J. Plant Physiol. 160. 283 – 292 (2003)
 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/jpp
Electrolyte leakage and lipid degradation account for cold sensitivity in
leaves of Coffea sp. plants
Paula Scotti Campos1 *, Virgínia Quartin2, José Cochicho Ramalho3, Maria Antonieta Nunes3
1
Departamento de Fisiologia Vegetal – Estação Agronómica Nacional, Avenida da República, P-2784-505 Oeiras, Portugal
2
Faculdade de Ciências Agrárias – Universidade Agostinho Neto, P.O. Box 815, Luanda, Angola
3
Centro Estudos Produção Tecnologia Agrícolas – Inst. Inv. Científica Tropical, Tapada da Ajuda, Ap. 3014, P-1301-901 Lisboa, Portugal
Received May 21, 2002 · Accepted July 29, 2002
Summary
Five Coffea genotypes differing in their sensitivity to low positive temperatures were compared with
regard to the effects of chilling on membrane integrity, as well as their ability to recover from coldinduced injury upon re-warming. Membrane damage was evaluated through electrolyte leakage,
changes in membrane lipid composition and malondialdehyde (MDA) production in control conditions (25/20 ˚C, day/night), after a gradual temperature decrease period to 15/10 ˚C, after chilling
treatment (3 nights at 4 ˚C) and upon re-warming to 25/20 ˚C during 6 days (recovery). C. dewevrei
showed the highest electrolyte leakage at 15/10 ˚C and after chilling. This was due mainly to lipid
degradation observed at 15/10 ˚C, reflecting strong membrane damage. Furthermore, MDA production after chilling conditions indicated the occurrence of lipid peroxidation. A higher susceptibility of
C. dewevrei to cold also was inferred from the complete absence of recovery as regards permeability, contrary to what was observed in the remaining plants. Apoatã and Piatã presented significant
leakage values after chilling. However, such effects were reversible under recovery conditions. Exposure to cold (15/10 ˚C and 3 × 15/4 ˚C) did not significantly affect membrane permeability in Catuaí and
Icatú. Furthermore, no significant MDA production was observed even after chilling treatments in
Apoatã, Piatã, Catuaí and Icatú, suggesting that the four genotypes had the ability to maintain membrane integrity and/or repair membrane damage caused by low temperatures. Apoatã, Piatã and, to
a lower extent, Catuaí, were able to cope with gradual temperature decrease through an enhanced
lipid biosynthesis. After acclimation, Piatã and Catuaí showed a lowering of digalactosyldiacylglycerol to monogalactosyldiacylglycerol ratio (MGDG/DGDG) as a result of enhanced DGDG synthesis,
which represents an increase in membrane stability. The same was observed in Apoatã after chilling,
in spite of phospholipids decrease. The studied parameters clearly indicated that chilling induced
irreversible membrane damage in C. dewevrei. We also concluded that increased lipid synthesis,
lower MGDG/DGDG ratio, and changes in membrane unsaturation occurring during acclimation to
low temperatures may be critical factors in maintenance of cellular integrity under chilling.
Key words: Coffea sp. – chilling – electrolyte leakage – malondialdehyde – membrane lipids – unsaturation
* E-mail corresponding author: [email protected]
0176-1617/03/160/03-283 $ 15.00/0
284
Paula Scotti Campos et al.
Abbreviations: DBI = double bond index. – DGDG = digalactosyldiacylglycerol. – DPG = diphosphatidylglycerol. – MDA = malondialdehyde. – MGDG = monogalactosyldiacylglycerol. – NL = neutral
lipids. – PC = phosphatidylcholine. – PE = phosphatidylethanolamine. – PG = phosphatidylglycerol. –
PI = phosphatidylinositol. – TFA = total fatty acids. – C16 : 0 = palmitic acid. – C16 : 1c = palmitoleic
acid. – C16 : 1t = 3-trans-hexadecenoic acid. – C18 : 0 = stearic acid. – C18 : 1 = oleic acid. – C18 : 2 =
linoleic acid. – C18 : 3 = linolenic acid
Introduction
Membranes are dynamic structures that support numerous
biochemical and biophysical reactions. They are also major
targets of environmental stresses (Leshem 1992). Chilling impairments mainly consist of alteration of metabolic processes,
decrease in enzymatic activities, reduction of photosynthetic
capacity and changes in membrane fluidity among others
(Dubey 1997). Such changes are frequently related to an increase in membrane permeability, affecting membrane integrity and cell compartmentation under stress conditions. Increased rates of solute and electrolyte leakage occur in a variety of chilled tissues and have been used to evaluate membrane damage following chilling (Wright and Simon 1973, Simon 1974). Leakage points may result from the appearance of
membrane domains presenting different configurations due to
cold-induced changes in lipid phases (Leshem 1992), or from
damage of membrane, particularly as regards lipids (Harwood 1997). Poliunsaturated fatty acids, very abundant in galactolipids molecules, are the preferential substrate of peroxidative and hydrolytic enzymes (Sahsah et al. 1998). Malondialdehyde (MDA) is one of the final products of stressinduced lipid peroxidation of poliunsaturated fatty acids (Leshem 1987), and has been considered a marker for cold sensitivity (Jouve et al. 1993, Alonso et al. 1997, Queiroz et al.
