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

Abstract

The first step of peroxisomal fatty acid ␤-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 promotertrapped mutant in which ␤-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 mediumchain acyl-CoA substrate specificity. Analysis of ␤-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 ␤-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.

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

“…This was shown by the lack of any altered regulation of genes representative of the PLC, PLD, and GDPD pathways. Compensatory mechanisms could involve enzymes that play a dual role in metabolism and pathway regulation, such as PAH 1 and 2 (Eastmond et al, 2000;Craddock et al, 2015).Since the PLC pathway leads directly to the generation of PCho and PEth polar heads, this pathway could be affected in our mutant lines, contrary to other pathways. To verify this hypothesis, our overexpressors and mutants will need to be combined with other lines containing mutations in different lipid degradation routes (such as the PAP and PLD pathways).…”

“…This was shown by the lack of any altered regulation of genes representative of the PLC, PLD, and GDPD pathways. Compensatory mechanisms could involve enzymes that play a dual role in metabolism and pathway regulation, such as PAH 1 and 2 (Eastmond et al, 2000;Craddock et al, 2015).…”

Arabidopsis Mutants in Short- and Medium-chain Acyl-CoA Oxidase Activities Accumulate Acyl-CoAs and Reveal That Fatty Acid β-Oxidation Is Essential for Embryo Development

“…In acx3 seedlings, there is a large reduction (Ͼ95% of wild type) in medium-chain, acyl-CoA oxidase activity. However, the phenotype of acx3 is unaltered throughout the life cycle of the plant, and seedling lipid breakdown is not affected, most probably due to the overlapping substrate specificities of the ACX1 and ACX4 gene products (1). The aim1 mutant shows abnormal inflorescence development (8) indicating that ␤-oxidation may play a role in floral development.…”

“…MFP2 and ACX4 (15) both have putative PTS1s ending with the tripeptide Ser, Arg, Leu, which has been demonstrated to target proteins to the peroxisome (21). The ACX3 protein lacks motifs diagnostic of either PTS1 or PTS2, and its mode of import is unknown (1).…”

“…The ACX1 gene (Munich Information center for Protein Sequences (MIPS) 1 code At4g16760) is a medium-to longchain ACX with a substrate optimum of C14:0, 2 whereas the ACX2 gene (MIPS code At5g65110) has optimum activity with long-chain saturated and unsaturated acyl-CoAs (C14:0 to C20:0) (13). The ACX3 gene (MIPS code At1g06290/At1g06300) exhibits medium chain length substrate specificity (C8:0-C14:0) (1,14), and AtSCX (ACX4) (MIPS code At3g51840) exhibits short-chain (C4:0-C8:0) substrate specificity (15). Two additional ACX genes, ACX5 (MIPS code At2g35690) and ACX6 (MIPS code At1g06310) are present in the Arabidopsis genome.…”