ENHANCED TRANSLATION OF A CHLOROPLAST EXPRESSED RBCS GENE RESTORES SSU LEVELS AND PHOTOSYNTHESIS IN NUCLEAR ANTISENSE RBCS PLANTS

Authors: DHINGRA, AMIT - MOL. BIO/ORLANDO, FL; PORTIS JR, ARCHIE; DANIELL, HENRY - MOL. BIO/ORLAND, FL

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/11/2004
Publication Date: 4/20/2004
Citation: Dhingra, A., Portis Jr., A.R., Daniell, H. 2004. Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants. Proceedings of the National Academy of Sciences. 101:6315-6320.

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 chloroplast 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 explored 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, using transformed (nuclear) plants in which Rubisco expression was severely reduced because of reduced expression from the nuclear genes. 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 chloroplast gene at high levels, expressed Rubisco at almost the original wildtype level and exhibited nearly restored photosynthesis rates. These results have opened an avenue for using chloroplast engineering for the evaluation of foreign Rubisco genes in planta, that can eventually result in achieving efficient photosynthesis and increased crop productivity and this information will benefit scientists attempting improve photosynthesis by the genetic engineering of plants.

Technical Abstract: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a key enzyme that converts atmospheric carbon to food and supports life on this planet. Its low catalytic activity and specificity for oxygen leads to photorespiration, severely limiting photosynthesis and crop productivity. Consequently, Rubisco is a primary target for genetic engineering. Separate localization of the genes encoding for its small and large subunit in the nuclear and chloroplast genomes and a complex assembly process resulting in a very low catalytic activity of hybrid Rubisco enzymes, have rendered several earlier attempts of Rubisco engineering unsuccessful. Here we demonstrate, for the first time, that the RbcS gene, when integrated at a transcriptionally active spacer region of the chloroplast genome, in a nuclear RbcS antisense line and expressed under the regulation of heterologous (gene 10) or native (psbA) UTRs, results in the assembly of a functional holoenzyme, short-circuiting nuclear control of gene regulation. There was about 150-fold more RbcS transcript in chloroplast transgenic lines when compared to the nuclear RbcS antisense line, while the wild type has 7-fold more transcript. The small subunit protein levels in the gene10/RbcS and psbA/RbcS plants were 60% and 106% respectively of the wild type. Photosynthesis of gene10/RbcS plants was about double that of the antisense plants while that of psbA/RbcS plants was almost completely restored to the wild type rates. These results have opened an avenue for using chloroplast engineering for the evaluation of foreign Rubisco genes in planta, that can eventually result in achieving efficient photosynthesis and increased crop productivity.