1998).
Changes in the lipid composition of higher-plant membranes have been reported under different environmental
stress conditions and may have an adaptive value (Kuiper
1985, Pham Thi et al. 1989, Ramalho et al. 1998, Campos et
al. 1999). When exposed to chilling, plant cell membranes undergo changes in lipid and fatty acid composition in order to
maintain chloroplast function at low temperature (Harwood
1997, Routaboul et al. 2000). Such changes may result from
an increase in the proportion of highly unsaturated fatty acids
in galactolipids, such as linolenic acid (C18 : 3), during low
temperature acclimation. More unsaturated (low-meltingpoint) molecular species of phosphatidilglycerol (PG), determined by higher levels of its major fatty acid, trans-∆3-hexadecenoic acid (C16 : 1t), may also contribute to a decrease of
phase transition temperature of the total thylakoid lipid (Moon
et al. 1995, Harwood 1997), resulting in enhanced membrane
stability when temperature is reduced.
The exposure of tropical and subtropical plants, such as
coffee, to low temperatures constitutes a severe limitation to
their physiology and production. The aim of this study was to
compare the effects of a cold treatment on membrane integrity of five Coffea genotypes, and to analyse their degree of
recovery. We expect that evaluation of cold-induced changes
in electrolyte leakage and MDA production may give an indication of their ability to cope with chilling, and that membrane
lipid composition will help to elucidate the mechanisms
involved in different cold sensitivity.
Material and Methods
Growth conditions
Two year old plants of five Coffea genotypes were used: C. canephora cv. Apoatã, C. arabica cv. Icatú, C. arabica cv. Catuaí, C. dewevrei and cv. Piatã (C. dewevrei × C. arabica). Plants were placed in
a growth chamber (EDTU 700, Aralab, Portugal) and submitted to a
gradual temperature decrease (0.5 ˚C per day) from 25/20 to 15/10 ˚C
(day/night). During the following 3 nights, plants were submitted to
chilling temperature (4 ˚C) with that temperature maintained also during the first 4 h of the following morning (thus concomitantly with light).
Subsequently, plants were allowed to recover at 25/20 ˚C for 6 days.
The other growth conditions were: photoperiod: 12/12 hours (day/
night); RH: 70 %; photosynthetic photon flux density: 450 – 500
µmol m – 2 s –1 provided by a combination of sodium vapour (HQI-BT,
OSRAM, Germany) and halogen (Halolux Ceram, OSRAM) lamps.
Measurements were performed in the 2 top pairs of mature leaves
from 6 – 8 plants of each genotype at control conditions (25/20 ˚C), at
the end of the cold acclimation period (15/10 ˚C), after the chilling
treatment (3 × 4/10 ˚C) and after 6 days of recovery (Rec 25/20 ˚C).
Electrolyte leakage
Fifteen freshly cut leaf discs (0.5 cm2 each) were rinsed 3 times (2 –
3 min) with demineralised water and subsequently floated on 10 mL of
demineralised water. The electrolyte leakage in the solution was
measured after 22 h of floating at room temperature using a conductimeter (Crison 522, Crison Instruments, S.A., Spain). Total conductivity was obtained after keeping the flasks in an oven (90 ˚C) for 2 h.
Results were expressed as percentage of total conductivity.
Lipid peroxidation
For the measurement of lipid peroxidation, the thiobarbituric acid
(TBA) protocol to determine the MDA produced was followed (Cakmak and Horst 1991). The test was performed using thirty freshly cut
Membrane lipids and cold sensitivity in Coffea sp.
285
leaf discs (0.5 cm2 each). The amount of MDA was calculated from
the absorbance at 532 nm after subtracting the non-specific absorption at 600 nm. The extinction coefficient 155 mmol/L –1 cm –1 for MDA
was used.
Lipid analysis
For fatty acid and polar lipid analyses, the general procedure of Pham
Thi et al. (1985) was followed. Leaf samples were boiled for 2 min in
demineralised water in order to stop lipolytic activities. Lipids were extracted in chloroform/methanol/water (1/1/1, v/v/v) according to Allen
et al. (1966). Fatty acids were saponified and methylated with BF3
(Merck) using the method of Metcalfe et al. (1966) after the addition of
heptadecanoic acid (C17 : 0) as an internal standard. Fatty acid
methyl esters were analysed by gas-liquid chromatography (Unicam
610 Series Gas Chromatograph, Unicam Ltd., U.K.), equipped with a
hydrogen flame-ionisation detector. Separation was performed using
a fused silica capillary column (DB-Wax, J & W Scientific, U.S.A) with
0.25 mm i.d. × 30 m, coated with polyethylene glycol (Carbowax) at a
thickness of 0.25 µm. Column temperature was programmed to rise
from 80 to 200 ˚C at 12 ˚C min –1, after 2 min at the initial temperature.
Injector and detector temperatures were 200 ˚C and 250 ˚C, respectively. Carrier gas was hydrogen with a flow rate of 1 mL min –1, at a
split ratio of 1: 100 of the sample.
