Submitted to: Physiologia Plantarum
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/20/2007
Publication Date: 2/15/2008
Citation: Lopez, A., Yang, Y., Thannhauser, T.W., Li, L. 2008. Phytoene desaturase is present in a large protein complex in the plastid membrane. Physiologia Plantarum. 133:190-198. Interpretive Summary: Carotenoids are indispensable to plants and play a critical role in human nutrition and health. In spite of recent significant progress in our understanding of carotenoid biosynthesis in plants, we still lack fundamental knowledge on various aspects of this metabolic process. To produce crops enriched with carotenoid content, it is necessary to gain a better understanding of carotenoid biosynthesis. Carotenoid biosynthesis has long been suggested to be catalyzed by multienzyme complexes in association with plastid membranes. However, there is no definite evidence to support such hypothesis. In this study, we cloned a cauliflower gene encoding phytoene desaturase, one of the key enzymes involved in carotenoid biosynthesis, and made antibodies again it. Using phytoene desaturase protein as an indicator, we show that the carotenoid biosynthetic enzymes indeed are present in a large protein complex to form a multienzyme complex to catalyze carotenoid biosynthesis in plastid membranes, the site of carotenoid biosynthesis. This study provides new information for a better understanding of carotenoid biosynthesis in plants.
Technical Abstract: Phytoene desaturase (PDS, EC 1.14.99.-) represents one of the key enzymes in the carotenoid biosynthetic pathway and is present in nearly all types of plastids in plants. To further characterize PDS, we isolated the PDS cDNA from cauliflower (BoPDS) and confirmed its function by heterologous expression in a strain of E. coli containing a carotenoid producing plasmid. The BoPDS cDNA encodes a predicted mature protein of ~ 55 kDa. In comparison with PDS from a few other plant species, BoPDS exhibited a high enzyme activity in E. coli, and its expression in plastids was independent of carotenoid levels. Plastids were purified from tissues of different plant species including cauliflower curds, tomato fruits, carrot roots, and Arabidopsis leaves. By employing both Blue Native PAGE and SDS-PAGE approaches in conjunction with Western blot analysis, it was found that PDS in these plants existed in two forms. The plastid membrane form was present in a large protein complex of approximately 350 kDa, whereas the stroma version was in a ~660 kDa complex.