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Plant and Cell Physiology Advance Access originally published online on February 23, 2008
Plant and Cell Physiology 2008 49(3):420-432; doi:10.1093/pcp/pcn019
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Identification of Dynamin as an Interactor of Rice GIGANTEA by Tandem Affinity Purification (TAP)

Makoto Abe1, Masayuki Fujiwara1,2, Ken-ichi Kurotani1,3, Shuji Yokoi1,4 and Ko Shimamoto1,*

1Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101 Japan

*Corresponding author: E-mail, simamoto{at}bs.naist.jp; Fax, +81-743-72-5502.


  Abstract

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulate photoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 and Hd3a were identified as orthologs of GI, CO and FT, respectively, and are also important regulators of flowering. Although GI has roles in both flowering and the circadian clock, our understanding of its biochemical functions is still limited. In this study, we purified novel OsGI-interacting proteins by using the tandem affinity purification (TAP) method. The TAP method has been used effectively in a number of model species to isolate proteins that interact with proteins of interest. However, in plants, the TAP method has been used in only a few studies, and no novel proteins have previously been isolated by this method. We generated transgenic rice plants and cell cultures expressing a TAP-tagged version of OsGI. After a two-step purification procedure, the interacting proteins were analyzed by mass spectrometry. Seven proteins, including dynamin, were identified as OsGI-interacting proteins. The interaction of OsGI with dynamin was verified by co-immunoprecipitation using a myc-tagged version of OsGI. Moreover, an analysis of Arabidopsis dynamin mutants indicated that although the flowering times of the mutants were not different from those of wild-type plants, an aerial rosette phenotype was observed in the mutants. We also found that OsGI is present in both the nucleus and the cytosol by Western blot analysis and by transient assays. These results indicate that the TAP method is effective for the isolation of novel proteins that interact with target proteins in plants.

Keywords: Flowering - GI - Proteomics - Rice - Tandem affinity purification (TAP)

Abbreviations: ADL, Arabidopsis dynamin-like protein; CaM, calmodulin; CaMV, cauliflower mosaic virus; CBP, calmodulin-binding peptide; CCA1, CIRCADIAN CLOCK ASSOCIATED 1; CDF1, CYCLING DOF FACTOR 1; CO, CONSTANS, ELF4, EARLY FLOWERING 4; FKF1, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1; FT, FLOWERING LOCUS T; GFP, green fluorescent protein; GI, GIGANTEA; GIS, GLABROUS INFLORESCENCE STEM; LD, long days; LDP, long-day plant; LHY, LATE ELONGATED HYPOCOTYL; LUX, LUX ARRHYTHMO; PCL1, PHYTOCLOCK 1; RT–PCR, reverse transcription–PCR; SD, short days; SDP, short-day plant; SPY, SPINDLY; TAP, tandem affinity purification; TEV, tobacco etch virus; TOC1, TIMING OF CAB EXPRESSION 1; YFP, yellow fluorescent protein; ZTL, ZEITLUPE.

2Present address: Plant Science Education Unit, Laboratory of Plant Protein Analysis, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101 Japan

3Present address: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan

4Present address: Faculty of Agriculture, Iwate University, Morioka, 020-8550 Japan

(Received December 10, 2007; Accepted February 3, 2008)
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