Characterization of NADP-isocitrate dehydrogenase expression in a carrot mutant cell line with enhanced citrate excretion

Citrate in the cytosol is converted to isocitrate, by the action of aconitase, and then isocitrate is converted to 2-oxoglutarate by the action of NADP-specific isocitrate dehydrogenase (NADP-ICDH). This pathway is modified in a mutant carrot (Daucus carota L.) cell line [designated as insoluble phosphate grower (IPG): Koyama et al., 1992 Plant Cell Physiol. 33, 173-177], which releases large amounts of the citrate and grows faster than the wild-type cells in Al-phosphate medium. In the current study, the activities of aconitase in mitochondria and in crude extracts were similar in the mutant cells and the wild-type cells, suggesting that the activity of the cytosolic aconitase was similar among the cell types. By contrast, the NADP-ICDH activity in the crude extracts of the mutant cells was almost half that of the wild-type cells. Western blot analysis revealed that the cytosolic NADP-ICDH was the most abundant isoenzyme in both cell types. However the NADP-ICDH amount was lower in the mutant cells than that in the wild-type cells. Transcripts level of DcICDH1, which appeared to be a unique isoform encoding the cytosolic enzyme in carrot, was lower in the mutant cells than that of the wild-type cells. Thus, we inferred that the lower transcript level of the cytosolic isoform caused the lower NADP-ICDH activities in the mutant cells, compared with the wild-type cells.