JXB Advance Access originally published online on February 10, 2006
Journal of Experimental Botany 2006 57(4):897-909; doi:10.1093/jxb/erj075
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RESEARCH PAPER |
Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthase II (KASII) genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves
1Finnish Forest Research Institute, Rovaniemi Research Station, PO Box 16, FIN-96301 Rovaniemi, Finland
2Department of Biological and Environmental Sciences, Faculty of Biosciences, University of Helsinki, PL 56, FIN-00014 Helsinki, Finland
* To whom correspondence should be addressed. E-mail: francoise.martz{at}metla.fi
Received 30 May 2005; Accepted 25 November 2005
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Abstract |
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Temperate and boreal tree species respond to low positive temperatures (LT) or a shortening of the photoperiod (SD) by inducing cold acclimation. One of the metabolic consequences of cold acclimation is an increase in fatty acid (FA) desaturation in membrane lipids, which allows functional membrane fluidity to be maintained at LT. The molecular mechanisms of FA desaturation were investigated in leaves of birch seedlings (Betula pendula) during cold acclimation. Four genes involved in FA biosynthesis were isolated: a 3-ketoacyl-ACP synthase II gene (BpKASII) involved in the elongation of palmitoyl-ACP to stearoyl-ACP, and three
-3 FA desaturase genes (BpFAD3, BpFAD7, and BpFAD8) involved in the desaturation of linoleic acid (18:2) to 
-linolenic acid (18:3). BpFAD7 was the main -3 FAD gene expressed in birch leaves, and it was down-regulated by LT under SD conditions. LT induced the expression of BpFAD3 and BpFAD8 and a synchronous increase in 18:3 occurred in glycerolipids. Changes in the photoperiod did not affect the LT-induced increase in 18:3 in chloroplast lipids (MGDG, DGDG, PG), but it modulated the LT response detected in extra-chloroplastic lipids (PC, PE, PI, PS). A decrease in the proportion of the 16-carbon FAs in lipids occurred at LT, possibly in relation to the regulation of BpKASII expression at LT. These results suggest that LT affects the whole FA biosynthesis pathway. They support a co-ordinated action of microsomal (BpFAD3) and chloroplast enzymes (BpFAD7, BpFAD8) in determining the level of 18:3 in extra-chloroplastic membranes, and they highlight the importance of dynamic lipid trafficking. Key words: Cold acclimation, fatty acid, freezing resistance, linoleic acid, low temperature, omega-3 fatty acid desaturase, short photoperiod
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Trees and perennial plants growing in temperate and boreal zones have to face freezing temperatures, occasionally during the growing season and annually during winter. The process of increased freezing resistance after a period of low positive temperature is known as cold acclimation. Cold acclimation is acknowledged to be a multigenic trait, involving a number of biochemical and physiological changes such as changes in sugars, soluble proteins, proline and inorganic acids, membrane lipid composition, and the appearance of new proteins (Kaye and Guy, 1995
; Thomashow, 1999). The acclimation process in late summer includes two main stages (Weiser, 1970). First, a decrease in the photoperiod induces cessation of growth, development of dormancy and a moderate increase in freezing resistance. The second stage is triggered by low temperature (LT) and enables full freezing resistance and overwintering of the plant to survive low winter temperatures. LT alone can also induce freezing resistance in many cold-tolerant plant species and this induction is normally associated with adaptation to short-term cold periods rather than to seasonal changes. In such conditions, the aim is more to sustain metabolic activity during a short period than to organize plant survival over a long unfavourable period. In woody species a short photoperiod (SD) and LT can independently activate cold acclimation (Welling et al., 2002), but the combination of SD and LT results in a higher freezing resistance than exposure to SD or LT alone (Christersson, 1978; Li et al., 2002; Puhakainen et al., 2004).
Membranes are major sites of freezing injury and the process of cold acclimation allows their stabilization to prevent damage leading to cell death, as occurs in non-acclimated tissues. Membrane fluidity is directly affected by changes in temperature. Recent studies have demonstrated that membrane rigidification, coupled with cytoskeletal rearrangements, calcium influxes, and activation of MAPK cascades, triggers low temperature responses in alfalfa (Örvar et al., 2000; Sangwan et al., 2002). Plants grown at LT show an increased level of fatty acid (FA) unsaturation (Nishida and Murata, 1996). This increase allows the maintenance of a functional membrane fluidity and is essential for plant survival (Mikami and Murata, 2003; Los and Murata, 2004). Fluidity of membrane glycerolipids depends on the nature of the head group and the melting point of the FAs which, in turn, is determined by the unsaturation level and the length of the acyl chains. In plants, FA synthesis mainly occurs in plastids through the condensation of malonyl-CoA units catalysed by the 3-ketoacyl-ACP synthase (KAS), leading to the formation of palmitoyl-acyl carrier protein (16:0-ACP) and stearoyl-ACP (18:0-ACP). Three classes of KAS enzyme have been described: class III is involved in the first condensation of acetyl-CoA and malonyl-ACP, class I in the elongation of the carbon chain up to 16 carbons, and class II is involved in the last condensation steps from 14 and 16 carbons to 18 carbons (Harwood, 1996). Although the KAS enzymes catalyse only the first of the four reactions of each condensation step, they are believed to determine the substrate specificity and are probably rate-limiting enzymes in de novo FA biosynthesis. A class IV (similar to class II) has been described in Cuphea sp., but it appears to be specific to medium chain length FA, and reflects the high content of medium chain FAs in Cuphea seed oil (Dehesh et al., 1998). After its synthesis, 18:0-ACP is rapidly desaturated to oleoyl-ACP (18:1-ACP) in the chloroplast stroma by a soluble stearoyl-ACP desaturase. The 16:0 and 18:1 FAs are then utilized in two distinct pathways for the biosynthesis of membrane lipids: in the chloroplast (prokaryotic or chloroplastic pathway) or in the endoplasmic reticulum (ER) (eukaryotic or cytoplasmic pathway).
