incidence of storage disorders in apple Yogarant- nam and Sharples, 1982. Lin and Ehret 1991
showed that the shelf-life of greenhouse cucumber cv. Mustang could be improved by increasing
the concentrations of N, P, K, Ca, S and Mg in the nutrient solution, but it was not possible to
isolate a specific effect due to P from their data.
The effect of P on the postharvest physiology of fruit may be attributed to its role as a component
of phospholipids, a major constituent of cell membranes. Alterations in membrane composi-
tion occur naturally during fruit ripening and senescence and are also a mechanism by which
cells adapt to changing environmental conditions. For example, phospholipid content increased in
apple during storage at 0°C, purportedly as an adaptation to maintain membrane function at this
low temperature Lurie et al., 1987. Phosphorus deficient plant organs may be unable to make this
adaptation. It is commonly observed in fruit tis- sue that membrane function declines with ripen-
ing and senescence, and this occurs concomitant with a loss of phospholipids andor changes in
their constituent fatty acids. The most frequently reported change in fatty acid chemistry is an
increase in saturation at the expense of unsatura- tion Lurie and Ben-Arie, 1983; Lester and Stein,
1993; Palma et al., 1995; Rogiers et al., 1998a.
A few studies have indicated that P content can potentially affect the postharvest respiration rate
of fruit, which is often inversely related to storage and shelf-life Kader, 1987. Letham 1969 found
that higher levels of P fertilization resulted in higher P concentration in apple and that fruit P
content was negatively correlated with respiration rate. Another study of respiration in apple found
no relation between tissue P concentration and respiration rate Davenport and Peryea, 1989. In
this latter study, differences in tissue P were due to natural variation among fruits and the applica-
tion of CaCl
2
sprays, and were not as great as those induced by varied P nutrition.
Despite inconsistencies in the literature, it is evident that plant P nutrition affects fruit P status
and this in turn may have a significant influence on postharvest physiology and thus storability
and shelf-life of fruit. The experiments described herein were designed to induce differences in P
concentration of European seedless cucumber fruit so that the effects of fruit P-status on respi-
ration rate, membrane lipid chemistry and mem- brane function could be assessed after harvest.
2. Materials and methods
2
.
1
. Plant culture European seedless cucumber Cucumis sati6us
L., cvs. Carmen and Corona fruit were grown from seed in a mixture of coarse sand, fine sand
and loam 2:2:1, vvv in a research greenhouse at the University of Alberta, Edmonton under
natural light supplemented with high pressure sodium lamps Sylvania 400 W; 450 mmol m
− 2
s
− 1
, 1 m above bench; 16 h photoperiod, as described
previously Trimble
and Knowles,
1995b. To produce fruit containing relatively low and high P levels, plants were fertilized with 250
mL of 120 or 480 mg l
− 1
P KH
2
PO
4
three times per week, starting 26 days after planting. Phos-
phorus treatments thus consisted of 90 and 360 mg of elemental P per plant per week. Potassium
levels were held constant by adding KCl to the 90 mg P per week treatments. All other nutrients
were applied uniformly pH 6 to each pot by an automated fertigation system Harrow Fertigation
Manager, Labbate Control Systems, Leamington, ON according to nutrient levels recommended
for cucumber fruit production Adamson and Maas 1981; Mirza, 1990. Fertigation began 26
days after planting. Each plant initially received 1 l d
− 1
of nutrient solution containing 50 mg l
− 1
NO
3
– N, 5 mg l
− 1
NH
4
– N, 75 mg l
− 1
K, 40 mg l
− 1
Ca, 40 mg l
− 1
Mg, and 400 mg l
− 1
max S. The volume of fertilizer and concentration of
nutrients were gradually increased with plant growth, so that by 64 days after planting each
plant received 5.4 l per day of solution containing 200 mg l
− 1
NO
3
– N, 20 mg l
− 1
NH
4
– N, 275 mg l
− 1
K, 120 mg l
− 1
Ca, 40 mg l
− 1
Mg and 400 mg l
− 1
max S, as previously described Trimble and Knowles, 1995b. Micronutrients in the fertiga-
tion solution were maintained at constant levels 0.840 mg l
− 1
Fe, 0.547 mg l
− 1
Mn, 0.164 mg l
− 1
Zn, 0.200
mg l
− 1
B,
0.102 mg l
− 1
Cu, and 0.072 mg l
− 1
Mo through- out the study.
