JXB Advance Access originally published online on September 21, 2006
Journal of Experimental Botany 2006 57(14):3707-3715; doi:10.1093/jxb/erl120
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© 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
The role of abscisic acid and low temperature in chickpea (Cicer arietinum) cold tolerance. II. Effects on plasma membrane structure and function
1Department of Agronomy, NWFP Agricultural University, Peshawar, Pakistan
2Department of Biological Sciences, Quaid-E-Azam, University of Islamabad, Pakistan
3Plant Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical & Life Science, University of Glasgow, Glasgow G12 8QQ, UK
* To whom correspondence should be addressed. E-mail: p.dominy{at}bio.gla.ac.uk
Received 23 December 2005; Accepted 14 July 2006
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Abstract |
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The frost hardiness of many plants such as chickpea can be increased by exposure to low non-freezing temperatures and/or the application of abscisic acid (ABA), a process known as frost acclimation. Experiments were conducted to study the response over a 14 d period of enriched plasma membrane fractions isolated from chickpea plants exposed to low temperature and sprayed with exogenous ABA. Measurement of the temperatures inducing 50% foliar cell death (LT50), and subsequent statistical analysis suggest that, like many plants, exposure to low temperatures (5/–2 °C; day/night) induces a significant level (P <0.05) of frost acclimation in chickpea when compared with control plants (20/7 °C; day/night). Spraying plants with exogenous ABA also increased frost tolerance (P <0.05), but was not as effective as low temperature-induced frost acclimation. Both pre-exposure to low temperatures and pre-treatment with ABA increased the levels of fatty acid desaturation in the plasma membrane (measured as the double bond index, DBI). Exposure of chickpea plants to low temperatures increased the DBI by 15% at day 4 and 19% at day 14 when compared with untreated control plants. Application of ABA alone did not increase the DBI by more than 6% at any time; the effects of both treatments applied together was more than additive, inducing a DBI increase of 27% at day 14 when compared with controls. There was a good correlation (P <0.05) between the DBI and LT50, suggesting that the presence of more unsaturated lipid in the plasma membrane may prevent cell lysis at low temperatures. Both pre-exposure to low, non-freezing temperatures and pre-treatment with ABA induced measurable changes in membrane fluidity, but these changes did not correlate with changes in LT50, suggesting that physical properties of the plasma membrane other than fluidity are involved in frost acclimation in chickpea.
Key words: ABA, abscisic acid, low temperature, double bond index, LT50, plasma membrane fluidity
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Introduction |
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Chickpea (Cicer arietinum) is a major crop in many parts of the developing world. In many regions, including Pakistan, extensive irrigation is required to achieve high yields, but this results in increased soil salination that eventually leads to a depression in crop yield. In regions where rainfall is sufficient to support chickpea growth, such as the foothills of the Himalayas, production is limited by the inherent sensitivity of chickpea to freezing temperatures. Recently, the frost tolerance of several species in the Cruciferaceae has been improved by the application of biotechnology (Jaglo-Ottosen et al., 1998; Gilmour et al., 2000), and it is possible that a similar approach may improve the cold tolerance of chickpea. A better understanding of the processes that result in cold acclimation in chickpea is now required as this may lead to important agricultural and economic benefits in the developing world.
Exposure of cold hardy plants to low temperatures, or the application of abscisic acid (ABA) (Mohaptra et al., 1988; Xin and Li, 1992; Prasad et al., 1994) induces the cold acclimation process, and many cellular changes have been reported to occur. These include alterations in soluble carbohydrate content, changes in the cell protein profile, and changes in membrane lipid composition and degree of fatty acid saturation (Sakai and Larcher, 1987; Rochester et al., 1989; Hallgren and Oquist, 1990; Anderson et al., 1994; Dallaire et al., 1994; Prasad et al., 1994). Many of the soluble compounds synthesized during cold acclimation have been implicated in preventing or mitigating the deleterious effects of freeze-induced cellular dehydration (Gusta et al., 1996). In addition, changes to the structure of the plasmalemma have been reported to accompany the cold acclimation process, and these have been implicated in the observed changes in some biophysical properties of this membrane. Structural changes to the plasma membrane include the level of acyl lipid desaturation, subtle alterations in lipid class, and changes in the protein complement (Steponkus et al., 1988; Uemura and Steponkus, 1994). An increased level of acyl lipid desaturation is thought to lower the transition temperatures of lipid phase changes including the liquid crystalline-to-gel and lamellar to hexagonalII transitions (Williams, 1990).
