Molecular cloning and biochemical characterization of indole-3-acetic acid methyltransferase from poplar.
Indole-3-acetic acid (IAA) is the most active endogenous auxin and is involved in various physiological processes in higher plants. Concentrations of IAA in plant tissues are regulated at multiple levels including de novo biosynthesis, conjugation/deconjugation, and degradation. In this paper, we report molecular isolation and biochemical characterization of a gene PtIAMT1 from poplar encoding IAA methyltransferase (IAMT), which plays a role in regulating IAA homeostasis. PtIAMT1 was identified from the poplar genome based on sequence similarity to Arabidopsis IAMT. A full-length cDNA of PtIAMT1 was cloned from poplar roots via RT-PCR. Recombinant PtIAMT1 expressed in Escherichia coli was purified to electrophoretic homogeneity. Enzyme assays combined with GC-MS verified that PtIAMT1 catalyzes formation of methyl indole-3-acetate using S-adenosyl-l-methionine (SAM) as a methyl donor and IAA as a methyl acceptor. PtIAMT1 had a temperature optimum at 25 degrees C and a pH optimum at pH 7.5. Its activity was promoted by K(+) but inhibited by Fe(2+), Cu(2+) and Zn(2+). Under steady-state conditions, PtIAMT1 exhibited apparent K(m) values of 23.1 microM and 30.4 microM for IAA and SAM, respectively. Gene expression analysis showed that PtIAMT1 had the highest level of expression in stems, a moderate level of expression in young leaves, and a low level of expression in roots. Presence of PtIAMT1 transcripts in several organs suggests that PtIAMT1 is involved in development of multiple organs in poplar.
Zhao
Nan
N
Department of Plant Sciences, 252 Ellington Plant Sciences Building, University of Tennessee, 2431 Joe Johnson Drive, Knoxville, TN 37996, USA.
Guan
Ju
J
Lin
Hong
H
Chen
Feng
F
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D010940:Plant Proteins; D011994:Recombinant Proteins; D008780:Methyltransferases