Lipid classes were separated by thin layer chromatography on
G60 silicagel plates (Merck) in chloroform/acetone/methanol/acetic
acid/water (50/20/10/10/5, by vol.) according to Lepage (1967), and
subsequently in petroleum ether/diethyl ether/acetic acid (70/30/0.4,
by vol.), according to Mangold (1961). After spraying with primuline
0.01 % in 80 % acetone and visualisation under UV, the lipid bands
were scraped off, saponified and methylated as described above. Individual fatty acids and lipid classes were identified by comparison
with known Sigma standards. The value for total fatty acids corresponds to the sum of the individual fatty acid components.
Figure 1. Conductivity values (%) in leaf discs of five Coffea genotypes under control (25/20 ˚C), at 15/10 ˚C, after three nights of chilling
(3 × 15/4 ˚C) and re-warming (Rec 25/20 ˚C) conditions. Each value
represents the mean + SE (n = 3).
Statistical analysis
The data were analysed statistically using a two-way ANOVA, applied
to the various measured and calculated parameters, followed by a Tukey test for mean comparison between genotypes or temperature
treatments at a 95 % confidence level. Different letters in tables
express significant differences between genotypes for the same temperature (a, b, c) or between different temperature treatments for the
same genotype (r, s, t), with a and r representing the highest values.
Figure 2. Changes induced by cold in the amount of malondialdehyde
(MDA) in leaves of five Coffea genotypes under control (25/20 ˚C), at
15/10 ˚C, after three nights of chilling (3 × 15/4 ˚C) and re-warming (Rec
25/20 ˚C) conditions. Each value represents the mean + SE (n = 3).
Results
Electrolyte leakage
Conductivity measurements showed that after the gradual
temperature decrease period C. dewevrei was strongly affected (170 % increase), while small changes occurred in the
remaining genotypes (Fig. 1). C. dewevrei maintained high
leakage values after chilling treatment (3 nights at 4 ˚C) and
showed a poor recovery upon re-warming conditions. Chilling
induced an increase in membrane leakage also in Apoatã
and Piatã (110 and 140 % increases, respectively), but during
recovery plants returned to control values. Catuaí and Icatú
presented only a slight tendency to increase leakage values
after exposure to cold (15/10 ˚C and 3 × 15/4 ˚C), thus showing
a low impact on membrane permeability (Fig. 1).
Lipid peroxidation
MDA production was stimulated in C. dewevrei after the cold
treatment, reaching a 63 % increase after chilling and showing some degree of recovery upon re-warming (Fig. 2).
286
Paula Scotti Campos et al.
Among the other genotypes, only Catuaí showed a tendency
to have higher values after the temperature decrease period.
In Icatú, Apoatã and Piatã, MDA decreased (ca. 20 – 26 %)
with the cold conditions, being this reduction further enhanced after the period for recovery.
Table 1. Changes in fatty acid composition (mol %) and unsaturation
(DBI) of total lipids in five Coffea genotypes under control (25/20 ˚C),
at 15/10 ˚C, after three nights of chilling (3 x 15/4 ˚C) and rewarming
(Rec 25/20 ˚C) conditions. Different letters express significant differences between plants for the same temperature (a, b, c) or between
different temperatures for the same plant (r, s, t); a and r represent the
highest values.
Lipid analysis
T (˚C)
Under control conditions, Icatú and Apoatã presented the
highest (15 mg g –1 DW) and the lowest (9 mg g –1 DW) total
fatty acid (TFA) contents, respectively (Fig. 3). Exposure to
decreasing temperatures induced a significant TFA increase
in Apoatã (46 %) and Piatã (16 %), a similar tendency being
observed in Catuaí (18 % increase). On the contrary C. dewevrei presented a 36 % reduction and no changes were observed in Icatú, which remained quite unaltered along the
whole stress and recovery periods (Fig. 3). Cold treatment reduced TFA (24 – 28 %) in relation to the 15/10 ˚C values in Catuaí and Apoatã. In Piatã, lipid synthesis was stimulated gradually during the whole experiment, resulting in a TFA increase
of 44 % at the end of the recovery period in comparison to
control plants.
Icatú presented the most unsaturated membranes, as inferred from the highest double bond index (DBI) values, in
opposition to Piatã (Table 1). Exposure to a slow temperature
decrease resulted in lower DBI values in Catuaí (14 %) due to
an increase in more saturated fatty acids, namely palmitic
acid (C16 : 0), and decreases in oleic (C18 : 1) and linoleic
(C18 : 2) acids. A similar tendency was observed in C. dewevrei and Icatú, mainly as a result of decreases (7– 8 %) in linolenic acid (C18 : 3). Apoatã changed in the opposite direction
< C 16 : 0
25/20
15/10
3 × 15/4
Rec 25/20
C16 : 0
25/20
15/10
3 × 15/4
Rec 25/20
C16 : 1 (c + t) 25/20
15/10
3 × 15/4
Rec 25/20
C18 : 0
25/20
15/10
3 × 15/4
Rec 25/20
C18 : 1
25/20
15/10
3 × 15/4
Rec 25/20
C18 : 2
25/20
15/10
3 × 15/4
Rec 25/20
C18 : 3
25/20
15/10
3 × 15/4
Rec 25/20
DBI
25/20
15/10
3 × 15/4
Rec 25/20
Apoatã
Catuaí
C.