It has been shown in the Brassicaceae that higher levels of acyl lipid desaturation are under genetic control, and both the cytosolic and chloroplast pathways of lipid biosynthesis are involved (Williams et al., 1988; Johnson and Williams, 1989; Johnson-Flanagan et al., 1991). Whether low temperature-induced desaturation is due to the increased production of desaturase enzymes, modulation of the activity of these enzymes, or some other process is not clear. The presence of more double bonds (unsaturated fatty acids) is reported to maintain membrane fluidity at low temperatures by introducing bends or ‘kinks’ in the acyl chains, thereby inhibiting tight packing of adjacent lipid molecules (Vigh et al., 1998). Changes in phospholipid lipid composition, therefore, also play an important role in reducing the membrane transition temperature and significantly affect the temperature range in which membranes undergo deleterious phase transitions (Vigh et al., 1998).
In an attempt to gain a better understanding of the processes involved in frost damage and frost tolerance in chickpea, a series of experiments was undertaken to investigate the role of acclimation temperatures and the application of the phytohormone ABA on chickpea. Plants were exposed to cold acclimating treatments and the results of in vivo and in vitro experiments that assess some biophysical and biochemical properties of the plasma membrane isolated from cold-acclimated and non-acclimated leaf tissue are reported here.
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Materials and methods |
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Growth of plants
Seeds of chickpea (C. arietinum L. cv. CM72) were soaked in distilled water overnight and five seeds were placed in 1.0 l pots containing sand. The pots were kept at in a 20/7 °C day/night temperature regime with a 12 h photoperiod (220 µmol photons m–2 s–1 PAR). Plants were watered every week with 250 ml of half-strength Hoagland's solution, and standard agronomic practices were followed throughout the course of the experiment. Forty days after sowing, each pot was sprayed with either 10 ml of water (no treatment) or 10 ml of 10–4 M ABA±enantiomer mixture (Sigma A1049). The temperature in one growth cabinet was then lowered to 5/–2 °C day/night temperature and the photoperiod maintained at 12 h. These conditions were chosen as they are similar to those encountered in the Himalayan foothills of NWFP, Pakistan. Plants were then harvested at 44, 47, and 54 d after sowing (i.e. day 4, 7, and 14, respectively) and used for determining the extent of tissue damage (LT50), plasma membrane fatty acid composition, and plasma membrane fluidity.
Measurement of freezing tolerance (LT50)
Cold hardiness was assessed by measuring solute leakage from leaves, essentially as described by Guy et al. (1987) but with a few modifications. Leaves were detached and briefly washed in distilled water, immediately wrapped in damp tissue paper, and placed in disposable capped 15 ml tubes. These were placed in a controlled temperature cryo-bath and equilibrated for 30 min at 0 °C. A small piece of ice (
100 µl volume) was then placed in contact with the moist tissue paper to seed extracellular ice crystal formation. The temperature was then lowered at a rate of 2 °C h–1 and held for 30 min at each of the experimental temperatures (4, 0, –4, –8, –12, –16, and –20 °C). Three replicates were removed at each temperature and placed immediately at 4 °C overnight to thaw. A 10 ml aliquot of deionized water was then added to each tube, and these were then shaken for 4 h to extract electrolyte from lysed cells. The electrical conductivity of the solution was measured using a Jenway 4070 conductivity meter. The samples were then frozen to –196 °C overnight to induce total (100%) cell lysis, thawed rapidly, and the electrical conductivity determined again. From these data, plots of the percentage electrolyte leakage versus temperature were constructed and a four-parameter logistic model fitted to the data using the Sigma Plot 7 ‘Fit Curve’ routine. From these fitted regressions, the temperature (LT50) that results in 50% electrolyte leakage was determined. To determine whether the treatments produced significant effects, the data were arcsin transformed and an analysis of variance test was performed (General Linear Model, Minitab version 13). The main factors in the analysis were as follows: time (levels day 4, day 7, and day 14), temperature regime (levels control and low), and ABA application (levels + and –).
