This Article Abstract Full Text (PDF) Supplemental Data All Versions of this Article: 139/3/1217    most recent pp.105.068171v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Vergnolle, C. Articles by Ruelland, E. Search for Related Content PubMed PubMed Citation Articles by Vergnolle, C. Articles by Ruelland, E. Agricola Articles by Vergnolle, C. Articles by Ruelland, E.

CELL BIOLOGY AND SIGNAL TRANSDUCTION

The Cold-Induced Early Activation of Phospholipase C and D Pathways Determines the Response of Two Distinct Clusters of Genes in Arabidopsis Cell Suspensions^1,[w]

Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Formation de Recherche en Évolution 2846, Centre National de la Recherche Scientifique/Université Pierre et Marie Curie, F–94200 Ivry-sur-Seine, France (C.V., M.-N.V., J.-C.K., A.Z., E.R.); and Unité de Recherche en Génomique Végétale, Unité Mixte de Recherche, Institut National de la Recherche Agronomique 1165, Centre National de la Recherche Scientifique 8114, F–91057 Evry, France (L.T., J.-P.R.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In plants, a temperature downshift represents a major stress that will lead to the induction or repression of many genes. Therefore, the cold signal has to be perceived and transmitted to the nucleus. In response to a cold exposure, we have shown that the phospholipase D (PLD) and the phospholipase C (PLC)/diacylglycerol kinase pathways are simultaneously activated. The role of these pathways in the cold response has been investigated by analyzing the transcriptome of cold-treated Arabidopsis (Arabidopsis thaliana) suspension cells in the presence of U73122 or ethanol, inhibitors of the PLC/diacylglycerol kinase pathway and of the phosphatidic acid produced by PLD, respectively. This approach showed that the expression of many genes was modified by the cold response in the presence of such agents. The cold responses of most of the genes were repressed, thus correlating with the inhibitory effect of U73122 or ethanol. We were thus able to identify 58 genes that were regulated by temperature downshift via PLC activity and 87 genes regulated by temperature downshift via PLD-produced phosphatidic acid. Interestingly, each inhibitor appeared to affect different cold response genes. These results support the idea that both the PLC and PLD pathways are upstream of two different signaling pathways that lead to the activation of the cold response. The connection of these pathways with the CBF pathway, currently the most understood genetic system playing a role in cold acclimation, is discussed.

In parallel to these studies of cold-induced transcriptome changes, much interest has been devoted to signaling pathways transducing the cold signal within the plant cell. We have shown that a cold treatment induces an increase of phosphatidic acid (PtdOH) within the first minutes of cold exposure to Arabidopsis (Arabidopsis thaliana) suspension cells (Ruelland et al., 2002). This was due to the activation of a phospholipase D (PLD) activity, as shown by transphosphatidylation, a reaction typical of PLD. But PtdOH also resulted from the action of phospholipase(s) C (PLC) coupled with a diacylglycerol kinase (DAGK) activity, as shown by the monitoring of inositol tris phosphate and by the effect of a DAGK inhibitor. The activation of PLC and PLD pathways were dependent on external calcium (Ruelland et al., 2002). These observations led us to ask the following questions with respect to the role of the activation of PLC and PLD in the cold response. Did PLC and PLD activation lead to a gene response, and if so, was it a positive or negative effect, activating or switching off gene expression? Which genes were cold responsive via the activation of PLC or PLD? In Arabidopsis, PLD, PLC, and DAGK are multigene families containing 12, 7, and 7 members, respectively (Elias et al., 2002; Mueller-Roeber and Pical, 2002; Qin and Wang, 2002; Gomez-Merino et al., 2004). The isoforms of PLD, PLC, or DAGK responsible for the activity detected early after a temperature downshift are unknown, although some of them have been shown to be up-regulated in response to a temperature downshift. For instance, AtDAGK2, PLD, and PLD are up-regulated by cold (Welti et al., 2002; Gomez-Merino et al., 2004; Li et al., 2004), and plants mutated in PLD are impaired in the development of freezing tolerance (Li et al., 2004). However, the fact that an isoform is up-regulated in response to a stress does not mean that it is the one responsible for the early transduction of this stress. To overcome this problem, we have applied a pharmacological approach using Arabidopsis suspension cells as a model. PLC activity was inhibited by U73122. As for PLD activity, adding ethanol to the cell medium, we were able to shift PtdOH production by PLD toward the production of phosphatidylethanol. However, in this case, it is important to note that ethanol does not inhibit the PLD-catalyzed hydrolysis of phospholipids but, being a substrate, reduces the production of the physiological signaling product, PtdOH, while promoting the formation of a new phospholipid. We monitored transcriptome changes in response to a cold exposure in the presence of these agents modifying PLC and PLD pathway activities. In this way, we were able to identify gene clusters that could be considered as dependent either on PLC activity or on PLD-produced PtdOH for their cold response. These clusters were mainly characterized by a positive action of PLC or of PLD-produced PtdOH on cold response gene expression. Interestingly, it was found that pathways dependent on PLC activity or on PLD-produced PtdOH controlled the transcription of two different gene clusters. The role of the PLC and PLD pathways regarding the CBF regulon is discussed.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Time Course of Gene Induction in Arabidopsis Plantlets and Suspension Cells at 4°C

