{"created":"2021-03-01T06:09:06.778747+00:00","id":5265,"links":{},"metadata":{"_buckets":{"deposit":"8605e200-5680-456d-8430-bfeefe3c2493"},"_deposit":{"id":"5265","owners":[],"pid":{"revision_id":0,"type":"depid","value":"5265"},"status":"published"},"_oai":{"id":"oai:niigata-u.repo.nii.ac.jp:00005265","sets":["453:455","468:563:564"]},"item_6_alternative_title_1":{"attribute_name":"その他のタイトル","attribute_value_mlt":[{"subitem_alternative_title":"イネ糖タンパク質α-アミラーゼは分泌経路とゴルジ体を経由してプラスチド局在化する"}]},"item_6_biblio_info_6":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2011-03-23","bibliographicIssueDateType":"Issued"},"bibliographicPageEnd":"76","bibliographicPageStart":"1","bibliographic_titles":[{}]}]},"item_6_date_granted_51":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2011-03-23"}]},"item_6_degree_grantor_49":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"新潟大学"}]}]},"item_6_degree_name_48":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（農学）"}]},"item_6_description_4":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"Rice α-amylase isoform I-1 (AmyI-1), well-characterized secretory N-linked glycoprotein, was localized within the plastids, and proved to be involved in the degradation of starch granules in the organelles of rice cells. AmyI-1 is synthesized on endoplasmic reticulum (ER)-membrane-bound ribosomes, signal-sequence-dependent translocation of the ER, vesicular transport to the Golgi apparatus, oligosaccharide modification to the complex type, and exocytosis, all proceed according to the canonical secretory mechanism. Nuclear-encoded plastidial proteins are normally synthesized in the cytosol and post-translationally imported into the organelle. In most cases, precursor proteins are synthesized with an N-terminal pre-sequence called a transit peptide. The transit peptide is necessary for and also sufficient for plastidial targeting and translocation initiation. The transit peptide is recognized by Toc (translocon at the outer envelope of chloroplast) complex and Tic (translocon at the inner envelope of chloroplast) complex. A few plastidial glycoproteins have been reported, however, the mechanism of targeting to plastids. The aim of this research is to clarify the mechanism of a unique transport system involved in the plastid-targeting of rice AmyI-1 glycoprotein. The contents are as follows. AmyI-1 degrades starch granules in living rice cells In electron microscopic analysis, the amyloplasts of transgenic rice callus cells with constitutively high expression of AmyI-1 had slim starch granules with large interspaces in contrast to the presence of ripe starch granules in the amyloplasts of wild-type cells. Additionally, quantitative measurements showed that the starch content in the callus cells overexpressing AmyI-1 was reduced to approximately half of that in the wild type. These date showed that AmyI-1 degrade starch granules in living rice cells. AmyI-1 contains a plastid-targeting signal common to both rice and onion cells To characterize the nature of plastid localization of AmyI-1 in details, I performed transient expression of α-amylase isoforms (I-1, II-3, II-4, II-5 and II-6) fused with green fluorescent protein (GFP) using onion epidermal cells. AmyI-1-GFP fluorescence overlapped with the plastids visualized by WxTP-DsRed but unlike in the AmyI-1-GFP, the fluorescence of AmyII-3-, AmyII-4-, AmyII-5- and AmyII-6-GFP rarely merged with the plastid marker These results indicate that AmyI-1, but no other isoforms, possesses a plastid-targeting signal common to both rice and onion cells. Membrane trafficking is necessary for plastid targeting of AmyI-1 Expression of dominant negative or constitutive active mutants of ARF1 and SAR1, which are defective in GTPase cycling, inhibits the ER-to-Golgi traffic. The transport of AmyI-1-GFP into plastids was severely inhibited by the ARF1 and SAR1 mutants and the fluorescent proteins remained. These results strongly suggest that the membrane trafficking from the ER is necessary for the plastid targeting of these glycoproteins. Golgi-to-plastid traffic In contrast to mammalian Golgi apparatus, plant Golgi stacks show active and \"stop-and-go\" tumbling movements along actin microfilaments. The active and \"stop-and-go\" tumbling movement of Golgi stacks suggests that the Golgi stack itself serves as a cargo container. High-speed two-dimensional and three-dimensional time-lapse analyses using confocal laser scanning microscopy were performed. When ST-mRFP trans-Golgi marker and WxTP-GFP plastid marker were simultaneously expressed together with AmyI-1, which would activate Golgi-to-plastid traffic in onion cells, significant merging of ST-mRFP with the GFP-labeled plastids was observed. Moreover, close contact