PHYSIOLOGIA PLANTAKUM 76: 479^84, Copenhagen 1989

Correlation of reduced chilling injury with increased spermine

and spermidine levels in zucchini squash
George F, Kramer and Chiem Y, Wang

Kramer. G. F. and Wang, C. Y. 1989. Correlation of reduced chilling injury with

increased spermine and spermidine levels in zucchini squash. - Physiol. Plant. 76:
479-484.

The development of chilling injury symptoms in zucchini squash {Cucurbita pepo L.

cv. Ambassador) was reduced by preconditioning the fruit for 2 days at 10°C. In the
control group, held continuously at 2.5°C, increasing chilling injury correlated with
an increase in putreseine and decreases in spermidine and spenrsine. Preconditioning
led to a significant increase in spermidine and spermine levels, beginning after the
initiation of storage at 2.5°C and lasting for 5 days, after which the levels decreased
but remained elevated relative to the control. The elevation of polyamine levels by
direct treatment of the fruit with spermine prior to storage resulted in reduced
chilling injury, indicating that poiyamines may be involved in the protective mecha-
nism of preconditioning. We have also examined the extent of lipid peroxidation by
determining the levels of chloroform-soluble fluorescent products in the skin of the
fruit subjected to chilling. The chilling injurj- in the control group resulted in an
increase in the fluorescent products. The levels of fluorescent products were signif-
icantly reduced by the preconditioning treatment. These results may indicate that
polvamines can act to prevent chilling injury in squash by a mechanism which
involves protecting membrane lipids from peroxidation.
Key words - Chilling injur)'. Cucurbita pepo, fluorescent products, peroxidatioti.
polyamine, postharvest physiology, preconditioning, putrescioe, spermidine, sper-
mine, zucchini squash.

G.F. Kramer and C.Y. Wang (corresponding author). Horticultural Crops Quality
Lab., Agricultural Research Sennce, U. S. Dept of .Agriculture, Beltsville, MD 20705,
USA.

can protect microsotnal membranes from peroxidation

Introduction
Polyamines are potent inhibitors of many senescence-
related processes in a variety of plant species (Galston
and Kaur-Sawhney 1987). Much of this antisenescent
activity may be membrane related (Roberts et al. 1986).
A key process in plant senescence appears to involve
membrane deterioration, especially via the formation of
lipid hydroperoxides (Thompson et al. 1985). Poly-
amines can associate with the surface of membranes,
presumably through interactions with anionic compo-
nents of the metnbrane such as phospholipids (Ballas et
al. 1983, Roberts et al. 1986). This interaction serves to
stabilize the bilayer surface and may thus retard mem-
brane deterioration (Roberts et al. 1986). This hypothe-
sis is consistent with the observation that polyamines
jj, ^i^j.^ (jcitada et al. 1979). Polyamines also have rad-
ical-scavenging properties (Drolet et al. 1986). Protec-
tion of membranes from peroxidation by polyamines
could involve both their ability to interact with phos-
pholipids and their antioxidant activity.
Chilling injury, like senescence, is thought to involve
alteration of membrane structure (Wang 19S2, Raison
and Orr 1989). Raison atid Lyons (1970) proposed that
the primary event causing chilling injury is a phase
transition in the molecular ordering of membrane lipids.
The membrane phase transition would have many dele-
terious effects on the tisstie, including increases in mem-
brane permeability and alteration of the activity of
membrane proteins (Wang 1982).
Given the relationship between polyamines and

