Peach fruit acquired tolerance to low temperature stress by accumulation of linolenic acid and N-acylphosphatidylethanolamine in plasma membrane

Food Chemistry 120 (2010) 864–872

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Peach fruit acquired tolerance to low temperature stress by accumulation of linolenic acid and N-acylphosphatidylethanolamine in plasma membrane Changfeng Zhang a,b, Shiping Tian a,* a b

Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, PR China Graduate School of Chinese Academy of Sciences, Beijing 100039, PR China

a r t i c l e

i n f o

Article history: Received 28 April 2009 Received in revised form 27 September 2009 Accepted 13 November 2009

Keywords: Chilling injury Fatty acids N-acylphosphatidylethanolamine Peach fruit Plasma membrane

a b s t r a c t Peach fruit (Prunus persica L. cv. Beijing 33) did not show symptoms of chilling injury in 0 °C-Air or 0 °CCA, but did in 5 °C-Air after 21 d. The mechanisms by which 0 °C storage could activate chilling tolerance of peach fruit were investigated by analysing characteristics of plasma membrane. We found that peach fruit stored in 0 °C-Air and 0 °C-CA had much higher linolenic acid content and unsaturation degree of plasma membrane than did that in 5 °C-Air. In addition, the fruits stored in 0 °C-CA showed a higher membrane fluidity and membrane integrity than did that in 0 °C-Air, which was related to the accumulation of N-acylphosphatidylethanolamine (NAPE) of peach fruits stored in 0 °C-CA. Based on these results, it appears that a higher unsaturation degree of membrane lipid and NAPE accumulation are beneficial for maintaining membrane fluidity, leading to an enhanced tolerance of peach fruit to chilling stress. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Many plant species, especially those of tropical or temperate origin, are severely injured by exposure to low but not freezing, temperatures (Saltveit & Morris, 1990). Exposure of these chilling-sensitive plants to unfavourable low temperatures often results in numerous cellular and metabolic dysfunctions, such as altered respiration rates, impaired photosynthetic activity, and changes in membrane permeability (Allen & Ort, 2001). Peach fruit is also sensitive to low temperature stress and chilling injury (CI) occurs easily when it is exposed to the low temperature for long periods (Saltveit & Morris, 1990). In a previous experiment, we found that peach fruits stored at 5 °C for 21 d usually showed CI, but no CI symptom occurred at 0 °C (Zhang & Tian, 2009). In addition, peach fruits kept at controlled atmosphere (CA), with 5% O2 plus 5% CO2 showed a stronger resistance to low temperature stress (Wang, Tian, & Xu, 2005) and application of methyl salicylate (MeSA) could effectively enhance tolerance of peach fruits to CI (Meng, Han, Wang, & Tian, 2009). A better understanding of the processes of chilling tolerance in fruit is now required as this may lead to important agricultural and economic benefits.

* Corresponding author. Address: Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing 100093, PR China. Tel.: +86 10 62836559; fax: +86 10 82594675. E-mail address: [email protected] (S. Tian). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.11.029

A biochemical basis to explain the CI mechanism of plant has not yet been established. Lyons (1973) proposes the existence of a primary event that results in a series of secondary events which, in turn, results in the symptoms of CI. The cell membranes are likely sites of primary effects of chilling. Mikami and Murata (2003) reported that unsaturated fatty acid (UFA) level in membrane lipids was positively correlated with plant chilling tolerance. Genetic manipulation of the level of UFAs led to the eventual modification of the cold sensitivity of tobacco plants (Murata et al., 1992). In cyanobacteria, sensitivity to cold is also closely correlated with the level of unsaturation of membrane lipids (Tasaka et al., 1996). Aside from the interest given to the responses of the UFA level to low temperature stress, the role of an unusual phospholipid class, N-acylphosphatidylethanolamines (NAPEs), in membrane protection and stabilisation has received considerable attention (Hansen, Moesgaard, Hansen, & Petersen, 2000). This compound is characterised by the presence of a third fatty acyl residue linked to the N-atom of the phosphatidylethanolamine headgroup by an amide bond and shows a propensity to accumulate under various stress conditions involving degenerative membrane changes (Schmid, Schmid, & Natarajan, 1990). For instance, NAPEs synthesis was observed in cultivated potato cells submitted to anoxia stress and the capacity to increase its NAPE level may confer some additional protection to the cell (Rawyler, Arpagaus, & Braendle, 2002; Rawyler & Braendle, 2001). However, studies on NAPEs biosynthesis and its involvement in the response of plant tissues to low temperature stress have, so far, been rare.

