Abstract
The first step of peroxisomal fatty acid beta-oxidation is catalyzed by a family of acyl-CoA oxidase isozymes with distinct fatty acyl-CoA chain-length specificities. Here we identify a new acyl-CoA oxidase gene from Arabidopsis (AtACX3) following the isolation of a promoter-trapped mutant in which beta-glucuronidase expression was initially detected in the root meristem. In acx3 mutant seedlings medium-chain acyl-CoA oxidase activity was reduced by 95%, whereas long- and short-chain activities were unchanged. Despite this reduction in activity lipid catabolism and seedling development were not perturbed. AtACX3 was cloned and expressed in Escherichia coli. The recombinant enzyme displayed medium-chain acyl-CoA substrate specificity. Analysis of beta-glucuronidase activity in acx3 revealed that, in addition to constitutive expression in the root axis, AtACX3 is also up-regulated strongly in the hypocotyl and cotyledons of germinating seedlings. This suggests that beta-oxidation is regulated predominantly at the level of transcription in germinating oilseeds. After the discovery of AtACX3, the Arabidopsis acyl-CoA oxidase gene family now comprises four isozymes with substrate specificities that encompass the full range of acyl-CoA chain lengths that exist in vivo.
Highlights
Peroxisomal -oxidation is the primary pathway of fatty acid catabolism in plants
We identify a new acyl-CoA oxidase gene from Arabidopsis (AtACX3) following the isolation of a promotertrapped mutant in which -glucuronidase expression was initially detected in the root meristem
One of two inverse PCR products from a line with GUS expression in the root meristem (Rm 328) was found to be homologous to acyl-CoA oxidases. These enzymes catalyze the first step in the pathway of peroxisomal -oxidation and are known to be highly active in the root tips of maize [2]. This putative acylCoA oxidase was different from those previously identified in Arabidopsis, and the new gene and mutant were designated AtACX3 and acx3, respectively
Summary
Plant Material and Growth Conditions—A T-DNA-mutagenized A. thaliana (ecotype Wassilewskija) population consisting of 10,000 lines [27] transformed with the pGKB5 vector designed for promoter trapping [28] was screened for transformants exhibiting GUS expression in the roots. After 7 days of growth in a 16-h photoperiod (23 °C light/18 °C dark) seedlings were stained for GUS as described below. For subsequent analysis of line Rm 328, seeds were germinated in continuous light on 0.8% (w/v) agar plates containing half-strength Murashige and Skoog media [29] (plus 1% (w/v) sucrose where indicated) at 20 °C after 4 days of imbibition at 4 °C in the dark. Approximately 10,000 2-day-old seedlings were homogenized in 3 ml of a medium containing: 150 Tricine/KOH, pH 7.5, 1 mM EDTA, 0.5 M sucrose using an UltraTurrax (Janke and Kunkel KG). The primers used were GUS1, ACX3S, and ACX3A (5Ј-GAAACATCAGCAACTGCATTCAAC)
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