We first wanted to study the kinetics of gene induction by a cold shock in Arabidopsis cv Columbia suspension cells. We chose different genes that have been described as cold responsive in whole plants: ELIP1, amylase, SAG21, COR47, COR15A, LTI78, KIN1, HVA22, DREB1A/CBF3, DREB1B/CBF1, DREB1C/CBF2, and CZF1 (Gilmour et al., 1998; Seki et al., 2001; Fowler and Thomashow, 2002). We followed their expression in plantlets and in suspension cells 45 min and 4, 8, and 24 h after transfer from 22°C to 4°C (Fig. 1).

View larger version (96K): [in this window] [in a new window]   Figure 1. Gene expression in response to cold in Arabidopsis suspension cells and plantlets. Plants or cell suspensions cultivated at 22°C were exposed at 4°C for different time periods. RNA was isolated and submitted either to RNA-blot hybridization or to RT-PCR. For RNA-blot hybridization, gene-specific probes were used, and rRNA was used as a loading control. For RT-PCR, gene-specific primers were used, with the number of cycles optimized for each primer pair. S19 was used as a control.

At this stage, our results indicated that Arabidopsis suspension cells responded to cold at the level of gene expression and that the CBF regulon, the currently most understood cold response regulon, was induced.

Transcriptome Analysis after 4 h at 4°C

In order to get a broader view of gene induction/repression in response to a cold shock, we performed a microarray experiment with the complete Arabidopsis transcriptome microarray (CATMA) chip containing 24,715 probes, representing approximately 22,000 genes (Crowe et al., 2003; Hilson et al., 2004). RNAs were extracted from 6-d-old suspension cells either nonexposed to cold (22°C) or exposed to 4°C for 4 h. The RNAs from three independent biological repetitions were pooled and used as the matrix for the synthesis of fluorescent cDNA probes, permitting the comparison on the same slide with two independent two-color hybridizations (one dye-swap). The differentially expressed genes were identified by a paired t test on the log ratios. The raw P values were adjusted by the Bonferroni methods, which control the familywise error rate (FWER). With an FWER set at 5%, most of the probes, i.e. 24,059 probes (97.2% of total monitored probes), showed no changes in their transcript level. Nevertheless, 511 genes (2% of total monitored genes) could be considered as up-regulated, while 187 (0.8%) were down-regulated. When compared to other reports on gene expression in response to cold (Seki et al., 2001, 2002; Chen et al., 2002; Fowler and Thomashow, 2002; Kreps et al., 2002; Chinnusamy et al., 2003; Maruyama et al., 2004; Vogel et al., 2004), our study describes 100 new genes that were cold repressed and 324 new genes that were cold induced. The list of the differentially regulated genes is available as Supplemental Table I.

Genes that were either up-regulated or down-regulated in response to cold were classified according to the Munich Information Center for Protein Sequences (MIPS) functional catalog categories (Schoof et al., 2004), using the MIPS interface (http://mips.gsf.de/proj/thal/db/index.html; Fig. 2). Selected examples of cold-induced genes and cold-repressed genes are described in Tables I and II, respectively. In both cases, the most represented category was transcription (Fig. 2), indicating that after 4 h at 4°C, a major change in the transcriptome is still under way. The second most represented category differs between up-regulated and down-regulated genes, being metabolism and protein fate, respectively. The third most represented category was cellular communication in both cases, meaning that a readjustment of the cell signaling machinery is necessary for cold adaptation. The next gene categories that were up-regulated by cold exposure (in decreasing order) are protein fate, cell rescue, defense, and virulence, transport facilitation, energy, protein synthesis, cellular transport, cell fate, and cell cycle and DNA processing. For the genes that were down-regulated, the order is energy, metabolism, cell rescue, defense, and virulence, transport facilitation, cell fate, cellular transport, and protein synthesis.

View larger version (31K): [in this window] [in a new window]   Figure 2. Functional categories of the cold-induced genes or the cold-repressed genes. Genes that were up-regulated or down-regulated during a 4-h exposure at 4°C according to the microarray analysis were classified into functional categories using the MIPS interface (http://mips.gsf.de/proj/thal/db/index.html).