Due to different substrate specificities of the plastidial and microsomal 1-acylglycerol-3-phosphate acyltransferase (esterification of the second acyl group to the sn-2 position of the glycerol), glycerolipids synthesized by the chloroplastic pathway have a 16-carbon FA at the sn-2 position of the glycerol backbone, while those synthesized by the cytoplasmic pathway have a 18-carbon FA in the corresponding position (Murata and Tasaka, 1997). Further FA desaturation occurs by plastidial and microsomal isoforms of acyl-lipid membrane-bound FA desaturases (FAD): oleic acid (18:1) is successively desaturated to linoleic acid (18:2) and then to -linolenic acid (18:3) at positions 
12 and -3, respectively (Los and Murata, 1998; Tocher et al., 1998).
Three genes have been described in Arabidopsis to encode for the -3 FADs: one for a microsomal protein (FAD3), and two for plastidial proteins (FAD7 and FAD8) (Somerville and Browse, 1991; Iba et al., 1993; Yadav et al., 1993; Gibson et al., 1994). Complementation of the Arabidopsis fad7 mutant with the fad8 gene showed that both genes are functionally equivalent (Gibson et al., 1994).
Increased unsaturation of FAs during cold-acclimation has been shown to be due to increases in desaturase activities (Cheesbrough, 1989; Williams et al., 1992). In actual fact, the expression of plastid -3 desaturase genes is stress-inducible: wounding and light stimulate FAD7 in Arabidopsis (Nishiuchi et al., 1995) and parsley (Kirsch et al., 1997), respectively, while LT induces only FAD8 in Arabidopsis (Gibson et al., 1994) and maize (Berberich et al., 1998). The steady-state level of FAD7 mRNAs was not affected by LT in Arabidopsis (Iba et al., 1993) and wheat (Horiguchi et al., 1996), but expression of FAD3 was transiently induced upon LT exposure in etiolated hypocotyls of Brassica napus (Tasseva et al., 2004).
Analysis of several Arabidopsis mutants deficient in one or more FAD activities (reviewed by Wallis and Browse, 2002) and transgenic tobacco over-expressing FAD7 (Kodama et al., 1994), clearly showed that FA unsaturation is important for the temperature stress response and in chilling sensitivity. Taken together, all these data suggest that dienoic FAs are required for photosynthesis (McConn and Browse, 1998), while trienoic FAs are more specifically necessary for the biogenesis and maintenance of chloroplast at LT (Routaboul et al., 2000).
No data about the genetic regulation of FA composition by LT in a woody plant species are so far available. As a result, the mechanisms of such regulation were explored during cold acclimation in birch, an extremely frost-hardy tree species and economically the most important deciduous trees in Fennoscandia. In this paper, the isolation of birch -3 FAD and KASII genes is reported, as well as their expression in leaves during cold acclimation induced by a short photoperiod (SD), low temperature (LT), or a combination of both (SD+LT). As far as is known, these results represent the first report of the co-ordinated expression analysis of the three -3 FAD genes during the cold acclimation process in photosynthetic tissue. Changes in freezing resistance and FA composition in phospholipids and galactolipids are correlated with the gene expression data.
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Material and methods |
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Plant material
Silver birch seeds (Betula pendula, northern ecotype, Rovaniemi 66° 30' N, 25° 50' E, Finland) were germinated and sown in a peat–sand mixture and grown in a growth chamber with a 20 h photoperiod and a +16/+13 °C (day/night) temperature regime. Two-month-old birch seedlings were used for the experiments. Shorter photoperiods of 16 h (SD1) or 12 h (SD2) were tested. Low-temperature treatments were performed at +4/+2 °C (day/night) with a 20 h photoperiod. A treatment involving a combination of SD1 and LT was also tested: it consisted of 1 week of SD1 (+16/+13 °C, day/night), followed by 3 weeks under the same SD1 photoperiod, but at LT (named SD1+LT treatment). The whole experiment, including the three treatments (SD1+LT, LT, SD2), was replicated three times with three different batches of seed. Mature leaves were used for the analysis, and were always collected at the same time of day (middle of the photoperiod).
cDNA library screening
A 
ZAP II cDNA library prepared with polyA+ mRNA from birch (Betula pendula Roth.) leaves exposed to low temperature was provided by Professor T Palva, Faculty of Bioscience, University of Helsinki (Finland). Two screenings were performed with poplar EST clones homologous to the Arabidopsis FAD7 and KASII genes (clone nos C047P26U and CO41P80U, respectively, PopulusDB, Umeå University, Sweden: http://www.populus.db.umu.se), following the main manufacturer's instructions (-ZAP® II vector, Stratagene).
