Minisymposium 15: Photosynthesis

Abs # 28002: Enhanced translation of a chloroplast expressed RbcS gene restores SSU levels and photosynthesis in nuclear antisense RbcS plants

Presenter:	Daniell, Henry , daniell@mail.ucf.edu
Authors	Dhingra, Amit  (A)   Portis, Jr., Archi R (B)   Daniell, Henry  (A)
Affiliations:	(A): University of Central Florida, 4000 Central Florida Blvd, Dept. Mol Biol & Microbiol, Biomol Sci, Bldg # 20, Rm 336, Orlando, FL 32816 (B): Department of Crop Sciences and Department of Plant Biology, University of Illinois, Urbana, IL 61801
Web Site:	http://pegasus.cc.ucf.edu/~daniell/

Rubisco is the most prevalent enzyme on this planet; it fixes carbon dioxide but oxygenase activity severely limits photosynthesis and crop productivity. Rubisco consists of eight plastid-derived large subunits and eight nuclear-derived small subunits that are assembled into functional holoenzyme. Genetic manipulation of Rubisco via nuclear or plastid genetic engineering has resulted in either Rubisco deficient plants, inefficient assembly of heterologous Rubisco or transgenic plants unable to survive under ambient CO₂ conditions. In this study, RbcS cDNA was engineered into a transcriptionally active spacer region between trnI/trnA genes of the rrn operon, of the chloroplast genome of nuclear RbcS antisense line; this line contains low level of RbcS mRNA(12%), Rubisco protein (38%) and activity (20%) when compared to wild type. In our study, stable integration of RbcS cDNA into the chloroplast genome of the antisense line resulted in ~ 150-fold more RbcS transcript than the antisense line, while the wild type contained 7-fold more transcript. Two different 5’-untranslated regions (5’-UTR), a heterologous (T7 gene 10) or native (psbA) UTR were used to facilitate enhanced translation of the engineered RbcS cDNA. Qualitative analysis revealed that total soluble protein yield from chloroplast transgenic lines was much higher than the antisense line or the wild type lines as seen in GelCode Blue stained SDS-PAGE gels. Quantitative analysis revealed that the small subunit levels in chloroplast transgenic lines were 106% of the wild type demonstrating that Rubisco was successfully assembled from chloroplast derived large and small subunits. Most importantly, evidence for the functionality of chloroplast derived Rubisco was established by restoration of photosynthesis and phenotype in the antisense lines and their ability to survive under ambient CO₂ conditions. Successful complementation of the SSU deficiency opens new avenues for further research on mechanisms controlling Rubisco expression and assembly. Consequently, this approach should greatly facilitate future Rubisco engineering in order to achieve the long-cherished goal of increasing plant productivity.