Effects of Phospholipase Inhibitors

Primary alcohols can be used as substrates by PLD, in the so-called transphosphatidylation reaction. In the presence of a primary alcohol, PLD will use it instead of water, leading to the formation of a phosphatidylalcohol instead of PtdOH. Secondary or tertiary alcohols are not substrates of this transphosphatidylation reaction (Munnik et al., 1995). We checked the effect of ethanol and tertiary butanol (tertButOH) on the production of PtdOH by PLD after a cold shock. Phospholipids were labeled by [³³P]-orthophosphate, and radioactive ATP was quenched by an excess of nonradioactive phosphate added 15 min before the cold exposure. In such conditions, because ATP is no longer radioactive but the substrates of PLD are, radioactive PtdOH can only be produced by PLD (Ruelland et al., 2002). An exposure of 10 min at 0°C led to a 50% increase of the PtdOH level, reflecting PLD activity (Fig. 3A). When 0.9% (v/v) ethanol was added 15 min before the cold shock, the radioactive PtdOH level decreased to the level of nonstressed cells, while preincubation with 0.9% (v/v) tertButOH had no significant effect on PtdOH production (Fig. 3A). Concomitantly, when ethanol was present, the appearance of phosphatidylethanol in cells stressed at 0°C confirmed that the PLD pathway was active. Besides, because primary and secondary alcohols have been shown to be able to activate PLD activity, maybe via G protein activation (Munnik et al., 1995), we checked whether the concentrations used did not lead to PLD activation. Cells were labeled for at least 12 h with ³³Pi, allowing a good labeling of putative PLD substrates. We then added to the cell medium 0.1% (v/v) primary butanol, together or not with 0.9% (v/v) ethanol or 0.9% (v/v) tertButOH. Lipids were extracted 10 min after the addition of the chemicals, and separated. As a control, cells treated with 0.1% (v/v) primary butanol were exposed for 10 min to 0°C or to 0.4% (v/v) 2-phenyl-2-propanol, an alcohol known to activate PLD. The production of phosphatidylbutanol was measured as a marker of PLD activity via its transphosphatidylation reaction. Our results (Supplemental Fig. 1) clearly showed that no increase in PLD activity could be seen when cells were treated with 0.9% (v/v) ethanol or 0.9% (v/v) tertButOH. In response to cold, an increase in PLD was detected, as it was in the presence of 2-phenyl-2-propanol. The fact that 0.9% (v/v) ethanol did not activate any PLD activity was confirmed when we monitored the level of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) in treated cells. Cells were labeled for at least 12 h with ³³Pi, and 0.9% (v/v) ethanol or 0.9% (v/v) tertButOH was added to the cell medium. Lipids were extracted at different times after alcohol treatment and separated by thin-layer chromatography. PtdCho and PtdEtn were quantified and their levels normalized to that of phosphatidylinositol (Supplemental Fig. 2). For short times or longer times (up to 4h), no significant differences in PtdCho and PtdEtn levels could be detected, thus suggesting that no phospholipid hydrolysis was taking place.

View larger version (18K): [in this window] [in a new window]   Figure 3. Inhibition of PLC/DAGK and PLD pathways. A, Cells were labeled in the presence of 53 MBq L–1 [33P]-PO43– for 2 h at 22°C. Different concentrations of U73122 or U73343 were added 15 min before cold shock. After a 10-min exposure at 0°C, lipids were extracted and separated by thin-layer chromatography. Radioactive PtdOH was first expressed as a percentage of total radioactive phospholipids and then normalized to the relative radioactive PtdOH content in the experiment with no chemicals. B, Cells were labeled in the presence of 53 MBq L–1 [33P]-PO43– for 24 h at 22°C, after which the cells were supplemented with 3 mM nonradioactive PO43–. When necessary, 0.9% (v/v) ethanol or 0.9% (v/v) tertButOH was also added to the cell medium. Cells were submitted to a 10-min exposure at 0°C before lipids were extracted and separated using thin-layer chromatography. Radioactive PtdOH was expressed as a percentage of total radioactive phospholipids and then normalized to the relative radioactive PtdOH content in the experiment with no cold shock.

Microarray Analysis: Genes Depending on PLD-Produced PtdOH

RNAs were extracted from 6-d-old cells submitted to four different conditions: (1) no stress (22°C), (2) exposure at 4°C for 4 h, (3) exposure at 4°C for 4 h in the presence of 0.9% (v/v) tertButOH (hereafter, 4°C^tertButOH), and (4) exposure at 4°C for 4 h in the presence of 0.9% (v/v) ethanol (hereafter, 4°C^ethanol). The RNAs of three independent biological repetitions were pooled and reverse transcribed either with Cy3-dUTP or Cy5-dUTP to perform a two-color hybridization with the CATMA chip. One dye-swap (i.e. two hybridizations) was carried out for each of the two following combinations: 4°C versus 4°C^tertButOH and 4°C^ethanol versus 4°C^tertButOH.