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As compared with plant responses to chilling stress, there are a number of papers describing that the development of CI of fruit is caused by an imbalance between oxidative and reductive processes due to metabolic gas gradients inside the fruit (Franck et al., 2007; Wang et al., 2005). Accumulation of reactive oxygen species may induce loss of membrane integrity which becomes macroscopically visible through the enzymatic oxidation of phenolic compounds to brown-coloured polymers. However, plasma membrane involvement in the process of CI development has received little attention to date. In order to fully understand whether the biochemical and biophysical characteristics of plasma membrane lipids are correlated with chilling-resistance of peach fruit, we investigated the function of linolenic acid (C18:3) and unsaturation degree of the plasma membrane in enhancing tolerance of peach fruit to low temperature stress. Here, we present the first evidence that peach fruit acquired chilling-tolerance by accumulation of C18:3 and NAPEs in plasma membrane, and explain why peach fruit stored at 0 °C showed a higher chilling tolerance than did that at 5 °C. 2. Materials and methods 2.1. Fruit and treatments Peach fruits (Prunus persica L. cv. Beijing 33) were harvested at commercial maturity from an orchard in the Pinggu district of Beijing, China, and transported to the laboratory, within 2 h, after harvest. Fruit used for experiment were selected for uniform size and for the absence of physical injuries or infections. There were 360 fruits in each group. One group was stored in air at 5 °C (5 °CAir) and served as control. A second group was stored in air at 0 °C (0 °C-Air) and a third group was stored in controlled atmosphere (5% O2 + 5% CO2) at 0 °C (0 °C-CA). Fruits were placed in plastic boxes (40  30  25 cm), wrapped in polyethylene film bags (0.04 mm thickness, with 5 holes of 20 mm diameter on upper and side surfaces) to maintain relative humidity (RH) at approximately 95%. At 7 day intervals, CI incidence, CI index and electrolyte leakage of fruit were measured on the same sample consisting of 30 fruits per replicate (30  3) for each treatment. Then these samples were cut into small pieces and frozen in liquid nitrogen, then stored at 80 °C for other assays. 2.2. Evaluation of CI The CI incidence and CI index were estimated on flesh browning according to the method of Wang et al. (2005). The score of CI was assessed by measuring the browning area in each fruit based on the following scale: 0 = no browning; 1 = less than 1/4 browning; 2 = 1/4–1/2 browning; 3 = 1/2–3/4 browning area; 4 = more than 3/4 browning. The CI index was calculated from the following formula: (1  N1 + 2  N2 + 3  N3 + 4  N4)  100/(4  N), where N = total number of fruit measured and N1, N2, N3 and N4 were the numbers of fruit showing the different degrees of browning. 2.3. Isolation of plasma membrane Plasma membrane (PM) was isolated, as described by Quartacci, Cosi, and Navari-Izzo (2001), using the two-phase aqueous polymer partition system. Frozen peach fruits (120 g) were homogenised (using an ordinary kitchen homogenizer) for 3  20 s in 300 ml of an extraction medium consisting of 0.33 M sucrose, 80 mM Tris, 5 mM Bis-Tris Propane (BTP)-Mes, pH 8.9, 10 mM ascorbic acid, 5 mM dithiothreitol (DTT), 5 mM Na2-EDTA, 10% glycerol, 0.4% BSA, 0.4% casein, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.15% (w/v) polyvinylpolypyrrolidone (PVPP).

