Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana

Abstract

Among nine cDNA clones (named RD) corresponding to genes that are responsive to dehydration in Arabidopsis thaliana, two clones, RD19 and RD21, were analyzed further. Northern blot analysis revealed that both the RD19 and RD21 mRNAs were not induced by abscisic acid. Neither RD19 nor RD21 mRNA synthesis was responsive to cold or to heat stress. On the other hand, transcription of both the RD19 and RD21 mRNAs was strongly induced under highsalt conditions, which suggests that the genes corresponding to RD19 and RD21 may be induced by changes in the osmotic potential of plant cells. Putative proteins, RD19 and RD21, encoded by two of the RD cDNAs have amino acid (aa) sequences typical of the catalytic sites of cysteine proteinases (CysP). RD21 and RD19 appeared to contain signal peptides that function in protein secretion. RD21 contains an aa sequence similar to that of the C-terminal extension peptide. Phylogenetic tree analysis indicated that the putative RD21 and RD19 proteins are quite different types of CysP. Genomic Southern analysis revealed that each gene family contains at least two members, which do not cross-hybridize. The two genes corresponding to RD19 and RD21 ( rd19A and rd21A, respectively) were cloned and their structural analysis revealed the presence of two and four introns, respectively. The numbers and sites of introns differ between the genes, supporting our hypothesis that rd19A and rd21A belong to different subfamilies of genes encoding CysP. The transcription start points were determined by primer extension. Two conserved sequences were found in the promoter regions of the two genes.