title: Molecular characterisation and regulation of acyl-CoA oxidase 1 (ACX1) in Arabidopsis thaliana
Creators: name: Valentine, Lisa Gallagher
Publisher: name: University of Glasgow
Year: 2001
Subjects: subject: Plant sciences.

description: The metabolic status of a plant is fundamental to its ability to respond either to internal developmental signals or external environmental signals. The metabolic pathway of fatty acid beta-oxidation is of particular importance during the germination of oil seed plants since the conversion of lipids into carbohydrate is essential for post-germinative growth. The first step of fatty acid beta-oxidation is catalysed by the acyl-CoA oxidase enzyme. In the model oilseed plant Arabidopsis thaliana, four genes encoding acyl-CoA oxidase enzymes with different substrate specificities have been functionally characterised. ACXl is one member of this gene family which has been shown to be induced during germination (Hooks et al., 1999a). Interestingly, this particular member has also been shown to be induced under stress and wounding conditions. This study aims to further characterise The genome sequencing project was completed at the end of the year 2000 (The Arabidopsis Genome Initiative, 2000). An earlier publication detailing sequence from an area of chromosome IV (Bevan et al, 1998) was found to include the genomic sequence for ACXl, including the sequence of the putative promoter. Annotation of the published sequence and further sequence analysis revealed that there was a cluster of putative germination-induced genes within 10kb of ACXl. To investigate expression patterns of these putative genes, mRNA transcript levels were monitored at day 2 of germination using both gel blot northern analysis and 'electronic' northern analysis of transcript abundance in an Expressed Sequence Tag (E.S.T.) database produced from a cDNA library prepared during the course of this work. Transcript levels of these genes were too low in 2 day old seedlings to allow any conclusion about co-expression to be drawn. The implications of this level of control are discussed. From the published genomic sequence it was possible to design oligonucleotide primers to be used in a PCR reaction to amplify the putative ACXl promoter. This putative promoter was fused to the luciferase reporter gene which was then transformed into Arabidopsis by A. tumifaciens. This system allowed investigation of the developmental and stress induction of ACXl. This study shows that the ACXl promoter is induced during both germination and dark-induced senescence. Induction is also seen upon wounding of the transgenic plants. It can be concluded that ACXl is a gene of particular interest not only due to its important developmental role but also in its suggested role in the plant defence system. To elucidate the signalling pathways involved in the induction of the ACXl transcript, Arabidopsis cell culture was used as an easily manipulatable system. Northern analysis of ACXl expression revealed that during the period of rapid growth between subculturing, the metabolic demand for ACXl expression was dominant, resulting in constitutive expression from 24 hours. If acid treatnaent experiments were carried out 18 hours after subculturing it was possible to observe a rapid induction of ACXl. This induction was accompanied by an immediate alkalinisation of the cell culture media. Cold treatment of the cells also resulted in alkalinisation of the media possibly suggesting that the respective acidification of the cytosol may be an early stress response which preceeds ACXl expression. The cell culture system has proven to be valuable and should be useful in the future elucidation of the signalling pathways.