Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus

Plant Physiology and Biochemistry 42 (2004) 811–822 www.elsevier.com/locate/plaphy

Original article

Changes in the endoplasmic reticulum lipid properties in response to low temperature in Brassica napus Guergana Tasseva, Jacques Davy de Virville, Catherine Cantrel, François Moreau, Alain Zachowski * Physiologie Cellulaire et Moléculaire des Plantes (CNRS, UMR 7632), Case courrier 154, Université Pierre et Marie Curie, 4, place Jussieu, 75252, Paris cedex 5, France Received 13 April 2004; accepted 8 October 2004 Available online 29 October 2004

Abstract Cold is an abiotic stress known to induce changes in membrane lipid composition. However, there is only limited information on the differential reactivity to environmental temperature of distinct cellular compartments. Therefore, we focused our attention on the endoplasmic reticulum (ER) that was never studied in this respect in plants. The ER membranes of etiolated Brassica napus (oilseed rape) hypocotyls grown at low temperature (4 °C) has been shown to be enriched in polyunsaturated fatty acids and phosphatidylethanolamine (PtdEtn) compared to hypocotyls grown at 22 °C. Despite the significant changes in their lipid composition upon cold exposure, the ER membranes showed a very partial physico-chemical adaptation as determined by measurement of membrane fluidity parameters such as local microviscosity of acyl chains and lipid lateral diffusion. To investigate the implication of transcriptional regulations during cold acclimation, we compared the abundance of transcripts for genes related to the fatty acid and the phosphatidylcholine (PtdCho)/PtdEtn biosynthesis pathways between normal temperature (22 °C)-acclimated and cold temperature (4 °C)-treated seedlings, using heterologous cDNA-array technology based on the knowledge on the Arabidopsis genome. Our studies demonstrate that a putative stearoyl-ACP desaturase isogene (orthologous to At1g43800) was up-regulated in response to low temperature. © 2004 Elsevier SAS. All rights reserved. Keywords: Cold acclimation; Heterologous cDNA-array; Lipid unsaturation; Membrane fluidity; Stearoyl-ACP desaturase

1. Introduction Temperature is one of the main environmental factors that alters plant development. The most commonly observed mechanism of adaptation towards suboptimal growth temperature in plants, like in many prokaryotes and animals, corresponds to remodeling of membrane lipid composition in order to maintain membrane integrity and membraneassociated protein functions under a wide range of environ-

Abbreviations: ACP, acyl carrier protein; BSA, bovine serum albumin; CCT, phosphocholine cytidylyltransferase; DBI, double bond index; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ER, endoplasmic reticulum; FAD, fatty acid desaturase; MOPS, 3-(N-morpholino) propanesulfonic acid; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PMSF, phenylmethylsulfonylfluoride; PVP, polyvinylpyrrolidone. * Corresponding author. E-mail address: [email protected] (A. Zachowski). 0981-9428/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.10.001

mental temperatures. Considerable attention has been devoted to the phenomenon of ‘frost hardening’ because of its important agricultural implications. Cold acclimation is a complex process defined as a period of growth at low but nonfreezing temperatures (2–6 °C), which increases the freezing tolerance of plants [33]. Among other changes cold acclimation involves a marked increase in the unsaturation degree of fatty acids. The latter is supposed to be a major factor that influences membrane fluidity, i.e. the extent of disorder and molecular motion within a lipid bilayer. On the basis of mechanisms of thermal adaptation characterized in bacteria, it is generally accepted that temperature response implies important modifications in the fluidity of cell membranes, possibly resulting into homeoviscous adaptation of the membrane bilayer [31]. Despite a large number of data reporting changes in membrane lipid composition in response to temperature stresses, most of the analyses focused on total cellular membranes

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while little is known about the responses of specific cellular compartments, with some exceptions such as mitochondrial membranes [4,6,20], chloroplast [36], tonoplast [2] and plasma membrane [34,35,40]. While endoplasmic reticulum (ER) is the site of the eukaryotic pathway of fatty acid and lipid biosynthesis, the modifications of its membrane properties in response to cold stress have not yet been characterized in plants. Two distinct glycerolipid biosynthesis pathways occur in higher plants, namely the prokaryotic (plastidial) and the eukaryotic (cytoplasmic/extraplastidial) pathways (reviewed in [22]). The 16:0, 18:0 and 18:1 fatty acids are synthesized in the plastidial compartment where the acyl chains are covalently bound to a soluble acyl carrier protein (ACP) during the extension cycles. The 18:1 is further desaturated by the plastidial acyl-lipid desaturases, or alternatively exported to the ER for desaturation by the microsomal acyl-lipid desaturases. A crucial role in the distribution of de novo synthesized acyl groups between the two pathways is played by acyl-ACP thioesterases, enzymes hydrolyzing the newly formed acyl-ACPs into free fatty acids which are further exported to the eukaryotic lipid biosynthesis pathway (reviewed in [3]). Finally, the determination of the membrane phospholipid composition is completed in the ER compartment by the action of microsomal desaturases and acyltransferases. For these reasons, it is expected that changes which may occur in fatty acid and lipid composition of the ER membrane in response to cold are determinant and would affect all cellular membranes, including plastidial membranes, since a reversible exchange of lipids exists between the eukaryotic and the prokaryotic pathways [3,21]. In the present study we focused our attention on the ability of ER membranes of Brassica napus seedlings to adjust their lipid composition and dynamics during cold acclimation. In order to prevent plastidial contamination of the ER-enriched cell fractions, this study was performed on etiolated hypocotyls for which both a freezing-tolerant winter cultivar (cv. Tradition) and a freezing-sensitive spring cultivar (cv. ISL) of oilseed rape were analyzed. Here we describe important changes in the lipid composition of ER membranes during cold acclimation that are not for as much accompanied by significant modifications in the bilayer dynamics. Taking into consideration these results, we looked further into our study for other regulatory mechanisms by analyzing enzymes of the lipid metabolism at the transcriptional level. For this purpose, we applied heterologous cDNA-array methodology taking advantage of knowledge on Arabidopsis genome (The Arabidopsis Genome Initiative 2000). It has been proven that such methodology was reliable for the analysis of orthologous genes from Arabidopsis related species such as B. napus [9]. In our study, we were interested in Arabidopsis gene families involved in fatty acid elongation, desaturation and PtdCho/PtdEtn biosynthesis. Our data report the first evidence for a low temperature up-regulated putative stearoyl-ACP desaturase isogene in B. napus (orthologous to At1g43800). The significance of such transcriptional up-regulation affecting this metabolic step and

the involvement of other regulatory mechanisms are discussed.

