[Show abstract][Hide abstract] ABSTRACT: The similarity and cellular location of NADP+-malic enzyme (NADP-ME, EC 220.127.116.11) in developing fruit and other parts of the tomato (Lycopersicon esculentum Mill.) plant were investigated in order to clarify the role of the enzyme in metabolism. There appeared to be a single enzyme (native NW 260 kD, subunit MW 66 kD) in mature green tomato fruit; this was purified to a specific activity of 42.5 μmol mg−1 min−1 and apparent homogeneity on SDS-PAGE. Polyclonal antibodies raised against this protein achieved 90% precipitation of enzyme activity, but required purification in order to detect specifically the 66 kD protein on Western blots. The purified antibodies recognized a similar protein in tomato leaves, roots and stems. The specific activity of NADP-ME was at least nine times higher in supernatant than in chloroplast fractions in tomato fruit and leaf, consistent with the distribution of a cytoplasmic marker (alcohol dehydrogenase), but in contrast to a chloroplast marker (rubisco). The purified NADP-ME antibodies did not detect a 66 kD protein in chloroplast preparations. A partial NADP-ME cDNA isolated from a fruit library specifically hybridized with a 2.1 kb transcript in RNA preparations from tomato leaves and fruits at different stages of development. The existence of a minor, possibly chloroplastidic NADP-ME in tomato cannot be excluded; the major NADP-ME is a cytosolic protein which is present in all plant organs analysed.
[Show abstract][Hide abstract] ABSTRACT: Cells associated with veins of petioles of C3 tobacco possess high activities of the decarboxylase enzymes required in C4 photosynthesis. It is not clear whether this is the case in other C3 species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C4 acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.
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