Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression

Abstract

Transcription factor DREB2A interacts with a cis-acting dehydration-responsive element (DRE) sequence and activates expression of downstream genes involved in drought- and salt-stress response in Arabidopsis thaliana. Intact DREB2A expression does not activate downstream genes under normal growth conditions. A negative regulatory domain exists in the central region of DREB2A, and deletion of this region transforms DREB2A to a constitutive active form (DREB2A CA). We carried out microarray analysis of transgenic Arabidopsis-overexpressing DREB2A CA and found that the overexpression of DREB2A CA induces not only drought- and salt-responsive genes but also heat-shock (HS)-related genes. Moreover, we found that transient induction of the DREB2A occurs rapidly by HS stress, and that the sGFP-DREB2A protein accumulates in nuclei of HS-stressed cells. DREB2A up-regulated genes were classified into three groups based on their expression patterns: genes induced by HS, genes induced by drought stress, and genes induced by both HS and drought stress. DREB2A up-regulated genes were down-regulated in DREB2A knockout mutants under stress conditions. Thermotolerance was significantly increased in plants overexpressing DREB2A CA and decreased in DREB2A knockout plants. Collectively, these results indicate that DREB2A functions in both water and HS-stress responses.

Fig. 1.

Stress-inducible expression of the DREB2A gene and accumulation of the DREB2A protein. (A and B) Expression of DREB2A and DREB2B in response to HS (37°C), drought, NaCl, and H₂O₂. Total RNA was prepared from 3-wk-old Arabidopsis plants that had been heated at 37°C (37°C), incubated at 22°C (22°C), dehydrated (drought), transferred to hydroponic growth in 250 mM NaCl, transferred to hydroponic growth in 20 mM H₂O₂, and transferred to water (H₂O). Treatments were carried out for 30 min, 1 h, 5 h, and 10 h. Accumulation of the DREB2A and DREB2B mRNAs was analyzed by quantitative RT-PCR. Data represent means and standard errors of three replications. The expression level under control conditions was defined as 1.0 (C) Nomarski microscope images (a, c, e, and g) and confocal microscope images of sGFP fluorescence (b, d, f, and h) of the promoter:sGFP-DREB2A plants. The root (a–d) and leaf (e–h) tissues were observed under microscope before or immediately after incubation at 37°C for 1.5 h. [Scale bars, 20 μm (a–d) and 50 μm (e–h).]

Fig. 2.

Quantitative RT-PCR analysis of DREB2A up-regulated genes in response to HS or drought stress. Data represent means and standard errors of three replications. (A) Genes induced by both HS and drought. (B) Genes induced by HS. (C) Genes induced by drought (the highest expression level was set to 100).

Fig. 3.

Analysis of DREB2A knockout mutants. (A) A scheme of the Arabidopsis DREB2A gene. Exons (open boxes) and an intron (line) are indicated. The positions of the T-DNA insertions are shown (not to scale). (B–E) Expression of DREB2A (B), DREB2A downstream genes induced by both HS and drought (C), DREB2A downstream genes induced by HS (D), and DREB2A downstream genes induced by drought (E) was analyzed in wild-type and dreb2a plants by quantitative RT-PCR (the highest expression level was set to 100). Data represent means and standard errors of three replications.

Fig. 4.

HS-stress tolerance of the 35S:DREB2A CA and dreb2a plants. (A) One-week-old seedlings of the vector control or the 35S:DREB2A CA plants were treated at 22°C or 45°C for 1 h. (B) One-week-old seedlings of wild-type or dreb2a plants were preincubated at 37°C for 1 h and then treated at 22°C or 49°C for 1 h. After treatment, plants were grown under normal conditions for 1 wk. Percentages of surviving plants and numbers of surviving plants per total numbers of tested plants are indicated around the photographs. More than 15 plants were used per test, and each test was repeated three times. ∗, These plants had significantly lower survival rates than the wild-type plants (χ² test, P < 0.05). ∗∗, These plants had significantly higher or lower survival rates than the vector control or the wild-type plants (χ² test, P < 0.01).