The plants were trained to maintain a single leader, according to guidelines for a sequence
cropping production system Adamson and Maas, 1981; Mirza, 1990. Fruit from plants grown un-
der the two phosphorus regimes were harvested after reaching a minimum of 30 cm length and 42
mm diameter Canada No. 1 grade, which corre- sponded to a fresh weight of between 300 and 400
g. These fruit were then used to characterize the effects of P nutrition on fruit P content, mem-
brane lipid chemistry, membrane permeability, and whole-fruit respiration during storage at
23°C.
2
.
2
. Respiration Respiration of low- and high-P fruit was ini-
tially compared over a 16-day period at 23°C starting 1 day after harvest. Fruit cv. Carmen,
harvested 77 days after planting, were blocked for fresh weight and enclosed in 5.7 L plastic cham-
bers one fruit per chamber, six replicates con- taining inlet and outlet air-flow ports. The
chambers were attached to a flow-board which distributed a constant flow of humidified air 120
mL min
− 1
to each chamber. Fruit respiration was determined daily by quantifying CO
2
in 1 mL samples of chamber effluent. The CO
2
was ana- lyzed with a Hewlett Packard 5890A gas chro-
matograph GC
equipped with
a thermal
conductivity detector and a 2.4 m stainless steel column 3.2 mm o.d. packed with HaySep T. The
carrier gas He flow rate was 30 ml min
− 1
and the column was 100°C. Injector and detector port
temperatures were 140°C. A follow-up study was conducted to more
clearly characterize the differences in fruit respira- tion between low- and high-P fruits that were
identified within 4 days of harvest in the longer- term study above. Canada No. 1 grade fruit for
this latter study were harvested from 82-day-old plants cv. Corona, blocked for fresh weight and
immediately placed into chambers one fruit per chamber, 10 replicates in an open airflow system
at 23°C as above. The outflows from each cham- ber were connected via a manifold to an infra-red
gas analyzer LI-COR model LI 6262, Lincoln, Nebraska that was programmed to read CO
2
concentration from each chamber at 4 h intervals over the 4-day postharvest period. Respiration
rates of fruit from low- and high-P plants were calculated as the rate of CO
2
produced m mol min
− 1
kg
− 1
on a fresh weight basis 9 SE and plotted against time after harvest.
2
.
3
. Internal ethylene The concentration of ethylene in Canada No. 1
grade fruit from high- and low-P-grown plants cv. Corona was quantified at 70 h after harvest.
The fruit had been stored at 23°C 95 RH prior to ethylene extraction and analysis. Ethylene was
extracted from the fruit under vacuum, as de- scribed in Beyer and Morgan 1970. Gas chro-
matographic separation and quantification of ethylene was according to Rogiers et al. 1998b.
2
.
4
. Electrolyte leakage The effect of P nutrition on membrane perme-
ability was assessed in electrolyte leakage studies using
methods modified
from Parrish
and Leopold 1978 and Ross 1974. Leakage mea-
surements were done approximately 72 h after harvest on tissue from Canada No. 1 grade fruit
cv. Carmen from low- and high-P plants. Fruit had been held at 23°C 95 RH prior to the
leakage measurements. Tissue discs 2 mm thick, 6 mm diameter for leakage studies were prepared
from a 5 cm long cross-section from the middle portion of each fruit. The discs were cut from
cores 6 mm diameter, 5 cm long of mesocarp tissue from this section with a Braun food proces-
sor. Fifteen discs were randomly selected, rinsed briefly with distilled deionized H
2
O, and placed in 10 mL distilled deionized H
2
O. Conductivity of the surrounding solution was monitored with a
CDM 83 conductivity meter Radiometer, Copen- hagen at 0.5 – 1 min intervals over 30 min. The
tissue was then frozen with liquid N
2
, boiled for 10 min and the total conductivity recorded. Elec-
trolyte leakage was expressed as a percentage of total electrolytes in the tissue.
