The rice glup6 plays a crucial role in the same pathway responsible for the intracellular transport of proglutelins from the Golgi to the protein storage vacuoles(PSVs) as glup4.
The GLUP6 candidate belonged to a gene family containing the Vps9 domain and was classified as a GEF for the Rab5 group. The GLUP6 gene was expressed throughout plant development. Seeds of glup6, which belongs to the same class as the glup4/rab5 mutant, exhibit a protein pattern similar to glup4 in having elevated proglutelin levels and reduced levels of acidic and basic glutelin subunits, prolamines, and 26-kD a-globulin. The GDP/GTP recycling of GLUP4/Rab5 by GLUP6/GEF is a key regulatory process in the trafficking of storage proteins to the protein storage vacuoles (PSVs)and in the general maintenance of the endomembrane system in developing rice endosperm.
Wild Type VS. Mutant
- GLUP6 / GEF in Fukuda's research and OsVPS9A/GPA2 in Liu's research locate in the same gene locus.
- The glup6 mutant accumulates large amounts of proglutelin. Figure 1A shows the profiles of total seed protein samples from three independent glup6 mutant lines, EM939, EM1327, and EM1484, as well as from the wildtype. All three glup6 lines contained elevated amounts of the 57-kD proglutelin polypeptide compared with the wild type. The change in the proglutelin levels between the wild type and the glup6 mutants was readily discernible by immunoblot analysis (Fig. 1B). Figure 1C shows that the grain morphology is also significantly altered in the glup6 mutants. All three mutant lines exhibited a floury endosperm.
- Immunoblot analysis using an antibody raised against GLUP6/GEF revealed two reacting polypeptide bands in developing wild-type seeds with the bottom band missing in all glup6 lines (Fig. 2). The theoretical molecular mass of GLUP6/GEF is 53.8 kD, a value in close agreement with the measured molecular size of the bottom polypeptide band. The ubiquitous presence of the top polypeptide band in Figure 2 may be due to another GEF-related protein. As all glup6 lines contain nonsense mutations, even one close to the C terminus(EM1327), the lack of accumulated truncated polypeptides indicates that the polypeptide and the mRNA are unstable, the latter macromolecule likely degraded by nonsense-mediated mRNA decay.
- gpa2, a loss-of-function mutant of OsVPS9A, which encodes a GEF of OsRAB5A, accumulated uncleaved proglutelins.. gpa2 seeds accumulate proglutelins and develop abnormal endosperm.Notably, gpa2 mutant endosperms appeared floury (Figure 3D) in contrast to wild-type (Figure 3C). Scanning electron microscopic analysis revealed that the gpa2 endosperms comprised round and loosely packaged, compound starch granules (Figure 3F and 3H) instead of tightly packaged, crystal-like structures in wild-type (Figure 3E and 3G). Time course analysis demonstrated that gpa2 seeds weighed less than wild-type throughout the process of grain filling, and this difference became apparent at 6 days after flowering (DAF) a very early stage of seed development (Figure 3I).In short, gpa2 mutant was defective in seed development, and the intracellular transport of proglutelins might be disturbed in the gpa2 endosperm cells..
- The full-length open reading frame of Os03g0262900 was amplified with gene-specificprimers usingcomplementary DNA (cDNA) reverse transcribed from mRNA extracted from the immature seeds of the wild-type Taichung65 as a template. Sequencing analysis of the amplified single band demonstrated that the GLUP6 candidate gene possessed a coding sequence of 1,443 bp coding for 480 deduced amino acids spanning over five exons.
- In order to elucidate the function of GLUP6/GEF in the intracellular transport of proglutelins, the endosperm of glup6 lines was analyzed by immunofluorescence microscopy (Fig.4).In wild-type endosperm, protein bodies containing prolamines (protein body type I [PB-I]) and glutelins and a-globulins (PB-II) increased in size and number as the seeds developed (Fig. 4,Aand B). In glup6 lines EM939 and EM1327, one or more large distended structures reactive to glutelin antibodies were conspicuously observed close to the cell wall (Fig. 4, B and C).
In the rice endosperm cells, glutelins are synthesized on rough endoplasmic reticulum as proglutelins and are sorted to the protein storage vacuoles (PSVs) called protein body IIs (PBIIs), where they are converted to the mature forms.Dense vesicle (DV)-mediated trafficking of proglutelins in rice seeds has been proposed, but the post-Golgi control of this process is largely unknown. OsVPS9A and OsRAB5A may work together and play a regulatory role in DV-mediated post-Golgi proglutelin trafficking to PBII (PSV).
Labs working on this gene
- Faculty of Agriculture, Kyushu University, Fukuoka 812–8581, Japan
- National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan
- Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164
- Department of General Education, Yamaguchi Prefectural University, Yamaguchi 753–8502, Japan
- Graduated School of Science, University of Tokyo, Tokyo 113–0033, Japan; and Japan Science and Technology Agency, PRESTO, Saitama 332–0012, Japan
- 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, Beijing100081, China
- College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
- Ueda Y, Satoh-Cruz M, Matsusaka H, Takemoto-Kuno Y, Fukuda M, Okita TW, Ogawa M, Satoh H, Kumamaru T (2010) Gene-gene interactions between mutants that accumulate abnormally high amounts of proglutelin in rice seed. Breed Sci 60: 568–574
- Masako Fukuda;Liuying Wen;Mio Satoh-Cruz;Yasushi Kawagoe;Yoshiaki Nagamura;Thomas W. Okita;Haruhiko Washida;Aya Sugino;Sonoko Ishino;Yoshizumi Ishino;Masahiro Ogawa;Mariko Sunada;Takashi Ueda;Toshihiro Kumamaru. (2013) A Guanine Nucleotide Exchange Factor for Rab5 Proteins Is Essential for Intracellular Transport of the Proglutelin from the Golgi Apparatus to the Protein Storage Vacuole in Rice Endosperm. 162(2): 663-674.
- Feng Liu;Yulong Ren;Yihua Wang;Cheng Peng;Kunneng Zhou;Jia Lv;Xiuping Guo;Xin Zhang;Mingsheng Zhong;Shaolu Zhao;Ling Jiang;Haiyang Wang;Yiqun Bao;Jianmin Wan. (2013) OsVPS9A Functions Cooperatively with OsRAB5A to Regulate Post-Golgi Dense Vesicle-Mediated Storage Protein Trafficking to the Protein Storage Vacuole in Rice Endosperm Cells. Molecular Plant ，6(6)：1918-1932