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The homogenate was filtered through four layers of nylon cloth and centrifuged at 12,000g for 1 h. A microsomal pellet was obtained from the supernatant by centrifugation at 120,000g for 1 h. This pellet was suspended in a total volume of 10 ml in 0.25 M sucrose, 3 mM KCl, 5 mM potassium phosphate, pH7.8, and 9.0 g of the suspension was added to the 27.0 g aqueous two-phase polymer system to give a 36.0 g phase system with a final composition of 6.2% (w/w) dextran T500, 6.2% (w/w) polyethylene glycol 3350, 0.33 M sucrose, 3 mM KCl, 5 mM potassium phosphate, pH 7.8. The PM was further purified using a two-step batch procedure. The resulting upper phase was diluted 4-fold with 50 mM Tris–HCl (pH 7.5), containing 0.25 M sucrose, and centrifuged for 30 min at 120,000g. The resultant PM pellet was resuspended in the same buffer containing 30% ethylene glycol and stored at 80 °C for lipid analyses. All steps of the isolation procedure were carried out at 4 °C. To check the purity of the PM, the activity of the vanadate-sensitive ATPase as a marker enzyme was determined. NO3-sensitive ATPase activities and azide-sensitive ATPase were used as markers of mitochondria and tonoplast, respectively. The chlorophyll assay was performed to determine the level of contamination from chloroplast (Quartacci et al., 2001). The marker enzyme assay for plasma membrane (vanadate-sensitive ATPase activity) suggested a 24-fold enrichment in the final plasma membrane fraction compared with the microsomal fraction, whilst the proportion of tonoplast, mitochondria, and chloroplast contamination decreased. In addition, chlorophyll was not detected in the PM fraction. 2.4. Spin labelling and measurement of electron paramagnetic resonance (EPR) Plasma membrane fluidity was investigated by EPR, according to the method of Zhang and Tian (2009), with a slight modification. Stock solution of the fatty acid spin probes, 5- and 16-doxylstearic acid (5- and 16-DSA, Aldrich), was made up in ethanol (10 mg ml1). The probes were added to 200 ll samples of plasma membrane at a label to lipid ratio of 1:20 (v:v). After incubation for 1 h at ambient temperature and under nitrogen atmosphere, the sample was washed three times in 0.1 M potassium phosphate buffer, pH 7.0, by centrifugation at 200,000g for 30 min. Free spin probes were not detected in the supernatant following the third washing. For EPR analysis, the plasma membrane suspension was transferred to a 100 ll glass capillary tube, which was sealed and inserted into a quartz sample holder and put in the microwave cavity of the spectrometer. EPR measurements were performed on an ER-200D Bruker spectrometer. EPR spectra were obtained at Xband (9.80 GHz) with microwave power of 20 mW, modulation frequency 100 kHz and amplitude 1G The sweep time was 100 s and magnetic field scan 200 G. In the case of 5- and 16-DSA, the fluidity of the lipid chain can be estimated from the order parameter S and the rotational correlation time sc, respectively. 2.5. Extraction and analysis of plasma membrane lipids Lipids were extracted from the PM suspension by the addition of boiling 2-propanol followed by CHCl3: MeOH (2:1, v/v) containing butylhydroxytoluene (50 lg ml1) as an antioxidant. The solvent mixture was then washed with 0.2 volume of 0.88% KCl to separate the CHCl3 phase. The upper H2O phase was re-extracted with CHCl3, than the CHCl3 phases combined and dried under a stream of N2. The individual polar lipid was purified by twodimensional thin-layer chromatography (TLC) on activated silica gel plates (Silica gel 60, 0.25 mm thickness, Qingdao, China) according to the method described by Zhang and Tian (2009). Fatty acids of the individual polar lipid classes were identified and quantified by gas chromatography (GC) after conversion to

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the corresponding methyl esters by hot methanolic sulphuric acid. Samples were analysed on the LECO PegasusÒ IV GC-TOFMS fitted with a 30 m  0.25 mm  0.25 lm SUPELCO waxTM10 capillary column. Methylated fatty acids were separated, using a temperature programme (the initial column temperature of 140 °C was held for 5 min, increased by 4 °C per minute to 240 °C and held for 0 min, and then increased by 50 °C per minute to a final temperature of 255 °C for 5 min). Authentic methylated fatty acid (Sigma–Aldrich 47801) was used as external standard to identify and quantify peaks; corrections were made at this stage for losses using the C17:0 internal standard. The double bond index (DBI), a measure of the membrane lipid unsaturation, was calculated according to Zhang and Tian (2009) as follows: DBI = [(3  mol% C18:3) + (2  mol% C18:2)]/[(mol% C16:0) + (mol% C18:0) + (mol% C18:1)]. 2.6. Standard NAPE synthesis 1,2-Dipalmitoyl-sn-glycero-3-phospho (N-palmitoyl) ethanolamine (N-palmitoyl-DPPE) was prepared, basically as described by Térová, Petersen, Hansen, and Slotte (2005). Briefly, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine was dissolved in dichloromethane. A twofold molar excess of palmitic anhydride and a 10-fold excess of triethylamine were added to the solution. The mixture was stirred for 30 min at room temperature, then evaporated to dryness and dissolved in chloroform. The formed N-palmitoyl-DPPE was purified by a SUPELEAN LC-Si SPE 500 mg column using step-wise solvent gradients consisting of chloroform and methanol (100:0, 98:2, 95:5, 90:10 and 80:20 by volume, respectively). The N-palmitoyl-DPPE was almost completely eluted after the chloroform: methanol (90:10) step. To verify the purity and identity of the compounds, ESI-MS analysis was carried out according to the method of Rawyler and Braendle (2001). 2.7. Sample NAPE purification and ESI-MS analysis The total lipid extract in chloroform was loaded onto a SUPELEAN LC-Si SPE 500 mg column. After washing successively with 5 ml of chloroform, 7 ml of acetone:acetic acid (99:1, v/v), and 7 ml of chloroform:methanol (95:5,v/v), phospholipids were finally eluted with 7 ml of chloroform:methanol (1:1, v/v). The phospholipid fraction was then separated by one-dimensional TLC on a silica gel column developed with chloroform:methanol:14% aqueous ammonia (80:20:2, v/v). The fully resolved NAPE spot was scraped off and identified by ESI-MS according to the method of Rawyler and Braendle (2001). ESI-MS analysis was carried out in the negative mode with a Micromass Q-Tof micro platform. Sample cone and capillary voltages were 35 V and 1.2 kV, respectively, with a source temperature of 40 °C. Samples were introduced as approx. 50 lM solutions in dichloromethane/methanol/triethanolamine (80:20:1, v/v), at a rate of 10 ll min1. Synthetic N-palmitoyl-DPPE was used as a standard. 2.8. RNA isolation and Northern blot analysis Total RNA was isolated from the sample using the hot-phenol protocol described by Zhang and Tian (2009). For cDNA synthesis, forward (50 -ATTCTGAGGGTGTAAGAAGAATGCG-30 ) and reverse (50 AGGTTACCTACATCCTTGTCTCTGT-30 ) primers for PLDa were used according to the sequence published in GenBank (DY648783 and DY634453). Aliquots of 20 lg of total RNA per lane were separated on a 1.2% formaldehyde agarose gel and blotted onto a Hybond-N+ membrane (Amersham International Ltd.). Afterwards, blot was hybridised with the cDNA probe, labelled with [32P] dCTP to high specific activity by random priming (Prime-a-geneÒ labelling system U110, Promega Co.) according to the manufacturer’s instructions. After a 16 h hybridisation period, the blot was washed