between the two organelles was occasionally observed. Three-dimensional time-lapse imaging showed that even without AmyI-1 expression, the ST-mRFP-labeled Golgi adheres to the surface of plastids and stromules.","subitem_description_type":"Abstract"},{"subitem_description":"The incorporation of ST-mRFP into plastids still occurred, albeit at lower frequency. In addition to these, Golgi-to-plastid trafficking was noticeably prevented by the expression of either AtARF1 (T31N) or AtSAR1 (Q74L). Electron microscopic studies were carried out of suspension-cultured cells derived from transgenic rice seed (A3-1 line) with a constitutively high expression of AmyI-1 using high-pressure frozen/freeze-substituted techniques. In the electron microscopic images, it was frequently observed that small membrane vesicles, perhaps derived from the Golgi apparatus stayed by their side, adhered to the envelope membrane of plastids. Tight association of the Golgi stacks with the envelope membrane was also observed in the same cells. Furthermore, immunological staining of ultrathin sections with anti-AmyI-1 antibodies revealed the presence of AmyI-1 in both the Golgi apparatus and the plastids. Taken together, I strongly inferred that Golgi-to-plastid basal communication occurs under tranquil physiological conditions, and that the AmyI-1 co-expression enhances the flow of Golgi-to-plastid traffic and activates the communication between two organelles in the cell. Characterization of the plastid-targeting signal of AmyI-1 To identify the plastid-targeting signal of AmyI-1, the transient expression and localization of a series of carboxyl-terminal truncated AmyI-1-GFP fusion proteins was examined in bombarded onion cells. And that indicated the importance of the peptide region from Trp^301 to Gln^369 for plastid targeting of AmyI-1. I selected Trp^302, Thr^307 and Gly^354, unique amino acid residues in the region from 301 to 369, for the next site-directed mutagenesis experiments. The substitution of Ala or Leu for Trp^302 strongly arrested the targeting of AmyI-1-GFP into the plastids. The substitution of Val for Thr^307 or Asn for Gly^354 also arrested the plastid targeting of AmyI-1-GFP, as in the W302A and W302L mutations. Furthermore, the chimera proteins of AmyII-6(1-266):AmyI-1(256-369) and AmyII-6(1-266):AmyI-1(287-369) failed to localize in the plastids. These results indicate that conformational changes in the region from Trp^301 to G1n^369 influence the targeting ability.","subitem_description_type":"Abstract"}]},"item_6_description_5":{"attribute_name":"内容記述","attribute_value_mlt":[{"subitem_description":"新潟大学大学院自然科学研究科","subitem_description_type":"Other"},{"subitem_description":"平成23年3月23日","subitem_description_type":"Other"},{"subitem_description":"新大院博（農）甲第106号","subitem_description_type":"Other"}]},"item_6_description_53":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"新大院博（農）甲第106号","subitem_description_type":"Other"}]},"item_6_dissertation_number_52":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"13101甲第3524号"}]},"item_6_full_name_3":{"attribute_name":"著者別名","attribute_value_mlt":[{"nameIdentifiers":[{"nameIdentifier":"49997","nameIdentifierScheme":"WEKO"}],"names":[{"name":"古賀, 彩"}]}]},"item_6_publisher_7":{"attribute_name":"出版者","attribute_value_mlt":[{"subitem_publisher":"新潟大学大学院自然科学研究科"}]},"item_6_select_19":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_select_item":"author"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"Kitajima-Koga, Aya"}],"nameIdentifiers":[{"nameIdentifier":"49996","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-08-05"}],"displaytype":"detail","filename":"D_S_KA.pdf","filesize":[{"value":"14.1 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"本文","url":"https://niigata-u.repo.nii.ac.jp/record/5265/files/D_S_KA.pdf"},"version_id":"41fb9136-fe0d-45dc-a09e-267cddd3a0dd"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Rice glycoprotein α-amylase targeting to plastid from the Golgi apparatus through the secretory pathway","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Rice glycoprotein α-amylase targeting to plastid from the Golgi apparatus through the secretory pathway"},{"subitem_title":"Rice glycoprotein α-amylase targeting to plastid from the Golgi apparatus through the secretory pathway","subitem_title_language":"en"}]},"item_type_id":"6","owner":"1","path":["455","564"],"pubdate":{"attribute_name":"公開日","attribute_value":"2012-06-05"},"publish_date":"2012-06-05","publish_status":"0","recid":"5265","relation_version_is_last":true,"title":["Rice glycoprotein α-amylase targeting to plastid from the Golgi apparatus through the secretory pathway"],"weko_creator_id":"1","weko_shared_id":2},"updated":"2022-12-15T03:38:15.502221+00:00"}