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Biochemical and Biophysical Research Communications 330 (2005) 11941198

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Dierential impact of low temperature on fatty acid unsaturation

and lipoxygenase activity in gleaf gourd and cucumber roots q
Seong Hee Lee, Sung Ju Ahn, Yang Ju Im, Kyoungwon Cho, Gap-Chae Chung,
Baik-Ho Cho, Oksoo Han *
Department of Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, College of Agriculture and Life Sciences,
Chonnam National University, 300 Yongbong-dong, Buk-gu, Kwangju 500-757, Republic of Korea
Received 5 March 2005
Available online 24 March 2005

Abstract
Previous studies show that low temperature strongly induces suberin layers in the roots of chilling-sensitive cucumber plants,
while in contrast, low temperature produces a much weaker induction of suberin layers in the roots of the chilling-tolerant gleaf
gourd [S.H. Lee, G.C. Chung, S. Steudle, Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and
-tolerant gleaf gourd, J. Exp. Bot. 56 (2005) 985995; S.H. Lee, G.C. Chung, E. Steudle, Low temperature and mechanical stresses
dierently gate aquaporins of root cortical cells of chilling-sensitive cucumber and gleaf gourd, Plant Cell Environ. (2005) in press;
S.J. Ahn, Y.J. Im, G.C. Chung, B.H. Cho, S.R. Suh, Physiological responses of grafted-cucumber leaves and rootstock roots
aected by low root temperature, Scientia Hort. 81 (1999) 397408]. Here, the eect of low temperature on fatty acid unsaturation
and lipoxygenase activity was examined in cucumber and gleaf gourd. The double bond index demonstrated that membrane lipid
unsaturation shows hyperbolic saturation curve in gleaf gourd roots while a biphasic response in cucumber roots to low temperature. In gleaf gourd, the hyperbolic response in the double bond index was primarily due to accumulation of linolenic acid. Chilling stress also signicantly induced lipoxygenase activity in gleaf gourd roots. These results suggest that the degree of unsaturation
of root plasma membrane lipids correlates positively with chilling-tolerance. Therefore, studies that compare the eects of chilling
on cucumber and gleaf gourd may provide broad insight into stress response mechanisms in chilling-sensitive and chilling-tolerant
plants. Furthermore, these studies may provide important information regarding the relationship between lipid unsaturation and
lipoxygenase function/activity, and between lipoxygenase activity and water channeling during the response to chilling stress.
The possible roles of these processes in chilling tolerance are discussed.

2005 Elsevier Inc. All rights reserved.
Keywords: Chilling stress; Cucumber; Double bond index; Figleaf gourd; Lipoxygenase

Low temperature is the major factor limiting the

growth of warm-season plants. One of the adverse effects of low temperature on plants is altered functional
properties of cellular membranes in plant leaves and/
or roots. At the cellular and molecular level, low temq
Abbreviations: DBI, double bond index; EL, electrolyte leakage;
LOX, lipoxygenase; GC, gas chromatography; SDS, sodium dodecylsulfate.
*
Corresponding author. Fax: +82 62 530 2169.
E-mail address: oshan@chonnam.ac.kr (O. Han).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2005.03.098

perature can have a severe impact on membrane uidity, which also aects metabolic rate and protein
turnover [13]. Previous studies showed that low temperature has dierential eects on the lipid composition
of membranes in chilling-sensitive and chilling-tolerant
plants. For example, the proportion of unsaturated
fatty acids tends to increase in the thylakoid membrane,
mitochondrial membrane, and plasma membrane during low temperature stress [35]. Chilling stress often
causes peroxidation of membrane lipids, a type of membrane damage that is also caused by oxidative stress.