2. Results 2.1. Freezing tolerance assays By assessing the freezing tolerance, it was determined that B. napus light-grown hypocotyls are able to acclimate after 3 d at 4 °C and survive freezing temperatures ranging from –1 to –20 °C. Under these growth conditions, the freezing tolerance of cv. ISL was estimated as 12%, 10%, 6% and 0% of survival after freezing at –5, –10, –15 and –20 °C, respectively, whereas cv. Tradition light-grown hypocotyls showed 89%, 72%, 71% and 43% of survival following the same freezing treatments. By contrast, etiolated hypocotyls required an acclimation period of 13 d at 4 °C to recover after freezing treatments below –5 °C. In this respect, cv. Tradition has a great capacity to survive below –15 °C than cv. ISL, showing 9% and 0% of recovery, respectively, after treatment at –20 °C. 2.2. Purity of the ER-enriched cell fractions ER-enriched cell fractions were obtained from total microsomes (40,000 × g pellet) of etiolated rape hypocotyls by discontinuous sucrose gradient centrifugation. Theses fractions were analyzed for antimycin A-insensitive NADHcytochrome c reductase activity (ER-specific) and for other marker activities including vanadate-insensitive ATPase (tonoplast-specific), vanadate-sensitive ATPase (plasma membrane-specific) and Triton-stimulated UDPase (Golgi apparatus-specific) in order to determine their relative enrichment with respect to the initial homogenate or total microsomes. The ER-enriched fractions showed a high antimycin A-insensitive NADH-cytochrome c reductase specific activity (314.0 ± 65.6 nmol of reduced cytochrome c min– –1 1 1 mg protein ) and an enrichment of approximately twofold over total microsomes (156.8 ± 19.8 nmol of reduced cytochrome c min–1 mg–1 protein1). There was no trace of mitochondria since antimycin A-sensitive NADH-cytochrome c reductase activity was undetectable. Similarly, the Golgispecific latent UDPase activity was too low to be quantified. Some vanadate-insensitive ATPase (62.2 ± 1.8 nmol of phosphate min–1 mg–1 protein2) and vanadate-sensitive ATPase (44.1 ± 6.8 nmol of phosphate min–1 mg–1 protein2) activities were also detected in these cell fractions but their respective enrichment never exceeded 1.2- to 1.4-fold with respect to the initial homogenate (45.6 ± 8.0 and 36.3 ± 6.82, respectively). Moreover, the amount of galactolipids (mono- and 1

Average values of specific activities ± S.D. of four independent gradients are indicated. 2 Average values of specific activities ± S.D. of three independent gradients are indicated.

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di-galactosyl diglycerides) varied from 4% to 5%, reflecting a low level of plastidial membrane contamination. Cardiolipid, on the other hand, was present in amounts so low that it could not be quantified, confirming the absence of mitochondrial membrane contamination in these fractions. 2.3. Fatty acid composition of total lipids in ER membranes according to growth conditions Etiolated rape hypocotyls were collected after 4 d of growth at 22 °C (time 0 of cold treatment) and at d 3 and d 13 after transfer to 4 °C (short- and long-term coldacclimating hypocotyls). These stages of cold acclimation were chosen to prospect for changes correlated to progressive rearrangements of lipid composition (starting after 1 d of cold treatment) and to the fully established rearrangements (stationary “plateau” region of lipid changes starting after 10–12 d of cold treatment), as reflected by the kinetics of changes altering total cellular lipid composition (not shown). Other hypocotyls were grown for 30 d at 4 °C in the dark and are referenced as cold-acclimated. When hypocotyls were grown at normal temperature (22 °C), ER membranes from both cultivars showed a very similar fatty acid composition although cv. ISL contained slightly less 18:3 species (Table 1). For cold-acclimated hypocotyls, the observed differences between cultivars were restricted to the 16:0 to 18:2 ratio, with the presence of lower amount of 16:0 and greater amount of 18:2 in cv. ISL comparatively to cv. Tradition. Similarly, the global rate of unsaturations reflected by the double bond index (DBI) value was almost identical for both cultivars when grown at constant (either normal or low) temperature (Table 1). By contrast, when transferred to 4 °C, the adjustments in the fatty acid relative content did not have the same amplitude in cv. Tradition and cv. ISL, respectively. We used the fatty acid composition of hypocotyls germinated and grown at 4 °C as a reference composition for completely cold-acclimated membranes. Following this criteria, cv. Tradition showed a more rapid capacity of cold acclimation response in comparison with cv. ISL. Significant changes were already detectable after 3 d of exposure to 4 °C in cv. Tradition. For example,

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Table 2 Phospholipid to protein ratio (w/w) in ER membranes of etiolated rape hypocotyls. Phospholipid content was assayed on a lipid extract from ERenriched cell fractions. For treatment see legend of Table 1. Data correspond to the mean ± S.D. of three independent experiments Treatment 22 °C 3 d, 4 °C 13 d, 4 °C 4 °C

Phospholipid to protein ratio (w/w) cv. Tradition cv. ISL 1.12 ± 0.20 1.09 ± 0.19 1.20 ± 0.22 1.24 ± 0.14 1.44 ± 0.11 1.25 ± 0.20 1.59 ± 0.15 1.29 ± 0.03

concerning the 18:3 content evolution, roughly 23% of the expected change was reached after 3 d of low temperature treatment, and 83% after 13 d. The corresponding values for cv. ISL membranes were less important (10% and 35%, respectively), thus illustrating a slower change and/or a lesser rate of 18:3 biosynthesis. In cv. ISL seedlings, almost no marked changes occurred at the short-term acclimation period and the new composition observed in the long-term acclimating hypocotyls was still substantially different from the one existing in acclimated hypocotyls, especially for 18:2 and 18:3 species. In addition, the global rate of unsaturation for cv. ISL membranes from long-term coldacclimating hypocotyls remained significantly lower to that from cold-acclimated seedlings, whereas cv. Tradition showed a total adjustment of this parameter. The phospholipid to protein mass ratio in ER membranes was also distinctively affected between cultivars by the temperature shift (Table 2). In cv. ISL a variation of this parameter was already visible after short cold exposure but the magnitude of this change after long exposure remained limited to 20%. On the other hand, this ratio showed a slower change in cv. Tradition but its extent was quite important (up to 40% for seedlings grown at constant 4 °C versus seedlings grown at 22 °C). 2.4. Changes in the composition of main phospholipids in response to temperature The major phospholipids detected in ER membranes, phosphatidylcholine (PtdCho) and phosphatidylethanola-

Table 1 Fatty acid composition of total lipids in ER membranes from rape hypocotyls grown under different temperature conditions. B. napus seedlings were grown at 22 °C for 4 d and then cold-treated at 4 °C for 3 or 13 d in the dark. Other seedlings were dark-grown at 4 °C for approximately 30 d so as to obtain the same hypocotyl elongation as for seedlings grown at 22 °C for 4 d. DBI was calculated as 2 × [(% of 18:1) + 2 × (% of 18:2) + 3 × (% of 18:3)]/100. The term ‘VLCFA’ includes fatty acids with chain length superior to 18 carbons. Data are mean of results obtained with three independent experiments and deviation of values was within ±10% Cultivar Tradition

