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
3
.
1
. Tissue P le6els, total lipid and phospholipids Plants grown with the two P fertilization
regimes produced fruit with significantly different P concentrations on a dry weight basis Table 1
and insets to Figs. 1 – 3. The absolute values differed for the two cultivars but in general the
four-fold lower P nutrition level resulted in 1.5 – 3.1-fold lower P concentrations in fruit. The high
P fertilization rate used in this study 360 mg per
Table 1 Phosphorus P levels in fruit tissue, total lipid, phosphatidylcholine PC, and phosphatidylethanolamine PE fractions of
European seedless cucumber fruit cv. Carmen, as affected by P nutrition. Canada No. 1 grade fruit see Section 2 were harvested from both treatments and P-levels were quantified dry weight basis after profiling whole-fruit respiration rates over a 16-day
postharvest interval at 23°C see Fig. 2
a
Phosphorus content mmol kg
− 1
Fruit Tissue P Nutrition mg per plant per week
Total lipid PC
PE 11.5
90 1.90
63 1.29
360 13.7
c,
198
d,
2.40
b,
2.01
c, a
Data are average of 6 fruit. Differences between P nutrition levels were significant at:
b,
P50.07;
c,
P50.05;
d,
P50.01 levels.
week is consistent with that recommended for greenhouse cucumber production in soilless cul-
ture systems in Alberta Mirza, 1990. Previous studies showed insignificant increases in plant
growth and yield with P applications above this rate Trimble, 1993; Trimble and Knowles,
1995b. Typical P deficiency symptoms, consisting of delayed fruit set and localized interveinal le-
sions of desiccated leaf tissue, were noted on the low-P plants late in development; however, no
visible symptoms of P deficiency were evident on the fruit. In fact, while the low-P plants had
produced 26 fewer marketable fruit than the high-P plants by 92 days after planting, the
Canada No. 1 grade fruit from both treatments were outwardly indistinguishable at harvest. This
is relevant from a practical standpoint in that an effect of P-nutrition on fruit physiology after har-
vest would likely impact relative storability and quality and, in the absence of visible symptoms,
fruit could not be sorted accordingly.
The concentration of P in the lipid fraction of fruit at the end of the postharvest period de-
pended on P fertilization level Table 1. Total lipid P in fruit from low-P plants was only 16
less than that in fruit from high-P plants, which contained three times the total P of low-P fruit on
a dry weight basis. Lipid P in the low-P grown fruit accounted for 18.3 of the total tissue P,
compared with only 6.9 in the high-P grown fruit. These results indicate that, when fruit are
produced under low levels of P nutrition, lipids receive priority for P over other P pools, and thus
P content of the lipid fraction is not reduced to the same extent as total P levels would indicate.
Concentrations of the two membrane phospho- lipids, PC and PE, were quantified via P analysis
after separation from the crude extract, and were dependent on the level of P nutrition. These two
phospholipids accounted for 28 and 32 of total lipid P in fruit from low- and high-P plants,
respectively. Fruit from high-P plants contained
Fig. 1. Time course of electrolyte leakage from mesocarp tissue taken from Canada No. 1 grade European seedless cucumber
fruit cv. Corona grown under low and high levels of P nutrition. Following harvest, fruit were held at 23°C for 3 days
prior to leakage measurements. Leakage is expressed as a percentage of total electrolytes within the tissue. F-value for
the interaction of P nutrition and time was significant at the 0.01 level. Data are the average of 4 fruit 9 standard error
which, for clarity, is only shown every 4 min. Inset shows total P concentrations in fruit dry weight basis.
Difference between P treatments was significant at the 0.01 level.
Fig. 2. Postharvest changes in respiration rates CO
2
evolu- tion of Canada No. 1 grade European seedless cucumber fruit
cv. Carmen grown under low and high levels of P nutrition. Fruit respiration was monitored in an open air-flow system at
23°C 95 RH. F-value for the interaction of P nutrition and time was significant at the 0.01 level. Data are the average of
6 fruit 9 standard error. Inset shows total P concentrations in fruit dry weight basis.
Difference between P treatments was significant at the 0.01 level.
3
.