Icatú
dewevrei
Piatã
9.7a/r
6.0a/s
6.4a/rs
5.9a/s
25.4b/r
23.5b/r
21.9b/r
24.7b/r
5.5a/r
4.1ab/rs
3.0b/s
3.4a/s
4.2b/r
3.5b/r
3.9b/r
3.8b/r
1.3a/r
1.1a/r
1.6a/r
1.1a/r
6.4a/r
9.0a/r
9.2a/r
9.3a/r
47.6a/s
52.8a/rs
54.0a/r
51.8a/rs
5.5a/s
6.8a/r
7.2a/r
6.3a/rs
6.2b/r
6.9a/r
8.2a/r
8.4a/r
22.1c/s
27.6ab/r
27.9ab/r
27.8b/r
4.3b/r
3.4b/r
3.9ab/r
3.8 a/r
6.7a/r
5.0ab/s
5.3ab/rs
5.3a/rs
1.7a/r
1.0a/r
1.0a/r
1.2a/r
10.1a/r
9.1a/r
9.4a/r
8.8a/r
47.7a/r
47.0ab/r
44.5b/r
44.6b/r
6.0a/r
5.0bc/rs
4.7b/s
4.7b/s
10.3a/r
7.8a/r
7.3a/r
7.4a/r
26.1b/r
29.4a/r
27.2ab/r
28.5b/r
5.6
a/r
4.4a/s
3.9ab/s
3.7a/s
3.1c/r
2.9b/r
2.5c/r
3.2b/r
1.2a/r
1.4
a/r
1.4a/r
1.3a/r
6.6a/s
11.0a/r
10.2a/r
10.6a/r
47.0a/r
43.2b/r
47.5ab/r
45.4a/r
5.5a/r
4.9bc/r
5.7b/r
5.1ab/r
9.6ab/r
8.8a/r
7.0a/r
7.8a/r
31.3a/rs
30.0a/s
30.3a/s
34.4a/r
5.5a/r
4.4a/s
4.1ab/s
3.3a/st
5.0b/r
4.2b/r
4.1b/r
4.7ab/r
1.7a/r
1.1a/r
1.1a/r
1.1a/r
9.0a/r
8.9a/r
8.8a/r
9.5a/r
38.0b/s
42.7b/rs
44.6b/r
39.2b/rs
3.8b/r
4.4c/r
4.6b/r
3.6b/r
5.7b/r
6.4a/r
7.6a/r
8.1a/r
21.3c/r
23.9b/r
23.9b/r
24.9b/r
3.8b/r
3.9ab/r
4.1a/r
3.8a/r
6.2ab/r
5.6a/r
6.0a/r
5.5a/r
1.2a/r
1.3a/r
1.1a/r
1.1a/r
8.7a/r
9.5a/r
8.7a/r
8.4a/r
53.1a/r
49.5ab/r
48.7ab/r
48.3a/r
6.6a/r
5.9ab/r
5.7b/r
5.5ab/r
DBI = [(C16 : 1 (c + t) + C18 : 1 + 2 × C18 : 2 + 3 × C18 : 3)/(C16 : 0 + C18 : 0)].
Figure 3. Changes induced by cold in the total fatty acids (TFA)
amounts of leaves in five Coffea genotypes under control (25/20 ˚C),
at 15/10 ˚C, after three nights of chilling (3 × 15/4 ˚C) and re-warming
(Rec 25/20 ˚C) conditions. Each value represents the mean + SE
(n = 3).
since a significant increase in DBI (23 %) was observed due
to more abundant C18 : 3. Similarly, Piatã showed higher
(15 %) values, although the increase was not significant. Chilling treatment further reduced unsaturation in Catuaí and Icatú
but not in C. dewevrei, where control values were re-established. After chilling conditions, unsaturation was maintained
in Apoatã and Piatã, and showed a slight reduction upon rewarming.
As regards lipid classes, the amounts of galactolipids
(MGDG, monogalactosyldiacylglycerol and DGDG, digalactosyldiacylglycerol) and phospholipids increased in Apoatã
Membrane lipids and cold sensitivity in Coffea sp.
Figure 4. Changes induced by cold in the amount of the main lipid
classes in leaves of five Coffea genotypes under control (25/20 ˚C), at
15/10 ˚C, after three nights of chilling (3 × 15/4 ˚C) and re-warming (Rec
25/20 ˚C) conditions. Each value represents the mean + SE (n = 3).
MGDG, monogalactosyldiacylglycerol; DGDG, digalactosyldiacylglycerol; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; DPG, diphosphatidylglycerol; NL, neutral lipids.