ISL

Treatment 22 °C 3 d, 4 °C 13 d, 4 °C 4 °C 22 °C 3 d, 4 °C 13 d, 4 °C 4 °C

DBI 3.06 3.28 3.65 3.55 2.92 3.06 3.22 3.59

C16:0 22.0 19.2 17.8 22.2 22.6 21.3 21.3 17.6

C18:0 3.5 3.3 2.2 3.3 5.0 4.5 4.5 3.6

C18:1 13.3 13.7 11.8 10.3 12.2 11.3 8.6 10.2

Fatty acid (mol%) 018:2 40.0 38.1 30.7 23.9 42.1 42.7 39.4 26.0

C18:3 20.0 24.6 36.4 39.8 16.5 18.8 24.5 39.1

VLCFA 1.2 1.1 1.1 0.5 1.6 1.4 1.7 3.5

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Table 3 PtdEtn to PtdCho mole ratio in ER membranes from etiolated rape hypocotyls. The lipid classes from ER-enriched cell fractions were separated on silica plates and the respective amounts of PtdEtn and PtdCho were estimated as mol% relatively to total ER lipid amount by the total fatty acid quantity in each of these phospholipids. For temperature treatment conditions, see legend of Table 1. Data correspond to the mean ± S.D. of three independent experiments

cv. Tradition cv. ISL

22 °C 1.08 ± 0.15 1.15 ± 0.22

3 d, 4 °C 1.11 ± 0.39 1.2 ± 0.22

mine (PtdEtn), accounted together for approximately 89–92% of total phospholipids. The analysis showed that the PtdEtn to PtdCho mole ratio was altered by cold treatment in both cultivars (Table 3). In hypocotyls grown at 22 °C, the PtdEtn to PtdCho ratio was almost identical for both cultivars, thus reflecting similar contents for both phospholipids at normal temperature. The PtdEtn to PtdCho mole ratio increased in the time course of cold acclimation. However after 13 d of cold treatment the increase of the PtdEtn to PtdCho ratio reached only 47% and 61% of the corresponding values determined for the cold-acclimated hypocotyls of cv. Tradition and cv. ISL, respectively. In cv. Tradition, the fatty acid compositions of PtdCho and PtdEtn in ER membranes from the long-term acclimating hypocotyls were almost identical to those of cold-acclimated hypocotyls (Fig. 1). By contrast, significant differences persisted in cv. ISL between the long-term acclimating hypocotyls and the cold-acclimated hypocotyls (Fig. 1). In particular, there was a trend to increase rather to decrease the 18:2 level in the time course of the acclimation. Thus, 18:2 species remained more abundant than 18:3 species in ER membranes from cv. ISL, contrarily to cv. Tradition, even if the 18:3 level increased in both cultivars by approximately two to threefold after long low temperature exposure.

Fig. 1. Temperature effects on fatty acid composition of the two major phospholipids in ER membranes of etiolated rape hypocotyls. Values are mean of three (hypocotyls acclimated to 22 °C and cold-acclimating hypocotyls) or two independent experiments (hypocotyls acclimated to 4 °C). Error bars represent standard deviations (n = 3) or intervals between values (n = 2). Long chain fatty acids are not figured as they accounted for less than 1% in PtdCho and less than 2% in PtdEtn. White bars: 4-d germinated etiolated seedlings grown at 22 °C; hatched bars: etiolated seedlings grown at 22 °C for 4 d and then transferred to 4 °C for 3 d; dotted bars: etiolated seedlings grown at 22 °C for 4 d and then transferred to 4 °C for 13 d; grey bars: 30-d germinated etiolated seedlings grown at 4 °C.

PtdEtn to PtdCho ratio 13 d, 4 °C 1.21 ± 0.10 1.41 ± 0.19

4 °C 1.36 ± 0.23 1.58 ± 0.31

2.5. Lipid dynamics in ER membranes Dynamic properties of ER membrane lipids were analyzed by measuring local microviscosity and lipid lateral diffusion. Local microviscosity at various depths of the lipid monolayer was probed by fluorescence depolarization of a set of anthroyloxy fluorophores substituted at different carbon positions of the acyl chain. Fig. 2 illustrates the fluorescence anisotropy as a function of the carbon position on the acyl chain, reflecting the degree of lipid order at different depths of the membrane bilayer. There was no evidence toward an adaptation of this fluidity parameter as a function of growth temperature since comparable lipid anisotropy

Fig. 2. Temperature effects on local microviscosity measured at different depths in ER membranes from dark-grown hypocotyls of rape cultivars. Local microviscosity was assayed on cv. Tradition (A) and cv. ISL (B) preparations by measuring the fluorescence anisotropy of anthroyloxy fatty acid derivatives with the fluorophore attached on different carbon atoms of the fatty acid chain. Measurements were carried out either at 22 °C (open symbols) or at 10 °C (crossed symbols) with ER membranes from 22 °Cacclimated hypocotyls (circles) or from 4 °C-acclimated hypocotyls (squares). Data are the mean of values obtained with two independent preparations. Intervals between the two experimental values were less than 5% of the mean (not represented).

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values were obtained at an equivalent measurement temperature with membranes purified from hypocotyls grown at 22 or 4 °C. There were no more significant changes for this parameter at any time point of cold exposure for the acclimating seedlings (data not shown). Lateral diffusion of lipids within the membrane bilayer was probed using a pyrene-labeled fatty acid at different temperatures between 10 and 28 °C (Fig. 3). The lipid lateral diffusion coefficient was slightly higher in cv. Tradition than in cv. ISL, as well for membranes from hypocotyls grown at 22 °C as for those from 4 °C-acclimated hypocotyls. The activation energy of this movement was almost identical for membranes submitted to different temperature treatments and was comprised between 19 and 21 kJ mol–1, as determined by the slope of an Arrhenius plot of the excimer to monomer fluorescence intensity ratio (data not shown). It has to be noted that growth at a lower temperature resulted in a slightly higher lateral diffusion of lipids in ER membranes, such change being more pronounced in cv. Tradition than in cv. ISL. Nevertheless, in no case the change could be indicative of a complete homeoviscous adaptation since its amplitude was far from compensating for the shift of 18° (22 – 4 °C). Compensation accounted for six degrees in cv. Tradition and three degrees in cv. ISL. When the same type of measurement was carried out with membranes from longterm acclimating hypocotyls, it indicated a trend for lateral diffusion reaching values obtained with cold-acclimated membranes. 2.6. Transcriptional regulations in response to low temperature treatment Changes appearing in lipid composition in response to growth temperature (unsaturation level, relative amounts of

Fig. 3. Temperature effects on lipid lateral diffusion in ER membranes from dark-grown hypocotyls of rape cultivars. The exchange frequency of 1-pyrenedodecanoic acid was measured at different temperatures for ER membranes from cv. Tradition (closed symbols) and cv. ISL (open symbols) either grown at 22 °C (circles), or at 4 °C (squares), or grown at 22 °C, then transferred to 4 °C and grown for 13 more days (diamonds). Curves are traced for thermally-acclimated samples (22- or 4 °C-acclimated) with continuous lines for cv. Tradition and dashed lines for cv. ISL. Data are mean of values obtained with three independent experiments. Deviations represented less than 10% of the mean.