Southern blot analysis
Genomic DNA was extracted according to Aldrich and Cullis (1993). Fifteen µg genomic DNA were digested with BamHI, EcoRI, XbaI (Fermentas), separated on a 0.8% agarose gel and transferred to Hybond-N+ membrane (Amersham). Gene copies were detected with DIG PCR-labelled probes (Roche) corresponding to the 5'-untranslated region (BpFAD7, BpFAD8) or to the full-length cDNA (BpFAD7, BpKASII), after high stringency washes (0.5x SSC, 0.1% SDS, 65 °C).
Gene expression analysis
Total RNAs were isolated according to Chang et al. (1993) and resuspended in formamide:H2O (1:1, v/v). RNAs extracted from the same time point in each of the three experimental replicates were equivalently mixed (by weight) to give only one sample, representative of the three experiments. Five µg of total RNA were separated on formaldehyde agarose gel and blotted on a nylon membrane (Roche). After transfer of the ethidium bromide-stained RNA, the membrane was observed under UV-light to monitor RNA loading. Detection was performed after high stringency washes (68 °C in 0.2x SSC, 0.1% SDS) using DIG-labelled RNA or DNA probes (Roche). BpFAD7 and BpFAD8 were specifically detected with probes corresponding to their 5'-untranslated region. BpFAD7 and BpKASII were detected with PCR DIG-labelled probes corresponding to the full-length cDNA.
Lipid and fatty acid analyses
Total lipids were extracted as described in Sutinen (1992). Galactolipids (GLs) and phospholipids (PLs) were extracted from the total lipid extract by column chromatography on silica gel (100–200 mesh). GLs were eluted with chloroform:acetone (1:1, v/v) and acetone (5 vol. and 12 vol., respectively). PLs were eluted with chloroform:methanol (1:1, v/v) and methanol (6 vols and 7 vols, respectively). The extracts were dried under a nitrogen stream at 42 °C and part of the GL and PL fractions was used for direct transmethylation. The other parts of the GL and PL fractions were used for separation of individual lipids by one-dimensional TLC (silica gel 60, Merck). TLC plates were prewashed with chloroform:methanol (2:1, v/v), air-dried and activated at 110 °C for 1 h. GLs were separated with the solvent system acetone:acetic acid:water (100:4:2, by vol.) and PLs with the solvent system chloroform:acetone:methanol:acetic acid:water (30:40:10:10:5, by vol.). Lipids were visualized by primuline spraying [5% (w/v) in acetone:water (8:2, v/v)] under UV-light and identified by co-migration with commercial lipid standards (Sigma). Fatty acid methyl esters were obtained by methylation with 0.6 N NaOH-methanol, neutralized with 0.6 N HCl and extracted twice with hexane. Heptadecanoate methyl ester (17:0) was added before transmethylation and used as an internal standard. Fatty acid methyl esters were separated by gas chromatography on a S-2330 column (Supelco) using the following conditions: 190 °C for 12 min, increase from 190–220 °C at 10 °C min–1, and 220 °C for 30 min.
Freezing resistance
Freezing resistance of the leaves was measured using the ion leakage method. Leaves were placed in 15 ml closed tubes and frozen in an alcohol bath at a cooling rate of 3.6 °C min–1. Each sample (three replicates) was frozen to six different temperatures, 3 °C apart, selected within the range +4 °C and –24 °C according to the progress of the experiment. At a selected temperature, the tubes were removed from the bath and stored in ice overnight for slow thawing. Distilled water was slowly infiltrated in the leaves and the conductivity of the water measured after 20 h shaking at 20 °C in the dark (initial conductivity). Tissues were autoclaved and ion conductivity was measured after 12 h shaking (final conductivity). The relative ion leakage was calculated as the (intial/final conductivity)x100, and the temperature causing 50% ion leakage (LT50) was estimated as the inflection point of the sigmoid curve of relative ion leakage versus temperature [controls=unfrozen samples (+4 °C)] (Sutinen, 1992).
Photochemical efficiency
Photochemical efficiency was measured on dark-adapted leaves with a Plant Efficiency Analyser (Hansatech) with a light intensity of 2400 µmol m–2 s–1 and at the same temperature as the growth temperature. A minimum of 30 measurements was made for each time point (10 measurements for each experimental replicate).
Sequence analysis
Sequence alignment was performed with ClustalX (Thompson et al., 1997) and phylogenic trees with MEGA2.1 (Kumar et al., 2001) using the Neighbor–Joining method with poisson correction and 1000 replications in the bootstrap test. BioEdit was used for DNA and protein sequence analysis.
Statistical analyses
The degree of association between the freezing resistance and the double bond index (16:1+18:1+2x18:2+3x(16:3+18:3)) measured in lipid fractions or individual lipids was calculated using the Pearson correlation and expressed as the correlation coefficient.