once for 20 min in 2  SSC, 0.1% SDS at 65 °C and twice for 10 min in 0.1  SSC, 0.1% SDS at 65 °C, and exposed to an autoradiography film at 80 °C for 48 h. Equal loading of samples of total RNA was identified by visualisation of rRNA that had been stained with ethidium bromide. 2.9. Lipid acyl-hydrolase (LAH) (EC 3.1.1.26) activity assay The LAH activity was assayed using monogalactosyldiacylglycerols (MGDG) as the substrate according to Matsuda and Hirayama (1979) with some modifications. The reaction mixture contained 1 mM MGDG, 0.4% (w/v) Triton X-100, and 0.1 Mcitrate-NaOH buffer (pH 6.0) in a final volume of 1.0 ml. After addition of the enzyme, the reaction mixture was incubated with shaking at 35 °C for 10 min. The fatty acids released from MGDG were extracted and determined with rhodamine 6 G reagent. 2.10. Determination of ATP contents Approximately 5 g of frozen tissue were homogenised in 2 ml of 1% (w/w) trichloroacetic acid (TCA) in water. Extraction took place on ice for 35 min. Samples were centrifuged at 10,000g for 10 min; 0.1 ml of supernatant was diluted with 9 ml of a freshly prepared 40 mM Tris–acetate buffer (pH 8.0). For determining ATP amounts in the extracts, a luciferin/luciferase kit (ENLITENÒ ATP Assay System, Promega) was used. Twenty five microlitres of sample, diluted in Tris–acetate buffer, were mixed with 25 ll of reagent from the kit in tubes. The light emission by the reaction was determined with a Veritas™ luminometer (Turner BioSystems, USA). 2.11. Statistical analysis All statistical analyses were performed by SPSS 11.0 (SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) was used to compare means. Mean separations were performed by Duncan’s multiple range test. Differences at P 6 0.05 were considered as significant. 3. Results 3.1. Development of chilling injury in peach fruit Chilling-injured peach fruit first showed a reddish-brown discoloration near to the stone, then this discoloured part later turns to a darker brown colour, often with grey–brown water-soaked areas extending from stone into flesh (Fig. 1A). The symptom appeared in the fruit stored at 5 °C for 21 d, and CI incidence reached 100% with CI index of 100% at 28 d after storage (Fig. 1B). There were no visible symptoms of CI in the fruit stored at 0 °C throughout the storage periods (Fig. 1). But 0 °C-CA storage could reduce CI incidence and CI index of peach fruits when they were translated to 25 °C in air for 3 d shelf time, as compared with the 0 °C-Air storage (data not shown). 3.2. Changes of plasma membrane fluidity EPR spectra of lipid-incorporated with 5- and 16-DSA spin, which labels at two different positions along the hydrocarbon chain, are shown in Fig. 2A and C, respectively. 5-DSA spin was recorded on the mobility of the double bilayer regions close to the polar lipid head groups, and 16-DSA spin recorded on the lipid mobility close to the core of the bilayer. Spectra of 5-DSA could be further analysed by determining S value, which is inversely related to membrane lipid fluidity. In comparison with that at the start of storage, S values of membrane lipid were lower in all sam-