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PtdCho and PtdEtn, see above) prompted us to study the transcriptional status of enzymes involved in various steps of fatty acid and phospholipid biosynthesis in response to cold stress and during cold acclimation. The steady-state mRNA levels of genes (listed in Table 4) functionally related to the 16:0 elongation and the C18 desaturation (prokaryotic and eukaryotic pathways), on the one hand, and the interconnected pathways of major phospholipid biosynthesis, on the other hand, were studied by heterologous hybridization. The B. napus seedlings were subjected to cold treatment for short periods of time (6 and 24 h) corresponding to cold stress and for longer periods of time (up to 13 d) corresponding to a cold acclimation period. As quantitative criteria, we considered as up- or downregulated by low temperature treatment a gene whose transcript level was at least twofold greater or lesser than the transcript level found in hypocotyls grown at normal temperature. Our data highlight a representative orthologous isogene belonging to the stearoyl-acyl carrier protein desaturase (stearoyl-ACP desaturase) family (At1g43800), which showed a significant increase of its transcript level in response to low temperature. This increase was well marked between 6 and 24 h of cold treatment and was six to eightfold in cv. Tradition (Fig. 4A) and two to threefold in cv. ISL (not shown). Its steady-state level was maintained superior to the control during at least 7 d of cold acclimation (data not shown). In cv. Tradition another B. napus ortholog of this family (At5g16230) showed a constant level of transcripts during the overall cold treatment. In cv. ISL hypocotyls no significant changes in transcript abundance occurred at any time after temperature shift (data not shown), except the single up-regulation described above for cv. Tradition. By contrast, in cv. Tradition hypocotyls changes in the steadystate mRNA level of several genes were detected (Fig. 4A). The FAD7 gene showed a very weak transcript level at normal as well as at low temperature growth conditions in etiolated B. napus hypocotyls. This gene has been described as light-inducible in wheat [13]. Concerning the two other genes involved in the plastidial pathway of fatty acid desaturation, the FAD6 transcripts were detectable only as traces while the FAD8 transcripts level slightly decreased at low temperature (Fig. 4A). By contrast to the desaturases involved in the prokaryotic pathway of the fatty acid metabolism, the steady-state mRNA levels of the microsomal omega-6 (FAD2) and omega-3 (FAD3) desaturase genes were sensibly high even at normal growth temperature. The steady-state level of the FAD3 transcripts was transiently higher between 6 h and at least 3 d of cold treatment than in the control (Fig. 4A), but returned to the initial level after 7 d of cold exposure. The FAD2 transcript steady-state level showed a remarkable decrease of about threefold during the short exposure to 4 °C and was restored to a relatively constant, and comparable to the control, level during the time course of the acclimation. Concerning all analyzed transcripts related to fatty acid and phospholipid metabolism, most of them were hardly

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Table 4 List of Arabidopsis thaliana gene families involved in fatty acid and phospholipid biosynthesis used for transcriptional analysis. Forward and reverse primers that served to amplify the Arabidopsis genomic fragments are shown. The corresponding PCR products were used to constitute macroarrays Product name Aminoalcoholphosphotransferase 1 (AAPT1) Aminoalcoholphosphotransferase 2 (AAPT2)

AGI number At1g13560 At3g25585

Forward primer 5′ → 3′ GTACTCGTCTCTTGATC CAAGTTCGTGTATGTTG

Reverse primer 5′ → 3′ CCAACAAGAAAGTAACC AAGCAACTAGAAGCTTC

Phosphocholine cytidylyltransferase (CCT1) Phosphoethanolamine cytidylyltransferase, putative (putative ECT) Phosphocholine cytidylyltransferase, putative (CCT2)

At2g32260 At2g38670 At4g15130

CGTAGACAGTATCCAAG AGATAATCCGTATTCTG GATGGGATTCAGCAACG

CGTAGACAGTATCCAAG TAGTATGTAGCATTTGG CCCTTTGTTGCTCACAT

Glycine max choline kinase (GmCK2p)-related Choline kinase, putative (putative CK) Ethanolamine kinase, putative (putative EK) Glycine max choline kinase (GmCK2p)-related Stearoyl-ACP desaturase, putative, mitochondrion (putative SACPD) Stearoyl-ACP desaturase, chloroplast (SACPD) (FAB2) Stearoyl-ACP desaturase, putative, chloroplast (putative SACPD) Stearoyl-ACP desaturase, putative, chloroplast (putative SACPD) Stearoyl-ACP desaturase, putative, chloroplast (putative SACPD) Stearoyl-ACP desaturase, chloroplast (SACPD) Stearoyl-ACP desaturase, chloroplast (SACPD)

At1g34100 At1g74320 At2g26830 At4g09760 At1g43800 At2g43710 At3g02610 At3g02620 At3g02630 At5g16230 At5g16240

TGGAACGCGACTCGTCG CTAGATTAGTAAGGATG CAAACTTGTACGGTATG GACTTCTCTCCAACTCT CCCACAGAATCTCAAAC GTCTCTCTTATTGCTTG CTTTAAGACTAACTAAC CAAGGGCAATGCAAACG GTAGCTGAGGGATATTG AGAGGAGAGAACACTCC GAAGGGACATTTAAGAG

GGGAAGAAGCAGTTAGC CAAAACCTTACGAGAAG AGTTCTAGGTCAGAGAG AACAAGAAGCTTCCTAC CTCCGGTGGCATGGTGT AAGAGAGACGATGATGG TTTCACTATTCAGTGCC ACATTGAGATCAACGCG AATGAGAGTGCATTGGG GAAATCGAAGATCTAAC AATGATCATCGCTGAAC

Omega-3 fatty acid desaturase, microsome (FAD3) Omega-3 fatty acid desaturase, chloroplast precursor (FAD7) Omega-6 fatty acid desaturase, microsome (FAD2) Fatty acid desaturase, putative (DES-1-LIKE) Omega-6 fatty acid desaturase, chloroplast precursor (FAD6) Temperature sensitive omega-3 fatty acid desaturase, chloroplast precursor (FAD8)

At2g29980 At3g11170 At3g12120 At4g04930 At4g30950 At5g05580

TCGACACATCTTCTCTC GACATAAGCGTGCGAAC ATGCCGCTTCTCTACTC TCCTCGCACACACATGC ATGAATGCTGTTAGTTC ATAGAAGACTGATTACG

CAAAGGAACCAGCTATC CTTCCTCCAATGGAGAC AGAGGCTGAGGGAGGAG GAACAAAACTAGACGTC ACACATAAGGCTTATTG CTCTCCGTGTCTTCCTC

Beta-ketoacyl-ACP synthase II, chloroplast (KASII, FAB1) Beta-ketoacyl-ACP synthase II, mitochondrion (KASII) Beta-ketoacyl-ACP synthase I, chloroplast (KASI)

At1g74960 At2g04540 At5g46290

GCGTCTCCACGCACGAT GCTCCTACAGTGCACGG CTGACGAACATAAGCTC

AACCCATAACAACATCG CATCAAATTCACCAGGG GTGTTAAGCTTATCACC

Phosphoethanolamine N-methyltransferase, putative Phosphoethanolamine N-methyltransferase, putative Phosphoethanolamine N-methyltransferase, putative Phosphoethanolamine N-methyltransferase 1 (NMT1)

