Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants

Abstract

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Figure 1.

Analysis of PHA produced in plants from ricinoleic acid and methyl vernolate. Transgenic plant line PHAC3.3, expressing a peroxisomal PHA synthase under the control of the CaMV 35S promoter, was grown in liquid medium supplemented with either oleic acid, ricinoleic acid, or methyl vernolate, all at 0.05% (v/v) final concentration. 3-Hydroxyacyl-CoA monomers are denoted by the prefix H. Values are ± sd. The amounts of PHA are expressed as micrograms of PHA per gram dry weight (dw) of plant material and are indicated at the top.

Figure 2.

Analysis of PHA produced in mature seeds of transgenic plants expressing the castor bean 12-hydroxylase. Line N10.3pC3.1 was transformed with the control vector, whereas lines N10.3NH1.1 and N10.3NH2.2 were transformed with a napin-hydroxylase construct. Values are ± sd.

Figure 3.

Analysis of PHA produced in mature seeds of transgenic plants expressing the C. palaestina 12-epoxygenase. Lines N4.1pC6.1 and N10.3pC3.1 were transformed with the control vector, whereas lines N4.1NE15.1 and N10.3NE6.2 were transformed with a napin-epoxygenase construct. Line N4.1pC6.1 is used as the control for line N4.1NE15.1, whereas line N10.3pC3.1 is used as the control for line N10.3NE6.2. Values are ± sd.