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Results |
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Isolation and characterization of
-3 fatty acid desaturase birch clonesA cDNA library made of cold-exposed birch (B. pendula Roth.) leaves was screened with a poplar EST probe selected for its homology with Arabidopsis FAD7. Three complete cDNAs, coding for proteins of 444, 455, and 386 amino acids (accession numbers AY135564-AY135566), were isolated. All of the three proteins contained the three His clusters typical of plant
-3 acyl-lipid desaturases (Los and Murata, 1998). Analysis with the TargetP Server (Emanuelsson et al., 2000) predicted a chloroplast location for two of the proteins (444 and 455 residues), while the chloroplast was excluded as a candidate location for the shortest protein (386 residues). The putative plastid protein isoforms shared 67–71% overall identity with Arabidopsis FAD7 or FAD8, and the shortest protein showed 69% overall identity with Arabidopsis FAD3. According to their sequence homologies with -3 FAD proteins of other plant species (Fig. 1A) and their expression profile in the birch leaves (see below), the isolated clones were named BpFAD7, BpFAD8, and BpFAD3. Generation of a phylogenic tree with FAD3, FAD7, and FAD8 protein sequences from several plant species showed that the three birch proteins are clearly different from each other (Fig. 1A). In contrast to an earlier report for Arabidopsis (Gibson et al., 1994), the birch BpFAD7 and BpFAD8 genes do not segregate together. Interestingly, FAD3 and FAD8 appeared to be closely related to the corresponding Glycine max sequences. The gene copy number of the -3 FAD genes was assessed in birch by Southern-blot hybridizations under high-stringency conditions. Probes corresponding to the 5' part of BpFAD8 and BpFAD7 were used, while the full-length cDNA was used to generate BpFAD3 PCR probes. None of the cDNA was cut by the restriction enzymes used and the results suggested that each of the three -3 FADs is present as a single copy in the birch genome (Fig. 2A, B, C).
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Isolation and characterization of a birch KASII cDNA
In a similar manner as for the isolation of the
-3 FAD cDNAs, a poplar EST clone homologous to Arabidopsis KASII was used to screen the same birch cDNA library. A clone of 841 bp was isolated (accession number AY845865), encoding for a partial protein of 177 amino acids and showing sequence homology with KASII genes from plants. A phylogenic tree was generated with several plant KAS protein sequences of classes I, II, and IV. All three classes were involved in a similar elongation reaction, but differed in the length of the carbon chain of their substrate. The tree confirmed that the isolated clone belongs to class II (Fig. 1B) and the clone was consequently named BpKASII. Perilla frutescens, and Glycine max KASII appeared to be the closest homologues of the partial birch sequence. Hybridization of genomic DNA with a BpKASII probe revealed one band after digestion by BamHI and XbaI (Fig. 2D). Two bands were detected after EcoRI digestion, which can be explained by the presence of at least one EcoRI restriction site in the BpKASII cDNA (one site present in the partial sequence). These results suggest that BpKASII is present as a single copy in the birch genome. By contrast, two copies have been found in the Perilla frutescens genome (Hwang et al., 2000).
Induction of freezing resistance by the different treatments
Similar to many woody species, northern birch ecotypes are highly sensitive to changes in temperature and photoperiod (Li et al., 2002, 2004), both of which induce the development of freezing resistance. Cold acclimation of 2-month-old birch seedlings was induced in growth chambers by decreasing the growth temperature [+16 °C to +4 °C (LT)] or shortening the photoperiod [20 h to 16 h (SD1) or 12 h (SD2)], or by a combination of the two (SD1 for 1 week followed by SD1+LT for 3 weeks). The freezing resistance was measured using the ion leakage method that reflects the plasma membrane integrity after a freeze/thaw cycle. The treatment that most effectively induced a rapid increase in freezing resistance (decrease in LT50) was LT (Fig. 3A). LT and SD2 induced an increase in freezing resistance with different rates, reaching a plateau at LT50 of about –10 °C after 2 weeks. With the SD1+LT treatment, the plateau was reached after 3 weeks (including 2 weeks at LT) at –12 °C.
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Changes in photochemical efficiency
The photochemical efficiency (Fv/Fm) was measured in the leaves at the same temperature as their growth temperature (Fig. 3B). LT induced a fast decrease in Fv/Fm within the first days of treatment. The results of exposure to SD2 and the first week of the SD1+LT treatment indicated that SD did not affect Fv/Fm. Pre-treatment of the leaves at SD appears to prepare the leaf tissues to sustain the following cold stress and helps in maintaining a certain photochemical efficiency for at least one week. The conditioning effect of the SD pre-treatment was also evident by the increase in freezing resistance under the SD conditions (Fig. 3A).
Changes in expression of the -3 FAD and KASII genes
Leaf RNAs were extracted at the same time points in each of three experimental replicates. All the time points from each replicated experiment were checked for expression of the BpFAD7, BpFAD8, BpFAD3, and BpKASII genes. Small differences were observed in the strength or the timing of gene induction between the three experimental replicates, but the expression profiles were all similar. In order to obtain the most correct overall picture, and in accordance with the results shown in Figs 3, 5, 6, and 7, it was decided to mix the corresponding RNA samples from each experiment equally. Northern-blot analysis of these mixed samples provided an average picture of the changes in gene expression during cold acclimation in birch leaves.
