Os03g0835800

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Annotated Information

Function

GPA3 encodes a plant-specific kelch-repeat protein that is localized to the trans-Golgi networks, DVs, and PSVs in the developing endosperm. In vitro and in vivo experiments verified that GPA3 directly interacts with the rice Rab5a-guanine exchange factor VPS9a and forms a regulatory complex with Rab5a via VPS9a.GPA3 acts synergistically with Rab5a and VPS9a to regulate DV-mediated post-Golgi traffic in rice[1].

Mutations

  • Phenotypic Characterization of the gpa3 Mutant

The gpa3 plants exhibited no visible abnormalities before the grainfilling stage, such as plant height, tiller number, and heading date.After fertilization, the gpa3 mutant exhibited a markedly slower grain-filling rate (Figure 1A) and eventually produced a shrunken and floury endosperm (Figure 1B),Scanning electron microscopy analysis revealed that the wild-type endosperm was filled with densely packed, polyhedral starch granules, but endosperm of the gpa3 mutant was packed with round, irregularly arranged compound starch granules (Figure 1C)[1].SDS-PAGE and immunoblot analyses revealed that the accumulation of prolamins was comparable in the dry seeds of the wild type and the gpa3 mutant. However, the gpa3 mutant seeds exhibited increased accumulation of 57-kD proglutelins, accompanied by reduced accumulation of the mature acidic and basic subunits as well as a-globulins, compared with wild-type seeds (Figures 1D and 1E),In addition, immunoblot analysis using isoform specific antibodies revealed increased accumulation of proglutelins for all glutelin subfamilies(GluA, GluB, and GluC; [2]), accompanied by decreased accumulation of their respective acidic subunits in the gpa3 mutant (Figure 1F), suggesting that a defect in a regulatory factor rather than glutelin structural genes may be responsible for the gpa3 mutant phenotypes(Figure 1)[1].

Figure 1. Phenotypic Analyses of the gpa3 Mutant.[1]
  • Abnormal Deposition of Glutelins and a-Globulins in the gpa3 Mutant

As shown in Figures 2A and 2B, Coomassie blue staining showed that the storage proteins were most abundant in the subaleurone layer of endosperm in both the wild type and the gpa3 mutant.numerous glutelin- and a-globulin–containing protein granules distributed near the cell periphery in the gpa3 mutant endosperm but not in wild-type endosperm (Figures 2C to 2F)(Figure 2)[1].

Figure2. Light and Immunofluorescence Microscopy Images of Protein Bodies in the Subaleurone Cells of the Wild Type and the gpa3 Mutant.[1]

Expression

the phenotypes of the gpa3 mutant were restricted to endosperm, GPA3 expression was detected in all tissues examined, including root, stem, leaf, leaf sheath, panicle, and endosperm, with highest accumulation in the leaf(Figure 3G).During endosperm development,the expression of GPA3 was low at an early stage, then peaked at;18 DAF, and slowly decreased after 21 DAF (Figure 3H).Thus,GPA3 encodes a plant-specific kelch-repeat protein and that it is broadly expressed inmultiple organs and tissues.

Figure3. Expression Analysis of GPA3.[1]

Evolution

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Localization

To determine the subcellular localization of the GPA3 protein,P35S:GPA3-GFP was transiently expressedin Arabidopsis protoplasts.As expected, GFP itself was distributed evenly in the cytoplasm and the nucleus, whereas the GPA3-GFP fusion protein was mainly localized to the cytoplasm and to punctate compartments in the cytosol. Moreover,we observed colocalization of various combinations of GPA3 fused with either fluorescent protein (GFP or mCherry) or a Flag tag at the N terminus or C terminus in Arabidopsis protoplasts.To determine the nature of these punctate compartments, we coexpressed GPA3-GFP and fluorescent marker proteins characteristic for the Golgi apparatus[3] , the TGN[4] and the PVC[5] . As shown in Figures 4A to 4C, the punctate compartments of GPA3-GFP were obviously distinct from the Golgi but partially overlapped with the TGN and PVC. Furthermore, correlation analysis using the Pearson-Spearman correlation (PSC) plugin for ImageJ revealed strong correlation between GPA3-GFP and the PVC marker , but the correlation between GPA3-GFP and the TGN marker appeared to be weaker.Furthermore, immunoelectron microscopy analysis with anti-GFP antibodies revealed that GPA3-GFP was targeted to the TGN, DVs, and PBIIs (Figure 4D). Together, these data indicated that GPA3 is localized to various post-Golgi compartments irrespective of the cell types(Figure 4).

Figure 4. Figure 4. Subcellular Localization of GPA3 in Arabidopsis Protoplasts and Developing Subaleurone Cells.[1]

Labs working on this gene

  • State Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China
  • National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • School of Life Sciences, Centre for Cell and Developmental Biology, Chinese University of Hong Kong, New Territories, Hong Kong 999077, China
  • College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China

References

<references>

Miao, Y., Yan, P.K., Kim, H.,et al.Localization of green fluorescent protein fusions with the seven Arabidopsis vacuolar sorting receptors to prevacuolar compartments in tobacco BY-2 cells[J].Plant Physiol,2006,142: 945–962.

Structured Information

  1. 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Yulong Ren,Yihua Wang,Feng Liu,et al.GLUTELIN PRECURSOR ACCUMULATION3 Encodes a Regulator of Post-Golgi Vesicular Traffic Essential for Vacuolar Protein Sorting in Rice Endosperm[J].Plant cell,2014,26: 410–425.
  2. 2.0 2.1 Takemoto, Y., Coughlan, S.J., Okita,et al.The rice mutant esp2 greatly accumulates the glutelin precursor and deletes the protein disulfide isomerase[J]. Plant Physiol,2002, 128: 1212–1222.
  3. 3.0 3.1 Tse, Y.C., Mo, B., Hillmer,et al.Identification of multivesicular bodies as prevacuolar compartments in Nicotiana tabacum BY-2 cells[J]. Plant Cell,2004, 16: 672–693.
  4. 4.0 4.1 Lam, S.K., Siu, C.L., Hillmer,et al.Rice SCAMP1 defines clathrin-coated, trans-Golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells[J]. Plant Cell ,2007b,19: 296–319.
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