman 1984; Hyodo et al., 1985 and subse-
quently inhibits the accumulation of ACC Riov and Yang 1982; Hyodo et al., 1985. If however
the first step of the pathway, binding of ethylene to the EBPs Ecker, 1995, is interceded with by
treating P. digitatum-infected fruit with 1-MCP, it is unclear what the outcome would be with re-
spect to the evolution of stress ethylene and the progression of disease through the host tissue.
The aim of this research was to provide some answers to this question.
2. Materials and methods
2
.
1
. Fruit source, inoculation and disease assessment
Grapefruit Citrus paradisi were collected from a local grove on a regular basis between October
and December, washed under hot water to re- move residual crop protectants, and air dried
prior to treatment.
On day 0, 20 min prior to fumigation, fruit were infected by piercing the flavedo to a depth of
2 mm and inoculating with a 5 ml spore suspen- sion 1 × 10
6
spores ml
− 1
water of P. digitatum + INF. Controls were inoculated with sterile
water − INF and all fruit were then incubated in the presence or absence of 1-MCP at 25°C for
4 days Section 2.2. Disease progression in each fruit was assayed by measuring the average lesion
diameter on day 2, 3 and 4. Two lesion diameters each intersecting the point of inoculation and
perpendicular to each other were recorded per fruit.
2
.
2
. Fruit fumigation To fumigate fruit with 1-MCP + MCP, the
active gas was produced by adding 4 ml of buffer- ing agent 50 potassium hydroxide, 50 sodium
hydroxide to 100 mg of 1-MCP Biotechnologies for Horticulture, Inc., Waltersboro, SC. Solution
was stirred briefly and immediately placed into a steel chamber 71 cm × 41 cm × 71 cm contain-
ing the fruit. The top of the chamber was covered with a gasket and a steel lid which were both
clamped in place day 0. After 16 h at 25°C, fruit were removed from the chambers day 1 and left
to stand in the dark for 24 h, 95 RH. Fumiga- tion was then repeated day 2 as described for a
further 16 h, after which the fruit were left to stand day 3 for 24 h, before analysis commenced
day 4. Control chambers − MCP contained 4 ml of buffering agent.
2
.
3
. Fruit color measurements Peel color three readings per fruit was
recorded using a Minolta Chromameter Model CR-300, Minolta Camera Corp., Osaka, Japan
on day 0, 2 and 4 of the 4-day incubation period Section 2.2. The Commission Internationale de
l’Eclairage a and b color index scale was em- ployed and expressed as a ratio of ab as
described by McDonald et al. 1997. An increas- ing ‘ab’ ratio, from − 0.55 to − 0.10, indicated
increased yellow color with a ratio of − 0.10 being considered the equivalent of mature colored
grapefruit.
2
.
4
. Ethylene determination Ethylene evolution from whole fruit was moni-
tored six fruit per treatment by incubating indi- vidual fruit in 1.75-l jars. After 90 min at 25°C, a
1-ml sample was extracted from the headspace of the jar via a rubber septum and injected into a gas
chromatograph Hewlett Packard, Model 5880A equipped with an alumina packed column and a
flame ionization detector. Results are expressed in nmol ethylene kg
− 1
h
− 1
. To determine ethylene production from flavedo,
discs excised from healthy tissue at increasing distances + 5, + 30, + 55 mm in front of the
lesion were sealed in 130-ml jars seven disks per jar per treatment with seven fruit assayed per
treatment which contained a disc of Whatman No. 1 filter paper pre-soaked with 1 ml of water.
After 90 min at 25°C, a 1-ml sample was extracted from the headspace of the jar via a rubber septum
and injected into the gas chromatograph for anal- ysis. Control discs were excised at the + 5 mm
distance from fruit inoculated with sterile water. Results are given in nmol ethylene kg
− 1
h
− 1
.
2
.
5
. ACC determination Flavedo discs 1.5 g fresh wt. were macerated
in liquid nitrogen and resuspended in 80 ethanol 2 ml g
− 1
. Samples were extracted with shaking 150 rev. min
− 1
at 25°C, after which the mixture was spun at 5000 × g for 15 min and the superna-
tant collected on ice. This extraction was repeated twice, the supernatants pooled, and the volumes
recorded. After reducing to near dryness at 50°C, extracts were brought to a volume of 3 ml with
H
2
O and centrifuged at 28 500 × g for 5 min. To remove extracted pigments, 200 ml of chloroform
was added and the volume spun at 28 500 × g for 5 min. The organic phase was decanted off and to
precipitate any protein present, 200 ml of phenol chloroformisoamyl alcohol 25:24:1 was added
and the suspension spun at 28 500 × g for 10 min. ACC in the aqueous phase was assayed by the
method of Lizada and Yang 1979 using 250 mM HgCl
2
and a 1.5-ml sample for injection into the gas chromatograph. Each sample was analyzed in
triplicate with an internal standard containing 4 mM ACC. All steps were performed at 4°C and
five fruit per treatment were tested. Recovery of ACC was typically between 60 and 80 with
ACC obtained, expressed as mmol kg
− 1
.