Isolation of enriched plasma membrane fractions
Fractions enriched in plasma membrane were prepared from 10 g of chickpea leaves by isopycnic sucrose gradients (essentially as described by Briskin et al., 1987), followed by aqueous two-phase separation (as described by Larsson et al., 1987). The two-phase separation method was optimized by adjusting the dextranT 500/PEG 3350 composition from 5/5% to 7/7% (Larsson et al., 1987). A phase composition of 6.2/6.2% (w/w) was found to be optimal and this was chosen for all subsequent preparations. Enrichment for plasma membrane was established by measuring the specific activity of vanadate-sensitive ATPase activity, a marker for the plasma membrane P-type H+-ATPase (Palmgren, 1990); protein levels were determined by a micro Lowry method (Petersen, 1977). The following marker enzyme assays were performed (Briskin et al., 1987) to determine the level of contamination from other organelles: nitrate-sensitive ATPase activity (tonoplast), cyanide-insensitive NADPH cytochrome c reductase (endoplasmic reticulum), cytochrome c oxidase (mitochondria), and chlorophyll (chloroplast).
Isolation and quantification of fatty acids
The methods for isolation and quantification of acyl lipids are described elsewhere (Christie 1982). Aliquots of enriched plasma membrane fractions containing 200 µg of protein were added to 5 ml of 0.1 M KCl and 20 ml of chloroform:methanol (2:1, v:v) containing 10 mg l–1 butylated hydroxytoluene (BHT). A 5 µg aliquot of C-15 fatty acid was added as an internal standard to establish the extent of losses during the extraction procedure (Sigma, P6125). The mixture was shaken for 5 min and then allowed to stand overnight at 4 °C. Samples were then centrifuged at 500 g for 5 min to separate the phases, the upper aqueous phase was re-extracted with chloroform:methanol (6:1, v:v), and the two lower phases pooled and evaporated to dryness with nitrogen. The lipids were resuspended in 0.6 ml of toluene and methyl esters prepared by heating for 2 h at 70 °C with 1.2 ml of methanol containing 1% sulphuric acid. A 3 ml aliquot of sodium chloride (5%) and 3 ml of hexane were then added, the upper organic phase was collected and evaporated to dryness in a stream of nitrogen, and the residue was resuspended in 100 µl of chloroform. Samples were analysed on a Packard 430 gas chromatograph fitted with a 25 mx0.25 mm id WCOT PC-Wax 58 CB capillary column (Chrompack) connected to a flame ionization detector. Methylated fatty acids were separated using a temperature programme (2 min at 60 °C, rise of 15 °C min–1; 10 min at 210 °C). Authentic methylated fatty acid (Sigma 18920) were used as external standards to identify and quantify peaks; corrections were made at this stage for losses using the C-15 internal standard. The double bond index (DBI), a measure of the degree of fatty acid desaturation, was calculated as described elsewhere (Cyril et al., 2002).
