| The rice endosperm is the major sources of the nitrogen and carbon that are necessary for plant growth in subsequent generations and a favorite biopharming host for the recombinant protein expression. Four types of proteins, i.e., glutelin, globulin, prolamin, and albumin, account for90%of the storage proteins were found in the rice endosperm. In the rice endosperm, glutelin and globulin localize to the protein storage vacuole (PSV) named protein body â…¡ (PB-â…¡), while prolamin is stored in protein body â… . The mechanism of intracellular protein sorting was considered to depend on the vacuolar sorting determinant (VSD) or the signals created by tertiary structure, and their corresponding receptors. Neither vacuolar sorting determinant has been reported for those storage proteins, nor the mechanism of trafficking route in rice endosperm cells. By sequential deletion and site-directed mutation, we studied the mechanism of glutelin sorting and trafficking in rice endosperm cells and tobacco B Y-2cells. Besides, the membrane and global proteomics of transgenic rice endosperm cells expressing recombinant PDI-IGF was investigated. The results show as below.1, The glutelin oteins, i.e., glutelin, globulin, prolamin, and albumin, account for90%of the storage proteins were found in the rice endosperm. In the rice endosperm, glutelin and globulin localize to tsed in rice endosperm cells. GlutN and GlutM were found in protein body â… , while the GlutC in protein body â…¡.2, The GlutC was further divided into three fragments GlutC-1, GlutC-2and GlutC-3. Then three fusion protein GlutC-1:GFP, GlutC-2:GFP and GlutC-3:GFP were expressed in rice endosperm cells. The microscopy results show the GlutC-1:GFP and GlutC-2:GFP were sorted into the protein body â…¡,but the GlutC-3:GFP into the protein body â… .A six amino acids sequence was found both in GlutC-1and GlutC-2.3, By size exclusion chromatography method, the GlutC:GFP and OsRMR3were found in a680kDa sorting complex, while no complex in the GlutM.GFP extracts. By Co-IP assay, it was confirmed the OsRMR3and GlutC:GFP were in the same complex. Furthermore, OsRMR3was found to interact with the GlutC or Glut, but not GlutM by BiFC assay.4, To investigate the function of OsRMR3in OsVSD-containing protein sorting, transient expression in BY-2cells was employed. The fusion protein was colocalized in the MVB but not TGN in the cells expressing Glut:GFP or GlutC:GFP when co-expressed with OsRMR3. However, GlutM:GFP was found in the TGN but not MVB, when whether co-expressed with OsRMR3or not. Thus, the OsVSD would be the prerequisite for sorting into MVB and the presence of OsRMR3could significantly increase the trafficking efficiency.5, The leucine or arginine replaced by glycine would lead to the sorting function polished, while leucine play a more critical role on that, indicating the function of those two residues for OsVSD function.6, Based on the results, three routes in rice endosperm cells were proposed. The first one is the VSD-dependent pathway, in this route the protein would be sorted into protein body II; The other pathway is VSD-independent pathway, protein synthesized in ER would be sorted into the ER derived protein body I. The last trafficking route could be independent of the storage vacuole pathway. In this route, endogenous or recombinant proteins are sorted into the intracellular space in storage organs in plant cell. This trafficking route has frequently been observed in transgenic endosperm cells overexpressing recombinant proteins7, In the rice endosperm cells highly expressing PDI-IGF, the subcellular structure changed greatly, which was caused by the ER stress.8, By the iTRAQ proteomics analysis to the total protein of immature rice endosperm cells,11significant different proteins were found in highly expression line, including six up-regulated and five down-regulated proteins; In the middle expression level line, there were12significant different proteins, while eight proteins were up-regulated, four proteins were down-regulated; And18proteins were significantly changed in the line with lowest expression level, with five proteins up-regulated and13down-regulated. 9, For the membrane proteins of immature rice endosperm cells,20proteins were significantly changed in the highly expression line, with11proteins up-regulated and nine down-regulated; In the middle expression line,18proteins were significantly changed, with eight proteins up-regulated and10down-regulated; And in the lowest expression line, the significant different protein, up-regulated and down-regulated protein are23, six and17, respectively.10, By investigating the transgenic rice expressing the recombinant proteins, we found the high level expressing protein would greatly affect the subcellular organelle structure, while low expressing protein hardly have effect in rice endosperm cells. Besides, the exogenous proteins lead to slight changes to the proteome, just by changing a few chaperones and translation factors. This result can be an evidence for safety genetic modified cereal for biopharming. |
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