287
after a gradual temperature decrease (Fig. 4). After chilling,
DGDG amounts in this genotype further increased (75 %) in
relation to control values, while the main phospholipids
showed significant reductions in relation to values reached
after 15/10 ˚C period. Therefore, a 27 % reduction of MGDG/
DGDG ratio was observed in this genotype due to increased
DGDG synthesis, in spite of the decrease induced by cold in
total lipids (Fig. 3). After the gradual temperature reduction
period, DGDG increased in Piatã (52 %) and Catuaí (23 %),
causing a reduction of MGDG/DGDG ratio of 41 % and 20 %,
respectively (Fig. 4). Chilling induced MGDG synthesis (34 %
increase) in Piatã, while no significant changes occurred in
phospholipids. In Catuaí, chilling caused the reduction of
MGDG (32 %) and DGDG (16 %) amounts. The TFA reduction
observed in C. dewevrei at 15/10 ˚C was the result of a significant decrease in the two main lipid classes (MGDG, 41 %;
DGDG, 28 %). In this genotype, all phospholipids were
reduced except phosphatidylcholine (PC). The decrease was
particularly drastic (54 – 59 %) in phosphatidylglycerol (PG)
and phosphatidylinositol (PI). This reduction was maintained
during more severe cold treatment, and enhanced in phosphatidylethanolamine (PE). PC amounts were stable throughout the whole treatment, but showed an unusual increase (2.3
fold) during recovery and in relation to control values. PE
amounts also tended to increase (33 %) during recovery and
in relation to values observed under chilling. Icatú did not
present significant changes in lipid class contents (Fig. 4),
which agrees with the same tendency observed in TFA and
with the absence of significant changes in all studied fatty
acids and DBI (Table 1).
As concerns the fatty acids composition of each lipid class
(Tables 2, 3), special attention must be given to galactolipids
unsaturation due to their high C18 : 3 percentage (Table 2). In
control conditions, Piatã presented the lowest content of
C18 : 3 in MGDG (82 %), in accordance with the lowest DBI
values of TFA (Table 1). These values remained lower in Piatã
than in the other genotypes. A slight increase in the proportion of MGDG in response to 15/10 ˚C and chilling treatments
was observed in Piatã, Catuaí and Apoatã. In MGDG C18 : 3
tended to decrease in all genotypes under re-warming conditions when compared with the values obtained under low
temperatures. No significant changes were observed in Icatú,
and C. dewevrei presented a small decrease after acclimation, while under chilling it recovered to higher values than
observed in control plants.
As for C18 : 3 percentage in DGDG (Table 2), Piatã, Catuaí
and C. dewevrei belong to the group of genotypes presenting
lower values (between 55 % and 60 %), in contrast with Icatú
and Apoatã (around 66 %). During gradual temperature decrease (15/10 ˚C) or chilling exposure, small changes were induced in Catuaí, Icatú and Apoatã, but Piatã showed a 31 %
increase in the proportion of C18 : 3 in DGDG after chilling. As
concerns C. dewevrei, C18 : 3 in DGDG showed a gradual increase during cold treatments and following recovery (Table 2).
288
Paula Scotti Campos et al.
Table 2. Fatty acid composition (mol %) of galactolipids in five Coffea genotypes under control (25/20 ˚C), at 15/10 ˚C, after three nights of chilling
(3 × 15/4 ˚C) and rewarming (Rec 25/20 ˚C) conditions. MGDG: monogalactosyldiacylglycerol; DGDG, digalactosildiacylglycerol. Statistical analysis is shown for the most representative fatty acids. Different letters express significant differences between different temperatures for the same
plant (r, s, t); r represents the highest values.
Apoatã
MGDG
DGDG
Catuaí
MGDG
DGDG
C. dewevrei
MGDG
DGDG
Icatú
MGDG
DGDG
Piatã
MGDG
DGDG
T (˚C)
C16 : 0
C16 : 1 c
C18 : 0
C18 : 1
C18 : 2
C18 : 3
25/20
15/10
3x15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
4.2s
4.7s
3.7s
6.1r
21.8r
22.3r
23.1r
23.3r
5.5r
3.8r
4.9r
4.6r
25.8r
23.7r
23.0r
22.4r
3.9s
5.5rs
4.0s
6.7r
28.2r
23.7rs
21.8s
20.1s
5.4r
5.7r
6.0r
5.9r
22.6s
30.0r
21.4s
19.0s
9.3s
9.1s
9.1s
17.1r
27.6r
29.7r
13.2s
28.3r
0
0
0.4
0
0.2
0
0
0
0
0
0
0.3
0.4
0.3
0.3
0.3
0
0
0
0
0
0
0.3
0
0
0
0
0
0.8
1.4
2.3
1.4
0
0
0
0
2.1
1.8
1.0
0
1.7
0.5
0.4
0.6
8.2
2.4
3.8
5.3
2.1
1.3
1.6
0.7
7.1
3.1
5.7
3.7
1.9
1.5
1.2
0.9
5.3
2.5
3.2
1.4
0.9
0.7
0.4
1.3
5.4
4.6
3.5
4.7
1.3
2.2
1.4
0.6
5.9
5.6
5.2
3.8
0.7
1.2
0.4
1.1
1.0
5.4
3.0
2.2
0.7
0.5
0.6
1.3
0.9
3.0
1.2
3.5
0.4
0.3
0.3
1.0
1.3
3.0
3.4
2.3
1.7
1.3
0.7
0.8
3.0
1.1
2.6
2.5
2.3
0.8
1.4
1.0
2.5
1.7
1.3
2.8
3.3r
3.2r
3.0r
3.8r
3.3s
2.8s
7.4r
6.5r
3.9r
2.8r
3.8r
3.5r
9.6r
9.1r
7.9r
7.7r
2.7r
3.6r
2.9r
3.9r
6.8rs
10.9r
6.5rs
2.0s
3.0rs
2.9rs
2.7s
3.2r
1.6r
1.1r
1.5r
1.2r
5.0r
4.0r
3.9r
4.0r
2.9r
3.4r
1.3s
2.4rs
90.0r
90.3r
92.2r
88.4r
65.5rs
67.1r
62.7s
62.7s
87.9r
91.7r
89.1r
89.7r
56.2s
60.7rs
61.9r
62.5r
91.1r
89.0r
91.7r
87.5r
58.4s
59.9s
64.7s
74.3r
89.0r
89.5r
90.1r
88.9r
66.7rs
61.8s
68.8r
71.2r
82.2r
83.9r
84.2r
77.4r
59.0s
57.8s
78.1r
62.7s
DBI = [(C16 : 1 c + C18 : 1 + 2 × C18 : 2 + 3 × C18 : 3)/(C16 : 0 + C18 : 0)].
