Mutants of Arabidopsis thaliana defective in the acquisition of tolerance to high temperature stress

Abstract

The ability of organisms to acquire thermotolerance to normally lethal high temperatures is an ancient and conserved adaptive response. However, knowledge of cellular factors essential to this response is limited. Acquisition of thermotolerance is likely to be of particular importance to plants that experience daily temperature fluctuations and are unable to escape to more favorable environments. We developed a screen, based on hypocotyl elongation, for mutants of Arabidopsis thaliana that are unable to acquire thermotolerance to high-temperature stress and have defined four separate genetic loci, hot1-4, required for this process. hot1 was found to have a mutation in the heat shock protein 101 (Hsp101) gene, converting a conserved Glu residue in the second ATP-binding domain to a Lys residue, a mutation that is predicted to compromise Hsp101 ATPase activity. In addition to exhibiting a thermotolerance defect as assayed by hypocotyl elongation, 10-day-old hot1 seedlings were also unable to acquire thermotolerance, and hot1 seeds had greatly reduced basal thermotolerance. Complementation of hot1 plants by transformation with wild-type Hsp101 genomic DNA restored hot1 plants to the wild-type phenotype. The hot mutants are the first mutants defective in thermotolerance that have been isolated in a higher eukaryote, and hot1 represents the first mutation in an Hsp in any higher plant. The phenotype of hot1 also provides direct evidence that Hsp101, which is required for thermotolerance in bacteria and yeast, is also essential for thermotolerance in a complex eukaryote.

Figure 1

Hypocotyl elongation phenotype of Arabidopsis seedlings of wild type (Col-O) and four mutants (hot1–4). After growth for 2.5 days in the dark at 22°C, seedlings were either maintained at 22°C; treated at 38°C for 90 min; at 45°C for 2 h; or at 38°C for 90 min followed by 2 h at 22°C and then 45°C for 2 h. Seedlings were returned to 22°C for 2.5 days and then photographed. Groups of three seedlings that received the same treatment, as indicated above the panel, are shown. hot1, -2, and -4 seedlings are F₃ progeny of a single backcross to the wild-type parent. hot3 seedlings are from the M₃ generation, but show an identical phenotype to mutants segregating in the F₂ progeny of a backcross (not shown).

Figure 2

hot1 has a missense mutation in a conserved residue in the second ATP-binding domain of Arabidopsis Hsp101. The upper line shows a segment of the lower arm of chromosome 1 with simple sequence-length polymorphism markers nga 280 and nga111. The number of recombinants recovered between these markers and the hot1 mutation, per chromosome screened, are indicated in parentheses. The second line shows an expansion of the region adjacent to nga111 with the position of bacterial artificial chromosome F9E11, whose T7 end sequence is identical to ≈570 bp the Hsp101 gene, including ≈200 bp of coding region. The third line shows the structure of the Hsp101 gene and neighboring putative esterase gene. Boxes indicate exons and hatched boxes indicate the ATP-binding domains of Hsp101. Position of the E → K mutation in hot1 is indicated.

Figure 3

Glu-637 is conserved in divergent members of the Hsp100/ClpB family [Arabidopsis AtHsp101 (U13949), Synechococcus PCC7942 ClpB (U97124), E. coli ClpB (M29364), and S. cerevisiae Hsp104 (M67479)] and the ClpA family [E. coli ClpA (M31045)] of proteins. Relevant regions from the second ATP-binding domain of each protein are aligned. Residues identical to Hsp101 are indicated with dashes. A gap was introduced in E. coli ClpA to optimize the alignment.

Figure 4

Transformation of the hot1 mutant with an Hsp101 genomic clone reverts the thermotolerance phenotypes. Wt, untransformed Col-O; hot1, hot1 mutant; hot1 +101, mutant transformed with Hsp101 genomic clone; hot1 +vect, mutant transformed with vector only. (A) The 2.5-day-old, dark grown seedlings were treated at 38°C for 90 min followed by 2 h at 22°C and then 2 h at 45°C. Seedlings were returned to 22°C in the dark and photographed 2.5 days later. (B) The 10-day-old, light grown seedlings treated as in A and photographed 5 days later. (C) Seeds imbibed on plates at 4°C for 3 days were grown for 3 days at 22°C in the dark (Upper) or heated at 45°C for 2 h, returned to 22°C, and photographed after 3-days growth in the dark (Lower).

Figure 5

Hsp101 and small Hsps accumulate normally in mutant seedlings after heat stress. Total proteins from 2.5-day-old dark grown seedlings were isolated either before (C) or after treatment at 38°C for 90 min, followed by 2 h at 22°C (H). Proteins were analyzed by Western blotting with either Hsp101 or Hsp17.6 antibodies.