Functional analysis of the early steps of carotenoid biosynthesis in tobacco

Carotenoids contribute to energy transduction in the light harvesting complexes and serve in protection from excess light fluence. Because of the importance of carotenoids, the genes encoding enzymes of carotenoid biosynthesis in higher plants are potential targets for herbicides. To obtain further insight into tobacco carotenoid biosynthesis and to investigate and prioritize potential herbicide targets in the pathway, the effects of changed phytoene synthase (PSY) and phytoene desaturase (PDS) gene expression were studied in transgenic tobacco (Nicotiana tabacum Petit Havana SR1) plants. Genes for both enzymes were cloned from tobacco, and surprisingly two functional PSY genes were found. Transgenic tobacco plants constitutively expressing these genes in both sense and antisense orientations were examined regarding phenotype, carotenoid content and transcript levels of carotene biosynthesis genes. Overexpression of either psy gene resulted in severe phenotypic effects including dwarfism, altered leaf morphology, and pigmentation. A correlation among phenotype, transcript level, and metabolic profile was demonstrated by comparison of hemizygous and homozygous plants from the same transformation event. Antisense expression of PSY and PDS also caused lethal phenotypes. Transcript levels of other carotene biosynthesis genes remained unaltered in the transgenic mutant. Phytoene accumulated in plants expressing antisense RNA to pds. However, elevated levels of phytoene were detected suggesting an increase in metabolic flux into this pathway.