S.H. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 11941198

Peroxidation of unsaturated lipids has been mentioned

as a possible cause of increased membrane rigidity in
tropical and sub-tropical plants exposed to low temperature stress [6,7]. Lipoxygenase (LOX, EC 1.13.11.12) is
thought to play a primary role in generating peroxidative damage in membrane lipids in common bean
[6,8], and reducing sugar that accumulates in potato tubers stored at low temperature may also alter membrane
permeability [9].
Another factor in low temperature stress could be the
development of hydraulic resistance. For example, coldinduced hydraulic resistance could lead to water imbalance in new shoots in chilling-sensitive plants [7]. Major
parameters that aect root hydraulics are anatomy, water
ow across the root cylinder, and the structure and activity of water channels in root cells. The activity of water
channels in the plasma membrane of root cells aects
cell-to-cell water ow, and it has been suggested that gating of water channel activity plays an important role in
determining root hydraulic resistance [10,11]. Since lipid
peroxidation, water channel activity, and membrane uidity are very sensitive to changes in temperature and the
composition of the plasma membrane, the level of saturation of plasma membrane fatty acids is a crucial determinant of low temperature stress. Several reports indicate
that increased unsaturation of membrane fatty acids correlates with chilling-tolerance [4,6,9]. However, these results are controversial and additional data on the
mechanisms by which low temperature could alter membrane lipid composition are needed.
Cucurbita is a species that grows particularly well at
relatively low temperatures. Thus, Cucurbita have been
widely used as rootstocks for Cucumis, allowing for successful production of cucumbers during the cold season
[12]. Earlier studies suggest that a comparative study of
the chilling-induced physiological response of cucumber
(Cucumis sativus L.) and gleaf gourd (Cucurbita cifolia Bouche) could provide signicant insight into mechanisms of chilling-resistance [7,12,13]. Because
cucumber and gleaf gourd may represent typical chilling-sensitive and chilling-tolerant species, respectively,
this could provide an ideal model system for studying
the chilling-sensitivity and chilling-tolerance. The present study analyzes chilling-induced changes in fatty acid
composition and lipoxygenase activity in the roots of
chilling-sensitive (cucumber) and chilling-tolerant (gleaf gourd) species. Membrane uidity was measured
using the double bond index (DBI). The results are
interpreted with respect to the possible involvement of
lipoxygenase in mechanisms of chilling-tolerance.

Materials and methods

Plant materials and growth conditions. Seeds of cucumber (C. sativus L. cv. Chung-Jang, Heung-Nong Seed, Korea) and gleaf gourd

1195

(Cu. cifolia Bouche, Sakada Seed, Japan) were germinated for 23

days at 23 C in the dark on lter paper soaked with tap water. After
germination, seedlings were transferred to containers (5 L with 8
seedlings per container) with aerated 1/5 strength Cooper solution [14]
in a climate chamber (day/night cycle: 12/12 h; temperature: 23/21 C;
and light intensity: 300 lmol m 2 s 1 PAR). The nutrient solution was
replaced frequently to avoid excessive depletion of nutrients. Cucumber and gleaf gourd seedlings used in the experiment were 1420 and
714 days, respectively, including the time for germination. At these
ages, root systems of cucumber and gleaf gourd were comparable in
size (length of root systems: cucumber, 450500 mm; gleaf gourd,
400500 mm). A cooler was used for low root temperature conditions
of 6 C for 7 days. A climate chamber was used when necessary.
Fatty acid analysis. Total lipids were extracted from plasma membranes of root systems according to Ryyppo et al. [15]. Roots were
ground into a powder in liquid nitrogen and the powder was mixed with
isopropanol. Then methanol, chloroform, and butylated hydroxytoluene (5 mg mL 1 chloroform) were added and the suspension was mixed
for 90 min at 4 C. After removal of water-soluble contaminants by
Folch wash, total lipids were fractionated into neutral lipids and
phospholipids by silica gel chromatography. The phospholipids were
converted to methyl esters by adding 0.6 M methanolic sodium
hydroxide and neutralized with 0.6 M hydrochloric acid. The methyl
esters of fatty acids were extracted twice with hexane and separated on a
gas chromatograph (GC-17B, Shimadzu) equipped with a Rtx-1 column
(Resteck) and a ame ionization detector (Shimadzu). Initially, column
temperature was maintained at 145 C for 2 min and then subjected to a
step gradient of 145160 C 5 C min 1, 160 C for 1 min, 160170 C
2 C min 1, and 170 C for 90 min. The injector and detector temperatures were 225 and 275 C, respectively. DBI was calculated according
to the formula of Wismer et al. [16]: DBI = [(2 %C18:2) +
(3 %C18:3)]/[(%C16:0) + (%C18: 0) + (%C18:1)].
Northern blot analysis. Total cellular RNA was extracted and
Northern blot analysis was conducted according to standard procedures.
Northern blot hybridization was performed for 20 h at 68 C in prehybridization solution supplemented with 10% dextran sulfate. The LOX
cDNA from Zea mays (GenBank Accession No. AF271894) was labeled
with [c32P]dCTP (Amersham pB10205) by nick translation (BRL,
9160SB), and BamHI and KpnI fragments of the LOX cDNA were used
as a probe [17]. The membrane was washed twice at room temperature in
0.1 SSC, 0.1%(w/v) SDS for 10 min and then once at 42 C in 0.1 SSC,
0.1%(w/v) SDS for 30 min. The Northern blot was visualized using a
BAS 1500 image system (Fuji Photo Film, Tokyo, Japan).
Lipoxygenase assay. Roots of cucumber and gleaf gourd were
frozen in liquid nitrogen and pulverized using mortar and a pestle. The
powdered sample was suspended in 50 mM potassium phosphate, pH
6.0, centrifuged at 12,000g for 10 min, and the supernatant was removed for activity assay. Linolenic acid or linoleic acid were used as
substrates. Substrate solution was prepared as described previously
[18]. The reaction was started by mixing the substrate solution with
enzyme, stopped by adding ethyl alcohol to aliquots of the reaction
mixture at 1, 4, 5, 6, and 7 min, and the mixture was diluted with 60%
ethyl alcohol. The absorbance was measured at 234 nm.