2
. Fatty acid profiles In light of the effects of P nutrition on the
concentrations of total lipid and phospholipids in fruit, it was of interest to determine whether fatty
acid saturation indices were also affected. Five major fatty acids in different lipid pools in low-
and high-P fruit were quantified and the DBI reported Table 2. The DBI of total fatty acid
and unesterified fatty acid fractions in low-P fruit were 21 and 30 lower than those in high-P fruit,
respectively. As fatty acids are integral compo- nents of phospholipids, changes in their chemistry
can alter the physical attributes of membranes. In particular, high levels of acyl-group unsaturation
are important in conferring increased membrane fluidity and function Yoshida and Uemura,
1984, especially during chilling Lynch and Steponkus, 1987. In addition, unesterified fatty
acids are substrates for peroxidation, a process that can hasten postharvest deterioration. It is
typical for ripening and senescing fruit tissues to undergo qualitative changes in the acyl composi-
tion of membrane lipids. Lester and Stein 1993 determined that the ratio of saturated:unsaturated
fatty acids in muskmelon plasma membrane in- creased with maturation of the fruit. A similar
change in fatty acids occurred in the microsomal membrane fraction of apple during ripening
Lurie and Ben-Arie, 1983. While DBI of the PC fraction in our cucumber fruit was not affected by
P nutrition, DBI of the PE fraction of low-P fruit was 18 lower than that of high-P fruit Table 2.
Phosphorus nutrient stress thus affected increased saturation of fatty acids in a major phospholipid
pool, reflecting a change in the molecular species of fatty acids in membranes of the harvested
cucumber fruit.
Though speculative, it is possible that the higher level of unsaturation in the unesterified
fatty acid pool Table 2, relative to the phospho- lipids, is a result of the positional specificity of a
particular phospholipase. The lysophospholipid thus formed would not be included in the pool of
native PC or PE Table 1 resolved by thin layer chromatography. This hypothesis could be tested
by a more detailed examination of the phospho- lipid and lysophospholipid pools of low- and
high-P fruit.
Fig. 3. Postharvest changes in respiration rates CO
2
evolu- tion of Canada No. 1 grade European seedless cucumber fruit
cv. Corona grown under low and high levels of P nutrition. Fruit respiration was monitored in an open air-flow system at
23°C 95 RH. F-value for the interaction of P nutrition and time was significant at the 0.01 level. Data are the average of
10 fruit 9 standard error. Inset shows total P concentrations in fruit dry weight basis.
Difference between P treatments was significant at the 0.01 level.
26 more PC and 56 more PE than fruit from low-P plants. Such a difference in phospholipid
content may negatively impact membrane func- tion, as has been shown in some plant tissues
during aging and senescence Suttle and Kende, 1980.
3
.
3
. Electrolyte leakage Since phospholipids and their constituent fatty
acids are important components of membranes, we tested the possibility that the low-P-induced
changes in concentration and saturation of phos- pholipids affected membrane integrity in the har-
vested fruit. Changes in membrane integrity that effect increased permeability can readily be
demonstrated by measuring leakage of electrolytes from tissue Ross, 1974; Parrish and Leopold,
1978; Barber and Thompson, 1980. Tissue from low-P fruit leaked electrolytes at a faster rate
initially, and lost a greater percentage of total electrolytes over a 30 min interval, than that from
high-P fruit Fig. 1. After 30 min, tissue from low-P fruit had leaked 19 more total electrolytes
than tissue from high-P fruit. Since the concentra- tion of electrolytes i.e. maximum conductivity
on a fresh weight basis was equal in tissue from the low- and high-P fruit, the greater efflux of
electrolytes from low-P tissue is indicative of re- duced membrane integrity. Moreover, the higher
membrane permeability was in close agreement with the 18 lower DBI in the PE fraction of
low-P fruit Table 2. Negative correlations be- tween permeability and DBI of membrane lipids
have been shown during aging of vegetative tis- sues and with changes in fruit development. For
example, aging of potato tubers during cold stor- age was accompanied by a progressive increase in
the degree of saturation of membrane lipids and this correlated with a significant decline in mem-
brane integrity Knowles and Knowles, 1989. Leakage of electrolytes from muskmelon fruit tis-
sue increased with ripening, correlating with an increase in the degree of saturation of plasma
membrane phospholipid fatty acids Lester and Stein, 1993. Our study is the first to demonstrate
that P nutrition can affect membrane function permeability
of harvested
cucumber fruit
through an effect on membrane phospholipid con- centration and saturation level. If the basal
metabolic rates of fruit are altered by P content, then P nutrition could potentially affect the rate
of deterioration of fruit after harvest.
3
.
4
. Fruit respiration Respiration rates of low- and high-P fruit cv.
Carmen were compared as a general indication of overall metabolic rate. Fruit harvested at 77 days
after planting from low-P plants had a 22 higher P B 0.01 respiration rate 6.0 mmol min
− 1
kg
− 1
than fruit from high-P plants 4.9 mmol min
− 1
kg
− 1
when averaged over the 16-day postharvest interval Fig. 2. Respiration rates declined over
the 16-day period, such that rates at day 1 were 153 higher than those at day 16, regardless of
the level of P nutrition P B 0.01. However, low- P plants produced fruit that experienced a climac-
teric in respiration within 5 days of harvest. At its maximum 72 h postharvest, respiration of low-P
fruit was 35 greater than that of high-P fruit. The increase in respiration was unique to the
low-P fruit.