Among the 698 genes that were differentially regulated in response to a temperature downshift, 93 showed a transcript level difference between 4°C^tertButOH and 4°C^ethanol and may be considered as responding to cold via PLD-produced PtdOH (Fig. 4). These genes are listed in Supplemental Table II. Of these 93 genes, 73 were up-regulated in response to cold, while 20 were down-regulated. Of the 73 genes that were up-regulated at 4°C, 72 (i.e. 99%) had a lower amount of transcripts at 4°C in cells treated with ethanol when compared to tertButOH, thus showing an inhibitory effect specific of primary alcohols. A single gene had a higher transcript level at 4°C when comparing ethanol with tertButOH-treated cells, thus showing an inducing effect of ethanol versus tertButOH. Of the 20 genes that were down-regulated at 4°C, 15 (i.e. 75%) had higher amounts of transcripts at 4°C when cells had been preincubated with ethanol compared to a preincubation with tertButOH, thus showing an inhibitory effect of ethanol versus tertButOH on this repression. Only 5 of the 20 genes (i.e. 25%) had lower transcript amounts at 4°C when cells had been preincubated with ethanol compared to tertButOH, thus showing an inducing effect of primary alcohols on the repression. When taken together, it appears that 87 genes out of 93 genes (i.e. 94%) showed a repressing effect of ethanol versus tertButOH on their response to cold (Fig. 4).

View larger version (23K): [in this window] [in a new window]   Figure 4. Determination of the genes downstream of the PLD pathway. Among the 861 genes that showed transcript level changes in response to 4°C versus 22°C or in response to 4°Cethanol versus 4°CterButOH, 93 showed transcript level changes in response to both 4°C versus 22°C and to 4°Cethanol versus 4°CterButOH. These genes are potentially cold regulated via the PLD pathway. Of these 93 genes, 20 were down-regulated by the cold treatment, while 73 were up-regulated.

Microarray Analysis: Genes of the PLC Pathway

RNA was extracted from 6-d-old cells that had been submitted to four different conditions: (1) no stress (22°C), (2) exposure at 4°C for 4 h, (3) exposure at 4°C for 4 h in the presence of U73122 (hereafter, 4°C^U73122), and (4) cells exposed to 4°C for 4 h in the presence of U73343 (hereafter, 4°C^U73343). RNA extracted from three independent biological repetitions was pooled and reverse transcribed in the presence of Cy3-dUTP or Cy5-dUTP to perform a two-color hybridization with the CATMA chip. One dye-swap (i.e. two hybridizations) was made for each of the two following combinations: 4°C versus 4°C^U73343 and 4°C^U73122 versus 4°C^U73343. Among the 698 genes that were differentially regulated in response to a temperature drop, we considered the genes showing a transcript level difference between 4°C^U73122 and 4°C^U73343 (Fig. 5). However, some of these genes also showed a difference in transcript levels between 4°C^U73343 and 4°C. Since U73343 was dissolved in a dimethyl sulfoxide (DMSO)/tertButOH mix, the difference in transcript levels between 4°C^U73343 and 4°C could indicate a solvent effect on gene expression. Therefore, the effect of U73122 versus U73343 could be attributed to an effect of the solvents and not to a PLC-dependent cold activation. These genes were not considered in our analysis. The 68 remaining genes showed a transcript level difference between 4°C^U73122 and 4°C^U73343 and between 4°C and 22°C. These genes may be cold-regulated via a PLC pathway. They are listed in Supplemental Table IV. Of these 68 genes, 48 were up-regulated in response to cold, while 20 were down-regulated. Of the 48 up-regulated genes, 45 (i.e. 94%) showed lower transcripts levels at 4°C when cells had been preincubated with U73122 compared to U73343, thus showing an inhibitory effect of U73122 on the induction. All of the 20 genes that were down-regulated at 4°C had higher transcript amounts at 4°C when the cells had been preincubated with U73122 compared to U73343, thus showing an inhibitory effect of U73122 versus U73343 on the repression. When considered together, it appeared that 65 out of the 68 genes (i.e. 96%) showed a repressing effect of U73122 versus U73343 on their response to the cold treatment.

View larger version (25K): [in this window] [in a new window]   Figure 5. Determination of the genes downstream of the PLC/DAGK pathway. Among the 1,172 genes that showed transcript level changes in response to 4°C versus 22°C or in response to 4°CU73122 versus 4°CU73343, 591 genes showing a transcript level change in response to 4°CU73343 versus 4°C were removed from the analysis. Of the remaining genes, 68 showed transcript level changes in response to 4°C versus 22°C and in response to 4°CU73122 versus 4°CU73343. These genes are potentially cold-regulated via the PLC pathway. Of these 68 genes, 20 were down-regulated by the cold treatment, while 48 were up-regulated.

Confirmation by RT-PCR and Northern-Blot Analyses

The results obtained by microarray analyses were confirmed on a selection of genes by using various agents acting on phospholipase pathways. For genes driven by PLD-produced PtdOH, we tested different concentrations of ethanol (0%, 0.3%, 0.9%, and 1.8% [v/v]) and 0.7% (v/v) tertButOH as a control. Northern blots or RT-PCR were carried out with SAG21, LTI78, HVA22, LTI30 (a Glu dehydrogenase encoding gene), CZF1, a gene encoding an AP2 domain containing protein, and WRKY33. A gene encoding a Myb factor, MYB73, which was not classified in the genes driven by PLD-produced PtdOH, was tested as a control; the gene (At3g04920) encoding the ribosomal protein S19 was used as a constitutive probe (Fig. 6). As expected, ethanol showed an inhibiting effect at 0.3% and at 0.9% (v/v). But for some genes, less inhibition was detected in the presence of 1.8% (v/v) ethanol (Fig. 6). This can be explained if the genes contain a cis-element in their promoter that is sensitive to G proteins, since they can be activated by this higher concentration of ethanol. The G protein activation would counterbalance the inhibiting effect of PLD by transphosphatidylation. The presence of ethanol had no effect on the expression of MYB73 and S19, as expected.

