Plant Cell Advance Online Publication
Published on February 9, 2007; 10.1105/tpc.106.045997
OPEN ACCESS ARTICLE
Received July 18, 2006
Returned for revision December 19, 2006
Accepted January 22, 2007
Arabidopsis Vacuolar Sorting Mutants (green fluorescent seed) Can Be Identified Efficiently by Secretion of Vacuole-Targeted Green Fluorescent Protein in Their Seeds
Kentaro Fuji 1, Tomoo Shimada 1, Hideyuki Takahashi 1, Kentaro Tamura 1, Yasuko Koumoto 1, Shigeru Utsumi 1, Keito Nishizawa 1, Nobuyuki Maruyama 1, and Ikuko Hara-Nishimura 1*
1 Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
* To whom correspondence should be addressed. E-mail: ihnishi{at}gr.bot.kyoto-u.ac.jp.
Two Arabidopsis thaliana genes have been shown to function in vacuolar sorting of seed storage proteins: a vacuolar sorting receptor, VSR1/ATELP1, and a retromer component, MAIGO1 (MAG1)/VPS29. Here, we show an efficient and simple method for isolating vacuolar sorting mutants of Arabidopsis. The method was based on two findings in this study. First, VSR1 functioned as a sorting receptor for  -conglycinin by recognizing the vacuolar targeting signal. Second, when green fluorescent protein (GFP) fusion with the signal (GFP-CT24) was expressed in vsr1, mag1/vps29, and wild-type seeds, both vsr1and mag1/vps29 gave strongly fluorescent seeds but the wild type did not, suggesting that a defect in vacuolar sorting provided fluorescent seeds by the secretion of GFP-CT24 out of the cells. We mutagenized transformant seeds expressing GFP-CT24. From  3,000,000 lines of M2 seeds, we obtained >100 fluorescent seeds and designated them green fluorescent seed (gfs) mutants. We report 10 gfs mutants, all of which caused missorting of storage proteins. We mapped gfs1 to VSR1, gfs2 to KAM2/GRV2, gfs10 to the At4g35870 gene encoding a novel membrane protein, and the others to different loci. This method should provide valuable insights into the complex molecular mechanisms underlying vacuolar sorting of storage proteins.
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