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Plant and Cell Physiology Advance Access originally published online on January 25, 2008
Plant and Cell Physiology 2008 49(3):375-385; doi:10.1093/pcp/pcn014
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Molecular Design of Photosynthesis-Elevated Chloroplasts for Mass Accumulation of a Foreign Protein

Yukinori Yabuta1,4,5, Masahiro Tamoi1,5, Kumiko Yamamoto1, Ken-ichi Tomizawa2, Akiho Yokota3 and Shigeru Shigeoka1,*

1Department of Advanced Bioscience, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara, 631-8505 Japan
2Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, 619-0292 Japan
3Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0101 Japan

*Corresponding author: E-mail, shigeoka{at}nara.kindai.ac.jp; Fax, +81-742-43-8976.


  Abstract

In order to increase production of a useful protein by the chloroplast transformation technique, it seems to be necessary to determine the upper limit for the accumulation of a biologically active foreign protein in chloroplasts and then improve photosynthetic capacity and plant productivity. Here we show that the stromal fractions of tobacco chloroplasts could accommodate an additional 200–260 mg ml–1 of green fluorescent protein in the stroma without any inhibition of gas exchange under various light intensity and growth conditions. The minimum amount of fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) limiting photosynthesis was then calculated. Analyses of the photosynthetic parameters and the metabolites of transformants into which FBP/SBPase was introduced with various types of promoter (PpsbA, Prrn, Prps2 and Prps12) indicated that a 2- to 3-fold increase in levels of FBPase and SBPase activity is sufficient to increase the final amount of dry matter by up to 1.8-fold relative to the wild-type plants. Their increases were equivalent to an increase of <1 mg ml–1 of the FBP/SBPase protein in chloroplasts and were calculated to represent <1% of the protein accumulated via chloroplast transformation. Consequently, >99% of the additional 200–260 mg ml–1 of protein expressed in the chloroplasts could be used for the production of useful proteins in the photosynthesis-elevated transplastomic plants having FBP/SBPase.

Keywords: Fructose-1,6-/sedoheptulose-1,7-bisphosphatase - Calvin cycle - Photosynthesis - Plastid transformation

Abbreviations: CBB, Coomassie brilliant blue; GFP, green fluorescent protein; FBPase, fructose-1,6-bisphosphatase; FBP/SBPase, fructose-1,6-/sedoheptulose-1,7-bisphosphatase; PFD, photon flux density; RMOP, regeneration medium of plants; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1.5-bisphosphate; SBPase, sedoheptulose-1,7-bisphosphatase.

4Present address: School of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture, Tottori University, Tottori 680 8553, Japan

5These authors contributed equally to this work.

(Received December 4, 2007; Accepted January 22, 2008)
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[Abstract] [Full Text] [PDF]


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