2
.
5
. Tissue phosphorus Whole fruit from the respiration studies and
mesocarp tissue from the electrolyte leakage stud- ies were lyophilized in preparation for P analysis.
The dried tissue was ground with a Wiley mill through a 40 mesh screen and 100 mg of tissue
was ashed overnight in a muffle furnace 550°C. The ash was digested in 1 mL HCl for 20 min,
diluted with 9 mL 0.72 N H
2
SO
4
and centrifuged 1640 × g, 30 min to settle any undigested mat-
ter. Total P of a 200 mL aliquot was determined using the methods of Serrano et al. 1976.
2
.
6
. Lipid extraction and analysis Freeze-dried, ground tissue of fruit from the
16-day respiration study was extracted for 30 s in 10 vol boiling isopropanol, and 60 s further after
the addition of 20 vol of CHCl
3
. The extract was filtered and re-extracted in 20 vol CHCl
3
:MeOH 2:1 vv. The combined filtrates were washed with
0.2 vol of 0.15 KCl and evaporated to dryness. An aliquot of lipid extract was concentrated
and spiked with phosphatidylcholine dipentade- canoyl and phosphatidylethanolamine diheptade-
canoyl as internal standards. The samples, along with an external standard of nonadecanoic acid,
were applied to thin layer chromatography plates of 0.5 mm silica gel G and developed in hex-
ane:Et
2
O:HOAc 70:30:1, vvv. The separated compounds were visualized with 0.05 2,7-
dichlorofluorescein in EtOH and the band corre- sponding to nonadecanoic acid was collected and
reserved for analysis as the unesterified fatty acid fraction. The phospholipids remaining at the
origin on the plates were eluted from the silica gel in CHCl
3
:MeOH:H
2
O 10:5:1, vvv, reapplied to another plate 0.5 mm silica gel H and developed
in CHCl
3
:MeOH:HOAc:H
2
O 50:25:7:3, vvvv. Narrow vertical lanes of the plates were sprayed
with molybdenum blue reagent for the identifica- tion of phospholipids Dittmer and Lester, 1964.
Ninhydrin and Dragendorf’s reagents Zweig and Sherma, 1972, in combination with authentic ex-
ternal standards, were used to verify the locations of PE and PC, respectively.
Unesterified fatty acids, PC, PE and an aliquot of crude extract were transesterified for gas chro-
matography by refluxing with 2 vv H
2
SO
4
in MeOH for 2 h. Fatty acid methyl esters were
recovered in hexane according to Christie 1989. These were analyzed on a Hewlett Packard 5890A
gas chromatograph using a flame ionization detec- tor and a 1.8 m × 3 mm stainless steel column of
10 SP2330 on 100120 mesh Chromosorb WAW. The operating temperature was 185°C
with a N
2
flow rate of 30 mL min
− 1
. The instru- ment was calibrated with authentic standards of
methylpalmitate 16:0,
methylstearate 18:0,
methyloleate 18:1, methylinoleate 18:2 and methylinolenate
18:3. Double
bond indices
DBI were then calculated for fatty acids in the total, unesterified, PC and PE fractions according
to the following equation Liljenberg and Kates, 1985:
[18:1 + 218:2 + 318:3] [16:0 + 18:0]
, where indicates mol percent.
To quantify lipid P, an aliquot of lipid extract was subjected to thin layer chromatography as
described above for the separation of lipid classes, but no internal standards were included. PC and
PE were located as before and transferred to a 30 mL digestion flask. A sample of crude extract was
applied to silica gel and removed without develop- ment for analysis of total lipid P. The fractions
were digested in 0.65 mL perchloric acid for 20 min. Phosphorus was analyzed according to
Rouser et al. 1969.
3. Results and discussion
