Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation

Abstract

We further investigated the role of the Arabidopsis CBF regulatory genes in cold acclimation, the process whereby certain plants increase in freezing tolerance upon exposure to low temperature. The CBF genes, which are rapidly induced in response to low temperature, encode transcriptional activators that control the expression of genes containing the C-repeat/dehydration responsive element DNA regulatory element in their promoters. Constitutive expression of either CBF1 or CBF3 (also known as DREB1b and DREB1a, respectively) in transgenic Arabidopsis plants has been shown to induce the expression of target COR (cold-regulated) genes and to enhance freezing tolerance in nonacclimated plants. Here we demonstrate that overexpression of CBF3 in Arabidopsis also increases the freezing tolerance of cold-acclimated plants. Moreover, we show that it results in multiple biochemical changes associated with cold acclimation: CBF3-expressing plants had elevated levels of proline (Pro) and total soluble sugars, including sucrose, raffinose, glucose, and fructose. Plants overexpressing CBF3 also had elevated P5CS transcript levels suggesting that the increase in Pro levels resulted, at least in part, from increased expression of the key Pro biosynthetic enzyme Delta(1)-pyrroline-5-carboxylate synthase. These results lead us to propose that CBF3 integrates the activation of multiple components of the cold acclimation response.

Figure 1

Transcript levels of CBF3 and target COR genes in transgenic plants overexpressing CBF3. Northern analysis of total RNA (20 μg for CBF3; 5 μg for the other genes) prepared from control Arabidopsis Ws-2 and B6 plants and from CBF3-expressing A40, A28, and A30 plants. Plants were either grown at 20°C (W) or at 20°C and then cold treated at 5°C for 7 d (C). eIF4a (eukaryotic initiation factor 4a) is a constitutively expressed gene used as a loading control (Metz et al., 1992). Upon long exposure, CBF3 transcripts can be detected in the total RNA samples prepared from cold-treated Ws-2 and B6 plants (not shown).

Figure 2

Protein levels of COR15am and COR6.6 in transgenic plants overexpressing CBF3. Western analysis of total soluble protein (50 μg) prepared from control Arabidopsis Ws-2 and B6 plants and from CBF3-expressing A40, A30, and A28 plants. Plants were either grown at 20°C (W), or grown at 20°C and then cold treated at 5°C for 7 d (C). Protein transfers were treated with antiserum made to recombinant COR15am and COR6.6 polypeptides (Gilmour et al., 1996).

Figure 3

Growth characteristics of CBF3-expressing transgenic plants. A, Non-transformed Ws-2 plants and transgenic CBF3-expressing A30 plants after 2 weeks growth at 20°C. B, Non-transformed Ws-2 and transgenic B6 “control” plants and transgenic CBF3-expressing A40, A30, and A28 plants after 15 d growth at 20°C. C and D, Non-transformed Ws-2 plants and transgenic CBF3-expressing A28 plants after 16 d growth at 20°C. E, Non-transformed Ws-2 plants and CBF3-expressing A30 and A28 plants after 5 weeks growth at 20°C. F, As in E except after 9 weeks of growth at 20°C.

Figure 4

Effect of CBF3 overexpression on plant freezing tolerance. A, Seedlings of control Arabidopsis Ws-2 plants and CBF3-expressing A30 plants were grown at 20°C on solid medium and then frozen at −2°C for 24 h followed by 24 h at −6°C. B, Control Arabidopsis Ws-2 plants and CBF3-expressing A40, A30, and A28 plants were grown at 20°C, and the freezing tolerance of leaves was measured using the electrolyte leakage test. C and D, Same as B except that plants were grown at 20°C followed by 7-d-cold acclimation at 5°C.

Figure 5

Effect of CBF3 expression on Pro levels. Free Pro levels were determined in leaf tissue from control Arabidopsis Ws-2 and B6 plants and CBF3-expressing A40, A30, and A28 plants grown at 20°C (warm), or plants grown at 20°C and cold-treated at 5°C for 7 d (7-d cold).

Figure 6

Effect of CBF3 expression on transcript levels of genes involved in Pro and sugar metabolism. Northern analysis of total RNA (20 μg for CBF3; 5 μg for other genes) isolated from control Arabidopsis Ws-2 and B6 plants and from CBF3-expressing A40, A30, and A28 plants. Plants were grown at 20°C then cold-treated at 5°C for the times indicated. The blots were hybridized with probes for CBF3, COR78, P5CS2, Suc synthase (SuSy), Suc-phosphate synthase (SPS), and eIF4a, a constitutively expressed gene used as a loading control (Metz et al., 1992).

Figure 7

Effect of CBF3 expression on levels of total soluble sugars. Total soluble sugars were determined for leaf tissue from control Ws-2 and B6 plants and CBF3-expressing A40, A30, and A28 plants grown at 20°C (warm) or plants grown at 20°C and cold treated at 5°C for 7 d (7-d cold).

Figure 8

Effect of CBF3 expression on the levels of specific soluble sugars. Total sugars were prepared from leaf tissue of control Ws-2 plants and CBF3-expressing A28 plants grown at 20°C (white bars) or at 20°C followed by 7 d at 5°C (black bars) and the levels of individual sugars determined by gas chromatography.