Long-chain acyl-CoA oxidases of Arabidopsis

Abstract

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coil gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent K(m) of approximately 5μM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.