It is proposed that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of fatty acids in Arabidopsis seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings. Double mutant seeds in which both genes are knocked out are dormant, and unable to utilise their stored lipid. Germination can be achieved by removing the seed coat and supplying sucrose. The seedlings are resistant to 2,4-dichlorophenoxybutyric acid (2,4DB), indicating a defect in peroxisomal β-oxidation. Beyond the seedling stage, double mutants also show arrested growth phenotype, and crucially are unable to produce seeds. The double mutant phenotypes can be restored by complementation with a cDNA encoding CSY with either its native PTS2 targeting sequence or a heterologous PTS1 sequence. These results suggest that peroxisomal CSY is not just a glyoxylate cycle enzyme but is also required for fatty acid respiration and to break seed dormancy. It is hypothesised that peroxisomal malate dehydrogenase (PMDH) serves to oxidise NADH produced by β-oxidation, and does not oxidise malate to provide oxaloacetate for the glyoxylate cycle. The Arabidopsis genome encodes eight putative NAD<sup>+</sup>-dependent malate dehydrogenase enzymes, two of which are predicted to be PMDHs. Double mutant seeds in which these two <i>PMDH </i>genes are knocked out are unable to establish as seedlings unless exogenous sucrose is supplied. In this respect the double mutant is similar to a range of β-oxidation mutants. Seedlings are impaired in breakdown of stored lipid and insensitive to 2,4-DB, showing that β-oxidation is defective. The metabolism of [2-<sup>14</sup>C]-acetate into sugars and organic acids is normal in double mutant seedlings, indicating that the glyoxylate cycle is still active.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:660730 |
| Date | January 2005 |
| Creators | Pracharoenwattana, Itsara |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15658 |
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