Occurrence of two acetoacetyl-coenzyme a thiolases with distinct expression patterns and subcellular localization in Tobacco

Abstract

Acetoacetyl-coenzyme A thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl-CoA units into acetoacetyl-CoA. By screening a tobacco (Nicotiana tabacum L.) cDNA library using a radish probe, we isolated two genes encoding an AACT. The deduced protein sequences, referred to as Nt AACT1 and Nt AACT2, were found to be 77% identical and to share high homologies with other AACTs. Here, we report that both NtAACTs exhibited distinct expression patterns in planta and that the enzymes localize to separate cell compartments, suggesting different metabolic functions. At its C-terminus, Nt AACT1 bears an SSL motif corresponding to a peroxisomal targeting signal of type 1. We present evidence that Nt AACT1 is readily imported into glyoxysomes by demonstrating invivo colocalization of the chimeric proteins, GFP- Nt AACT1 and RFPSKL, in transiently transformed tobacco BY-2 cells. The importance of the SSL motif was stressed by the absence of glyoxysomal delivery of the fusion proteinlacking the tripeptide. In contrast, Nt AACT2 is clearly a soluble enzyme, with no obvious targeting signal to ade fi ned cell membrane compartment. Its participation in isoprenoid biosynthesis via the mevalonate pathway wasattested by potato virus X-induced gene silencingexperiments in Nicotiana benthamiana plants. Silencing of endogenous NbAACT2 was shown indeed to trigger a 50% reduction in the levels of sterol biosynthetic precursors. A spatiotemporal study of expression patterns of both genes in planta indicated that NtAACT2 was expressed throughoutthe plant, but more effectively in young tissues. In contrast, NtAACT1 displayed a signi fi cant expression onlyin senescent leaves and early stages of germination and appears to be functional in the glyoxysomal/peroxisomal b -oxidation of fatty acids, catalyzing the last step.