At1g48600 At1g73600 At3g17990 At3g18000

GGAACAGAGGAAGAGAG TCCCTGGTCTTACTCTC CTCTCTCCCCTCTCTTG AACCAGCCTTGTTTAGG

GATTTTCAAGTCTGGAG CATATGAGTCATTCTCG CATCGACTCAGAAGGTG CCGATGCACACCTCTCC

Acyl-ACP thioesterase, putative, chloroplast (FATB1) Acyl-ACP thioesterase, chloroplast (FATA1) Oleoyl-ACP hydrolase-like, chloroplast (FATA-like) Palmitoyl-protein hydrolase-like, chloroplast Palmitoyl-protein hydrolase-like, chloroplast Palmitoyl-protein hydrolase-like, chloroplast Palmitoyl-protein hydrolase-like, chloroplast Palmitoyl-protein hydrolase-like, chloroplast Palmitoyl-protein hydrolase-like, chloroplast Palmitoyl-protein hydrolase-like, chloroplast

At1g08510 At3g25110 At4g13050 At3g60340 At4g17470 At4g17480 At4g17483 At5g47330 At5g47340 At5g47350

ATCCTCCTGCTAGTAGC CTTCTTCACCTCGGACC CCTCGGTCTCATCGTCG TACCTCTGTGATAAATC ACACAAGTAAACCGTTC GTAAGATCAGTAGATAC GACTCACACATAACGTA CTTGGCCGCAACTTCCA GAAGCAGAGCGTTAATG GGCCACAACTTCCAACC

CACCAGCATGTCAGAAC GCGAGAGTAGCCAAACC CCGGAGACGATCCGCCG AAGACAGAATGCTGTTG AGAAGGAGTTGTGTCGG AGAAGGAGTTGGTTCGG AATAGATGAGGAAGAAG CTTCTTCTACATGTCCC ACTCTTCTACATGGCCC TAGTCACTTTGCACGTC

Glycerol-3-phosphate acyltransferase, chloroplast (GPAT) (ATS1) 1-Acylglycerol-3-phosphate acyltransferase, putative, microsome (putative LPAAT) 1-Acylglycerol-3-phosphate acyltransferase, putative, microsome (putative LPAAT) 1-Acylglycerol-3-phosphate acyltransferase-like protein, microsome (putative LPAAT) 1-Acylglycerol-phosphate acyltransferase, hypothecal microsome (putative LPAAT)

At1g32200 At1g51260

TGACTCTCACGTTTTCC TCTGGTAATGTTGTTGG

GGAATCGAACAATCTCC GAAGGAACAATGCAGTG

At1g75020

GGGAACTTAAAGGTGGC

AACACGTTTCTCAACCG

At3g57650

GTTCTATCATGGGCATG

ATGTTTGGATCTGTGAG

At3g18850

TCGTATCAACCTGACCC

TGTGAAGTTGTGAACTG

NADH-cytochrome b5 reductase (Nb5R) (ATCBR) NADH-cytochrome b5 reductase, putative (putative Nb5R) NADPH-ferrihemoprotein reductase, putative NADPH-ferrihemoprotein reductase (ATR1) NADPH-ferrihemoprotein reductase (ATR2)

At5g17770 At5g20080 At3g02280 At4g24520 At4g30210

GAACCCTAGATCGTCAG TCTCAGAGATCTAAACC TCGAATATGAAGTTGGC GAATCCTCAAACCCTGG GTCAACCTCCATGATCG

TAAGAAGTACGAGGACC TCGTCTGATTGAATATG TCGTCGAGGTGAAGCGG GAAGATAGAGACTCTAG GTAACTTTCTTACGCCC

Tubulin alpha-5 chain-like (TUBA)

At5g19770

ATTTAGGTCGGTTAAGC

GCAACAACGCAAAAGAC

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Fig. 4. Cold treatment effects on mRNA steady-state levels of enzymes involved in the fatty acid and major phospholipid biosynthesis pathways in etiolated hypocotyls from B. napus cv. Tradition. (A) Transcript abundance profile by reverse Northern analysis of genes involved in the 16:0 elongation and the C18-fatty acid unsaturations; (B) transcript abundance profile of genes showing a cold-repressed expression (except the ECT isogene). A small scale DNA array analysis was performed on B. napus hypocotyls that were grown in the dark at 22 °C for 4 d (time 0 of the cold treatment) and then transferred to 4 °C for 6, 24 h (cold stress responses), 3 or 13 d (cold acclimation responses). The results presented in this figure correspond to two independent experiments referring to the short periods of cold treatment (cold stress) and to the longer periods of cold treatment (cold acclimation), respectively. Hypocotyls grown at 22 °C for 4 d served as a control for the short-term of cold treatment. Hypocotyl elongation was used as developmental control for the long-term cold-treated seedlings (see ‘Section 4’). The mRNA steady-state level of the alpha-tubulin At5g19770 gene was used to normalize the amounts of transcripts between separate arrays. Similar results were obtained in three separate experiences.

affected (levels varying by a factor inferior to 2) throughout the cold treatment. However, some genes showed significant variations of their mRNA steady-state levels corresponding to a low temperature down-regulation. We observed a low temperature down-regulation for the orthologs of two putative isogenes of the palmitoyl-protein hydrolase family. The steady-state mRNA levels of these orthologous genes, namely the At4g17480 ortholog(s) and the At5g47350 ortholog(s), were 2.5- and 3.5-fold lower at 6 h of cold treatment, respectively (Fig. 4B). Another studied gene family includes the microsomal NAD(P)H cytochrome (b5 or P450) reductases associated with the desaturase activities. Among them, one of the two NADH-cytochrome b5 reductase isogenes showed a 2.5-fold decrease in its mRNA steady-state level at 6 h of cold treatment (Fig. 4B). Concerning the biosynthesis of PtdCho and PtdEtn, the orthologs of both phosphocholine cytidylyltransfearse isogenes, AtCCT1 and AtCCT2, responded with a 2- to 2.5-fold decrease in their mRNA levels after 6 h of cold treatment, as well as orthologs of two putative isogenes of choline kinases (At1g34100 and At1g74320) which denoted a 2.5-fold and twofold reduced mRNA levels, respectively (Fig. 4B).

3. Discussion In order to investigate the response of the ER membranes to cold acclimation, at the level of the lipid composition and

dynamics, we isolated ER-enriched cell fractions from B. napus hypocotyls grown at normal temperature (22 °C), low temperature (4 °C), and in parallel, from cold-treated hypocotyls (transferred from 22 to 4 °C). We also searched for the possible involvement of transcriptional regulations during this adaptive process. Analyses were carried out with two chilling-resistant cultivars differing in their sensitivity with respect to freezing temperatures in attempt to discern causative changes in ER membrane lipid composition and dynamics related to cold acclimation. Several recent works on animals demonstrated homeoviscous adaptation over periods of cold exposure as short as a few hours. Changes in the behavior of membranes within 6 h after the beginning of cold treatment have been described in the chilling-resistant plant Arabidopsis [25]. In dark-grown hypocotyls studied in this work, the acclimation periods are several times longer as observed through the long-term evolution of changes in the composition and the dynamic properties in ER membranes both spread over 7–13 d-long periods. The longer time required for cold acclimation may be attributed to the specific nature of this plant organ but also more probably to the absence of light during the overall growth of seedlings. Freezing tolerance assays confirmed this hypothesis since light-grown hypocotyls required only 3 d of acclimation at 4 °C whereas etiolated hypocotyls required a longer acclimation period of 13 d at 4 °C to survive freezing temperatures below –5 °C whatever the rape cultivar.