The other fatty acids also showed small changes in response to cold treatments, but C18 : 0 presented a general
tendency to decrease in galactolipids, especially in DGDG of
all genotypes except Icatú (Tables 2, 3).
Another important fatty acid is the trans-∆3-hexadecenoic
acid (C16 : 1t), which is specifically found in chloroplast PG.
In control conditions higher amounts of C16 : 1t were observed in Catuaí, Icatú and C. dewevrei (31%), which showed
Membrane lipids and cold sensitivity in Coffea sp.
289
Table 3. Fatty acid composition (mol %) of phospholipids in five Coffea genotypes under control (25/20 ˚C), at 15/10 ˚C, after three nights of chilling (3 × 15/4 ˚C) and rewarming (Rec 25/20 ˚C) conditions. PC: phosphatidylcholine; PG, phosphatidylglycerol. Statistical analysis is shown for the
most representative fatty acids. Different letters express significant differences between different temperatures for the same plant (r, s, t); r represents the highest values.
Apoatã
PC
PG
Catuaí
PC
PG
C. dewevrei
PC
PG
Icatú
PC
PG
Piatã
PC
PG
T (˚C)
C16 : 0
C16 : 1 t
C18 : 0
C18 : 1
C18 : 2
C18 : 3
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
25/20
15/10
3 × 15/4
Rec 25/20
53.5r
47.0r
46.5r
50.3r
29.4r
23.2s
22.1s
26.2r
46.1r
45.1r
46.1r
47.2r
20.3r
22.2r
19.1r
22.1r
54.0r
52.5r
52.9r
44.5s
38.4r
32.1r
31.8r
38.2r
45.7s
45.7s
46.6s
60.6r
17.5s
17.5s
14.0s
23.5r
50.5s
54.8rs
57.0r
57.6r
33.2r
26.6s
26.3s
35.4r
–
–
–
–
16.4s
10.9t
19.8rs
26.5r
–
–
–
–
40.8r
43.8r
34.3s
32.6s
–
–
–
–
31.4r
30.2r
30.1r
27.8r
–
–
–
–
35.3r
40.9r
36.8r
38.4r
–
–
–
–
7.2r
7.6r
10.6r
7.0r
9.2
7.3
4.4
4.7
11.2
3.1
13.5
6.1
8.9
7.8
7.5
7.9
7.4
4.9
8.7
7.8
6.6
3.3
3.8
3.9
6.4
4.7
5.8
4.4
7.6
5.7
6.3
4.4
4.5
4.2
4.5
5.5
5.9
4.7
5.3
4.9
5.7
5.3
4.7
4.2
1.6
3.4
5.0
2.9
6.7
8.4
0.5
10.2
1.9
1.7
1.5
2.8
9.1
4.0
9.1
10.1
1.7
4.7
4.0
3.6
7.4
13.2
13.2
12.8
4.3
3.3
2.3
2.5
14.1
13.1
13.9
10.3
5.1
4.0
2.7
3.1
6.3
6.1
6.0
5.8
5.0r
5.8r
6.2r
5.7r
11.0rs
15.5r
18.1r
7.2s
7.6r
7.2r
7.1r
8.6r
8.9r
9.8r
8.4r
6.2r
8.0s
10.6s
6.9s
18.1r
4.6r
6.7r
6.3r
4.9r
4.1s
6.7r
6.7r
4.2s
4.8s
8.2r
8.3r
5.6rs
5.9r
4.9r
4.9r
5.6r
23.1r
20.9r
20.0r
19.4r
30.7s
36.6r
38.0r
36.5r
25.4s
39.0r
26.0s
23.8s
35.6rs
38.2r
37.7r
33.6s
13.5s
15.4s
20.5r
21.2r
29.8r
29.0r
32.4r
29.8r
11.8r
13.2r
13.7r
12.0r
38.4r
38.7r
38.1r
28.3s
23.8r
16.2s
22.5r
16.6s
32.7r
31.7r
30.2r
28.8r
24.6s
33.6r
32.5r
28.2rs
DBI = [(C16 : 1 t + C18 : 1 + 2 × C18 : 2 + 3 × C18 : 3)/(C16 : 0 + C18 : 0)].
values of 41 %, 35 % and 31 %, respectively (Table 3). The
lowest values were observed in Apoatã (16 %) and Piatã
(7 %). After initial temperature decrease (15/10 ˚C) this fatty
acid increased in Icatú (16 %) and Catuaí (7 %), and under
chilling conditions decreased 10 and 22 %, respectively, in relation to 15/10 ˚C values. In contrast, Apoatã showed a decrease (33 %) at 15/10 ˚C, followed by strong increases after
chilling and recovery. At this time, plants presented values
290
Paula Scotti Campos et al.