Fluorescence polarization measurement
The methods employed were essentially those of Aricha et al. (2004). Aliquots of 1.5 ml of the enriched plasma membrane fractions (100 µg protein ml–1) were incubated with 2 µM 1,6-diphenyl-1,3,5-hexatriene (DPH) for 1 h at 25 °C with gentle stirring to intercalate the fluorescent probe into the lipid bilayer. DPH fluorescence polarization was monitored by excitation at 385 nm (15 nm silt width) and emission at 450 nm (20 nm silt width) using a Perkin Elmer luminescence LS-55 spectrometer fitted with a PE L2250100 polarizer accessory. The degree of fluorescence polarization (P) was calculated using the following equation:
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are the linearly polarized emissions oriented parallel and perpendicular, respectively, to the plane of the polarized excitation beam. |
Results |
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Assessment of cold-induced injury
The effects of ABA application and exposure to acclimating temperatures on the susceptibility of shoots to low temperature damage were assessed by monitoring solute leakage from excised leaves. To establish if there were significant differences between the treatments, the data were first arcsin transformed, and an analysis of variance test performed on logistic regressions of these data. Transformation of the data is necessary as the measurements are constrained between 0% and 100%, and an analysis of variance test on untransformed data, therefore, is invalid. The logistic model used (see legend to Fig. 1) fitted the data well, and all model parameters were highly significant. In addition, both of the main experimental factors (temperature and ABA application) produced a highly significant (P <0.001) change in solute leakage, and a significant (P <0.05) interaction was found between the main factors. The conclusions are that both acclimation temperature and ABA application produce significant changes in low temperature-induced damage to chickpea leaf tissue, and that these effects are not merely additive.
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Figure 1 presents the solute leakage curves for untransformed data, and lines of best fit were calculated using a logistic model. From these curves, the temperature that produced 50% cell lysis (LT50) was determined for three replicate curves for each treatment at each time. The average of these three values for each treatment is presented in Table 1. As these values were derived from untransformed data, it is not strictly valid to perform a test for significance between these treatment means. For this reason, the LT50 values were determined from similar plots of the arcsin cell leakage curves, and an analysis of variance test was performed on these data (factor ABA, 0 M and 10–4 M; factor temperature, control and low regime; factor time, 4, 7, and 14 d); a Duncan's multiple range test was then used to establish between-treatment differences (Snedecor and Cochrane, 1980). Table 1, therefore, presents the average LT50 values for untransformed data, and the significance determined from arcsin LT50 values.
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When kept at control temperatures, by day 4 the application of ABA caused a significant decrease (P <0.05) in the LT50 value of leaves cooled to –4 °C and –8 °C, but below this temperature no significant (P >0.05) effect of ABA was found (compare control –ABA versus control +ABA; Fig. 1a). As a result, the LT50 of treated plants decreased from –7.2 to –9.7 °C (Table 1). Plants exposed to the low temperature regime for 4 d were more cold hardened than those kept in the control regime (Fig. 1a), and this was reflected in their lower LT50 values of –12.8 °C (low –ABA) and –13.5 °C (low +ABA; Table 1). No significant differences (P >0.05) were found between treated and untreated samples (±ABA) after 4 d exposure to the low temperature regime (Fig. 1a, Table 1). Below –16 °C, there were no clear differences in the cold hardiness of plants exposed to the four treatments (Fig. 1a).
Solute leakage was also monitored at day 7 (data not presented) and day 14 (Fig. 1b), and broadly similar results were obtained at these times. The LT50 values for both treated and untreated control plants did not change with time (Table 1). It was noted, however, that in treated controls (control +ABA), the sigmoidal response to low temperature disappeared after 4 d, and a more linear response was observed. The loss of this sigmoidicity at day 7 and 14 appears to render tissue at least as sensitive as untreated controls (control –ABA) at temperatures down to –10 °C (Fig. 1b, Table 1).
The LT50 values for plants exposed to the low temperature regime were substantially lower than those of plants kept in the control temperature regime (Table 1). By day 14, the LT50 values of untreated plants (low –ABA) had declined significantly (P <0.05) from those of controls (control –ABA), from –9.5 to –13.6 °C, and a similar difference was found at day 7. Plants kept in the same low temperature regime and treated with ABA (low –ABA versus low +ABA) showed an increased level of cold hardening by day 4 (–13.5 °C versus –12.8 °C), and this difference became significant (P <0.05) and more pronounced with time (–13.6 °C versus –17.3 °C by day 14).
