Arabidopsis Hsa32, a novel heat shock protein, is essential for acquired thermotolerance during long recovery after acclimation

Abstract

Plants and animals share similar mechanisms in the heat shock (HS) response, such as synthesis of the conserved HS proteins (Hsps). However, because plants are confined to a growing environment, in general they require unique features to cope with heat stress. Here, we report on the analysis of the function of a novel Hsp, heat-stress-associated 32-kD protein (Hsa32), which is highly conserved in land plants but absent in most other organisms. The gene responds to HS at the transcriptional level in moss (Physcomitrella patens), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa). Like other Hsps, Hsa32 protein accumulates greatly in Arabidopsis seedlings after HS treatment. Disruption of Hsa32 by T-DNA insertion does not affect growth and development under normal conditions. However, the acquired thermotolerance in the knockout line was compromised following a long recovery period (>24 h) after acclimation HS treatment, when a severe HS challenge killed the mutant but not the wild-type plants, but no significant difference was observed if they were challenged within a short recovery period. Quantitative hypocotyl elongation assay also revealed that thermotolerance decayed faster in the absence of Hsa32 after a long recovery. Similar results were obtained in Arabidopsis transgenic plants with Hsa32 expression suppressed by RNA interference. Microarray analysis of the knockout mutant indicates that only the expression of Hsa32 was significantly altered in HS response. Taken together, our results suggest that Hsa32 is required not for induction but rather maintenance of acquired thermotolerance, a feature that could be important to plants.

Figure 1.

Hsa32 was responsive to HS from lower to higher plants. Hsa32 transcript levels in Arabidopsis (A) and rice (B) seedlings were revealed by northern blot and in P. patens (C) by RT-PCR. Plants were either without HS treatment (N) or treated at the indicated temperatures for various times. The transcript levels of Arabidopsis Hsp21, rice OsHsp16.9C, and P. patens sHsp were shown as positive controls. Ribosomal RNA (A and B) or actin (C) was shown to ensure equal loading of samples.

Figure 2.

Hsa32 protein accumulates in response to HS and declines during recovery. A, Immunoblotting analysis of Hsa32, sHsp-CI, and Hsp90 in Arabidopsis 3-d-old seedlings during and after HS treatment at 37°C for indicated time. In each lane, 50 μg of protein was loaded. Rubisco large subunit stained by Amido black was shown to ensure equal loading. Calculated molecular mass of each protein based on mobility was indicated. B, Semiquantification of Hsa32, sHsp-CI, and Hsp90 levels of immunoblots according to conditions described in A. The relative amount of each protein was expressed as percentage of the highest signal detected after normalized by the level of Rubisco. The data were means of two biological repeats. Error bars represent sd.

Figure 3.

Suppression of HS induction of Hsa32 by T-DNA insertion and RNAi. The Arabidopsis wild type (wt), Hsa32 T-DNA knockout mutant (hsa32-1), and RNAi lines (C057-2, -10, -11, and -16) seedlings were treated at 37°C for 1 h and recovered for 3 h, then harvested for protein extraction. Hsa32 and sHsp-CI levels were revealed by immunoblotting. In each lane, 50 μg of protein was loaded. Rubisco large subunit stained by Amido black was shown to ensure equal loading. The numbers below indicate the relative amount of Hsa32.

Figure 4.

Disruption of Hsa32 by T-DNA insertion leads to heat-sensitive phenotype after a long recovery. The phenotypes of the wild-type (wt) and hsa32-1 seedlings were revealed after treatment by different HS regimes schematically shown on the right of each section except B. The times of HS treatment at 44°C in B and C were 160 and 220 min, respectively. The arrowheads indicate the end of seed imbibitions. In B, phenotype of Hsp101 (At1g74310) T-DNA knockout plants (hsp101) was shown as a comparison. The survival rates of wild-type and hsa32-1 lines in C were 43% and 44%, respectively. In G, older seedlings were employed for the thermotolerance test. The plants in A to G were photographed 6 to 11 d after HS treatment. Seedlings of each section were grown on the same plate and reorganized for presentation. H shows the progression of phenotypes of representative wild-type (top) and hsa32-1 (bottom) seedlings 0 to 6 d after treatment by the same HS regime shown in D. The bar represents 1 mm.

Figure 5.

Heat sensitivity of Hsa32 RNAi lines after a long recovery. The 3-d-old seedlings of the wild type (wt), hsa32-1, and RNAi (C057-2, -10, -11, and -16) lines were subjected to acclimation at 37°C for 1 h and recovered for 2 d, challenged at 44°C for 60 min, then recovered at 22°C for 8 d before the photograph was taken.

Figure 6.

Acquired thermotolerance decays faster in the absence of Hsa32. The wild type, hsa32-1, RNAi lines C057-10 and -16, and hsp101 seedlings were first conditioned at 37°C for 1 h then without (A) or subject to (B) severe HS treatment after recovery for the indicated times as shown schematically at the right. Then, the elongation of hypocotyl during 2.5 d of recovery was measured as indicated. The relative hypocotyl length in B was expressed as percentage of the numbers in A. Error bars represent sd and are based on data in four separate duplicates of five seedlings of each line. The arrowheads indicate end of seed imbibitions.

Figure 7.

Comparison of HSR in the wild-type and hsa32-1 plants by microarray analysis. Relative changes in Arabidopsis gene expression were studied in the hsa32-1 knockout mutant after HS treatment (37°C 2 h) by employing Arabidopsis ATH1 genome array. Expression signals (based on log₂) of 1,306 HS-responsive genes (see “Materials and Methods” for definition) in the HS-treated wild-type plants were plotted against those of the HS-treated hsa32-1 mutant as illustrated by the scatterplot. The signal of Hsa32 was indicated by an arrow. Lines of 3-fold difference in expression between the wild-type and mutant plants were shown to assist reading. The data were the means of two biological replicates for the wild type and mutant.