62 % higher than controls (Table 3). Piatã and C. dewevrei
showed stable values along the experiment.
Discussion
Leakage, permeability and membrane damage
As regards membrane leakage, although an increased permeability occurred in Apoatã and Piatã plants submitted to
chilling, these plants presented a return to control values after
re-warming (Fig. 1). Catuaí and Icatú, which were much less
affected by low temperature, also showed a recovery of leakage values, suggesting the presence of reversible damages
mainly resulting from changes in the biophysical properties of
the membrane. Thylakoids and plasma membrane are considered the primary site of attack during chilling injury (Leshem 1992). As a consequence of temperature lowering, membrane lipids commonly undergo phase transitions, i.e., liquidcrystalin or fluid to gel or solid, which temporarily affect membrane permeability during transient periods of temperature
decrease (Simon 1974, Leshem 1992). Irreversible permeability changes may occur when certain lipids aggregate to form
an inverted structure with hexagonal packing simmetry,
called HexII phase, which disrupts the membrane bilayer
causing an increased permeability of plasma membrane to
water and solutes upon re-warming (Uemura et al. 1995, Xin
and Browse 2000). In thylakoids, membrane heterogeneity
due to different lipid configuration domains also increases
permeability (Webb and Green 1991, Leshem 1992).
Leakage points may also result from damage of membrane
components. A higher susceptibility of C. dewevrei to cold,
evidenced by the strong leakage increase during chilling,
was related with MDA production (Fig. 2), which indicates the
occurrence of lipid peroxidation. Furthermore, no recovery
occurred as regards leakage, contrary to what was observed
in the remaining plants, indicating the presence of irreversible
membrane damage in C. dewevrei. This was further supported by galactolipids degradation readily observed after a
gradual temperature decrease (Fig. 4), reflecting strong thylakoid damage. These results are in agreement with previous
reports of photosynthesis impairment and photobleaching induced by cold in this plant, associated with smaller contents
of lipidic components of thylakoid membranes, namely dissipation pigments (carotenoids) and chlorophylls (Ramalho et
al. 2002).
Apoatã, Piatã and, to a lesser extent, Catuaí, showed an
enhanced lipid biosynthesis during the cold acclimation period (Figs. 3, 4). Gross membrane biogenesis is usually
observed when plant growth occurs under low temperature
(Harwood 1997). This may contribute to counterbalance
subsequent lipid degradation and enhanced permeability
induced by chilling, particularly in the case of Apoatã and
Piatã. Increased lipid amount in the dry weight biomass was
previously observed by our team in response to other stres-
ses, such as photoinhibition for Coffea (Ramalho et al. 1998)
and drought for Vigna and Arachis (Campos et al. 1999, Lauriano et al. 2000). Lowering of MGDG/DGDG ratio as a result
of enhanced DGDG synthesis occurred in Piatã and Catuaí
after the gradual temperature decrease (15/10 ˚C). The same
was observed in Apoatã after chilling in spite of phospholipid
decrease. The reduction of this ratio (MGDG/DGDG) through
DGDG synthesis may represent an increase in membrane
stability. Indeed, the proportion of DGDG, a bilayer-forming
lipid, is likely to be correlated to physical properties of thylakoid membranes. High DGDG was reported to give better
control of ionic permeability in the chloroplasts, minimizing
changes in lipid environment and hence preserving activity of
membrane proteins (Navari-Izzo et al. 1995, Webb and Green
1991). This does not seem to be the case in C. dewevrei,
where galactolipids drastically decreased during acclimation,
and a reduction of MGDG/DGDG ratio was the result of a
stronger MGDG degradation, possibly an indicator of a
higher susceptibility to cold as was reported previously for
drought in Vigna plants (Sahsah et al. 1998, Campos et al.
1999).
Phosphatidylcholine (PC) and phosphatidylethanolamine
(PE) are the main phospholipids of plasmalemma and mitochondria (Simon 1974). Higher PC and PE amounts observed
in C. dewevrei after recovery were responsible for higher total
lipid amounts, probably reflecting some degree of membrane
repair. An increase in phospholipid amounts, and particularly
PC, has been described frequently as a result of low positive
temperature treatments (Pham Thi et al. 1989, Jouve et al.
1993, Harwood 1997). If the ratio of PC to PE increases in a
given membrane, such as happens during recovery of C.
dewevrei, fluidity in a cold environment is expected to be
improved since the phase transition temperature for PC is
about 13 ˚C lower than for PE for equivalent species (Harwood
1997). Conversion of PE into PC is easy through methylation,
which could be a mechanism to achieve a better performance under cold conditions. In the case of C. dewevrei these
late changes were inefficient in coping with early induced
membrane injury, and particularly with irreversible damage in
chloroplast membranes, as inferred from galactolipids
decreases.