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period (P > 0.05) (Table 1). The DBI of plasma membrane lipids in the fruits stored at 0 °C was significantly higher than that at 5 °C after 21 or 28 d (P < 0.05) (Table 1). 3.4. Occurrence and identification of NAPE in plasma membrane lipid of peach fruit

Fig. 1. Symptoms of CI in peach fruit at 28 d after storage (A), and CI incidence and CI index (B) of peach fruit stored in 5 °C-Air (black column) for 21 or 28 d, respectively. Both the CI incidence and CI index of peach fruits in 0 °C-Air or 0 °C-CA were zero. Data are the means ± SE of three replications.

ples at day 7 (Fig. 2B), suggesting that plasma membrane fluidity was enhanced in the early stage of storage. With prolonged storage time, the S values of plasma membrane increased in all samples; however, the plasma membrane fluidity of peach fruits stored at 5 °C was significantly lower (higher S value) than that kept at 0 °C (P < 0.05) (Fig. 2B). Higher fluidity of plasma membrane was found in the fruits stored in 0 °C-CA than 0 °C-Air after 21 and 28 days (P < 0.05) (Fig. 2B). Spectra of 16-DSA could be characterised by sc value for the spin probe motion by assuming that the motion was in the fast regime and isotropic. The sc value, which is inversely related to plasma membrane fluidity, was significantly longer in 5 °C peach fruits than in 0 °C (P < 0.05) peach fruits (Fig. 2D), indicating more hindered dynamics of the spin probe in the lipid bilayer of the fruits at 5 °C. In addition, the sc value of plasma membrane proved to be significantly longer in the fruits at 0 °C-Air than in 0 °C-CA in the later stage of storage (P < 0.05) (Fig. 2D).

3.3. Changes of fatty acids composition in total polar lipid from plasma membrane To investigate the relationship between plasma membrane fluidity and membrane lipid composition, fatty acid composition and DBI of plasma membrane lipid from the various storage conditions are shown in Table 1. The C18:3 level of plasma membrane in peach fruits stored at 0 °C was significantly higher (P < 0.05), but the levels of palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2) were significantly lower than those kept at 5 °C (P < 0.05). No significant difference of the C18:3 level in plasma membrane lipid was found in peach fruits stored at 0 °C during this

When the phospholipids of plasma membrane were fractionated by TLC, a spot, near the front of the plate, was observed in 0 °C-CA peach fruit (Fig. 3A, lane 3). However, this low polarity compound could not be found in the phospholipid fraction of 5 °C-Air or 0 °C-Air peach fruit after 21 or 28 d (Fig. 3A, lanes 1 and 2). This compound had a slightly higher mobility than had N-palmitoyl-O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-ethanolamine (Fig. 3A, lane 4). A building block analysis of this compound revealed a fatty acid to phosphorus molar ratio close to three, so we suspected it to be NAPE. The purified phospholipids were then analysed by ESI-MS in the negative mode. The instrument response was first checked with synthetic N-palmitoyl-O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)-ethanolamine. This standard NAPE gave a single [MH] (molecular mass minus proton) peak at m/z 928.94 (Fig. 3B, upper spectra), in full agreement with the value of 929.74 computed from the sum of its constituents (Table 2). Fig. 3B shows that the high mass region of the spectrum (from m/z 925–985) presents several molecular ions that are perfectly consistent with a single phospholipid class showing the general NAPE structure. After protonation, the m/z value of all peaks in Fig. 3B differed by less than 0.3‰ from their expected NAPE masses (Table 2, columns 3 and 4). Altogether, these data allow one to assign an unequivocal NAPE identity to this group of molecular ions. From the m/z values reported in Fig. 3B, several NAPE species could be identified, which were divided in three groups according to their number of acyl carbons (Table 2). Without any consideration of positional specificity of acyl residues, group I was made of 16/16/16 species, group II of 16/16/18 species and group III of 16/18/18 species. As shown in Fig. 3B, a group can be constituted of peaks with different m/z values, called subgroups. More than one NAPE molecular species may be present in a subgroup because several combinations of acyl residues can give the same m/z value. For instance, subgroup III-b can be made up of two individual NAPE species, 16:0/18:2/18:2 and 16:0/18:1/18:3 (Table 2), that all collected at m/z 976.942 (Fig. 3B). Subgroup III-c can be made up of two individual NAPE species, 16:0/18:0/18:3 and 16:0/18:1/18:2 (Table 2), that all collected at m/z 978.942 (Fig. 3B). Other subgroups (I, II-a, II-b and III-a) contained a single NAPE molecular species (Table 2). In addition, no difference in NAPE species or their relative abundance was found in 0 °C-CA peach fruit after 21 and 28 d (Table 2). 3.5. The relative contents and DBIs of individual lipids separated from total polar lipids in plasma membrane We separated digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), phosphatidyl choline (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositols (PI) and free fatty acid (FFA) from plasma membrane of peach fruits (Fig. 4A). The relative contents (mol%) of PC, PE and FFA are shown in Fig. 4C. The FFA level was highest in the fruits stored in 0 °C-CA, but lowest in 5 °C-stored fruits. The PC and PE levels in 5 °C-stored peach fruits were significantly lower than in 0 °C-stored fruits (P < 0.05). However, there were no differences in the PC and PE levels between the fruits stored in 0 °C-Air and 0 °C-CA (P > 0.05) (Fig. 4C). The major fatty acids in PC, PE and FFA from all samples were C16:0, C18:0, oleic acid (C18:1 n9), C18:2 and C18:3 (Fig. 4B). The DBI of FFA was highest