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Different time 0 samples were collected and analysed in each experiment. The use of different northern blots for the analysis of the different set of samples (SD1+LT/LT, SD2) is an additional explanation of the apparent inconsistent gene expression levels on control samples.
In the control leaves (+16 °C, long days), BpFAD7 was highly expressed while BpFAD8 and BpFAD3 were barely detectable (Fig. 4). No significant changes were noticed under SD conditions (SD1 and SD2), but the situation clearly changed when the temperature decreased. BpFAD8 and BpFAD3 were both cold-induced, but with different kinetics: BpFAD3 exhibited a rapid but transient response to LT, while BpFAD8 mRNAs were still detected after 2 weeks. BpFAD3 was previously reported to be constitutively expressed in birch leaves under cold stress (Martz et al., 2003). However, the results were obtained using full-length RNA probes which appeared to be non-specific. DNA probes were used later and the cold-induction of BpFAD3 in birch, as well as the expression profiles of BpFAD7 and BpFAD8, were confirmed by quantitative RT-PCR (not shown). LT itself did not significantly affect the expression of BpFAD7 after 2 weeks but, in combination with SD (SD1+LT), it progressively down-regulated BpFAD7 expression (Fig. 4: LT 0 d and 14 d in comparison with SD1+LT 7 d and 21 d). BpKASII mRNAs were detected in the birch leaves. However, the SD conditions did not significantly affect its expression, but LT slightly stimulated the gene expression after 1 week of treatment (Fig. 4). Leaves newly developed at LT were also analysed (Fig. 4: LT, 14diamond)), but only for gene expression due to the small amount of material available. Expression of BpFAD7 and BpFAD8 was similar in leaves developed at +4 °C than in leaves developed at +16 °C and exposed to LT for 2 weeks. By contrast, BpKASII, and mainly BpFAD3, were more expressed in the new LT-developed leaves compared with the leaves developed at +16 °C and cold-stressed for 2 weeks.
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Leaf glycerolipids were extracted and analysed for their FA composition in order to gain a better understanding of the role played by these changes in gene expression during cold acclimation.
FA composition in glycerolipids
Total leaf lipids were extracted and the galactolipid (GL) and phospholipid (PL) fractions were separated on a silica column. Both fractions were first analysed for their FA composition and further separated by TLC to purify the main lipids. Only FAs of 16 and 18 carbons were taken into account in calculating the FA composition: FAs of more than 18 carbons represented about 1% and 2% of all the FAs in the GL and PL fractions, respectively.
Galactolipids
GL fractions from several control samples were analysed and 18:3 was found to represent the major FA with 83.9% (mol%). Minor FAs were 16:0 (7.7%), 18:2 (3.4%), 16:3 (2.7%), 18:1 (1.3%), while 16:1 and 18:0 each represented less than 0.5% (mol%). LT induced changes in FA composition in the GL fractions. Three FAs were mainly affected within the first week at LT: 18:3 increased with concomitant decreases of 18:2 and 16:0 (Fig. 5). Similar ranges were observed after 1 d at LT, with or without a SD pretreatment.
Monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) were purified from the GL fractions. Compared with DGDG, MGDG was characterized by a higher 18:3 (89.7% versus 78.8%) and 16:3 content (4.2% versus 2.1%) but a lower 16:0 content (1.5% versus 14.5%). DGDG is mainly synthesized from ER-derived MGDG (Kelly and Dörmann, 2004), which explains its lower 16:3 content compared with MGDG. DGDG was more affected than MGDG at LT. The increase in the proportion of 18:3 at LT was relatively regular in both lipids, but was higher in DGDG than in MGDG. An increase in 18:3 was also detected under SD conditions, especially in DGDG. In all conditions, the increase in 18:3 was mainly compensated by a decrease in 16:0 in DGDG. The main 16-carbon FA in MGDG is 16:3. Its relative content slightly decreased after several days at LT, but not under SD conditions.
Phospholipids
Each PL had a different FA composition and the major FAs were 18:2, 18:3, 16:0, and 18:1, 16:1 only being found in phosphatidyglycerol (PG) (Fig. 6). In PG, which is synthesized almost exclusively in the chloroplast envelope, changes in 18:3 showed a similar pattern as that observed in DGDG under all the conditions tested (including SD2), although the increase in 18:3 was higher than that measured in DGDG during the first few days at LT. The proportion of 16:1 increased only during the first 3 d at LT. Compensating changes were found in 18:3 and (18:1+18:2) on the one hand, and in 16:0 and 16:1 on the other hand.