Received 14 December, 1988; revised 26 April, 1989

Phvsiol. Plam. 76, .1TO9

membrane protection and between chilling injury and
membrane damage, the possible connection between
polyamines and chilling injury has been of great in-
terest. Chilling injury in a variety of fruit results in
significant increases in putreseine levels (McDonald and
Kushad 1986, Wang and Ji 1988), These results are
consistent with the observation that putreseine increases
in plants in response to a number of stresses, including
water stress (Wang and Steffens 1985), acid treatment
(Young and Galston 1983) and osmotic shock (Flores
and Galston 1982), Such results do not indicate whether
the increase in putreseine is a protective response or
whether putreseine itself is the cause of the stress-in-
duced injury (McDonald and Kushad 1986), However,
other results indicate that polyamines may actually pro-
tect plants from chilling injury. The cold hardening of
several plant species correlates with increases in poly-
amines (Guye et al, 1986, Kushad and Yelenosky 1987,
Nadeau et al, 1987), Also, the reduction of chilling
injury in squash and senescence in Chinese cabbage and
apples by controlled atmosphere storage is coupled with
increased polyamine levels (Wang 1988, Wang and Ji
1988, Kramer et al, 1989), These results are consistent
with the suggestion that polyamines preserve membrane
integrity, resulting in increased cell viability during
chilling (Guye et al, 1986),
A by-product of lipid peroxidation is the accumu-
lation of chloroform-soluble fluorescent products (Tap-
pel 1975), These compounds have an excitation maxi-
mum in the region of 340-380 nm and an emission
maximum in the region of 440-480 nm (Tappel 1975,
Wilhelm and Wilhelmova 1981), The accumulation of
such compounds has been measured during the senes-
cence of cotyledon membranes (Thompson et al, 1985)
and leaf chloroplasts (Wilhelm and Wilhelmova 1981),
and during the ripening of fruit (Maguire and Haard
1975), These fluorescent products comprise a family of
compounds resulting from the reactions of the break-
down products of peroxidized lipids with free amino
groups resulting in a characteristic Schiff base structure
(-N=CH-CH=CH-NH-) (Tappel 1975), Free radical
damage may also be involved in chilling injury (Omran
1980, Patterson et al, 1984), This hypothesis would also
predict membrane damage to be of significance, since
unsaturated fatty acids are easily oxidized and lipid
peroxidation is an auto-catalytic (chain-reaction) proc-
ess (Droillard et al, 1987), The activity of the HjOj-
scavenging enzyme catalase decreases during chilling
injury in both the leaves and fruit of plants (Omran
1980, Patterson et al, 1984, MacRae and Ferguson 1985,
MacRae et al, 1986), Initial catalase levels can also be
used to rank the chilling sensitivity of plants in closely
related subgroups, with the more tolerant cultivars con-
taining the highest catalase levels (MacRae et al, 1986),
Increased free radical production during carnation se-
nescence is correlated with the loss of membrane integ-
rity (Droillard et al, 1987), demonstrating the deleteri-
ous effects of such compounds. The prevention of chill-
480
ing injury in fruit by antioxidant treatment (Wang and
Baker 1979) also supports this free radical hypothesis.
Thus, chilling injury may involve a chain of events,
which commences with the disruption of membrane
structure, leading to the initiation of lipid peroxidation.
The objective of the present study was to test whether
reduction of chiUing injury by temperature precondi-
tioning in squash could involve increased polyamine
levels with a concomitant reduction of lipid peroxida-
tion products.
Abbreviations - FW, fresh weight; HPLC, high pressure liquid
chromatography.
Materials and methods
Zucchini squash {Cucurbita pepo L, cv. Ambassador)
fruits were freshly harvested from a local farm near
Beltsville, MD, USA, Samples were selected for their
uniformity of size (16-22 cm in length) and were ran-
domly divided into two lots. The first group (control)
was placed in storage at 2,5°C, The second lot was
preconditioned at ]0°C for the first 2 days of storage and
then moved to 2,5°C for the remainder of the study,
Polyamine treatment of the fruit involved the use of
pressure infiltration. The squash fruits were immersed
in 10 mM spermine (Sigma; adjusted to pH 7 with
NaOH) and subjected to 0.7 kg cm~^ of air pressure for
3 min.
Samples were taken on various days throughout the
storage period. Three squash were chosen at random
from each group being sampled. The degree of chilling
injury, as judged by the extent of surface pitting, was