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The most common change in response to downward shift of environmental temperature in bacteria [37] and ectotherm organisms [10] is the enhanced level of fatty acid unsaturations. Accordingly, the major modification observed in ER membranes submitted to cold treatment was the accumulation of the most unsaturated fatty acid in plant membranes, namely linolenic acid (18:3). The 18:3 content increased approximately twofold upon long cold exposure in ER from both cultivars. Significant increases in the linolenic acid level in response to chilling have been already reported for mitochondrial membranes from various chill-hardened plant species [20]. The same authors reported a positive correlation between linolenic acid content in polar lipids and chill resistance of plants. In cold-acclimated hypocotyls from both cultivars compared to normal temperature-acclimated hypocotyls, the increase in 18:3 level was mainly accompanied by an important decrease in the 18:2 content, thus arguing in favor of an enhanced linoleate desaturase activity triggered by low temperature. However, when normal temperaturegrown seedlings were subjected to cold treatment, striking differences were observed between these two cultivars in their respective membrane compositional changes. According to the freezing sensitivity of cv. ISL, we observed slower changes and some discrepancies in the fatty acid composition from the long-term cold-exposed hypocotyls referring to the cold-acclimated hypocotyls. The fatty acid composition analyses suggest the existence of a less efficient mechanism of thermal acclimation in cv. ISL affecting the conversion of 18:2 to 18:3 and resulting in a less efficient 18:3 synthesis. In addition to the unsaturation rate modifications, there was a change in the PtdEtn to PtdCho mole ratio with an enrichment of ER membranes in PtdEtn species during cold exposure. However, after 13 d of stay at 4 °C, the PtdEtn to PtdCho mole ratio has not yet reached the complete cold acclimation values obtained with 4 °C-acclimated hypocotyls, thus suggesting a partial acclimation response for this parameter in both cultivars. The precise involvement of changes in the relative amount of lipid polar heads in response to cold stress still remains controversial. Several studies reported an increased proportion of PtdCho among other phospholipids in low temperature treated plants. However, the relative increase of PtdEtn over PtdCho content that we have observed seems to be a common mechanism of response to downward shift of temperature in etiolated plant tissues, such as dark-grown soybean hypocotyls [6]. It is well known that PtdCho serves as metabolic precursor for the biosynthesis of plastidial galactolipids, such as monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol (reviewed in [22]) and that it serves as substrate for acyl-lipid desaturases in the ER. Routaboul et al. [27] showed that biogenesis and maintenance of chloroplasts under the long-term cold exposure are dependent on the trienoic fatty acid level of membranes. Therefore, PtdCho would play an important role in the rapid recovery of chloroplastic membrane functions under cold stress conditions, thus explaining the selective increase in its amount

over other phospholipid species. In dark-grown hypocotyls, the efficient acclimation to low temperature would not require enhanced synthesis of PtdCho species over other phospholipids. The impact of low temperature treatment on lipid dynamics depends on the type of lipid motion. No important changes in the bilayer microviscosity were detected between 22 and 4 °C-acclimated hypocotyls. This result is in contradiction with the concept that an increase in the unsaturation rate of phospholipid acyl chains is associated with a decrease in membrane microviscosity [30]. However, this lack of sensitivity to temperature shift may be explained by the extremely low activation energy of local acyl chain motions [30]. Consequently, local microviscosity parameter seemingly does not contribute to cold adaptation in ER membranes. It is usually accepted that increase in the fatty acid unsaturation level and also in the lipid to protein ratio enhance lipid lateral diffusion. In agreement with this concept, we observed that lipid diffusion in ER membranes of coldacclimated hypocotyls was higher in comparison to those of hypocotyls grown at normal temperature. However, with respect to a 100% compensation of low temperature effect on membrane fluidity stated by the homeoviscous adaptation theory [31], lateral diffusion compensation reached only 33% and 16% in cv. Tradition and cv. ISL, respectively, despite of the important increase in the content of 18:3, which is considered as a major regulator of membrane fluidity. A part of the explanation for the lack of a most efficient fluidity recovery under cold exposure may reside in the fact that two other trends were observed in Brassica hypocotyls, namely a relative increase in the PtdEtn content over the PtdCho content and an elevated phospholipid to protein mass ratio. According to several authors, PtdEtn would rather order than fluidize the lipid bilayer (reviewed in [10]), thus potentially counteracting the disordering effect due in part to the enhanced content in 18:3 fatty acids. At a functional level, it is conceivable that the nature and the extent of modifications in lipid dynamics would depend on the type of cellular membrane considered. It is probable that a partial membrane fluidity compensatory response of cold acclimation could be sufficient to ensure the required level of ER membrane-associated functions. Moreover, we must consider that other regulatory mechanisms modulate the lipid metabolic enzymatic activities probably in conjunction with this partial compensatory effect on the level of membrane fluidity. Therefore, we also investigated the transcriptional status of enzymes involved in lipid biosynthetic pathways associated to the ER or the plastid compartments. Highly duplicated genomic sequences have been reported for the desaturase multi-gene families in B. napus, a tetraploid (amphidiploid) species, where copy number varies between 4 and 8 [28]. Thus, a single orthologous hybridization signal obtained in reverse Northern analyses could be assigned to the transcript levels of at least four distinct genes in B. napus corresponding to a single Arabidopsis ortholog. It is

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not clear whether all or few of these duplicated genomic sequences, that may have diverged one from another during evolution, correspond to functional gene copies, or alternatively to pseudogenes. Additionally, alterations in either the rate of transcription or the stability of the respective transcripts might be responsible for the differences observed in the steady-state mRNA levels. Reverse Northern analyses made on both cultivars revealed an accumulation of putative stearoyl-ACP desaturase transcripts in response to low temperature. These transcripts are encoded by genes orthologous to At1g43800. Despite many efforts, we failed however to detect such an accumulation by using Northern blotting. Such discrepancy between the two techniques is likely due to a difference in their respective sensitivities as already suggested [32,38]. Depending on the genes, these authors observed that DNAarrays may exceed Northern blots in sensitivity. The level of stearoyl-ACP desaturase activity measures the rate of fatty acid de novo synthesis and it is a key determinant of the overall level of fatty acid unsaturation since in plants it is the unique enzyme that introduces the first double bond in C18-fatty acid chains. Since etiolated hypocotyls need active fatty acid and phospholipid biosynthesis for organ expansion even during cold exposure, it is conceivable that higher amounts of this enzyme are required to sustain the important flow of de novo fatty acid biosynthesis for cell elongation. It has been evidenced that a stearoyl-ACP desaturase (FAB2) activity plays a major role in cell expansion and overall plant growth since fab2 mutant plants displaying elevated 18:0 levels also possessed a stunted morphology [17]. The dwarf phenotype of fab2 mutants was corrected at elevated temperatures [18], therefore it has also been proposed that FAB2 plays a determinant role in temperature adaptation by adjustment of fatty acid pattern of membrane lipids and modulation of membrane fluidity. In chilling-lethal (chs) Arabidopsis mutants, the transcript accumulation of FAB2 (At2g43710) showed a defect upon moderate (13 °C) low temperature exposure [24] thus suggesting that the correct expression of this isogene can be important for the transition to low temperatures. These observations explain why the stearoyl-ACP desaturase represents a potential target for low temperature-induced regulation of the unsaturation rate. Although a significant increase of the polyunsaturated fatty acid content during cold exposure of Brassica hypocotyls, the oleate desaturase and linoleate desaturase genes (FAD2/At3g12120, FAD3/At2g29980) were regulated only in a transient manner in response to low temperature. Previous reports on both monocots and dicots revealed that genes encoding the microsomal oleate and linoleate desaturases were not transcriptionally regulated by chilling exposure [11,14,15,23]. Instead, a post-transcriptional regulation induced noticeable increase in the steady-state amount of the FAD3 enzyme in wheat [14]. Moreover, the level of the FAD2 transcript has been reported to be present several times in excess compared to the amount needed for sufficient

