Journal Article

Visualization of Plastid Nucleoids In situ Using the PEND–GFP Fusion Protein

Kimihiro Terasawa and Naoki Sato

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 4, pages 649-660
Published in print April 2005 | ISSN: 0032-0781
Published online April 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci070
Visualization of Plastid Nucleoids In situ Using the PEND–GFP Fusion Protein

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Plastid DNA is a circular molecule of 120–150 kbp, which is organized into a protein–DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND–green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND–GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.

Keywords: DNA-binding protein; Green fluorescent protein; PEND protein; Plastid division; Plastid nucleoid; Real-time imaging; cpDNA, chloroplast DNA; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; PEND, plastid envelope DNA-binding; SiR, sulfite reductase

Journal Article.  7273 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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