evaluated 5 h after transfer of squash from storage
chambers to room temperature by rating on a scale of 0
to 4: 0, no abnormality; 1, trace; 2, slight; 3, moderate;
4, severe chilling injury. Two 2,0 g skin samples were
removed from each fruit and immediately frozen. Sam-
ples were stored at — 80°C prior to analysis.
Polyamine analysis
Extracts for polyamine analysis were prepared by ho-
mogenizing 2,0 g tissue in 15 ml 5% (v/v) perchloric acid
using a Polytron homogenizer (Brinkman), 1,6-Hexane-
diamine [500 nmol (g fresh weight)"'] (Sigma) was add-
ed as an internal standard. The prob'^ was rinsed with
another 15 ml aliquot of 5% perchloric acid which was
then added to the first aliquot. The homogenate was
then centrifuged at 47 000 g for 20 min. The supernatant
was removed and used for polyamine analysis,
Polyamines were analyzed using high pressure liquid
chromatography (HPLC) with methods similar to those
of Smith and Davies (1985), Dansylation was perfomed
by mixing 400 jxl (18,5 mM in acetone) dansyl chloride
(Sigma) and 150 \i\ saturated sodium bicarbonate with
200 |xl of extract. After incubation overnight at room
temperature, 200 \i\ (0,43 M) proline were added and
the incubation was continued for 1 h. After centrifu-
Physiol. Plant..76, 1989
gation for 10 min in a microcentrifuge (Beckman), the nitrogen. The fluorescent products were resuspended in
pH of the supernatant was checked and adjusted with 2 ml chlorofom , and 200 yd methanol was added. The
HCl close to neutral (pH 6 to 8) as necessary. Samples fluorescence was measured with a Perkin-Elmer 650-
(100 yd) of the supernatant were used for HPLC analy- lOS spectrofluorometer using an emission wavelength of
sis, HPLC was performed on a system consisting of two 460 nm and an excitation wavelength of 345 nm. The
6000A pumps (Waters, Milford, MA, USA) pro- instrument was standardized with 0,1 ug m r ' quinine
grammed with a 720 System Controller (Waters), Sam- sulfate (Sigma) in 0,1 M sulfuric acid.
ples were injected using a Waters U6K injector onto a
reverse-phase C-18 column (Supelco 25 cm LC-18 with
a Supelguard LC-18 5 ^m guard column). Samples were
eluted from the column at a flow rate of 1,5 ml min'' Figure preparation
with a programmed solvent gradient of 0, 100, 0; 15, 0, The figures were prepared using SigmaPlot software
100; 19, 0, 100, where the first number is the time (min), (Jandel Scientific), The traces presented are computer-
the second number is the % buffer A (60:40, v/v, meth- generated regression lines.
anol/water) and the third number is the % buffer B
(100% methanol), Elution was completed in 19 min,
Eluates were detected by a 650-1 OS spectrofluorometer
(Perkin-Elmer, Norwalk, CT, USA) using an excitation
Results
wavelength of 365 nm and an emission wavelength of
510 nm. Data were collected and analyzed using a NEC Eifeet of temperature preconditioning on ciiilling injury
APCIV PowerMate 2 computer system equipped with a Figure 1 shows the average chilling injury index of the
Maxima 820 Chromatography Workstation (Dynamic fruits removed on various days. All fruits appeared
Solutions), Polyamines were quantified by the compari- normal without any symptoms of chilling injury after 2
son of peak areas with those of standards. days of exposure to 2,5°C, Traces of pitting were found
on the skin of fruits from the control group after 3 days,
followed by rapid development of chilling injury symp-
Analysis of fluorescent products
toms during days 3-11, In severe cases, the entire fruit
Extracts for the fluorescent product assay were pre- was covered with numerous sunken areas. The precon-
pared by homogenizing 2,0 g of skin tissue in 100 mM ditioning treatment significantly reduced chilling injury
sodium phosphate, pH 7,6, The homogenate was ex- in the fruit. These squash fruits did not develop moder-
tracted as described by Fletcher et al, (1973), Five ml of ate or severe chilling injury until after 15 days.
the homogenate were added to 5 ml chloroform:metha-
nol (2:1, v/v) and vortexed vigorously. The mixture was
centrifuged at 171 g for 10 min to separate the phases. Effect of chiUing injury on polyamine levels
Two ml of the chlorofom phase were applied to a 1,5 ml Samples of skin tissue were used to analyze the poly-
silica column (poured in a Pasteur pipet) which had amine levels via HPLC, Figure 2 shows the correlation
been equilibrated with chloroform. The column was
washed successively with 6 ml chlorofom , 5 ml isopro-
panol and 6 ml methanol:water (10:1, v/v). The metha-
nol-water fraction was collected and evaporated under 1.6
0.50
T /*
1.4 V - 0.45 - 0,10
4.0
T T