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18:2 biosynthesis in Arabidopsis [23]. Our results demonstrate that the cold-induced accumulation of 18:3 in the ER membranes of etiolated Brassica hypocotyls seems not to be due to enhanced mRNA level of the microsomal omega-3 desaturase. This suggests that some more direct regulatory mechanisms, such as enhanced translation or enhanced enzymatic activities, are involved in ER lipid composition changes in response to cold. In our study, two putative 16:0-protein hydrolases (orthologous to the At4g17480 and the At5g47350) showed a low temperature down-regulation in rape hypocotyls. This observation suggested a potential role played by palmitoylprotein hydrolases in controlling the membrane lipid composition under cold exposure. We also focused our attention on the PtdCho and PtdEtn interconnected biosynthesis pathways. The phosphocholine cytidylyltransferase (CCT) activity is considered as the ratelimiting step in the biosynthesis of PtdCho. In cv. Tradition we observed a down-regulation of both Brassica CCT orthologs instead of an increase in their transcript level. Moreover, two orthologous isogenes of putative choline kinases (At1g34100 and At1g74320), enzymes responsible for the first committed step in the biosynthesis of PtdCho via the nucleotide pathway, were also transiently down-regulated by cold. We did not observe a low temperature regulation for anyone of the isogenes of the N-methyltransferase family, corresponding to the methylation pathway for PtdCho biosynthesis. Genes corresponding to the PtdEtn biosynthesis pathway were not altered by cold exposure. These results denote that the increased PtdEtn to PtdCho mole ratio in ER membranes during growth at low temperature was correlated with a balance between a globally unaffected PtdEtn biosynthesis pathway and a partially down-regulated at the transcriptional level PtdCho biosynthesis pathway. In summary, we have demonstrated important lipid rearrangements of ER membranes in response to low temperature in B. napus hypocotyls that were accompanied by partial adaptive changes in the membrane physical properties. Additionally, putative stearoyl-ACP desaturase isogene(s) was identified via a reverse Northern analysis as cold-responsive in the dark-grown rape hypocotyls. Our data deserve future studies on the regulation of this stearoyl-ACP desaturase gene family as well as investigations on its biological role in the acquisition of cold tolerance using reverse genetic approaches.

4. Methods 4.1. Plant material, growth conditions and evaluation of freezing tolerance Two cultivars of oilseed rape (B. napus L.) were used in this work, a freezing-tolerant cultivar, cv. Tradition, and a freezing-sensitive cultivar, cv. ISL (cv. ISL/97/2/P). Seeds were germinated on moist vermiculite in a growth chamber

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in darkness at 22 °C for 4 d. The etiolated hypocotyls were then either left at 22 °C or transferred to 4 °C for further growth in the dark for up to 13 d. Hypocotyls grown for 5 and 7 d at 22 °C were at a similar developmental stage as estimated by the length and weight of the hypocotyl as hypocotyls transferred at 4 °C for 3 and 13 d, respectively. Such hypocotyls grown at 22 °C were used as developmental controls in transcriptome analysis. Other hypocotyls were grown continuously at 4 °C for 30 d. For freezing tests, etiolated hypocotyls acclimated for 3 or 13 d at 4 °C as well as light-grown hypocotyls acclimated for 3 d at 4 °C were treated as described by Wanner and Juntilla [39]. The freezing tolerance was assessed by visual monitoring of the hypocotyl survival and plantlet growth during the next 7 d at 22 °C. 4.2. Isolation and purification of ER membranes Dark-grown B. napus hypocotyls collected at the desired day of growth were ground in a Waring blendor (5 s at low speed, then 10 s at high speed) with homogenization buffer (3 ml g–1 of fresh weight) containing 20 mM 3-(Nmorpholino) propanesulfonic acid (MOPS)-KOH, pH 7.5, 300 mM mannitol, 1 mM ethylenediaminetetraacetic acid (EDTA), 4 mM cysteine, 0.6% polyvinylpyrrolidone (PVP), 0.1% bovine serum albumin (BSA), 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was filtered through two layers of Miracloth and subjected to differential centrifugations at 3500 × gmax for 15 min, 18,000 × gmax for 30 min and 40,000 × gmax for 90 min. The resulting microsomal pellet was gently resuspended in 3 ml of buffer containing 20 mM MOPS–KOH, pH 7.3, 250 mM mannitol, 1 mM EDTA, 5 mM dithiothreitol (DTT), 0.6% PVP, 0.2% BSA, 0.09% ascorbic acid, 0.5 mM PMSF. Membrane suspension was loaded onto three 26 ml discontinuous sucrose density gradients containing 20 mM MOPS, pH 7.3. Gradients were composed of 1 ml of 37% (w/v), 5 ml of 30%, 10 m of 26% and 10 ml of 12% sucrose. After centrifugation at 55,000 × gmax for 45 min using a swinging bucket rotor (preparative Beckman ultracentrifuge), the ER-enriched membrane fraction (crude ER fraction) was collected from the 12%/26% sucrose interface, diluted with 10 mM MOPS–KOH (pH 7.3) buffer containing 1 mM EDTA and 10 mM KCl (buffer A), and subjected to centrifugation at 100,000 × gmax for 35 min in a fixed angle rotor. The pellets of ER membranes-enriched fractions were resuspended in a minimum volume of buffer A and yield was estimated to approximately 1 mg of total ER proteins obtained from 100 g (fresh weight) of etiolated hypocotyls. Aliquots were immediately used for enzyme assays and lipid extraction. The remaining fractions were stored at –20 °C before fluorescence measurements. 4.3. Enzyme assays For measurement of the ATPase activity, aliquots of membranes (80 µg of proteins) were incubated at 25 °C in 0.9 ml

