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United States Department of Agriculture

Agricultural Research Service

Title: Molecular Cloning and Biochemical Characterization of Indole-3-acetic Acid Methyltransferase from Poplar (Populus trichocarpa)

Authors
item Zhao, Nan - UNIVERSITY OF TENNESSEE
item Guan, Ju - UNIVERSITY OF TENNESSEE
item Lin, Hong
item Chen, Feng - UNIVERSITY OF TENNESSEE

Submitted to: Phytochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 21, 2007
Publication Date: May 29, 2007
Citation: Zhao, N., Guan, J., Lin, H., Chen, F. 2007. Molecular Cloning and Biochemical Characterization of Indole-3-acetic Acid Methyltransferase from Poplar (Populus trichocarpa). Phytochemistry. 68:1537-1544.

Interpretive Summary: Indole-3-acetic acid (IAA) is a plant hormone that controls various physiological processes in plants. Concentrations of IAA in plant tissues are regulated at multiple levels. We report identification and biochemical characterization of a gene from poplar (Populus trichocarpa) encoding IAA methyltransferase (IAMT), an enzyme involved in regulating the level of IAA in plant tissues. Poplar IAMT (PtIAMT1) was identified from the poplar genome based on sequence relatedness to Arabidopsis IAMT. The PtIAMT1 gene was cloned from poplar leaves via RT-PCR. Recombinant PtIAMT1 expressed in E. coli was purified. Enzyme assays combined with gas chromatography-mass spectrophotometery (GC-MS) verified that PtIAMT1 functions as a methyl donor and IAA serves as a methyl acceptor. The enzymatic properties of PtIAMT1 were also characterized. Optimal activity of PtIAMT occurred at pH 7.5. PtIAMT activity was promoted by K+ but inhibited by Fe2+, Cu2+ and Zn2+. Gene expression analysis showed that PtIAMT1 has the highest level of expression in stems, a moderate level of expression in young leaves, and a low level of expression in roots. The presence of PtIAMT1 RNA transcripts in several organs suggests that PtIAMT1 is involved in multiple biological processes in poplar.

Technical Abstract: Indole-3-acetic acid (IAA) is the most active endogenous auxin involved in various physiological processes in higher plants. Concentrations of IAA in plant tissues are regulated at multiple levels including de novo biosynthesis, degradation, and conjugation/deconjugation. In this paper, we report identification and biochemical characterization of a gene from poplar (Populus trichocarpa) encoding IAA methyltransferase (IAMT) that also plays a role in regulating homeostasis of IAA. Poplar IAMT (PtIAMT1) was identified from the poplar genome based on its sequence relatedness to Arabidopsis IAMT. A full-length cDNA of PtIAMT1 was cloned from poplar leaves via RT-PCR. Recombinant PtIAMT1 expressed in E. coli was purified to electrophoresis homogeneity. Enzyme assays combined with GC-MS verified that PtIAMT1 catalyzes the formation of methyl indole-3-acetate using S-adenosyl-L-methionine as a methyl donor and IAA as a methyl acceptor. In addition to using IAA as substrate, PtIAMT1 showed activity, although at a lower level, with another endogenous auxin, indole-3-butyric acid. PtIAMT1 has a pH optimum at pH7.5. Its activity can be promoted by K+ but inhibited by Fe2+, Cu2+ and Zn2+. Under steady-state conditions, PtIAMT1 exhibited Km values of 23 'M and 30 'M for IAA and SAM, respectively. Gene expression analysis showed that PtIAMT1 has the highest level of expression in stems, a moderate level of expression in young leaves, and a low level of expression in roots. The presence of PtIAMT1 transcripts in several organs suggests that PtIAMT1 is involved in multiple biological processes in poplar.

Last Modified: 7/30/2014
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