No Induction of ?-oxidation in leaves of Arabidopsis that over-produce lauric acid

Abstract

Leaves from transgenic Brassica napus L. plants engineered to produce lauric acid show increased levels of enzyme activities of the pathways associated with fatty acid catabolism (V.A. Eccleston and J.B. Ohlrogge, 1998, Plant Cell 10: 613-621). In order to determine if the increases in enzyme activity are mirrored by increases in the expression of genes encoding enzymes of beta-oxidation, which is the major pathway of fatty acid catabolism in plants, the medium-chain acyl-acyl carrier protein (ACP) thioesterase MCTE from California bay (Umbellularia california) was over-expressed under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis thaliana (L.) Heynh. Arabidopsis was the most suitable choice for these studies since gene expression could be analyzed in a large number of independent MCTE-expressing lines using already well-characterized beta-oxidation genes. Levels of MCTE transcripts in leaves varied widely over the population of plants analyzed. Furthermore, active MCTE was produced as determined by enzymatic analysis of leaf extracts of MCTE-expressing plants. These plants incorporated laurate into triacylglycerol of seeds, but not into lipids of leaves as shown by gaschromatographic analysis of total fatty acid extracts. The expression levels of the beta-oxidation and other genes that are highly expressed during developmental stages involving rapid fatty acid degradation were measured. No significant difference in gene expression was observed among MCTE-expressing plants and transgenic and non-transgenic controls. To eliminate the possibility that post-translational mechanisms are responsible for the observed increases in enzyme activity acyl-CoA oxidase activity was also measured in leaves of MCTE-expressing plants using medium and long chain acyl-CoA substrates. No significant increases in either medium- or long-chain acyl-CoA oxidase activities were detected. We conclude that endogenous beta-oxidation is sufficient to account for the complete degradation of laurate produced in rosette leaves of Arabidopsis expressing MCTE.

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

“…It can be concluded that medium-chain acyl-CoA oxidase activity exerts little control over the breakdown of storage lipid in germinating seeds. Long-chain acyl-CoA oxidase activity is about 8-fold less than that of medium-chain acyl-CoA oxidase in wild type seedlings (Table I) and leaves (26). If acyl-CoA oxidase plays a significant role in the control of peroxisomal ␤-oxidation (7,8), then long-chain acyl-CoA oxidase is likely to predominate.…”

“…In the majority of cases where crops have been genetically engineered to produce novel fatty acids, the accumulation of these products is much lower than that required for commercial exploitation (23,24). It is believed that in these plants the novel fatty acids are synthesized at significant rates but are subsequently degraded by peroxisomal ␤-oxidation (25,26). Moreover, Eccleston and Ohlrogge (25) provide evidence that in the case of plants producing medium-chain fatty acids, medium-chain acyl-CoA oxidase activity is up-regulated to facilitate this degradation.…”

Postgerminative growth and lipid catabolism in oilseeds lacking the glyoxylate cycle

“…In the absence of a mechanism for transporting acetyl-CoA, a similar process could be responsible for the movement of carbon skeletons derived from ␤-oxidation of fatty acids out of the peroxisome. The fact that plant cells do respire lipids also is implied by the fact that the capacity for ␤-oxidation is present in many tissues that lack the glyoxylate cycle (16,33).…”

Arabidopsis Lipins, PDAT1 Acyltransferase, and SDP1 Triacylglycerol Lipase Synergistically Direct Fatty Acids toward β-Oxidation, Thereby Maintaining Membrane Lipid Homeostasis

“…Because the rate of FA degradation was strongly enhanced in leaves of the tgd1-1 mutant ( Figure 9C; Fan et al, 2013a), we sought to determine the enzymatic activity of acyl-CoA oxidase (ACOX), which catalyzes the first and rate-limiting step in the b-oxidation cycle (Aoyama et al, 1994) and is a reliable indicator of peroxisomal b-oxidation capacity (Eccleston et al, 1996;Hooks et al, 1999). Compared with the wild type, there was no significant difference in ACOX activity with long-chain acyl-CoA as substrate in leaves of tgd1-1 (Supplemental Figure 9A).…”