Arabidopsis vacuolar sorting mutants (green fluorescent seed) can be identified efficiently by secretion of vacuole-targeted green fluorescent protein in their seeds.
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 beta-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 approximately 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.
Fuji
Kentaro
K
Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Shimada
Tomoo
T
Takahashi
Hideyuki
H
Tamura
Kentaro
K
Koumoto
Yasuko
Y
Utsumi
Shigeru
S
Nishizawa
Keito
K
Maruyama
Nobuyuki
N
Hara-Nishimura
Ikuko
I
D052179:Antigens, Plant; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D005916:Globulins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055314:Seed Storage Proteins; D012639:Seeds / Q000378:metabolism* / Q000648:ultrastructure; D030262:Soybean Proteins / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D052179:Antigens, Plant; D029681:Arabidopsis Proteins; C106564:ELP protein, Arabidopsis; D005916:Globulins; C515611:Mag1 protein, Arabidopsis; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; D033921:Vesicular Transport Proteins; C013894:beta-conglycinin protein, Glycine max; D049452:Green Fluorescent Proteins