y /-
3.5 1.2 © / 0.40 -
0.08
3.0
Control
1.0 - /
0.35 -
OJ 2.5
* 0,06
, E 2.0 : / T
\
i : c
(D
O
c
o
0.8

0.6
0,30 -
CJ 1.5 1- o
0) 0.04 •
1.0 c 0.4 • I 0.25 -
Preconditioned
0.5 - 'E
a
, Putrescine'
t 0'0 1 2 3 0 1 2 3 4 1 2 3 4
10 12 16 18
Cl index
Days of Storage
Fig. 2. Relationship of polyamine content of skin to the chilling
Fig, 1, Developmentof chilling injury with time. Control group injury index of individual fruits in the control group. Means ±
placed at 2,5°C at the beginning of the first day. Precondi- SE (n=45 for chilling injury = 0; 10,1; 13, 2; 21, 3; 13, 4), The
tioned group stored at 10°C for the first 2 days, then moved to correlation coefficients were: putrescine, 0.934; spermidine,
2,5°C for the remainder of the experiment. Means ± SE (n=6). -0.945; and spermine,-0,952,

Physiol. Plant. 76, 1989 481

 2,0
1 I 1—1—1—1—1—1—•—1—•—1—'—I—1—1—1— between chilling injury and the polyamine levels in the
Control i T skin of the control group of squash. The level of putres-
1,8
cine increased by > 5-fold, while the concentration of
1,6 spermidine decreased 2-fold and spermine decreased
j]
1,4 4-fold (Fig, 2),
•5 1,2
E Efi'ect or temperature preconditioning on polyamine levels
1,0
<u 0,8 wv V Preconditioned In order to determine whether the protection from chill-
"escir

0,6
--A • ing injury resulting from preconditioning could involve
polyamines, we determined the effect of this treatment
D 0,4,
< on the levels of putrescine, spermidine and spermine in
a. 0,2 the fruit. Figure 3 shows the effect of the treatment on
0,0 .. . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 .
the putrescine concentration in the skin, Putrescine in-
0 2 4 6 8 10 12 14 16 18 creased during the treatment and continued to increase
Day after transfer to chilling temperatures at the same rate
Fig. 3. Effect of temperature preconditioning on putrescine as in the control (Fig. 3), The spermidine levels in the
content. Means ± sii (n=6). preconditioned squash increased between days 2 and 7
(Fig, 4), After day 7, the levels began to decrease, but
remained about 1,5- to 2-fold higher than the control.
Preconditioning also significantly increased spermine
levels in the skin (Fig, 5), In the treated fruits, spermine
levels rose between days 2 and 7. The levels fell after
day 7 but remained higher (2- to lO-fold) than the
control. The effect of preconditioning on spermine was
virtually identical to the effect of this treatment on
spermidine levels. These results show that the increased
resistance to chilling injury in the preconditioned
squash was correlated with higher spermine and spermi-
dine levels in the skin tissue.

CL
^ 0,15 4,0
1 1 .

T T
3,5
^ ^
8 10 12 U 16 18 3,0 ^ Y
Day
Fig, 4, Effect of temperuture preconditioning on spermidine ^2,5 Untreated yi i^-^ /-]
content. Means ± SE (n=6). ,E 2,0
-
^ 1,5 -
1,0 v^ y ^ Treated -
0.5

0,30
I 0.0 i
\x::^A^^^^ —1 1
A,
1 1 1

1 . • ' • ' • '

g~0,25
(
•11 0.30

S" 0.25
L.
®\ ®
B. '

So,2O \ J- Preconditioned - cn
\ J Treated
- ' 0.20
0 \ 5
:3.0,15 3_ 0.15 -
\ i T T • •
Sperfnme

= 0.10 ®
E

^^ J ' -
p
-•
0

unircQtsQ ^^^~~~~—-—___
S, 0.05
n -—~—
Contro — • — _ ^

0.00 1 1 1 1 1 1 1 \ 1 I 1 1 1 1
0
b
01

Days of Storage
D

D
0

0 2 4 6 8 10 12 14- 16 18 Fig. 6. Effect of spermine treatment on chilling injury and

Day spermine content of skin. The squash fruits were treated with
Fig. 5. Effect of temperature preconditioning on .spermine 10 mM spermine prior to storage at 2.5°C. A, effect on chilling
content. Means ± ST. (n=6). injury; B, effect on skin spermine levels. Means ± SE (n=3).