View larger version (58K): [in this window] [in a new window]   Figure 6. Effect of inhibitors of the PLD pathway on gene induction in response to cold. RNA was isolated from 5-d-old cell suspensions cultivated at 22°C with or without exposure for 8 h at 4°C. In the case of a cold exposure, when indicated, the cell medium was supplemented with 0.3% (v/v), 0.9% (v/v), or 1.8% (v/v) of ethanol or with 0.7% (v/v) of primary butanol or 0.7% (v/v) of tertButOH. RNA was submitted either to RNA-blot hybridization or to RT-PCR. For RNA-blot hybridization, gene-specific probes were used, and rRNA was used as a loading control. For RT-PCR, gene-specific primers were used, with the number of cycles optimized for each primer pair. S19 was used as a control.

View larger version (58K): [in this window] [in a new window]   Figure 7. Effect of inhibitors of the PLC pathway on gene induction in response to cold. RNA was isolated from 5-d-old cell suspensions cultivated at 22°C with or without exposure for 8 h at 4°C. In the case of a cold exposure, when indicated, the cell medium was supplemented with U73122 (60 µM), U73343 (60 µM), or edelfosine (150 µM). RNA was submitted to RT-PCR using gene-specific primers. The number of cycles was optimized for each primer pair. S19 was used as a control.

In order to determine whether some genes were dependent on both pathways, we compared the list of the cold response genes that depended either on PLC activity or on the production of PtdOH by PLD. Among the cold-induced genes, only seven were activated via both PLC and PLD (Table V). This means that among the genes that were cold induced via PLC, 83% were not cold activated via PLD-produced PtdOH and that 89% of the genes that were cold-induced by PLD-produced PtdOH were not cold activated via PLC. For the genes that were cold repressed, only one gene was repressed via both PLC activity and PLD-produced PtdOH. This strongly suggests that PLC activity and PLD-produced PtdOH are upstream pathways that regulate two different clusters of genes and that the signaling pathways triggered by the activation of PLC in response to cold and by PtdOH production by PLD in response to cold are distinct.

The CBF/DREB1 pathway is currently the best understood genetic system playing a role in cold acclimation. A number of studies have looked for CBF/DREB1 target genes by over-expressing these proteins (Kasuga et al., 1999; Seki et al., 2001; Fowler and Thomashow, 2002; Maruyama et al., 2004; Vogel et al., 2004). We searched whether our gene candidates being driven by PLD-produced PtdOH or by PLC activity have been described as CBF targets. For genes up-regulated by cold, we found that 17 out of 511 (3.3%) are mentioned as CBF target genes in at least one of the reference studies (Supplemental Table I). When the 72 genes dependent on PLD-produced PtdOH for their cold activation are considered, the proportion of CBF targets rises to 12.5% (9 out of 72 genes). On the other hand, for PLC-dependent genes, only 3 out of 42 have been described as CBF target genes, i.e. 7.1%. No obvious overrepresentation can be concluded from these results on so few genes. Therefore, we checked the cold expression of DREB1A in the presence or absence of agents disturbing PLD (Fig. 8) and PLC (data not shown) pathways. An inhibitory effect of primary alcohols can be seen while no inhibitory effect of U73122 or edelfosine could be detected.

View larger version (30K): [in this window] [in a new window]   Figure 8. Effect of inhibitors of the PLD or PLC/DAGK pathways on the cold induction of the DREB1A gene. RNA was isolated from 5-d-old cell suspensions cultivated at 22°C with or without exposure for 8 h at 4°C. In the case of a cold exposure, when indicated, cell medium was supplemented with 0.3% (v/v), 0.9% (v/v), or 1.8% (v/v) ethanol or with 0.7% (v/v) tertButOH or with 0.7% (v/v) nButOH. RNA was submitted to RT-PCR using primers specific for DREB1A. S19 was used as a control.

