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 carotaL.) 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 wildtype cells, suggesting that the activity of the cytosolic aconitase was similar among the cell types. By contrast, the NADPICDH 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.