It was noted that at both day 7 and day 14 a sharp discontinuity in the response of the low temperature-exposed plants occurred between 0 °C and –4 °C. For this reason, the values for 0 °C were not included in these regressions of the low temperature-treated plants only. Comparison of the leakage values obtained after incubation at 4 °C for 2 h showed no significant difference between any of the treatments; values ranged between 3% and 7% (data not presented). Significant (P <0.05) damage occurred to plants kept in the control temperature regime however, when plants were incubated at 0 °C (Fig. 1). The conclusion is that exposure to cold acclimating temperatures (7 °C day/–2 °C night) prevents solute leakage even at 0 °C where frost damage is minimal.
Studies on enriched plasma membrane fractions
Enriched plasma membrane fractions were prepared by centrifugation of leaf microsomal fractions through sucrose density gradients, followed by aqueous two-phase partitioning in dextran T-500 and PEG 3350 (see Materials and methods). To confirm that these procedures produced consistent, enriched plasma membrane fractions, marker enzyme assays for different organelles were performed on samples at each stage of the preparation. Table 2 presents a summary of these marker enzyme assays for a control –ABA, day 4 sample; similar results were obtained for other preparations. The marker enzyme assay for plasma membrane (vanadate-sensitive, Mg2+-dependent ATPase activity) suggested a 27-fold enrichment in the final plasma membrane fraction compared with the microsomal fraction, whilst the proportion of tonoplast, endoplasmic reticulum, mitochondria, and chloroplast contamination decreased. Subsequently, enriched plasma membrane fractions were prepared from all samples using these methods, and fold enrichment for each determined using the vanadate-sensitive, Mg2+-dependent ATPase marker assay. No consistent significant difference (P >0.05) was found in the fold enrichment of plasma membrane marker enzyme activity between any of the samples; values ranged from 23–37-fold. Similarly, neither acclimation temperature nor ABA treatment produced a consistent change in the specific activity of the plasma membrane marker (data not presented). These results confirm that plasma membrane fractions of similar quality can be isolated from chickpea shoot tissue exposed to each of the treatments.
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Fatty acid composition and cold tolerance
In some studies, increased frost resistance has been correlated to changes in the lipid composition of the plasma membrane which decrease the extent of freeze-induced cell rupture. To investigate further the basis of ABA-induced and low temperature-induced increased frost resistance in chickpea leaves, total lipid was extracted from the enriched plasma membrane fractions and the degree of acyl lipid desaturation estimated by capillary gas chomatography.
Table 3 presents a summary of the changes in acyl lipid composition of the enriched plasma membrane fractions isolated from the various treatments. The application of ABA to plants kept at control temperatures did not result in any major changes in the abundance of the major acyl lipid classes, although oleic acid levels increased and palmitic acid decreased over the 14 d period. When exposed to low temperatures, however, application of ABA produced a time-dependent major decrease in linoleic acid and a major increase in linolenic acid.
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An analysis of variance test on the effects of treatment on the DBI (a measure of the degree of acyl lipid desaturation) indicated that both main factors (temperature and ABA) had a significant (P <0.001) effect (Table 3). Over the day 4 to day 14 period, the DBI of untreated controls (control –ABA) increased from 1.18 to 1.29, suggesting a developmental increase in the level of desaturation. Application of ABA to plants kept at control temperatures did not produce a major change in the DBI at any time (control –ABA versus control +ABA). In contrast, low acclimation temperatures had a significant effect (P <0.05) on the DBI of untreated plants (control –ABA versus low –ABA) producing increases of 15% at day 4 and 19% at day 14 (Table 3). Comparison of the effects of ABA application on acyl lipid DBI of plants exposed to low acclimation temperatures (low –ABA versus low +ABA) also showed significant (P <0.05) changes. There was an increase in desaturation of 4–5% at day 4, and 7% at day 14 (Table 3).
