Characterization of the NADP-malic enzymes in the woody plant Populus trichocarpa

Plant NADP-malic enzyme (NADP-ME, EC 1.1.1.40) participates in a large number of metabolic pathways, but little is known about the NADP-ME family in woody plants or trees. Here, we characterized the tree Populus trichocarpa NADP-ME (PtNADP-ME) family and the properties of the family members. Five NADP-ME genes (PtNADP-ME1-PtNADP-ME5) were found in the genome of Populus. Semi-quantitative RT-PCR analysis show that the transcription levels of PtNADP-ME1 in lignified stems and roots are clearly higher than in other tissues, and PtNADP-ME2, PtNADP-ME3, PtNADP-ME4 and PtNADP-ME5 are broadly expressed in various tissues. PtNADP-ME gene expression was found to respond to salt and osmotic stresses, and NaCl salts upregulated the transcripts of putative plastidic ones (PtNADP-ME4 and PtNADP-ME5) significantly. Further, the NADP-ME activities of Populus seedlings increased at least two-fold under NaCl, mannitol and PEG treatments. Also, the expression of PtNADP-ME2 and PtNADP-ME3 increased during the course of leaf wounding. Each recombinant PtNADP-ME proteins were expressed and purified from Escherichia coli, respectively. Coomassie brilliant blue and NADP-ME activity staining on native polyacrylamide gels showed different oligomeric states of the recombinant PtNADP-MEs in vitro. Noticeably, the cytosolic PtNADP-ME2 aggregates as octamers and hexadecamers while the plastidic PtNADP-ME4 resembles hexamers and octamers. The four PtNADP-ME proteins except for PtNADP-ME1 have high activities on native polyacrylamide gels including different forms for PtNADP-ME2 (octamers and hexadecamers) or for PtNADP-ME4 (hexamers and octamers). High concentrations of NADP substrate decreased the activities of all PtNADP-MEs slightly, while the malate had no effect on them. The kinetic parameters (V (max), K (m), K (cat), and K (cat)/K (m)) of each isoforms were summarized. Our data show the different effects of metabolites (influx into tricarboxylic acid cycle or Calvin cycle) on the activity of the individual PtNADP-ME in vitro. According to phylogenetic analysis, five PtNADP-MEs are clustered into cytosolic dicot, plastidic dicot, and monocot and dicot cytosolic groups in a phylogenetic tree. These results suggest that woody Populus NADP-ME family have diverse properties, and possible roles are discussed.