2
.
6
. Assay of ACC synthase ACS
acti6ity Flavedo discs 3 g were homogenized Brink-
mann Homogenizer, Model PT 1035, Westbury, NY for 30 s in a buffer 2 ml g
− 1
containing 100 mM potassium phosphate, pH 8.0, 5 ammo-
nium sulfate, 5 mM pyridoxal phosphate, 4 mM DTT plus 3 PVPP. The extract was filtered
through a layer of Miracloth Calbiochem, La Jolla, CA and centrifuged at 10 000 × g for 10
min. The supernatant was brought to 90 saturation
with ground
ammonium sulfate,
stirred for 90 min at 4°C and the suspension centrifuged at 25 000 × g for 10 min. Pelleted ma-
terial was resuspended in 3 ml of incubation buffer 100 mM potassium phosphate, 0.1 mM
DTT and 4 mM pyridoxal phosphate, pH 7.0. For desalting, the sample was loaded onto an
Econo-Pac 10DG chromatography column Bio- Rad, Hercules, CA and eluted with 4 ml of
incubation buffer. Protein content was determined on this extract according to Lowry et al. 1951
with BSA as standard. ACC synthase EC 4.4.1.14 activity was determined in a reaction
mixture containing 3.67 ml desalted flavedo ex- tract, 375 ml incubation buffer and 450 ml of 500
mM S-adenosyl-
L
-methionine AdoMet. After a 3-h incubation at 30°C, protein was eliminated
from the mixture by adding 3.75 ml of phenol chloroformisoamyl alcohol 25:24:1 followed by
vortexing and then centrifuging for 10 min at 28 500 × g. The aqueous phase was decanted and
clarified by centrifuging at 28 500 × g for 10 min; ACC formed was assayed by the method of
Lizada and Yang 1979 using 50 mM HgCl
2
and a 1.5-ml sample of headspace gas for injection
into the GC. Each sample was analyzed in tripli- cate and contained one internal standard spiked
with 4 mM ACC. All steps were performed at 4°C and each treatment was assayed three times. En-
zyme activity was expressed as mmol of ACC formed kg
− 1
protein h
− 1
.
2
.
7
. RNA extraction Total RNA was extracted from 1 g flavedo
disks using the guanidine – phenol – chlorofrom method described by Strommer et al. 1993 with
slight modification. Tissue was ground to a pow- der under liquid N
2
, resuspended in RNA extrac- tion buffer 4 M guanidium isothiocyanate, 25
mM sodium citrate, 0.5 sarcosyl, 10 ml b-mer- captoethanol ml
− 1
extraction solution. The aqueous phase was extracted a second time with
phenol – chloroform and RNA precipitated with an equal volume of isopropanol at − 20°C. RNA
was pelleted by centrifugation at 10 000 × g for 10 min. The RNA pellet was redissolved in 500 ml
water, mixed with an equal volume of 4 M LiCl, and allowed to precipitate at 0°C for 1 h. The
precipitated RNA was pelleted by centrifugation at 16 000 × g for 10 min, washed 2 × with 70
ethanol, air dried and dissolved in water.
2
.
8
. Probe labeling The ACS gene sequence, cloned and described
by Mullins et al. 1999, was labeled with digoxi- genin using the PCR digoxigenin DIG probe
synthesis kit Boehringer Mannheim, Boehringer, Germany following the manufacturer’s instruc-
tions.
2
.
9
. Northern blot analysis Total RNA 10 mg was fractionated in 1.5
agarose gel containing 2 formaldehyde. Follow- ing electrophoresis, the gels were equilibrated in
1 × NaPO
4
– EDTA, pH 7 prior to transfer to nylon membrane. RNA was transferred by capil-
lary in 1 × NaPO
4
– EDTA, pH 7 onto positively charged nylon membrane Boehringer Mannheim
and following transfer, RNA was UV-crosslinked to the membrane. Membranes were prehybridized
in DIG Easy Hyb Boehringer Mannheim for 4 h at 50°C. DIG-labeled probe was denatured by
boiling for 10 min, syringe filtered 0.45 mM and then added to the hybridization solution to give a
final concentration of 25 ng ml
− 1
. Hybridization was carried out overnight at 50°C. Following
hybridization, the membranes were washed twice in 2 × SSC + 0.1 SDS at room temperature for
15 min and then twice in 0.1 × SSC + 0.1 SDS at 55°C for 15 min. Probe, hybridized to the
target, was detected using the DIG chemilumines- cent detection system Boehringer Mannheim fol-
lowing
the manufacturer’s
instructions. All
Northern blot experiments were completed in duplicate.
3. Results
