Promoter Trapping of a Novel Medium-chain Acyl-CoA Oxidase, Which Is Induced Transcriptionally during Arabidopsis Seed Germination

Peter J Eastmond,Mark A Hooks,Dawn Williams,Peter Lange,Nichole Bechtold,Catherine Sarrobert,Laurent Nussaume,Ian A Graham

doi:10.1074/jbc.m004945200

Peter J Eastmond, Mark A Hooks + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m004945200

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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

EXPERIMENTAL PROCEDURES

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)

RESULTS

Substrate carbon chain length

DISCUSSION