By contrast with the response observed in chloroplast-synthesized lipids (MGDG, DGDG, PG), a different LT response occurred in phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol+phosphatidylserine (PI+PS) in the LT and the SD1+LT treatments. In the LT treatment, an increase in the proportion of 18:3 was found during the first few days (in PC, PE, PI+PS), and again after the first week (in PC and PE) at LT. In the SD1+LT treatment, a significant increase in 18:3 was not detected during the first few days at LT, but only after the first (in PC) or the second (in PE) week at LT (the high standard deviation bars in PE on the first day at LT makes this point unreliable). The amount of 18:3 did not significantly change in PI+PS during the SD1+LT treatment. The FA composition of PC was also modulated under the SD2 conditions, with a slight decrease in 18:3 during the first week of the treatment. Globally, when the FA composition was modified, all four FAs were affected and a decrease in 16:0 generally occurred when 18:3 was increasing. PC was characterized by an increase in 18:1 before the increase in 18:3.
C16 FAs versus C18 FAs
Changes in the length of the FA carbon chains became clear when the sum of all 16-carbon (C16) FAs was plotted as a function of time (Fig. 7): a specifically cold-induced decrease in the amount of C16 FAs was found in all the glycerolipids except PG, in which both SD1 (first week of SD1+LT treatment) and SD2 had a similar effect to LT. In PC and PE (partly in PI+PS) it was especially clear that SD (SD1 or SD2) had no effect on the abundance of C16 FAs.
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Discussion |
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Gene isolation and induction of cold acclimation in birch
The protein sequences of
-3 FADs are highly conserved, and this conservation has earlier allowed the isolation of -3 FAD cDNAs from plant species using heterologous probes (Yadav et al., 1993; Hamada et al., 1994; Berberich et al., 1998). In this work, three birch -3 FAD and one KASII cDNA clones were isolated from a cDNA library using poplar EST probes. According to their homology with the Arabidopsis FAD3, FAD7, and FAD8 and their expression profiles in birch leaves, the three birch -3 FAD clones were named BpFAD3, BpFAD7, and BpFAD8, respectively. Genome analysis suggested that each of the genes analysed in this work exist as a single copy in the birch genome (Fig. 2). The BpFAD8 sequence was used for homology searches in the birch EST library recently created at the University of Helsinki (73 881 ESTs, T Palva et al., Helsinki): among the 15 ESTs showing significant homology with BpFAD8, eight ESTs corresponded to microsomal and plastidial forms of the 12 FAD (FAD2, FAD6), and the other seven ESTs all corresponded to one of the -3 FAD gene described in this work. However, the presence of any other -3 FAD gene cannot be ruled out in the birch genome.
Cold acclimation was induced in the birch leaves by decreasing the growth temperature (LT) or the photoperiod (SD2), or a combination of both (SD1+LT). The development of cold acclimation was estimated by measuring freezing resistance using the ion leakage method that reflects the level of plasma membrane damage after a freeze/thaw cycle. Different rates of increase in freezing resistance were induced by the different treatments, and LT was the most effective treatment to induce a fast increase in the freezing resistance (Fig. 3A). Irrespective of the treatment applied, a stable level of freezing resistance was reached after 2 weeks. An additive effect of SD+LT was found, as shown by the higher level of freezing resistance at the end of this treatment. Similar levels of freezing resistance were obtained by Li et al. (2002, 2004) in birch leaves grown at LT under different photoperiods. Many studies have reported correlation between the level of FA unsaturation and the chilling sensitivity of plants, and also shown that FA unsaturation is clearly involved in the temperature stress response (Nishida and Murata, 1996; Wallis and Browse, 2002). In birch leaves, the double bond index [16:1+18:1+2x18:2+3x(16:3+18:3)] measured in the PL fractions (PC, PE, PG, PI+PS) rather than in the GL fractions (MGDG, DGDG, sulpholipids) correlated with changes in the freezing resistance induced by LT (r= –0.84 and –0.57, respectively). At the level of the individual glycerolipids, the double bond index of PG, PC, PE, DGDG, but not of MGDG, showed a similar correlation (r= –0.75, –0.71, –0.73, –0.82, and –0.33, respectively).
Expression of the -3 FAD genes and regulation of FA unsaturation at LT
BpFAD7 was the main -3 FAD gene expressed in the non-acclimated birch leaves, while BpFAD3 and BpFAD8 were barely detectable (Fig. 4). A similar situation has been observed in Arabidopsis, where only FAD7 was expressed in expanding leaves and FAD3 expression was restricted to the shoot meristem during vegetative growth (Matsuda et al., 2001).
Analysis of non-acclimated birch leaves showed that each purified glycerolipid had a different FA composition. Using the extraction procedure described in the Materials and methods, sulpholipids were present in the GL fractions, but they were not further purified and analysed for their FA composition in this study. Since they remain minor glycerolipids, these results for the total lipid composition can be compared with the results previously described in birch (Selstam and Hällgren, 1989). The lipid composition determined in non-acclimated leaves of B. pendula was comparable with that reported in B. pubescens or B. tortuosa (Selstam and Hällgren, 1989), with about 40% MGDG, 30% DGDG, 15% PC, 6% PE, 6% PG, and 3% PI+PS (mol%).
Transcription of BpFAD7 was not affected by a 2-week LT treatment under long day conditions, but it was down-regulated when LT was combined with SD (Fig. 4), a simulation of outdoor conditions in the autumn. A similar SD potentiation of the LT response was observed in up-regulation of a dehydrin gene in birch, possibly by the activation of different signal transduction pathways by SD and LT (Puhakainen et al., 2004). Expression of BpFAD3 and BpFAD8 was induced after the downward shift in temperature. Interestingly, the expression profiles of the two genes were different: BpFAD3 was rapidly and transiently induced for few days, while BpFAD8 showed a constitutive up-regulation.