Results
Chilling-induced change in plasma membrane DBI in
gleaf gourd and cucumber roots
We previously reported that low temperature
strongly induces suberin layers in the endodermis of
cucumber roots, but it induces much weaker suberization in gleaf gourd roots [10,11]. Here, the eect of

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S.H. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 11941198

low root temperature on plasma membrane lipid composition, particularly the unsaturation level, was investigated by isolating the plasma membrane fraction and
measuring DBI as a function of time at low temperature.
The results are shown in Fig. 1. DBI increased to a rapid
saturation with hyperbolic kinetics in gleaf gourd; no
associated change in electrolyte leakage (EL) was observed in gleaf gourd leaves (Fig. 1A). In contrast,
the eect of low temperature on DBI in cucumber roots
was complex and biphasic (Fig. 1B); in addition, low
temperature caused a strong increase in EL of cucumber
leaves, indicating that exposure to cold was associated
with signicant membrane damage. These data strongly
suggest that a gradual increase in unsaturated plasma
membrane lipids may play a major role in chilling-tolerance. In chilling-sensitive plants such as the cucumber,
biphasic changes in DBI may reect an initial phase in
which cold-induced damage occurs, followed by a second phase in which the consequences of plasma membrane damage are evident. These phases may
correspond to early and late periods of plant stress response observed previously [13].

Ratio of C18-fatty acids to C16-palmitic acid in gleaf

gourd and cucumber roots as a function of time at 6 C
The fact that DBI saturates with hyperbolic kinetics
in chilling-tolerant gleaf gourd might indicate inhibition of fatty acid synthesis and/or activation of fatty
acid desaturase. Fig. 2 shows the ratio of C18 fatty acids
(C18:3, C18:2, C18:1, and C18:0) to palmitic acid
(C16:0) in gleaf gourd and cucumber roots as a function of time at 6 C. The results clearly indicate that
an increase in linolenic acid (C18:3) is largely responsible for the cold-induced increase in DBI in gleaf gourd.
More importantly, the increase in C18:3 in gleaf gourd
roots is accompanied by a decrease in C18:0 lipid (Fig.
2A). This suggests that fatty acid desaturases may convert C18:0 to C18:3 through C18:1 and C18:2 intermediates; this reaction may play an important role in
maintaining or increasing unsaturation in plasma
membrane lipids in chilling-tolerant plants exposed to
chilling stress. Similar eects were not observed in chilling-sensitive cucumber roots (Fig. 2B).
Low temperature-induced activation and expression of
LOX in gleaf gourd and cucumber roots
Lipoxygenase carries out peroxidation reactions on
plasma membrane lipids, which could increase the level

Fig. 1. DBI of chilling-tolerant gleaf gourd (A) and chilling-sensitive

cucumber (B) roots as a function of time at 6 C. Plasma membranes
were isolated from plant roots at the indicated time points and DBI
was calculated as described under Materials and methods. Data for EL
were adapted from Ahn et al. [12].

Fig. 2. Ratio of C18-fatty acids to C16-palmitic acid as a function of

time at 6 C. (A) Figleaf gourd; (B) cucumber. The C18/C16 ratio was
calculated as described under Materials and methods.

S.H. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 11941198

Fig. 3. Eect of low root temperature on lipoxygenase specic activity

in the roots of gleaf gourd and cucumber. Plants were maintained at
6 C for the indicated number of days. Lipoxygenase activity was
measured as described under Materials and methods.

Fig. 4. Northern blot analysis of lipoxygenase mRNA in the roots of

gleaf gourd and cucumber. Plants were maintained at 6 C for the
indicated number of days, and total RNA was isolated and analyzed as
described under Materials and methods.

of lipid unsaturation and increase membrane uidity.