A plot of the DBI versus the corresponding LT50 for the samples is presented in Fig. 2. There is a significant negative correlation between the DBI and LT50, suggesting that an increase in the amount of unsaturated lipid may give rise to the observed increases in freezing tolerance presented in Fig. 1.
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Membrane fluidity measurements
It is widely reported that an increase in the degree of unsaturation in membrane lipid results in an increase in membrane fluidity at low temperatures, and this helps to prevent freeze-induced membrane rupture. To investigate whether the decrease in LT50 reported above correlates with changes in membrane fluidity, fluorescence polarization measurements were made on enriched plasma membrane fractions isolated from the leaves of treated and untreated chickpea plants. The plasma membrane preparations were incubated with the fluorescent probe DPH, and fluorescence polarization was measured as the samples were cooled from 10 °C to –15 °C (see Materials and methods).
Membrane fluidity decreased with decreasing assay temperature in all samples (Fig 3a, b). At an assay temperature of 10 °C, fluidity appeared to be least (high P-values) in samples prepared from plants kept in the control temperature regime, although ABA application increased fluidity to some extent. Samples prepared from plants exposed to the low temperature regime showed a considerably greater degree of fluidity (low P-values) at an assay temperature of 10 °C, but this increased fluidity was rapidly lost with decreasing assay temperature so that there was little difference between all samples below –4 °C. Unfortunately, the present experimental set-up did not allow us to cool below –15 °C to determine the temperature of the liquid crystalline-to-gel phase transition which would have been manifest as a sharp increase in P.
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These findings may be summarized as follows. Low temperatures and exogenous ABA application both increase plasma membrane fluidity in chickpea leaves above –4 °C of frost, but at between –4 °C and –16 °C, the temperature range where large differences in LT50 were observed, no major differences were observed.
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Discussion |
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The experiments reported here have produced some interesting observations on the role of ABA in the frost acclimation process.
The electrolyte leakage experiments (Fig. 1) show that a significant degree of cold hardiness is manifest after only 4 d of exposure to acclimating temperatures, but ABA application does not further enhance frost resistance. In contrast, 4 d after ABA application to non-acclimated plants, a partial cold hardening to mild frost conditions (0 °C to –10 °C range) was observed. At this time, all plants were equally sensitive to deep frost conditions (–16 °C and below; Fig. 1a). A significantly longer period of time was required to achieve improved cold hardening to deep frost conditions, and exogenous ABA does provide added protection (Fig. 1b). It appears that cold acclimation is a two-stage process in chickpea. Adaptation to mild frost conditions occurs within a few days, but adaptation to deep frost takes several days longer to manifest. ABA is partly involved in the adaptation to mild frost conditions, but further experiments using a range of exogenous ABA concentrations will be required to establish whether full adaptation to mild frost can be achieved by the application of ABA alone. Survival from exposure to deep frost is improved by the application of ABA but, as stated above, this effect takes longer to manifest.
To investigate further the acclimation process in chickpea leaf tissue, the biochemical composition and biophysical properties of enriched plasma membrane fractions from treated plants were studied. In a parallel study, changes in the proteome of whole chickpea leaves that accompany the low acclimation temperature and ABA treatment have been identified. These differences were identified by two-dimensional gel electrophoresis, a powerful technique that unfortunately is not well adapted for resolving hydrophobic membrane proteins. It is unlikely, therefore, that the differentially abundant proteins resolved with this technique reflect changes in the protein composition of the plasma membrane. New, state-of-the-art quantitative techniques will be required to resolve differences in this membrane fraction (e.g. isotope-coded affinity tagging followed by two-dimensional liquid chromatography/mass spectrometry); unfortunately, these methods were not available at the time of this study.