In relation to the cold-induction of the -3 FAD gene expression, the relative amount of 18:3 FAs increased in the lipids. However, this increase differed between the lipid species and was not regular (Figs 5, 6). The lipids most affected by the downward shift in temperature were MGDG, DGDG, and PG, MGDG being the least and PG the most modified. Two phases of 18:2 desaturation were distinguished: a rapid phase during the first few days of LT exposure and a second phase occurring the second week of the LT exposure. Both 18:2 desaturation phases occurred in chloroplast lipids (MGDG, DGDG, and PG) under long- and short-day conditions. They were also found in extra-chloroplastic lipids (PC, PE) under long-day conditions but not under SD conditions, where only the second and late phase of 18:2 desaturation was detected. Induction of BpFAD3 and BpFAD8 was similar under long- or short-day conditions during the first few days at LT, which cannot explain the down-regulation of the first phase of 18:2 desaturation detected under the SD conditions in extra-chloroplastic lipids. In chloroplast lipids (GL fractions, PG), the second phase of 18:2 desaturation appeared to be more important in the LT than in the SD1+LT treatment. This may be due to the continuously high expression of BpFAD7 during the LT compared to the SD1+LT treatment. FAD7 and FAD8 have been shown to be functionally equivalent (Gibson et al., 1994), but no data are available about the catalytic activity of FAD7 at LT.
The relative amount of 18:3 FAs increased significantly in DGDG and PG after 1 week under SD1 or SD2 conditions. The steady-state amount of the BpFAD7 mRNAs increased slightly after 1 week of the SD2 but not the SD1 treatment, which suggests that BpFAD7 was not involved in this increased unsaturation. Compared with what was observed at LT, no significant induction of BpFAD8 or BpFAD3 was detected under SD conditions (Fig. 4). Although the presence of another birch -3 FAD gene cannot be excluded (see above), these results suggest that either a post-transcriptional regulation of the -3 FAD gene(s), or mechanisms other than a direct unsaturation of 18:2, are involved in the SD-induced regulation of the trienoic FA content in birch leaf membranes.
Modulation of the lipid 18:3 FA content in the birch leaves appeared to be relatively low compared with the strong gene induction at LT, especially of BpFAD8. One reason for this discrepancy is that the FA composition was expressed as mol% of total FAs, which does not take into account changes in the amounts of lipid. In actual fact, an increase in the amounts of PLs and GLs by about 50% of their control value occurred in birch leaves after 2 weeks at LT, but not under SD conditions (data not shown). Another reason could be regulation at the post-transcriptional level of the -3 FAD genes. The existence of a post-translational regulation of FAD8 was recently demonstrated by the role of the C-terminal end of FAD8 in the heat instability of the protein (Matsuda et al., 2005). Cold activated the transcription of FAD3 in Brassica napus (Tasseva et al., 2004), but also induced an increase in the steady-state amount of the FAD3 protein (Dyer et al., 2001). Similarly, cold increased the translation or the protein stability of FAD3 in wheat (Horiguchi et al., 2000).
The proportion of 18:3 FAs was increasing after 2 weeks at LT in PC and PE (second phase of 18:2 desaturation), and the expression profiles of BpFAD8 and BpFAD3 suggest that the plastidial enzymes rather than the microsomal enzyme were involved in this late unsaturation increase. A role of the plastidial enzymes in the 18:3 content of extra-chloroplastic lipids has been previously described in Arabidopsis. Analysis of several fad mutants of Arabidopsis, a 16:3 plant, showed that mutations of chloroplast desaturase (fad6, fad7) also affected the extra-chloroplastic lipids (Browse et al., 1986, 1989). By contrast, chloroplastic lipids were not affected in mutants of microsomal desaturases (fad2, fad3), and the chloroplast -3 FAD activity can even partially complement the mutation in leaves (Miquel and Browse, 1992; Browse et al., 1993). In particular, labelling kinetics with leaf lipids in the fad3 Arabidopsis mutant suggested that the 18:3 present in PC was the result of 18:2 lipids being transferred to the chloroplast, desaturated by the chloroplast desaturase, and then returned to the ER and other extra-chloroplastic lipids (Browse et al., 1993). Taken together, these analyses of Arabidopsis mutants showed that microsomal and chloroplast enzymes (FAD3, FAD7, FAD8) co-operate in determining the level of 18:3 FA in extra-chloroplastic membranes, and results of this study suggest a similar regulation system in birch leaves at LT.
The subcellular location of FA desaturases in plastids remained unclear up until the 18:2 desaturase activity was detected in isolated envelope membranes in spinach (Schmidt and Heinz, 1990). Proteomic analysis of the chloroplast membrane of Arabidopsis confirmed the location of FAD6 and FAD7 in the inner membrane (Ferro et al., 2003). In this context, these results also illustrate the importance of dynamic lipid trafficking from the chloroplast envelope to the extra-plastidial and thylakoid membranes.
