NEW HAVEN, CONNECTICUT
Yale University

The root and leaf isoforms of NADP-dependent malic enzyme are encoded by distinct genes
--Tausta, SL; Nelson, T

NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, the chloroplastic leaf isoform of NADP-ME is a component of the C4 pathway which delivers carbon dioxide to ribulose bisphosphate carboxylase. A second NADP-ME isoform of uncertain function is found in maize roots and can be distinguished from the leaf isoform by molecular weight, pI, Km, and pH activity profiles. To assign these distinct enzymatic activities to specific genetic/physical loci, we obtained isoform-specific clones by screening two root cDNA libraries with the previously cloned leaf NADP-ME cDNA (Rothermel and Nelson, J. Biol. Chem. 264:19587-19592, 1989).

Two distinct cDNAs (94% DNA identical) encoding root NADP-ME isoforms were identified, indicating that the root NADP-ME is encoded by one or two separate genes different from the leaf NADP-ME gene. The two root ME cDNAs are 85% identical at the nucleotide level and 80% identical at the amino acid level to the leaf ME cDNA.

Both root cDNAs contain putative chloroplast transit peptide coding sequences. However, there are no chloroplasts in the root and the root NADP-ME has C3-like enzymatic characteristics and most C3 NADP-MEs are cytosolic (Nishikido and Wada, Biochem. Biophys. Res. Commun. 61:243-249, 1974). To test the functionality of these sequences, we used a chloroplast import assay. One full length root cDNA was transcribed and translated using a coupled reticulocyte lysate system (TNT kit: Promega, Madison WI). The resulting (35S)Met labeled protein was subjected to a chloroplast import assay using isolated pea chloroplasts (Bruce, Perry, Froehlich and Keegstra. In SB Gelvin, RA Schilperoort. eds, Plant Molecular Biology Manual, vol J1. Kluwer Academic Publishers, pp 1-15, 1994 ). The preprotein (73 kD) was imported and the chloroplast transit peptide cleaved off resulting in a 68 kD protein band (Figure 1), suggesting the root NADP-ME is plastid localized.

We utilized the leaf- and root-specific ME cDNAs to start to determine genetic map positions. Currently, there are three known loci associated with ME activities or coding sequences. A locus, me1, has been mapped to chromosome 3 (bin 3.08) and is associated with an electrophoretically defined isoform of NADP-ME which accumulates in coleoptiles, but does not hybridize with our ME probes under stringent conditions (Goodman et al., Genetics 96:697-710, 1980). The leaf ME cDNA sequence has been mapped to the gene locus me3 on chromosome 3 (bin 3.03) (Rothermel and Nelson, 1989; Keith et al., Plant Physiol. 101:329-332, 1993). A third locus, me2, is found on the long arm of chromosome 6 (bin 6.05) and is probably one of the root MEs (Rothermel and Burr). Because of their extreme sequence similarity, it has been difficult to map the two root NADP-MEs separately. It is intriguing that the C4 (leaf) ME is encoded by a gene embedded in a block of chromosome 3 homeologous to a block on chromosome 8 and a non-C4 (root) ME gene appears to be on a block of chromosome 6, which is also homeologous to chromosome 8 (Helentjaris, MNL 69:67, 1995). If the second root cDNA corresponds to another ME gene, it seems likely that it will be found on chromosome 8.

Figure 1. Chloroplast import of the root ME preprotein. 1) Translational product from the full length root ME cDNA clone pRC14. Sample loaded on the gel represents 3% of the total in vitro reaction product. 2) Intact purified chloroplasts representing 12% of the original import reaction. 3) Intact purified chloroplasts treated with the protease thermolysin and representing 12% of the original import reaction. Thermolysin treatment caused the disappearance of the preprotein, but not the processed protein, indicating the 68 kD protein is imported into and protected by the chloroplasts. 


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