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Plant and Cell Physiology Advance Access originally published online on October 6, 2009
Plant and Cell Physiology 2009 50(11):1933-1949; doi:10.1093/pcp/pcp138
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use distribution, and reproduction in any medium, provided the original work is properly cited.

Ectopic Overexpression of The Transcription Factor OsGLK1 Induces Chloroplast Development in Non-Green Rice Cells

Hidemitsu Nakamura1,3,*, Masayuki Muramatsu2, Makoto Hakata1,4, Osamu Ueno2,5, Yoshiaki Nagamura1, Hirohiko Hirochika1, Makoto Takano2 and Hiroaki Ichikawa1,2,*

1Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan
2Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan
3Department of Applied Biological Chemistry, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan

*Corresponding authors: Hidemitsu Nakamura, E-mail, hnakamura{at}pgr1.ch.a.u-tokyo.ac.jp; Fax, +81-3-5841-8025; Hiroaki Ichikawa, E-mail, hichkw{at}affrc.go.jp; Fax, +81-29-838-7073.


  Abstract

For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development.

Keywords: Chloroplast development • FOX hunting system • GARP transcription factor • OsGLK1 • Oryza sativa • Rice

Abbreviations: ALA, 5-aminolevulinic acid; CAO, chlorophyllide a oxygenase; Chl, chlorophyll; DIG, digoxigenin; FL-cDNA, full-length cDNA; FOX, full-length cDNA overexpresser; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GltX, glutamyl-tRNA synthetase; HemA, glutamyl tRNA reductase; HemL, glutamate-1-semialdehyde aminotransferase; Hyg, hygromycin B; ICS, isochorismate synthase; NEP, nuclear-encoded plastid RNA polymerase; PEP, plastid-encoded plastid RNA polymerase; PSI, photosystem I; PSII, photosystem II; RT–PCR, reverse transcription–PCR; SAM, shoot apical meristem; SAR, systemic acquired resistance; VB, vascular bundle; VBS, vascular bundle sheath.

4Present address: National Agricultural Research Center, Joetsu, Niigata, 943-0193 Japan.

5Present address: Laboratory of Plant Production Physiology, Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan.

(Received August 31, 2009; Accepted September 28, 2009)
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