Unsaturation and fluidity
The unsaturation of membrane lipids is considered to be one
of the most critical parameters for the functioning of biological
membranes (Raison 1980). It is noteworthy that in Apoatã and
Piatã the overall membrane unsaturation given by DBI tended
to be higher after the gradual temperature decrease period
(Table 1). Such an increase was probably due to a higher
C18 : 3 percentage and to a decrease in the more saturated
fatty acids in the newly synthesized lipids. It may also depend
on compositional changes resulting from lipid turnover, although the latter are usually regarded as emergency re-
Membrane lipids and cold sensitivity in Coffea sp.
sponses to a sudden lowering of environmental temperature
(Harwood 1997). That could compensate for the decrease in
the fluidity of membrane lipids that is brought about by the
downward shift in temperature contributing to maintaining the
activity of membrane-bound enzymes and, hence, metabolic
activity under chilling (Raison 1980, Nishida and Murata
1996). The increase of lipid content observed in C. dewevrei
after chilling was accompanied by a re-establishment of unsaturation values similar to those of control plants mainly due
to a higher linolenic acid percentage in DGDG. The latter may
further enhance activity of peroxidative enzymes, as inferred
from MDA production, which is the result of membrane lipid
degradation and a clear marker of senescence (Leshem
1987, Jouve et al. 1993, Alonso et al. 1997). Therefore, a low
rate of repair mechanisms associated with strong cold damage account for a high chilling susceptibility in C. dewevrei.
On the contrary, it should be emphasized that increased
unsaturation observed after acclimation in Apoatã and Piatã
was not associated with higher MDA production, which is
considered the final product of stress-induced lipid peroxidation of poliunsaturated fatty acids. On the contrary, in these
plants, as well as in Catuaí and Icatú, MDA amounts tended
to decrease during the whole experiment. Lower MDA values
found at 15/10 ˚C (Icatú, Apoatã, Piatã) or chilling (Catuaí)
suggest that efficient anti-oxidative mechanisms might avoid
lipoperoxidation in these genotypes.
Thylakoid phosphatidilglycerol (PG) is also important in determining temperature sensitivity of different species (Murata
et al. 1992). The level of its major fatty acid, trans-∆3-hexadecenoic acid (C16 : 1t) plays an important role in chilling resistance, probably because it decreases phase transition temperature of the total thylakoid PG, and contributes to maintenance of fluidity under decreasing temperatures (Moon et
al. 1995, Harwood 1997). An increase of C16 : 1t in PG of
Apoatã under chilling (Table 3) might have contributed to
enhancement of membrane unsaturation, in spite of PG degradation (Fig. 4). Furthermore, it is expected that the abundance of C16 : 1t in PG would affect the efficiency of photosynthetic processes since it is positively correlated with oligomerization of LHCII, the major energetic antenna system of
the thylakoid membranes (Krupa and Baszynski 1989, Garnier et al. 1990). However in cold tolerant cereals, low temperature-induced decreases in C16 : 1t accompanied a shift from
oligomeric to monomeric LHCII, which could reflect a mechanism for regulating energy distribution within the photosynthetic apparatus to counteract the potentially deleterious
effects of low temperature-induced photoinhibition (Huner et
al. 1989). A similar mechanism could be related to a certain
degree of cold injury avoidance observed in Catuaí and Icatú
as regards photosynthetic parameters (Ramalho et al. 2002).
There is a large body of evidence on the increase of membrane unsaturation under low temperature conditions (Williams et al. 1996, 1997) and its importance in chilling tolerance. Fatty acid desaturation during chilling acclimation is involved in conferring low-temperature tolerance e.g., to young
291
tobacco leaves (Kodama et al. 1995). Unsaturation of chloroplast lipids stabilizes the photosynthetic machinery against
low-temperature photoinhibition in transgenic tobacco plants
(Moon et al. 1995). However, it was also demonstrated that an
overall reduction in the level of unsaturation of chloroplasts
lipids enhances thermal tolerance in a mutant of Arabidopsis
(Kunst et al. 1989). Decreased lipid unsaturation may reflect a
lower susceptibility to peroxidation and, hence, contribute to
preservation of membrane integrity and function. This mechanism was previously suggested to represent an adaptive feature of chloroplast membranes against photooxidative stress
in Catuaí (Ramalho et al. 1998). Furthermore this genotype
presented an excellent recovery when control temperatures
were re-established. Icatú also presented a good ability to
maintain membrane integrity under cold conditions, evidenced by stable lipid amounts and small permeability
changes. Such stability may be partly achieved by lowering
membrane unsaturation, as a result of turn-over, which would
explain the significant MDA decrease after gradual temperature lowering up to 15/10 ˚C. Although a loss of membrane
fluidity is inherent to decreasing lipid unsaturation, the concomitant reduction of susceptibility to lipoperoxidation should
not be excluded as being critical to preserving cell metabolism under decreasing temperature, constituting another way
to achieve cold acclimation.
Acknowledgements. We are grateful to Vera Silva and Fátima Silva
for help in lipid analysis. This work was supported by PRAXIS/PCNA/C/BIA/110/96 project, with the contribution of BARTOLOMEU
DIAS CNDP and PRODEP program grants.
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