Fig. 1.

Identification of the CAA39 gene. (A) Constitutive luciferase activity and phenotype of the flu AAA:LUC⁺ caa39 and the flu AAA:LUC⁺ parental line. Seedlings were grown in continuous light on Murashige and Skoog agar plates for 5 d and 10 d. Bioluminescence (LUC), corresponding visible images (Light), and Trypan blue (TB) staining of cell death are presented. (Scale bars, 0.5 cm.) (B) Relative transcript levels of the AAA-ATPase gene (AAA) and the Luciferase reporter gene (LUC) in cotyledons of 5-d-old and 10-d-old seedlings of the flu AAA:LUC⁺ caa39 mutant and the flu AAA:LUC⁺ parental line. Transcript levels were determined by quantitative RT-PCR. Results represent mean values of two biological replicates ± SE. (C) Map-based cloning of CAA39. Initial mapping analysis revealed genetic linkage of CAA39 to markers located on top of chromosome V. Fine mapping localized the caa39 mutation in a 100-kb region covered by BAC clones F9G14 and F15A17. (D) A single G-to-A nucleotide substitution was found in the second exon of the At5g28020 locus in the caa39 mutant, resulting in a Pro-337 to Leu amino acid exchange. Filled boxes indicate exons, lines indicate transcribed regions. (E) Complementation of caa39. AAA-ATPase expression, morphological phenotype and Trypan blue staining of cell death in 10-d-old seedlings, (Left) and morphological phenotype of 35-d-old mature plants (Right) of flu, flu caa39, and flu caa39 complemented with the wild-type copy of the At5g28020 gene. (Scale bars, 0.5 cm.)