Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fattyacid beta-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings

3-ketoacyl-CoA thiolase (KAT) (EC: 2.3.1.16) catalyses a key step in fatty acid beta -oxidation. Expression of the Arabidopsis thaliana KAT gene on ch romosome 2 (KAT2), which encodes a peroxisomal thiolase, is activated in ea rly seedling growth. We identified a T-DNA insertion in this gene which abo lishes its expression and eliminates most of the thiolase activity in seedl ings. In the homozygous kat2 mutant, seedling growth is dependent upon exog enous sugar, and storage triacylglycerol (TAG) and lipid bodies persist in green cotyledons. The peroxisomes in cotyledons of kat2 seedlings are very large, the total peroxisomal compartment is dramatically increased, and som e peroxisomes contain unusual membrane inclusions. The size and number of p lastids and mitochondria are also modified. Long-chain (C16 to C20) fatty a cyl-CoAs accumulate in kat2 seedlings, indicating that the mutant lacks lon g-chain thiolase activity. In addition, extracts from kat2 seedlings have s ignificantly decreased activity with aceto-acetyl CoA, and KAT2 appears to be the only thiolase gene expressed at significant levels during germinatio n and seedling growth, indicating that KAT2 has broad substrate specificity . The kat2 phenotype can be complemented by KAT2 or KAT5 cDNAs driven by th e CaMV 35S promoter, showing that these enzymes are functionally equivalent , but that expression of the KAT5 gene in seedlings is too low for effectiv e catabolism of TAG. By comparison with glyoxylate cycle mutants, it is con cluded that while gluconeogenesis from fatty acids is not absolutely requir ed to support Arabidopsis seedling growth, peroxisomal P-oxidation is essen tial, which is in turn required for breakdown of TAG in lipid bodies.