The freezing tolerance of the plasma membrane can be increased by artificial enrichment of the fraction with mono- or di-unsatured species of phosphatidylcholine (PC) using protoplast–liposome fusion techniques (Steponkus et al., 1988). Of the PC species tested in their study, 18:2/18:2-PC and 18:3/18:3-PC were the most effective; fusion with saturated species had neither a positive or a negative effect. It has also been reported that the levels of cis-desaturation increase when plants are exposed to low temperature; studies with cyanobacteria, protozoa, and plants confirm roles for acyl lipid desaturation in cold adaptation (Cossins, 1994; Nishida and Murata, 1996; Tasaka et al., 1996; Samala et al., 1998; Cyril et al., 2001, 2002). For example, when cyanobacteria were transformed with a desaturase, a significant decrease in the level of membrane lipid saturation was observed which also increased low temperature tolerance and restored normal membrane function (Wada et al., 1990). This desaturation of fatty acids is reported to decrease the liquid crystalline gel phase transition temperature resulting in a viscosity-induced dysfunction of the fluid membrane (Lyons, 1973; Raison, 1986; Raison and Orr, 1986; Lynch and Thompson, 1988; Vigh et al., 1998).
The results confirm that acyl lipid desaturation increases with increased frost tolerance (LT50 values). It was found that application of ABA alone induces significant changes in the acyl lipid composition of the plasma membrane, but only when ABA was applied in combination with exposure to low temperatures was an increase in acyl lipid desaturation observed. It is conceivable that application of ABA alone will also effect an increase in plasma membrane desaturation, but that the critical level of exogenous ABA required to achieve this was not found in the present studies; further experiments using a wider range of ABA concentrations may confirm this. Alternatively, some other cold-induced factor in addition to ABA may be required. A significant negative correlation was found between the LT50 values of acclimated plants and the corresponding DBI (Fig. 2), giving further support to the notion that an increase in plasma membrane acyl lipid desaturation leads to the observed increase in frost hardiness. In the present studies, however, the deep frost tolerance observed at day 14 post-treatment was accompanied by a major decrease in the level of linoleic acid (18:02) and a major increase in linolenic acid (18:03); the fraction of other acyl lipids, including the fully saturated forms, remained relatively unaffected. It is conceivable that deep frost tolerance of the plasma membrane results from a specific reduction in linoleic acid and an increase in linolenic acid, rather than from a general non-specific desaturation.
Studies on the temperature-dependent changes in membrane viscosity of the enriched plasma membrane fractions demonstrated that up to 14 d post-treatment, pretreatment with both low acclimation temperatures and ABA increased membrane fluidity, particularly at temperatures above 0 °C. When the fluidity of these membrane fractions was assessed at temperatures of –5 °C and below, the temperatures at which significant tissue damage occurs (Fig. 1), no major differences were observed. No sharp discontinuities in the polarization thermograms were observed over the –15 °C to 10 °C temperature range, suggesting that no major lipid phase changes occurred in any of the samples. These results suggest that cooling the shoots of chickpea plants to –15 °C does not induce any major changes in either the phase properties or the viscosity of the plasma membrane bilayer that could account for the ABA- and low acclimation temperature-induced changes in leaf tissue frost damage (LT50). The possibility cannot be discounted that DPH partitions into microdomains or patches within the membrane and does not, therefore, report the overall behaviour of the membrane. Other structural changes in the structure of the plasma membrane (e.g. heaxagonalII phase) may occur below 0 °C that do correlate well with the electrolyte leakage measurements, but further experimentation will be needed to assess this. The conclusion is that the level of plasma membrane acyl lipid desaturation correlates well with membrane viscosity at temperatures above 0 °C, and also correlates well with the extent of tissue damage induced below 0 °C. Frost hardiness in chickpea, however, does not appear to be closely related to plasma membrane viscosity.
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Abbreviations |
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ABA, abscisic acid; DBI, double bond index; DPH, 1,6-diphenyl-1,3,5-hexatriene; LT50, temperature inducing 50% foliar cell death..
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References |
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