The possibility that LOX-catalyzed lipid peroxidation
plays a role in the response to chilling stress was examined by measuring LOX activity in gleaf gourd and
cucumber roots as a function of time at low temperature. Fig. 3 shows that LOX activity increased in gleaf
gourd roots and reached a maximum after two days at
6 C. In contrast, increase in LOX activity was not observed in cucumber roots. Transcription from the LOX
gene also increased and reached a maximum after 3 days
at 6 C in gleaf gourd roots; no increase in LOX transcription was observed in cucumber roots (Fig. 4).
Therefore, LOX activity may play an important role in
the mechanism of chilling-tolerance in gleaf gourd
roots. It is possible that activation of LOX protein
and/or induction of LOX gene expression may be characteristic of chilling-tolerant species.

Discussion
The present study shows that low root temperature
has dierential eects in the roots of chilling-tolerant gleaf gourd and chilling-sensitive cucumber. In particular, this study focuses on the impact of chilling stress
on LOX activity and DBI in plasma membrane lipids.

1197

Data presented here demonstrate that DBI increases to

saturation in the gleaf gourd, while DBI has a biphasic
response to chilling stress in cucumber roots. The response in cucumber roots is associated with apparent
membrane damage and increased EL. Because membrane uidity generally increases with increasing
amounts of unsaturated lipid, a gradual saturation in
DBI could contribute to chilling-tolerance. In contrast,
heat stress drastically decreases membrane lipid unsaturation in bentgrass roots [3]. These data suggest that
membrane lipid composition is an important determinant of plant tolerance to both chilling stress and heat
stress.
Fatty acid hydroperoxides produced by LOX are
metabolized by a variety of enzymatic systems. Linolenic acid (C18:3) is a precursor for biosynthesis of jasmonic acid, a signaling molecule that activates plant
defense mechanisms. Since the chilling-induced increase
in DBI observed in gleaf gourd is largely attributable
to increase in the concentration of linolenic acid (Fig.
2), it is unlikely that a signicant amount of linolenic
acid is used for synthesis of jasmonic acid in response
to low temperature stress. This implies that jasmonic
acid-dependent signaling may not play a signicant role
in chilling-tolerance. In this case, LOX activity induced
by chilling stress in gleaf gourd could utilize linoleic
acid as a primary substrate instead of linolenic acid.
This is consistent with the existence of multifunctional
lipoxygenase with diverse substrate specicity [18,19].
Because all lipoxygenase isozymes in gleaf gourd appear to be induced by chilling stress (Fig. 3), and this increase precedes transcriptional activation of specic
LOX genes (Fig. 4), it seems possible that LOX enzymes
are activated at least in part by post-translation modication. However, it should be emphasized that the role
of LOX in stress response is complex [9]. LOX-dependent depletion of polyunsaturated fatty acids from the
plasma membrane would decrease DBI, while LOX-dependent peroxidation of membrane lipids would increase DBI. The former possibility is not consistent
with data presented here and would contribute to chilling-sensitivity instead of chilling-tolerance. This apparent contradiction could be explained if a major LOX
substrate in low temperature-stressed plants is a dienoic
fatty acid such as linoleic acid rather than a trienoic acid
such as linolenic acid. In this case, increased LOX activity would increase DBI and increase membrane uidity.
Furthermore, Fig. 2 suggests that C18:0 lipids decrease
while C18:3 lipids increase in chilling-stressed gleaf
gourd roots; this may be a mechanism to balance cellular levels of octadecanoids.
Reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radicals, increase in the early
stage of chilling stress. Previous studies provided cytochemical evidence that more hydrogen peroxide accumulates in low temperature-stressed cucumber roots

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S.H. Lee et al. / Biochemical and Biophysical Research Communications 330 (2005) 11941198

than in gleaf gourd roots [7]. A high concentration of

hydrogen peroxide is likely to produce toxic free radicals
via Fenton chemistry, which in turn causes homolysis of
polyunsaturated fatty acids. This mechanism would explain some of the adverse eects of low temperature
on chilling-sensitive plants. Furthermore, our recent
study indicated that activation energies of water ow
at the cellular level are high in cucumber, but low in gleaf gourd [10,11]. Thus, there is an inverse correlation
between LOX activity and the level of hydrogen peroxide in cucumber (low LOX, high hydrogen peroxide)
and gleaf gourd (high LOX, low hydrogen peroxide),
respectively. These data are likely important in
understanding the relationship between LOX activity,
membrane lipid unsaturation, and water ow in
chilling-stressed plants and in evaluating the importance
of these factors in chilling-tolerance.
Acknowledgments
This study was performed during the sabbatical year
of 2003 in Chonnam National University and supported
by Agricultural Plant Stress Research Center (Grant
R11-2001-09202003-0) in part.
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