The changes concerning the transcript levels of PLC, PLD, and DAGK genes (Supplemental Table VI) were extracted from the different comparisons of our microarray data. The nomenclature used for these genes are from Mueller-Roeber and Pical (2002), Qin and Wang (2002), and Gomez-Merino et al. (2004). For PLC genes, only AtPLC2 showed a difference in transcript levels in one of our comparisons, with the transcript level at 22°C^U73343 being higher than at 22°C. For DAGK genes, only AtDAGK7 showed a difference in transcript levels, with transcript abundance being higher at 4°C^U73343 compared to 4°C. For PLD genes, two genes, AtPLD1 and AtPLD2, showed differences in transcript levels in a single comparison. Both genes had fewer transcripts at 22°C^U73343 than at 22°C and fewer transcripts at 22°C^tertButOH when compared to 22°C. Besides, AtPLD2 transcript levels were higher at 4°C than at 22°C as well as at 4°C with respect to 4°C^tertButOH. This clearly reveals that under our conditions, PLC and DAGK genes were not induced by cold at a level higher than the cutoff designed for the microarray experiment. On the other hand, a single PLD gene (PLD2) was found to be cold induced.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The fact that a temperature downshift induced the expression of genes known to be cold regulated in Arabidopsis plantlets in Arabidopsis cell suspensions allowed us to consider this system as a good model for a genome-wide analysis of gene expression in response to a cold shock.

Analysis of Transcriptome Changes

The monitoring of cold-stress-induced changes within the transcriptome led us to identify 100 new cold-repressed and 324 new cold-induced genes. The fact that these genes had not been described in a similar microarray studies with the Arabidopsis Affymetrix GeneChip AtH1 (Vogel et al., 2004) could be due to differences in the model used: i.e. suspension cells versus plantlets. However, it could also reflect differences in the gene sets probed in the two arrays. All in all, the analyses of the genes either up-regulated or down-regulated (Supplemental Table I) by cold offer a good picture of the challenge faced by plant cells during a temperature downshift and an insight into the mechanisms activated to cope with this stress. For instance, after a 4-h exposure at 4°C, it can be seen that the first functional category of up-regulated genes is transcription, and it is also the first functional category of down-regulated genes. This indicates that, in response to cold, a complex pattern of gene induction is triggered that results in a succession of transcription waves. This will lead to new communication webs as shown by the induction, and also repression, of kinases, calcium-dependent kinases, protein phosphatases, or calcium binding proteins. In parallel to this, a total change in metabolism is triggered, with nonphotosynthetic sources of energy being up-regulated. This correlates with metabolomic studies where the increased levels in organic acids of the citric acid cycle suggest an up-regulation of respiration (Cook et al., 2004; Kaplan et al., 2004). Conversely, photosynthesis is shut down, as shown by the repression of genes encoding photosystem II and chlorophyll a/b-binding proteins. This adjustment of light-harvesting antenna size is believed to be a photoprotective mechanism against the production of activated oxygen species. Indeed, the induction of pathways leading to the synthesis of flavonoids, carotenoids, or polyamines (Supplemental Table I) can be correlated with the cells experiencing a photooxidative stress because these classes of molecules have been implicated in photoprotection (Harvaux and Kloppstech, 2001; He et al., 2002). The fact that cells experience oxidative stress during a temperature downshift has been confirmed by the induction of genes encoding molecules involved in detoxification, such as lignols (Rice-Evans et al., 1997), ferritin, peroxidase, or -glutamylcysteine synthetase. Finally, our transcriptome analysis indicates that Arabidopsis cells in suspension can activate during a temperature downshift the synthesis of molecules that contribute to cold and to freezing tolerance, like amino acids, which can be seen as compatible solutes (genes involved in amino acid metabolism are indeed disturbed by a cold exposure; Supplemental Table I), or sugars, that can have a protective role for membranes (Uemura et al., 2003). Proteins, like dehydrins of the cold-responsive/late embryogenesis abundant family, are also induced in our suspension cells. They are thought to protect membranes (Steponkus et al., 1998; Puhakainen et al., 2004). Finally, changes in lipid metabolism, such as induction of desaturases, can also contribute to the adaptive response of the cells by counteracting the cold-induced rigidification of membranes.

Since the changes in the transcriptome found under our conditions are in good agreement with the conclusions of previous transcriptomic and metabolomic studies, it was concluded that the cell suspension model could be used to investigate the role of the early activation of the PLC and PLD pathways in cold response.