Regulation of the carbon chain length of FAs
Birch was classified as an ‘18:3 plant’ by Mongrand et al. (1998), which means that MGDG is synthesized mainly by the cytoplasmic pathway. The low content of 16:3 in MGDG described in this study (Fig. 5) and by Selstam and Hällgren (1989) shows that the chloroplastic pathway functions in birch leaves albeit at a low level. After a brief increase during the first day of LT exposure, the proportion of 16:3 in MGDG decreased (Figs 5, 7). A down-regulation of the chloroplastic pathway would explain the decrease in 16:3. The down-regulation of the chloroplastic pathway at LT has been previously described in tomato (Yu and Willemot, 1997), possibly as a consequence of the down-regulation at LT of the plastidial glycerol-3-phosphate acyltransferase (GPAT), which controls the entry of the chloroplastic pathway. This hypothesis is supported by the fact that there was no decrease in 16:3 under SD conditions in birch (Fig. 5).
Although 16:3 is the main C16 FA in MGDG, it is a minor C16 FA in birch galactolipids. Both 16:0 and 16:1 (trans-16:1 in PG) FAs are high melting point FAs, and decreasing their proportion in the lipids has consequences on the physical properties of the membranes (Wu and Browse, 1995). During cold acclimation in birch leaves, the relative amount of C16 FAs (16:0+16:1+16:3) decreased in MGDG, DGDG, PC, and PE more specifically during the LT stress (Fig. 7). A similar trend has been observed in other plant species at LT (Uemura and Steponkus, 1994; Samala et al., 1998; Falcone et al., 2004). The regulation of the C16 to the C18 FAs ratio in lipids can be controlled either at the level of their synthesis by the KASII enzyme (production of 16:0-ACP or 18:1-ACP) as shown in the fab1 Arabidopsis mutant (Wu et al., 1994), or at the level of their use for lipid synthesis. LT only slightly stimulated the expression of BpKASII after 1 week at LT (Fig. 4). No data are available about a possible post-transcriptional regulation of KASII in plants. This enzyme remains one possible step for the control of the C16 to C18 FAs ratio in plants, as it is in bacteria. In E. coli, synthesis of C18 FAs increases at LT at the expense of C16 FAs and the KASII enzyme is involved in this regulation through increases in its intrinsic activity and not through a de novo enzyme synthesis (Garwin and Cronan, 1980; Garwin et al., 1980). The first reaction in lipid synthesis is acylation in the sn-1 position of the glycerol backbone catalysed by the GPAT, present in the stroma of the chloroplast and in the ER membrane (reviewed by Murata and Tasaka, 1997). Two types of plastidial GPAT with different substrate specificities (16:0-ACP or 18:1-ACP) have been described in chilling-resistant and -sensitive plants, but very little is known about the microsomal GPAT. In connection with the redistribution of FAs that were detected in PC, PE, and DGDG at LT in birch, an 18:3 plant, the presence of different isoforms of microsomal GPAT with different substrate specificities and expression patterns would be interesting to study.
This work shows for the first time the possible contribution of different -3 FAD and KASII genes in modulating membrane FA unsaturation at LT and under SD conditions in a highly cold-tolerant woody plant. As far as is known, this is the first report of co-ordinated expression analysis of several -3 FAD genes under LT stress in a photosynthetic tissue. A redistribution of all acyl chains in glycerolipids was observed and not only an increase in 18:3 with a corresponding decrease in 18:2. Such a redistribution was not observed in the fad7 or fad3 Arabidopsis mutant or in transgenic plants displaying modified expression of -3 FAD genes (Murakami et al., 2000), which shows that the whole FA and lipid biosynthesis pathways are affected at LT, but probably under SD conditions as well. Modulation of the FA composition by high temperature in Arabidopsis, an 16:3 plant, led Falcone et al. (2004) to a similar conclusion. Due to the specific location of the -3 FAD enzymes, these results also highlight the importance of the transfer of lipids: from the chloroplast to the thylakoid membranes, but also between extra-plastidial and chloroplast membranes.
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Acknowledgements |
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This work was supported by the European Science Foundation (Plant Adaptation Programme, to FM), the Finnish Centre for International Mobility (to FM), the Marie Curie Individual Fellowship Programme (contract QLK5-CT-2000-52074, to FM), the Academy of Finland (grant 76237, to SK), the NorFA network ‘Temperature Stress and Acclimation in Plants’, and the Finnish Forest Research Institute, Rovaniemi Research Station. We are grateful to Dr Richard Bligny and Dr Annikki Welling for the critical reading of the manuscript. We particularly thank Dr Minna Turunen for her invaluable help in obtaining funding from the Finnish Centre for International Mobility. The help from Dr Anna Palmé in generating the phylogenic trees is gratefully appreciated.
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Present address: Chemistry Department, University of Oulu, PL 3000, FIN-90014 University of Oulu, Finland. 
Present address: Finnish Forest Research Station, Muhos Research Station, Kirkkosaarentie 7, FIN-91500 Muhos, Finland.
Abbreviations: ACP, acyl carrier protein; DGDG, digalactosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.
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