In order to identify genes that are critical for the ABA-dependent stress response in the resurrection plant Craterostigma plantagineum, a gene was isolated with homology to class 3 variable substrate aldehyde dehydrogenases (ALDH). The C. plantagineum gene Cp-ALDH constitutes a novel class of plant ALDHs. In a search for corresponding genes from Arabidopsis thaliana, Ath-ALDH3 and Ath-ALDH4 were isolated, showing 70% and 80% similarity to Cp-ALDH. Phylogenetically, the Cp- and Ath-ALDH3 and -ALDH4 proteins are closely related to aldehyde dehydrogenases from bacteria and mammalian species and are separated from known plant ALDHs and betaine-aldehyde dehydrogenases (BADH). Cp-ALDH transcript and polypeptide are up-regulated in vegetative tissues and callus in response to dehydration or ABA-treatment. Ath-ALDH3 expression was induced in response to dehydration and ABA treatment, while Ath-ALDH4 is constitutively expressed at a low level. Recombinant Cp-ALDH protein oxidizes nonanal, propionaldehyde and acetaldehyde, with Km values of 2.2 microm, 0.27 mm and 3.23 mm, respectively, in an NAD-dependent manner. Immunogold electron microscopy shows that Cp-ALDH is localized in plastids.