of buffer containing 10 mM MOPS–KOH, pH 7.3, 10 mM KCl, 1 mM EDTA, 3 mM MgSO4 in the presence or absence of 0.1 mM vanadate. The reaction was started with the addition of 1 mM ATP and aliquots were taken out at different times. Reaction was stopped and liberated phosphorus was assayed according to Anner and Mossmayer [1]. For Tritonstimulated UDPase, aliquots of membranes were incubated as above but the buffer contained 1% TritonX-100 to permeabilize Golgi vesicles. Reaction was started by the addition of 1 mM UDP and treated as described above. The NADHcytochrome c reductase activity (in the presence or absence of antimycin A and cyanide) was determined according to Hodges and Leonard [12] as the reduction of cytochrome c in a 10 mM phosphate buffer, pH 7.2 containing 60 µM cytochrome c. In order to determine the ER NADH-cytochrome reductase activity, 1 mM KCN and 16 µM antimycin A were added to the assay solution 3 min prior measurement. The reaction was initiated by the addition of NADH at 0.2 mM final concentration and the assay was performed at 25 °C. 4.4. Lipid analysis Lipids were extracted according to Folch et al. [7] from the ER-enriched fractions of B. napus etiolated hypocotyls and they were separated into representative lipid classes by two-dimensional silica gel thin layer chromatography (20 × 20 cm; Merck) as described by Lepage [16]. Lipid classes were subjected to methanolysis at 70 °C for 60 min in 3 ml of 2.5% sulfuric acid (v/v) in methanol. The resultant fatty acid methyl esters were extracted with 3 ml of hexane and analyzed by gas chromatography (Varian) on a fused-silica capillary column. Quantification of fatty acids was carried out using methylheptadecanoate (C17) as an internal standard, which was introduced before methanolysis. 4.5. Phosphorus and protein analysis Lipid phosphorus content was determined according to Rouser et al. [26]. Protein content was assayed according to Lowry et al. [19] using BSA as a standard. 4.6. Fluorescence measurements The lateral diffusion of the excimer-forming probe, 1-pyrenedodecanoic acid (Molecular Probes, Inc., Eugene, OR) was determined in purified ER membranes according to Galla and Luisetti [8] at different temperatures from 10 to 28 °C. Aliquots of membranes equivalent to 100 nmol of phospholipids were suspended in 1.5 ml of a medium containing 10 mM MOPS, pH 7.3, 10 mM KCl, 1 mM EDTA. Successive additions of the probe were carried out to give a molar ratio of probe to phospholipids varying from 0.001 to 0.13. The ratio of excimer fluorescence intensity to monomer fluorescence intensity (emission maxima at 473 and 378 nm, respectively) is a measure of the collision rate between phospholipid molecules. Local microviscosity was assayed using

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stearic and palmitic acid analogs substituted with anthroyloxy moieties on different carbon positions along the fatty acid chain (2-, 3-, 6-, 7-, 9-, or 12-anthroyloxy stearic acid and 16-anthroyloxy palmitic acid, Molecular Probes). These analogues were embedded in ER membranes at a molar ratio of probe to phospholipid of 0.02. The fluorescence depolarization of anthroyloxy-substituted fatty acids embedded in ER membranes was measured either at 10 and 22 °C after its addition to ER-enriched fraction at ambient temperature for 20 min. Excitation and emission wavelengths were set at 368 and 440 nm, respectively. All measurements were performed on a Varian spectrofluorimeter. 4.7. Arabidopsis macroarray preparation and cDNA labeling 4.7.1. BLAST searches and macroarray preparation To retrieve nucleotide sequences corresponding to fatty acid and phospholipid biosynthesis genes, we initially did searches in the Arabidopsis Information Resource database (www.arabidopsis.org) using enzyme names. Genomic sequences with a corresponding characterized protein function were used for BLASTN searches to retrieve additional plant sequences for glycerolipid biosynthesis genes. Top parts of the BLAST outputs were saved to constitute exhaustive multi-gene families. We systematically performed sequence alignments in order to choose specific region for each gene. The gene families analyzed in this work are listed in Table 4 with the corresponding forward and reverse primers used to amplify genomic fragments by standard PCR reaction. Alpha-tubulin isogene (At5g19770) was used as an internal standard for normalization of the data [29]. PCR reactions were made in 100-µl volume containing 1 µM of each primer, 0.2 mM of each desoxynucleotide, 10 mM PCR buffer (containing 15 mM MgCl2), 2.5 units of Taq DNA Polymerase (Qiagen) and approximately 2–10 ng of Arabidopsis ecotype Columbia genomic DNA. The reactions were run on a thermoblock system (PE Biosystems) and comprised one step of denaturation at 94 °C for 3 min, 40 cycles of 1 min at 94 °C, 1.25 min at 53 °C and 1.5 min at 72 °C. Terminal extension was carried out for 10 min at 72 °C. The purity of the PCR products was assessed after agarose gel electrophoresis and their identity was checked by restriction analysis. Aliquots (280 ng) of each PCR product was denatured in 0.4 M NaOH DNase-free water solution and arrayed on a Nytran SuperCharge transfer membrane (Schleicher&Schuell) previously wet with 0.4 M NaOH using a dot blotter (Fisher brand). The DNA fragments were fixed on membranes at 80 °C for 2 h. The arrays were then stored at 4 °C until use. 4.7.2. RNA extraction and probe synthesis Total RNA was extracted from 2 g of hypocotyls using a modified phenol extraction procedure including an additional precipitation step to remove contaminating polysaccharides. This was done by the addition of 1/30 vol. of 3 M Na acetate and 0.1 vol. of absolute ethanol to the resuspended

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Li acetate pellet. Poly(A)+ RNA were isolated from 100 µg of total RNA using PolyATract mRNA Isolation System III following the manufacturer’s instructions (Promega). Preparation of 32P-labeled probes of cDNA was performed as follows: 200 ng of lyophilized poly(A)+ RNA was solubilized in 6 µl containing 22 µM of 18-mer oligo-dT primer. The mixture was incubated at 70 °C for 10 min, chilled on ice, and mixed in a total reaction volume of 20 µl containing 1× First-Strand buffer, 0.5 mM each of dATP, dTTP, dGTP, 3 µM of dCTP, 10 mM DTT, 2.2 MBq of [alpha 32P]-dCTP and 200 units of Superscript™ II reverse transcriptase (Invitrogen). The reaction was run at 42 °C for 60 min and the cDNA products denatured at 95 °C for 5 min. Messenger RNA was digested with 2 units of RNaseH (Promega, Madison, WI) at 37 °C for 20 min.

4.7.3. Hybridization and quantification Labeled cDNAs were diluted in a buffer according to Church and Gilbert [5] so as to calibrate each probe to the same radioactive concentration (10 × 106 cpm ml–1). The prehybridization (1 h) and the hybridization (16 h) were performed in a hybridization oven (PI Applied Biosystems) at 60 °C. Following hybridization, membranes were washed twice in 2× SSC, 0.1% (w/v) SDS for 10 min, then twice in 1× SSC, 0.1% (w/v) SDS for 15 min. Membranes were then placed on 3 MM Whatman paper to remove excess humidity, wrapped in saran and then exposed to a phosphor screen overnight. Quantification was done using ImageQuant technology. Expression values of two controls per experiment corresponding to hypocotyls maintained at 22 °C were averaged and fold changes were calculated by dividing cold-treated expression value by the corresponding developmental control expression value as well as by the averaged control expression value. To define genes as low temperature regulated we used the criteria that twofold changes or greater must be reproducible for these two control expression values, the corresponding developmental control as well as the averaged controls of the same experiment.

Acknowledgements This work was supported by grants from the Centre National de la Recherche Scientifique (CNRS), the Ministère de l’Education Nationale, de la Recherche et des Technologies, and the Université Pierre et Marie Curie (UMR 7632). We are grateful to Dr. Luc Richard for many useful suggestions and helpful discussions. We thank Serasem (La Chapelle d’Armentières, France) for kindly providing us with the rapeseeds used in this study.

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