The cold-induced early activation of phospholipase C and D pathways determines the response of two distinct clusters of genes in Arabidopsis cell suspensions

Abstract

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.

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.

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).

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.

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°C^ethanol versus 4°C^terButOH, 93 showed transcript level changes in response to both 4°C versus 22°C and to 4°C^ethanol versus 4°C^terButOH. 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.

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°C^U73122 versus 4°C^U73343, 591 genes showing a transcript level change in response to 4°C^U73343 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°C^U73122 versus 4°C^U73343. 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.

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.

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.

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.