Identification of two chilling-regulated 1-aminocyclopropane-1-carboxylatesynthase genes from citrus (Citrus sinensis Osbeck) fruit

Diurnal change in the temperature below or above 12.5 degrees C hastens the degreening of citrus peel and elicits the phytohormone ethylene production in citrus fruit. Ethylene triggers the degradation of chlorophyll and synt hesis of carotenoids in citrus peel. To investigate if ethylene is required for the degreening of citrus peel elicited by low temperatures, we studied the chilling-regulated gene expression of ACC synthase, one of the key enz ymes catalyzing ethylene biosynthesis. We isolated and characterized a chil ling-inducible 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) ge ne, CS-ACS1, and a chilling-repressible gene, CS-ACS2, from citrus peel. Th e CS-ACS1 transcript 1.7 kb in length encodes a polypeptide of 483 amino ac ids (M-r 54 115, pI 6.63), whereas the CS-ACS2 transcript of 1.8 kb encodes a polypeptide of 477 amino acids (M-r 53 291, pI 6.72). Both genes showed a rapid but transient induction (within 2.4 h) of transcripts upon rewarmin g after the chilling (4 degrees C) treatment. After 24 h of incubation at r oom temperature, CS-ACS1 mRNA diminished to an undetectable level, whereas the CS-ACS2 mRNA regained its basal level of expression attained prior to t he chilling treatment. Chilling-induced ethylene production and ACC accumul ation were also observed upon rewarming. Both genes were also induced by th e wound stress (excision). The protein synthesis inhibitor cycloheximide su per-enhances the accumulation of both ACS transcripts at room temperature. Molecular analysis of the 3.3 kb genomic DNA of CS-ACS1 revealed that this gene consists of three introns and four exons. The intron 3 is exceptionall y large (1.2 kb) and shares significant homology with mitochondrial DNA, su pporting the intron-late theory.