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Fig. 2. Examples of EPR spectra of the membrane lipid-incorporated 5-DSA (A) and 16-DSA spin (C), and dynamic changes of the fluidity of hydrophobic core (B, 5-DSA spin labelling) and hydrophobic regions nearer to hydrophilic face (D, 16-DSA spin labelling) of plasma membranes in peach fruit stored in 5 °C-Air (—d—), 0 °C-Air (—s—) and 0 °C-CA (—.—). Data are the means ± SE of three replications.

Table 1 The fatty acid profile of polar lipids extracted from enriched plasma membrane fractions in peach fruit stored in different conditions over the 21 d or 28 d periods. Storage time

Treatment

Fatty acid profile of polar lipids (mol %) C16:0

21 d

28 d

5 °C-Air 0 °C-Air 0 °C-CA 5 °C-Air 0 °C-Air 0 °C-CA

28.5 ± 0.83 24.6 ± 0.42 24.5 ± 0.51 31.2 ± 1.23 25.6 ± 1.03 24.0 ± 0.96

C18:0 a b b a b c

7.07 ± 0.33 2.07 ± 0.29 2.19 ± 0.08 11.1 ± 0.17 2.79 ± 0.11 2.16 ± 0.09

DBI C18:1

a b b a b c

21.6 ± 1.23 22.0 ± 1.23 22.3 ± 0.75 20.1 ± 1.26 22.1 ± 1.45 23.1 ± 1.49

C18:2 b ab a b ab a

17.3 ± 0.09 12.5 ± 0.05 12.3 ± 0.07 15.8 ± 0.52 12.4 ± 0.09 12.2 ± 0.89

C18:3 a b b a b b

25.5 ± 0.83 39.8 ± 0.52 39.1 ± 0.36 21.7 ± 0.87 37.2 ± 1.33 38.5 ± 1.39

b a a b a a

4.29 ± 0.83 6.24 ± 0.43 6.26 ± 0.71 3.74 ± 0.13 5.59 ± 0.49 6.23 ± 0.48

b a a b a a

The data are the averages and standard errors of values from three replicates per treatment. For each treatment, values in columns followed by different lowercase letters indicate significant differences at P < 0.05 level.

in 0 °C-CA-stored peach fruits, but lowest in 5 °C-stored fruits. The levels of DBI of PC or PE were significantly lower in 5 °C-stored fruits than in the 0 °C-stored fruits (P < 0.05) (Fig. 4D).

age time, LAH activity was significantly higher (Fig. 5A), but the ATP contents were significantly lower (P < 0.05) (Fig. 5B) in fruit stored in 0 °C-CA after 14 d than in fruits stored in 5 °C-Air or 0 °C-Air.