PLD Activity Is an Element of Cold Transduction Leading to Gene Response

The PLD(s) activated during a temperature downshift can form phosphatidylethanol in the presence of ethanol, thus leading to a decrease in the amount of PtdOH formed. This allowed for the determination of genes regulated by PLD-produced PtdOH by comparing data obtained after a cold stress applied in the presence or in the absence of this alcohol. However, it is important to note that ethanol does not inhibit PLD catalytic activity on phospholipids; therefore, the effect of ethanol on gene expression cannot be attributed to PLD activity but to PLD-produced PtdOH. Besides, the alcohol effect on cell metabolism is certainly not limited to an inhibition of the PLD pathway. Although primary alcohols can be substrates of transphosphatidylation by PLD, they can also activate G proteins and fluidize membranes. On the other hand, secondary alcohols are not PLD substrates, but they can activate G proteins and fluidize membranes. While tertiary alcohols are not substrates of PLD, they cannot activate G proteins, but they can fluidize membranes. This raises the question of which alcohol should be chosen to serve as a control of the primary alcohol used. Comparing the effects of primary alcohols versus secondary alcohols on cold response could lead to the removal of genes that are downstream of G protein activation. This is not the case when comparing the effects of primary alcohols versus tertiary alcohols. Nevertheless, since tertiary alcohols do not activate G proteins, the primary alcohol used had to have little effect on G protein, and ethanol has been reported to fulfill this condition (Kiss and Anderson, 1989; Munnik et al., 1995). Our control experiments show that neither 0.9% (v/v) ethanol nor 0.9% (v/v) tertButOH activate PLD activity, contrary to cold treatment. Therefore, we chose to compare the effect of ethanol and tertButOH on gene regulation by a cold shock. We showed that 87 genes, representing 94% of the genes that were cold regulated, displayed a transcript level difference between 4°C^ethanol versus 4°C^tertButOH, thus showing an inhibitory effect of the primary ethanol. As shown by ² analysis, this proportion reflects a specific inhibitory action of ethanol versus tertButOH that can be considered as the inhibitory action on PLD-produced PtdOH and the cluster of 87 genes considered as driven by PLD-produced PtdOH. The dependence of some of these genes on the production of PtdOH by PLD during cold response was confirmed by northern-blot or RT-PCR analysis. We tested different concentrations of ethanol. For most genes, higher concentrations of ethanol led to an increase in transcript levels. This must be seen as a G protein effect. This does not necessarily mean that these genes are dependent on G proteins for their cold response but that G protein activation can activate their transcription. Interestingly, the cluster of genes driven by PLD-produced PtdOH included genes that were induced by the cold as well as genes that were down-regulated by this treatment. This indicates that PLD activation, leading to PtdOH production, triggers a signaling pathway that leads to the activation or repression of genes. The production of PtdOH by PLD has a positive action, since its inhibition mainly inhibits the cold response. This is an important fact indicating that PLD is implicated in the activation of the cold response and not in the turning off of this response.

As a control, we tested the effects of 0.9% (v/v) ethanol and 0.9% (v/v) tertButOH on gene expression at 22°C. Very few genes showed a difference in transcript levels after a 90-min incubation with these alcohols. This suggests a minor role, if any, of a basal PLD activity on gene regulation in nonstressed cells.

In the microarray experiment, a single PLD gene, PLD2, was found to be up-regulated by cold exposure. It would be interesting to elucidate whether or not this isoform is responsible for the early production of PtdOH by a PLD activity.

PLC Activity Is an Element of Cold Transduction Leading to Gene Response

The identification of a cluster of genes that respond to cold via PLC was achieved by using U73122. This molecule acts on PLC, but we cannot rule out an action on other enzymes; therefore, its effect has to be compared to that of U73343, an analog with a significantly lower effect on PLC activity. When tested in response to a cold stress, increasing concentrations of U73122 led to a decreased production of PtdOH via the PLC/DAGK pathway, an effect that could not be reproduced with U73343. When RNA levels in the presence of U73122 during the cold shock were compared to RNA levels in the presence of U73343, we found an inhibition of the cold response genes in the presence of U73122. Indeed, 68 genes displayed a difference in transcript levels at 4°C in the presence of U73122 versus U73343, and 96% of these genes (65 genes) showed an inhibiting effect of U73422 on their cold response. Again ² analyses confirmed a specific inhibitory action of U73122 versus U73343 on PLC. Therefore, the cluster of 65 genes can be considered as PLC driven. In order to know whether this regulation at 4°C by PLC activity was in a way correlated with a regulation already present at ambient temperature, microarray experiments were performed monitoring the effect of U73122 and U73343 on gene expression at 22°C. Of the 65 genes, seven of them showed a similar sensibility toward these molecules at 22°C as at 4°C. We therefore discarded these genes, thus leaving 58 genes identified as cold regulated via PLC activity.

Several of these genes were tested by northern and RT-PCR analyses and were confirmed to be PLC dependent. Here again, among these genes, some were up-regulated, while others were down-regulated by cold, indicating that PLC activation triggers a signaling pathway that can lead to both an activation and a repression of genes. However, with respect to cold, PLC appeared to have a positive action, since its inhibition led to an inhibition by the cold response, indicating that PLC is implicated in the activation of the cold response and not in the turning off of this response.

In the control experiments at 22°C, many genes, 1,507, showed a difference in transcript levels in the presence of U73122 versus U73343. Does this mean that a basal PLC activity exists that controls the expression of genes in nonstressed cells? This cannot be ruled out. However, this might be an artifact due to the solvent (a mixture of DMSO and tertButOH) in which the U73122 and U73343 are dissolved. Indeed the effect of U73122 versus U73343 at 22°C might be effective not on the PLC-dependent basal expression of genes but on the expression regulated by DMSO (or tertButOH). This hypothesis is strengthened by the fact that 87% of the genes with transcript levels higher at 22°C^U73122 versus 4°C^U73343 also have lower transcript levels at 22°C^U73343 versus 22°C, suggesting that the effect at 22°C of U73122 versus U73343 mainly affects the gene expression disturbed by U73343 (certainly because of the solvents in which it is dissolved). Because at 4°C we did not consider the genes that showed a difference in transcript levels between 22°C^U73122 and 22°C^U73343, we are certain that the effects we observe at 4°C are cold induced and not caused by a regulation via the solvents.