482 Physio!. Plant. 76. 1989


26
CD
U 24
C
Control /
CD
O 22
cn
(D
/ ucts (Fig, 7), The reduction of chilling injury by this
/ treatment was thus correlated with reductions in the
o 20
/x
18 -
CD /
16
D Discussion
CD
Qi
14
12
- V ^ ^
i 1 1 Preconcjitioned
10 - -
n'f , r
0 2 4 6 8 10 12 14 16 18
Day
Fig. 7. Effect ol temperature preconditioning on chloroform-
soluble fluorescent products. Means + SE (n=3).
Effect of polyamine application on chilling injury
In order to determine directly whether polyamines
could be involved in the inhibition of chilling injury by
preconditioning, the effect of polyamine application to
the squash prior to chilling treatment was determined.
The fruits were pressure infiltrated with 10 mM spermi-
dine or spermine and stored at 2,5°C, Spermine treat-
ment delayed the development of chilling injury symp-
toms (Fig. 6A). Analysis of the skin of these fruits
demonstrated that the treatment elevated the tissue
spermine level by >2-fold (Fig. 6B), Spemidine treat-
ment had no effect on chilling injury. However, analysis
of the skin revealed that spermidine levels were the
same as in the non-treated control, indicating that this
polyamine did not penetrate into the tissue (data not
shown).
Measurement of fluorescent products
In order to determine whether the reduced chilling in-
jury and increased polyamines resulting from temper-
ature preconditioning might be correlated with protec-
tion of membranes from oxidative damage, the fluo-
rescent properties of chlorofom extracts of squash tissue
were examined. We observed that these extracts con-
tained compounds with similar fluorescent properties
(emission maximum at 440-460 nm, excitation maxi-
mum at 340-350 nm) to those described for other plant
fluorescent products (Maguire and Haard 1975, Wil-
helm and Wilhelmova 1981, Thompson et al, 1985),
However, the pigments in the skin chlorofom extract
effectively quenched this fluorescence, Fractionation on
silica columns separated these pigments from a fraction
Phvsiol. Plant. 76, 1989
of the fluorescent products (B. Whitaker, G, Kramer
and C, Y, Wang, unpublished data). Chilling injury in
the control group of squash was observed to increase
the amount of fluorescent products in this fraction of
skin extracts (Fig, 7). The preconditioning treatment
/ - prevented this increase in the levels of fluorescent prod-
levels of fluorescent products found in the skin of the
fruit.
^
Our results demonstrate that two treatments which sig-
nificantly increased polyamine levels (temperature pre-
conditioning and direct application of spermine) also
led to reduced chilling injury as compared to untreated
, 1 , (control) fruit. Chilling injury in squash resulted in an
increase in putrescine levels (5-fold) and decreases in
spermidine (2-fold) and spermine (4-fold) in the skin
(Fig, 2). The temperature-preconditioning regimen re-
sulted in 1,5- to 2-fold increases in spermidine (Fig, 4)
and 2- to lO-fold increases in spermine (Fig, 5) relative
to the control. These variable effects on the different
polyamines are significant in terms of their structure.
The relative effectiveness of polyamines as antisenes-
cent agents corresponds to the number of positive
charges per molecule; i.e,, spermine (tetramine) is
more effective than spermidine (triam.ine), which is
more effective than putrescine (diamine) (Galston and
Kaur-Sawhney 1987), We found that chilling stress re-
sults in a greater reduction of spermine than spermi-
dine. Also, the temperature preconditioning had a grea-
ter relative effect on spermine than spermidine and a
greater effect on spermidine than putrescine. The rela-
tive increases in the individual polyamines by the pre-
conditioning treatment were, thus, correlated with their
antisenescence activity.
These results suggest that polyamines can somehow
protect squash from chilling injury, Polyamines have
been proposed to inhibit chilling injury by lowering the
membrane phase transition temperature via fluidity ef-
fects (Guye et al, 1986), However, there is insufficient
evidence to equate a change in membrane fluidity with a
change in the phase transition temperature (Raison and
Orr 1989), The inhibition of chilling injury in squash by
polyamines may involve alteration of the physical prop-
erties of membranes, but determination of the effects of
polyamines on the phase transition temperature would
be necessary to substantiate such a suggestion.
Given the possible relationship between chilling in-
jury and oxidative damage to membranes, we examined
the effects of chilling injury on the levels of chloroform-
soluble fluorescent products in squash. The levels of
fluorescent products in skin increased with chilling in-
jury (Fig, 7), Temperature preconditioning significantly
reduced these levels of fluorescent products in the skin.
This may indicate that decreased lipid peroxidation is
483
associated with the reduction of chilling injury in the
treated fruit, Polyamines may therefore inhibit chilling
injury by retarding lipid peroxidation. This activity
could be a result of their membrane-binding and/or
antioxidant properties. The ability of antioxidant treat-
ment to increase the content of unsaturated fatty acids
and reduce injury during the chilling of cucumbers and
peppers (Wang and Baker 1979) and the ability of po-
lyamine treatment to protect tomato plants from injury
during ozone treatment (Ormrod and Beckerson 1986)
support this conclusion.
Acknowledgements - We would like to thank Jeffery Baker and
James Imlay for critical reading of the manuscript and Hilarine
Repace for technical assistance.
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