3.6. Characterisation of PLDa by Northern blot Changes in the levels of PLDa mRNA from peach fruit stored in different condition were analysed using a [32P] dCTP-labelled 701 bp of PLDa cDNA as the probe. The levels of PLDa transcripts were higher in peach fruits stored at 5 °C and attained a maximum at day 21, but were undetectable in the fruits kept at 0 °C during all storage periods (Fig. 4E). 3.7. Changes of LAH activity and ATP contents There were no significant differences in LAH activity or ATP contents in all fruits at 14 d of storage (P > 0.05). With prolonged stor-

4. Discussion The results obtained in this work highlight the role of plasma membrane composition and property in the tolerance of peach fruit to low temperature stress. Membrane fluidity is an important biophysical property that regulates membrane function by its effect on the orientation of integral membrane proteins and membrane permeability, and by its modulation of transmembrane transport processes (Los & Murata, 2004). EPR spectroscopy has been widely used for the quantitative assessment of the rotational mobility of lipid molecules and as a measure of molecular order within membranes (Berglund et al., 2002). Our results, obtained

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Fig. 3. One-dimensional TLC of the phospholipid fraction from plasma membrane lipid of peach fruit stored in 5 °C-Air (lane 1), 0 °C-Air (lane 2), 0 °C-CA (lane 3) and the standard NAPE (N-palmitoyl-DPPE) (lane 4) (A). Electrospray ionisation mass spectra (ESI-MS, negative mode) of the standard NAPE (N-palmitoyl-DPPE, upper spectra) and the putative NAPE class prepared from plasma membrane lipids of peach fruit stored in 0 °C-CA for 21 d (lower spectra) (B).

Table 2 ESI-MS identification, acyl composition and relative amounts of the main NAPE molecular species found in the plasma membrane lipid of peach fruit stored in 0 °C-CA for 21 d or 28 d. NAPE subgroup

m/z Value in negative mode

NAPE mass After proton addition

Formula weight

I II-a II-b III-a III-b

928.941 950.934 952.907 974.928 976.942

929.951 951.944 953.917 975.938 977.953

929.74 951.73 953.74 975.73 977.74

III-c

978.942

979.953

979.76

from the EPR spectroscopy, proved that plasma membrane fluidity was an important signal of CI in fruit, because peach fruit kept at

Acyl residues in each individual N2APE species (not positional) C16:0 C16:0 C16:0 C16:0 C16:0 C16:0 C16:0 C16:0

C16:0 C16:0 C16:0 C18:2 C18:2 C18:1 C18:0 C18:1

C16:0 C18:3 C18:2 C18:3 C18:2 C18:3 C18:3 C18:2

Relative abundance (% of total) 21 d

28 d

Peak height

Group

Peak height

Group

14.8 10.9 23.3 11.9 21.8

14.8 34.2

14.2 12.4 22.5 10.6 24.9

14.2 34.9

17.3

51.0

50.9

15.4

0 °C maintained a higher fluidity of plasma membrane than did that at 5 °C (Fig. 2) and thus led to some resistance to chilling stress

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Fig. 4. Examples of chromatogram of the plasma membrane lipid classes separated from each other by two-dimensional TLC (A) and examples of chromatogram of fatty acids in the plasma membrane lipid analysis using gas chromatograph–mass spectrometry (B). Changes in the relative content (in mol%) (C) and double bond index (DBI) (D) of FFA, PC and PE in the plasma membrane lipids, and the level of PLDa mRNA (E) in peach fruit stored in different conditions. A: 1 (unidentified), 2 (PI), 3 (PC), 4 (DGDG), 5 (PE), 6 (SQDG), 7 (PG) and 8 (FFA). B: 1(C16:0), 2 (C17:0, internal standard), 3 (C18:0), 4 (C18:1 n9), 5 (C18:2) and 6 (C18:3). C and D: 5 °C-Air (white pillar), 0 °C-Air (grey pillar) and 0 °C-CA (black pillar). E: 5 °C-Air (a), 0 °C-Air (b) and 0 °C-CA (c).

Fig. 5. Changes in the level of LAH activity (A) and ATP contents (B) in peach fruit stored in 5 °C-Air (—d—), 0 °C-Air (—s—) and 0 °C-CA (—.—).