To further document and characterize the effect of P nutrition on fruit respiration rates, a different
Table 2 Double bond indices DBI of fatty acids in various lipid fractions of Canada No. 1 grade European seedless cucumber fruit cv.
Carmen, as affected by P nutrition
a
Lipid fraction DBI of fatty acids P Nutrition mg per plant per week
PC PE
Total Unesterified
2.60 90
2.74 5.56
5.07 3.35
b
2.34 ns
d
7.21
c
360 7.03
c a
Data are from the same fruit described in Table 1.
b
Differences between P nutrition levels were significant at P50.07.
c
Differences between P nutrition levels were significant at P50.01 levels, respectively.
d
ns, not significant.
cultivar Corona was produced under similar cul- tural conditions and respiration was monitored at
4 h intervals for 100 h after harvest Fig. 3. Fruit with the lower P concentration from low-P plants
again showed a respiratory climacteric that lasted from 50 – 90 h after harvest. Low-P fruit had a
57 greater respiration rate than high-P fruit at the climacteric maximum about 70 h after har-
vest. The fruit grown with high P fertilization had higher tissue P Fig. 3, inset and did not
show a rise in respiration during this period. Phosphorus nutrition thus affected respiratory
metabolism of cucumber fruit after harvest. Since respiration and shelf-life of fruits are often in-
versely related Salunkhe and Desai, 1984; Kader, 1987, further research on the effects of P nutri-
tion on postharvest deterioration of cucumber fruit is warranted.
In addition to P-induced respiratory differ- ences, respiration rates of cucumber at harvest
can depend on fruit andor plant maturity. Fruit cv. Carmen harvested at 77 days after planting
respired at a greater rate than those harvested at 54 days after planting Trimble, 1993. When fruit
maturity was defined as days after anthesis, postharvest respiration rates of cv. Deliva fruit
increased with maturity, while shelf-life declined Kanellis et al., 1986. Similar to our results,
‘Deliva’ fruit harvested 25 or 30 days after anthe- sis underwent a peak in respiration a few days
after harvest, with the most pronounced peak in the more mature fruit Kanellis et al., 1986.
Although shelf-life was not determined in our study, it is reasonable to speculate that fruit with
higher respiration rates due to low tissue P would also exhibit a more rapid loss in postharvest
quality.
3
.
5
. Internal ethylene Like field cucumbers, European seedless green-
house cucumbers are harvested at a physiologi- cally immature stage and thus lack the ability to
complete many of the changes associated with full ripening. As such, cucumbers of commercial ma-
turity behave as nonclimacteric fruit Kanellis et al., 1986. If left to ripen on the plant, or if
harvested after attaining physiological maturity, cucumber fruit may indeed exhibit climacteric be-
havior Abeles et al., 1992. Nevertheless, to confirm that the low-P-related respiratory climac-
teric Figs. 2 and 3 was not induced by ethylene and associated with ripening, ethylene concentra-
tions were compared at 72 h after harvest coin- ciding with the respiratory peak in the low-P
fruit. The low- and high-P fruit contained 2.7 and 2.5 nmol L
− 1
ethylene, respectively, and the difference
was not
significant. Saltveit
and McFeeters 1980 noted that a burst of ethylene
could occur from processing cucumber anywhere from 5 – 25 days after harvest, but this increase in
ethylene production was not associated with a concomitant increase in respiration. The increase
in respiration rate of our low-P fruit was not associated with ripening and was likely a P stress
response. Phosphorus stress during growth either indirectly, through influencing the rate at which
fruits mature, or directly, through an effect on membrane composition and function, affects the
postharvest respiration of cucumber fruit.
In apple, respiration of harvested fruit did not correlate with fruit P over a wide range of tissue P
concentrations 164 – 618 mg kg
− 1
, dry weight basis Davenport and Peryea, 1989. However, in
a long-term study of the effects of N, P and K fertilization levels on postharvest respiration of
apple, Letham 1969 found that fruit with the highest P concentration had the lowest respiration
rate while those with the lowest P concentration had the highest respiration rate. Low-P apple fruit
initiated the climacteric sooner than high-P fruit, indicating that lower levels of tissue P were suffi-
cient to alter fruit developmental physiology. Fruit P concentrations in Letham’s study were
within the range of those reported in Davenport and Peryea’s work, but were induced as a result of
manipulation of P fertilization levels during production.
4. Summary and conclusions
