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Title: COMPLEMENTATION OF THE NUCLEAR ANTISENSE RBCS GENE INTO THE TOBACCO PLASTID GENOME

Author
item ZHANG, XING-HAI - FORMERLY ARS
item EWY, ROBERT - FORMERLY ARS
item WIDHOLM, JACK - CROP SCI UI URBANA
item PORTIS JR, ARCHIE

Submitted to: Plant and Cell Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/2/2002
Publication Date: 12/31/2002
Citation: ZHANG, X., EWY, R.G., WIDHOLM, J.M., PORTIS JR, A.R. COMPLEMENTATION OF THE NUCLEAR ANTISENSE RBCS GENE INTO THE TOBACCO PLASTID GENOME. PLANT AND CELL PHYSIOLOGY. 2002. V. 43. P. 1302-1313.

Interpretive Summary: Photosynthesis, the process by which plants use light energy from the sun to make carbohydrates for growth from carbon dioxide and water, occurs in the chloroplasts. Rubisco, which is located in the choloroplast and initiates photosynthetic carbon fixation, is composed of two polypeptides, with one gene for one polypeptide located in the chloroplast and many genes for the other located in the nucleus. In this report, we explore the possibility that a copy of the genes located in the nucleus could be placed in the chloroplast via chloroplast transformation and utilized to make Rubisco. If so, the Rubisco genes could be more easily replaced with foreign or genetically engineered genes to increase photosynthesis. Transgenic plants were obtained that expressed mRNA from the introduced gene at high levels, but no Rubisco protein attributable to the presence of the introduced gene or improvement in plant growth or photosynthesis was obtained. It appears that incorrect processing of the polypeptides synthesized from this gene occurs. This information will benefit scientists attempting improved photosynthesis by the genetic engineering of plants.

Technical Abstract: The small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (rbcL) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco expressing antisense rbcS coding DNA that results in low rbcS protein content [Rodermel et al. Cell 55:673-681 (1988)] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the rbcS protein or improvement in plant growth and photosynthesis. A limitation in efficient, correct processing and modifications of the plastid-synthesized rbcS protein might cause the rapid degradation of unassembled Rubisco subunits. We propose possible mechanisms regarding the fate of the plastid-synthesized rbcS and a working scheme for genetic engineering of higher plant Rubisco.