(Fig. 1). A similar result was found in our recent study dealing with total membrane lipid of peach fruit, and we considered that higher fluidity of the total membrane lipid could reduce lipid phase changes of membranes, resulting in increase in membrane integrity of peach fruit (Zhang & Tian, 2009). The changes in the biophysical properties of plasma membrane probably involve changes in lipid composition of peach fruit. This study found that a lower unsaturation degree (DBI value) of membrane lipid resulted in lower membrane lipid fluidity (Table 1;

Fig. 2; Fig. 4D). The result is in accordance with those reported by Szalontai, Kota, Nonaka, and Muratas (2003); they confirmed the dependence of membrane lipid fluidity on the UFAs level in cyanobacteria and plant chloroplasts. In addition, peach fruit stored at 0 °C showed a higher C18:3 level, concomitant with a lower C18:2 level than did fruit stored at 5 °C (Table 1), indicating that the higher C18:3 level was largely responsible for the higher DBI value of peach fruit at 0 °C. The extent of desaturation of individual fatty acids is regulated genetically and environmentally, and

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temperature is a critically important environmental factor that regulates the extent of desaturation. The cold-induced expression of genes for fatty acid desaturase increases the extent of desaturation of fatty acids at low temperature in cyanobacteria (Deshnium et al., 2000; Los & Murata, 1998). Dyer, Chapital, Cary, and Pepperman (2001) observed that the C18:3 level in Saccharomyces cerevisiae expressed the Brassica omega-3 fatty acid desaturase (FAD). This was enhanced in response to a decrease in temperature, and omega-3 FAD was regulated by temperature at the post-transcriptional level. In a previous study, we proved that the accumulation of C18:3 in the membrane of peach fruit was due to the enhanced mRNA level of the omega-3 FAD, and the omega-3 FAD catalysed the conversion of C18:2 to C18:3 in membrane lipids (Zhang & Tian, 2009). Additionally, the content of MDA, which is often used as an indicator of lipid peroxidation caused by oxidative stress (Smirnoff, 1995), in the fruits stored in 0 °C-Air or 0 °C-CA, is much lower than that in 5 °C-Air (data not shown). Therefore, we considered that the higher C18:3 level was also dependent on the lower peroxidation of C18:3 in peach fruit stored at 0 °C. Another important outcome of this work is that a marked accumulation of the NAPEs is found in peach fruit stored in 0 °C-CA, and not found in the other two samples (Fig. 3; Table 2). The accumulation of NAPEs may help maintain fluidity of plasma membrane, and explain why peach fruit stored in 0 °C-CA showed higher membrane fluidity than that in 0 °C-Air over the 21 day or 28 d periods (Fig. 2). NAPEs were first reported in the mid 1960s as a minor constituent in wheat flour and their occurrence soon became associated with seeds of higher plants (Chapman, 2000). Recent evidence has indicated that the NAPEs appeared to be biosynthesized as part of a protective mechanism in cell membranes, and NAPEs biosynthesis was observed after elicitor treatment of tobacco cells (Chapman, Conyers-Jackson, Moreau, & Tripathy, 1995) or after abscisic acid treatment of cotton seedlings (Chapman & Sprinkle, 1996). It has been shown that plant NAPE is synthesized exclusively by acylation of the amino group PE with FFAs (Rawyler & Braendle, 2001). Our results showed that the accumulation of NAPEs in peach fruit, under the 0 °C-CA condition, was related to increased availability of PE and FFAs (Fig. 4C). A higher PE level was maintained in peach fruit stored at 0 °C than at 5 °C (Fig. 4C) which was related to 0 °C condition repressing the Phospholipase D (PLDa) mRNA level (Fig. 4E). Stimulation of PLDa transcriptional level was shown in plants in response to treatments of ABA, light, fungal elicitors, bacterial pathogens and wounding injury (Wang, 2000). From the results reported here, new information is added to our understanding of the role of PLDa in CI. The higher FFAs level was positively related to the higher LAH activity in peach fruits stored in the 0 °C-CA condition, and the LAH activity may be activated by lower ATP contents (Figs. 4C and 5). Earlier studies showed that FFAs accumulated in leaf cells when they were subjected to stresses such as wounding (Conconi, Miquel, Browse, & Ryan, 1996), water deficit (Wilson, Burke, & Quisenberry, 1987), elicitor (Mueller, Brodschelm, Spannagl, & Zenk, 1993) and ozone treatment (Sakaki, Tanaka, & Yamada, 1994). Rawyler, Pavelic, Gianinazzi, Oberson, and Braendle (1999) reported that FFAs could be provided by LAH hydrolysing membrane lipids, and LAH activity was triggered whenever ATP production rate falls below a threshold level. In summary, we have demonstrated that lipid rearrangements of plasma membranes occur in response to low temperature, and this process is accompanied by adaptive changes in membrane biophysical properties. The C18:3 in membrane lipid plays a vital role in fruit acquiring chilling tolerance because the higher C18:3 level is largely responsible for the higher unsaturation degree of membrane lipid, thus maintaining membrane fluidity. Additionally, the accumulation of the NAPE is first found in peach fruit stored in 0 °C-CA, indicating that NAPEs may be beneficial for maintaining

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