A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis

Abstract

The expression of heat shock proteins (Hsps) induced by nonlethal heat treatment confers acquired thermotolerance (AT) to organisms against subsequent challenges of otherwise lethal temperature. After the stress signal is removed, AT gradually decays, with decreased Hsps during recovery. AT of sufficient duration is critical for sessile organisms such as plants to survive repeated heat stress in their environment, but little is known regarding its regulation. To identify potential regulatory components, we took a reverse genetics approach by screening for Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants that show decreased thermotolerance after a long recovery (2 d) under nonstress conditions following an acclimation heat treatment. Among the tested mutants corresponding to 48 heat-induced genes, only the heat shock transcription factor HsfA2 knockout mutant showed an obvious phenotype. Following pretreatment at 37 degrees C, the mutant line was more sensitive to severe heat stress than the wild type after long but not short recovery periods, and this could be complemented by the introduction of a wild-type copy of the HsfA2 gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant reduction in the transcript levels of several highly heat-inducible genes was observed in HsfA2 knockout plants after 4 h recovery or 2 h prolonged heat stress. Immunoblot analysis showed that Hsa32 and class I small Hsp were less abundant in the mutant than in the wild type after long recovery. Our results suggest that HsfA2 as a heat-inducible transactivator sustains the expression of Hsp genes and extends the duration of AT in Arabidopsis.

Figure 1.

Disruption of HsfA2 by T-DNA insertion led to a severe defect in AT, which could be complemented by the wild-type copy of the gene. A, The phenotypes of Arabidopsis seedlings of the wild type (wt), hsfA2-1, and two independent transgenic lines (C112-1 and -2, at T₂ generation) harboring transgenic AtHsfA2 and bar genes in the background of hsfA2-1 after severe HS challenge (44°C for 55 min) applied after a 2-d recovery at room temperature following a conditioning treatment at 37°C for 1 h. All the seedlings were germinated, conditioned at the 3-d-old stage, and challenged in the same plate. The photographs were taken 8 d after the challenge and reorganized for presentation. The survival rate after HS challenge and Basta-resistant rate were calculated on the basis of 50 seedlings and indicated at the bottom. B, RT-PCR analysis of the transcripts of HsfA2 in the lines mentioned above. RNA samples were purified from 5-d-old seedlings with (H) or without (C) HS treatment at 37°C for 1 h. The RT-PCR product of actin is shown as a loading control.

Figure 2.

hsfA2-1 plants were less tolerant than wild type to severe HS challenge after a long but not short recovery following acclimation treatment. A to F, The phenotypes of the wild type (wt), hsfA2-1, and T-DNA knockout lines of Hsa32 (hsa32-1) and Hsp101 (hsp101) plants were revealed after treatment by different HS regimes schematically shown on the right of each section. The arrowheads indicate the end of seed imbibitions. The plants were photographed 7 to 10 d after HS treatment. Seedlings of each section were grown on the same plate and reorganized for presentation. G, The progression of phenotypes of representative wild-type and mutant seedlings 0 to 6 d after the second HS treatment as shown in D. H, The morphology of roots of the wild-type and mutant lines at 0 or 6 d after the second HS treatment as shown in D. In this case, plants were grown vertically. Bar = 1 mm in both G and H.

Figure 3.

Ion leakage of the wild type and mutants. Seedlings 3 d old were conditioned at 37°C for 1 h, recovered at room temperature for 2 d, and then challenged by 44°C for 45 min. After the second HS treatment, the seedlings were allowed to recover at room temperature again for various times, from 0 to 24 h, before undergoing ion leakage analysis as described in “Materials and Methods.” Error bars represent sd based on data in three separate duplicates. The plants with asterisks had significantly higher ion leakage than wild-type plants (P < 0.05, independent Student's t test).

Figure 4.

AT declined faster in the absence of HsfA2 as revealed by quantitative hypocotyl elongation assay. The wild-type, hsfA2-1, hsa32-1, and hsp101 seedlings were first conditioned at 37°C for 1 h, then without (A) or subjected to (B) severe HS treatment at 44°C for 45 min after recovery for the indicated times as shown schematically at the right. The arrowheads indicate the end of seed imbibition. The positions of the top of hypocotyls were labeled right after HS treatment. The elongation of hypocotyls during 2 d of recovery in the dark was then measured. The relative hypocotyl length in B was expressed as a percentage of the numbers in A. Each data point represents the mean of 25 seedlings. Error bars represent sd based on data in five separate duplicates of the mean of five seedlings.

Figure 5.

Disruption of HsfA2 caused a slight reduction in basal thermotolerance. A, Etiolated seedlings 3 d old underwent direct, severe HS challenge at 44°C for 15 to 30 min or, in B, 46°C to 50°C for 5 min. After HS challenge, the seedlings were labeled for position of hypocotyls and then allowed to grow vertically for another 3 d in the dark. The elongation of hypocotyls after HS was expressed as a percentage of that of the nontreated control (C). Each data point represents the mean of 25 seedlings. Error bars represent sd based on data in five separate duplicates of the mean of five seedlings. The plants with asterisks had significantly lower values than wild-type plants (P < 0.05, independent Student's t test).

Figure 6.

Transcript and protein levels of several Hsp genes declined faster in hsfA2-1 than in wild type during recovery. A, Semiquantitative RT-PCR analysis of the mRNA levels of Hsp101, Hsa32, Hsp25.3-P, and Hsp18.1-CI in the wild-type (wt) and hsfA2-1 (m) plants during recovery, from 0 to 48 h, after HS treatment of 3-d-old seedlings at 37°C for 1 h. The RT-PCR products of actin were shown as a loading control. PCR cycles for Hsp101, Hsa32, Hsp25.3-P, Hsp18.1-CI, and actin were 30, 30, 30, 25, and 30, respectively. The control (C) samples were seedlings without heat treatment and collected at the same time as the treated samples at 48 h. The expected sizes of the PCR products are indicated at the right. B, Immunoblot analysis of the protein levels of Hsp90, Hsa32, and class I small Hsps detected with corresponding antibodies. The samples were the same as described in A, except the additional samples were collected at 72 h of recovery. In each lane, 50 μg of total protein was loaded. Rubisco large subunit stained by Amido black was shown to ensure equal loading. The control (C) samples were seedlings without heat treatment and collected at the same time as the treated samples at 72 h. Similar results were obtained from two biological repeats, with one shown here.

Figure 7.

Transcript level of Hsp genes was lower in hsfA2-1 than in wild type during prolonged HS. Semiquantitative RT-PCR analysis of the mRNA levels of Hsp101, Hsa32, Hsp25.3-P, and Hsp18.1-CI in the wild-type (wt) and hsfA2-1 (m) plants during prolonged HS, from 0 to 8 h, at 37°C. Total RNA was purified from 3-d-old seedlings after treatment. The RT-PCR products of actin were shown as a loading control. PCR cycle for Hsp101, Hsa32, Hsp25.3-P, Hsp18.1-CI, and actin was 30, 30, 25, 25, and 30, respectively. Similar results were obtained from two biological repeats, with one shown here.