Do PLC and PLD Activate the Same Transduction Pathways?

It is now obvious that the activation of PLC and PLD activities are elements in the transduction of the cold signal leading to cellular responses via downstream regulation of gene transcription. However, a remaining question is whether PLC and PLD activate two different pathways or if they coactivate a single pathway. Indeed, the same class of lipid molecules (PtdOH) is produced by both pathways: PLDs produce PtdOH, while PLCs produce InsP3 and diacylglycerol, which can be phosphorylated to PtdOH. Cellular targets of PtdOH are beginning to be unraveled (Anthony et al., 2004; Testerink et al., 2004; Zhang et al., 2004; Testerink and Munnik, 2005). Interestingly, the molecular species of PtdOH produced by each pathway is different, since the PtdOH produced by PLC/DAGK is mainly 16:0/18:2 and 16:0/18:3 species, while the PtdOH produced by PLD is composed of these species and additional species like 18:3/18:2 and 18:2/18:2 (E. Ruelland and A. Zachowski, unpublished data). Our results indicate that each pathway mainly activates different targets. Indeed, among the 107 genes classified as up-regulated by cold either via PLC activity or via PLD-produced PtdOH, only seven were both PLC and PLD dependent; among the 30 genes classified down-regulated by cold via PLC or PLD, only one appeared to be dependent on PLC activity or on PLD-produced PtdOH. One explanation could be possible differences in the composition of the molecular species of PtdOH produced by each pathway, with specific molecular species activating specific targets. However, PLCs also produce diacylglycerol and InsP₃, which may play a role in triggering distinct signaling cascades.

Physiological Relevance of PLC and PLD Activating Two Distinct Pathways

In this work, we have shown that PLC and PLD activation participate in the cold response of Arabidopsis suspension cells. However, what is the link between PLC and PLD activation and the genetic regulons that have already been documented? The most documented regulon is CBF/DREB, although it does not account for all cold-responsive genes. Vogel et al. (2004) estimated that at least 70% of the cold-responsive genes, including transcription factors, were not affected by the expression of CBF transcription factors. This implies the existence of other genetic regulons. In their microarray analysis, Vogel et al. (2004) identified six genes encoding transcription factors that were coordinately regulated with CBF2. These genes, which can be expected to be primary transcription factors that should drive the cold induction of target genes, were ZAT12 (At5g59820), RAV1 (At1g13260), MYB73 (At5g37260), ZAT10 (At1g27730), CZF1 (At2g40140), and CZF2 (At5g04340). Interestingly, we show that the inhibition of PLC activity also inhibited the cold induction of MYB73 and that the inhibition of the production of PtdOH by PLD also inhibited the induction of DREB1A, ZAT10, CZF1, and CZF2. This pleads for a major role of the PLC and PLD pathways in the cold response. However, concerning the link between PLD and the CBF regulon, it should be noted that although the cluster driven by PLD-produced PtdOH is enriched in CBF target genes, many identified CBF target genes were not found in this cluster. Furthermore, when an in silico promoter analysis was performed with PLD-driven genes, no overrepresentation of C-repeat elements could be detected (data not shown). This may indicate that PLD-produced PtdOH is going to induce a helper factor that is necessary for the action of CBF on some promoters but not on all promoters. Indeed, Maruyama et al. (2004) identified a conserved sequence (A/GCCGACNT) as a dehydration-responsive element core motif in direct downstream targets of DREB1A (CBF3). However, among the 13,434 Arabidopsis genes they monitored in their study, 531 contained the A/GCCGACNT motif. Many of these genes were not influenced by overexpression of DREB1A. This is an indication that this motif is a required cis-element for the expression of genes driven by DREB1A but that other elements, bound by unidentified factors, must be necessary for the expression of these genes. These helper factors are not necessarily the same for all CBF target genes, and one helper factor may be active in different genetic regulons. It is interesting to note that the overexpression of ZAT10, MYB73, or CZF2 did not lead to the expression of cold-responsive genes (Vogel et al., 2004), which suggests that these genes encode such putative helper factors. We propose that PLD and PLC activation is upstream from the cold activation of such factors, ZAT10 and MYB73, respectively.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Materials

Edelfosine, U73122, and U73343 were from Calbiochem-Novabiochem. The culture medium for Arabidopsis (Arabidopsis thaliana) suspension cells (Gamborg B5) was from Duchefa. [³³P]-orthophosphate was purchased from Amersham Biosciences. Edelfosine (15 mM in water), U73122 (10 mM in DMSO:tertButOH, 27:53, v/v), and U73343