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
3
.
1
. Peel color changes Peel color of non-fumigated, P. digitatum in-
fected fruit − MCP + INF changed from imma- ture green to mature yellow during the 4-day
incubation period. This was in contrast to +
MCP + INF treated fruit that retained their green color, as indicated by the ab ratio Fig.
1. The degreening of − MCP − INF fruit was clearly not as advanced as the − MCP + INF
treatment but still recorded a higher ab ratio than
the +
MCP − INF fruit.
Considering −
MCP − INF fruit were held in the absence of
any ethylene, their degreening seemed to be a diverse event, that could possibly be as a result of
the two successive 16-h confinement periods. In all treatments, degreening was most prominent
between the second and fourth day of the incuba- tion period Fig. 1.
3
.
2
. Disease progression Analogous with the changes recorded in peel
color was the progress of P. digitatum through the host tissue. After 2 days of incubation there was
no difference in lesion diameter among treatments Fig. 2. However, after an additional 24 h, lesion
diameters on the infected samples with or with- out 1-MCP treatment had increased fourfold.
During the next 24 h, lesion diameters had in- creased eightfold. 1-MCP fumigation of grapefruit
infected with P. digitatum did not induce any significant change in the susceptibility of the fruit
to the disease Fig. 2, or in its progression over the 4-day incubation period.
Fig. 2. Progression of P. digitatum infection in 1-MCP treated + MCP grapefruit. Fruit were uninoculated − or inocu-
lated + on day 0 and showed initial symptoms of infection by day 2. Lesion diameter was recorded on day 2, 3, 4 of the
4-day incubation period, during which the fruit were fumi- gated twice day 0 and day 2 and incubated at 25°C. Data are
presented as the mean lesion diameter of 20 fruit per replicate three replicates per treatment, four treatments per time pe-
riod. Columns sharing the same letter within each time period are not significantly different P B 0.05 by the Tukey Test.
Fig. 1. Influence of 1-MCP on development of peel color in P. digitatum-infected grapefruit. Average color values of four
treatments: − MCP + INF
, − MCP − INF , + MCP + INF and + MCP − INF . Over the 4-day
incubation period, fruit were fumigated twice and the ab ratio was determined on day 0, 2, and 4. Values are the
mean 9 S.E. of three replicates 10 fruit per replicate, three replicates per treatment.
3
.
3
. Ethylene and ACC biosynthesis Evolution of ethylene from whole grapefruit
was substantially increased following exposure to 1-MCP. Fumigation of grapefruit with 1-MCP
increased ethylene production from 0.0834 to 1.823 nmol kg
− 1
h
− 1
Fig. 3. This response was compounded when the fruit were infected with P.
digitatum. Infected fruit treated with 1-MCP pro- duced 6.276 compared to 1.06 nmol kg
− 1
h
− 1
for the non-fumigated infected fruit.
Ethylene production from flavedo discs Fig. 4, sampled at distances in front of the advancing
lesion, was similar to that for intact fruit. Com- pared to the − MCP − INF disc sample, flavedo
discs from + MCP − INF treated fruit synthe- sized 40 × more ethylene. In the − MCP + INF
samples, there was a significant decline P B 0.05 in ethylene production between samples taken 5
mm from the lesion and those taken at 30 and 55 mm, with no difference P B 0.05 between the
latter two
distances and
the corresponding
− MCP − INF controls.
For all sampling points + 5, + 30, + 55 mm of infected fruit, there was a difference P B 0.05
Fig. 5. Accumulation of ACC in flavedo tissue excised from 1-MCP fumigated grapefruit + MCP at increasing distances
in front of a P. digitatum lesion. Data are presented as the mean of five fruit per treatment. Columns sharing the same
letter are not significantly different P B 0.05 by the Tukey Test.
Fig. 3. Effects of 1-MCP fumigation and P. digitatum infection on whole grapefruit ethylene evolution. Ethylene readings
taken from six fruit per treatment and the data are presented as means 9 S.E.
between the + MCP and corresponding − MCP samples Fig. 4. While ethylene production by
both the −
MCP + INF and +
MCP + INF treated fruit declined with increased distance from
the lesion, the reduction was more pronounced within the − MCP + INF samples than within
the + MCP + INF samples.
Similarly, ACC, the immediate precursor of ethylene, accumulated more in + MCP + INF
treated fruit than in − MCP + INF fruit at all three distances from the infection front Fig. 5.
ACC production was highest for both treatments at + 5 mm but declined with increased distance
from the lesion.
3
.
4
. ACC synthase acti6ity and gene expression In − MCP fruit, P. digitatum infection induced
an increase P B 0.05 in ACS activity at 5 mm in front of the lesion compared to the uninfected
sample Fig. 6. At the 30- and 55-mm sampling points, infection did not significantly influence
enzyme activity in − MCP samples. 1-MCP fumi- gation of infected fruit amplified ACS activity
Fig. 4. Ethylene evolution from flavedo discs excised from 1-MCP treated grapefruit + MCP at increasing distances in
front of a P. digitatum lesion. Data are presented as the mean of six fruit per treatment. Any column sharing the same letter
are not significantly different P B 0.05 by the Tukey Test.
significantly P B 0.05 at all three distances ahead of the lesion compared to the corresponding
− MCP samples.
In the absence of 1-MCP, accumulation of ACS mRNA mirrored ACS activity Figs. 6 and 7.
The greatest abundance of ACS mRNA was ob- served at + 5 mm. ACS mRNA decreased sub-
stantially at + 30 mm and further still at + 55 mm. As with all of the previous experiments, the
consequences of exposure of the tissue to 1-MCP was clear. Transcript accumulation, with the ex-
ception of + 5 mm where it was possible that maximum transcript accumulation had already
been achieved, was increased substantially in both uninfected and infected samples in contrast to the
−
MCP treated samples. Furthermore, combining the data from Figs. 6 and 7 suggested the exis-
tence of post-transcriptional control of ACS in flavedo tissue, particularly when the stress of P.
digitatum infection was absent.
4. Discussion
