Long-chain acyl-CoA oxidases of Arabidopsis

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated b y screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxi dases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 a mino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptid e of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisom al targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtA CX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and bi ochemical analysis. AtACX1 was active with both medium-and long-chain satur ated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity wit h mono-unsaturated acyl-CoAs was slightly higher than with the correspondin g saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showe d maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl -CoAs was approximately twice as high as with the corresponding saturated a cyl-CoA, Both enzymes have an apparent K-m of approximately 5 mu M with the preferred substrate. Northern analysis was conducted to determine the expr ession patterns of AtACX1 and AtACX2 during germination and in various tiss ues of a mature plant. The two genes showed generally similar expression pr ofiles and steady-state mRNA levels in seedlings and mature tissues, but su btle differences were observed. Enzymatic analyses of plant extracts reveal ed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA ox idases specific for shorter-chain acyl-CoAs. Through expression of antisens e constructs of the individual genes, we were able to decrease long-chain o xidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and est ablished normally; however, reduced root growth appeared to be a general fe ature of antisense AtACX1 plants.