title: Characterization of the NADP-malic enzymes in the woody plant Populus trichocarpa.
abstract: 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.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, 26 Hexing Road, Harbin, 150040, China.
authors.forename: Qiguo
authors.initials: Q
authors.lastname: Yu
authors.forename: Jinwen
authors.initials: J
authors.lastname: Liu
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Wang
authors.forename: Jiefei
authors.initials: J
authors.lastname: Nai
authors.forename: Mengyan
authors.initials: M
authors.lastname: Lü
authors.forename: Xiying
authors.initials: X
authors.lastname: Zhou
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D004926:Escherichia coli; D019143:Evolution, Molecular; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050538:Malate Dehydrogenase (NADP+) / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008293:Malates / Q000737:chemistry; D010802:Phylogeny; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D055049:Salt Tolerance; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013312:Stress, Physiological
chemical_list: D008293:Malates; D010940:Plant Proteins; D011994:Recombinant Proteins; C030298:malic acid; D050538:Malate Dehydrogenase (NADP+)