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- Title
Characterization of the NADP-malic enzymes in the woody plant Populus trichocarpa.
- Authors
Yu, Qiguo; Liu, Jinwen; Wang, Zhifeng; Nai, Jiefei; Lü, Mengyan; Zhou, Xiying; Cheng, Yuxiang
- 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, K, K, and K/ K) 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.
- Subjects
NICOTINAMIDE adenine dinucleotide phosphate; BLACK cottonwood; REVERSE transcriptase polymerase chain reaction; GENETIC transcription in plants; GENE expression in plants; POLYACRYLAMIDE; PROTEINS
- Publication
Molecular Biology Reports, 2013, Vol 40, Issue 2, p1385
- ISSN
0301-4851
- Publication type
Article
- DOI
10.1007/s11033-012-2182-y