Plant Molecular Biology 45: 281–293, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 281 Analysis of carotenoid biosynthetic gene expression during marigold petal development Charles P. Moehs 1 , Li Tian 2 , Katherine W. Osteryoung 3 and Dean DellaPenna 2,* 1 Department of Biochemistry/MS330, University of Nevada, Reno, NV 89557, USA; 2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA ( * author for correspondence; e-mail: della_d@med.unr.edu); 3 Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824, USA Received 18 April 2000; accepted in revised form 21 September 2000 Key words: carotenoid, gene expression, marigolds, plastid division genes, Tagetes erecta Abstract Marigold (Tagetes erecta L.) flower petals synthesize and accumulate carotenoids at levels greater than 20 times that in leaves and provide an excellent model system to investigate the molecular biology and biochemistry of carotenoid biosynthesis in plants. In addition, marigold cultivars exist with flower colors ranging from white to dark orange due to >100-fold differences in carotenoid levels, and presumably similar changes in carbon flux through the pathway. To examine the expression of carotenoid genes in marigold petals, we have cloned the majority of the genes in this pathway and used these to assess their steady-state mRNA levels in four marigold cultivars with extreme differences in carotenoid content. We have also cloned genes encoding early steps in the biosynthesis of isopentenyl pyrophosphate (IPP), the precursor of all isoprenoids, including carotenoids, as well as two genes required for plastid division. Differences among the marigold varieties in the expression of these genes suggest that differences in mRNA transcription or stability underlie the vast differences in carotenoid synthesis and accumulation in the different marigold varieties. Introduction Carotenoids are a large family of C 40 isoprenoid pig- ments that are found in all higher plants as well as many bacteria and fungi. Over 600 different carotenoid structures have been identified (Starub, 1987). Carotenoids play essential roles in photosyn- thesis in the green tissues of higher plants. They have structural and functional roles in the light-harvesting antennae and serve as photoprotective compounds by quenching triplet chlorophyll and singlet oxygen de- rived from excess light energy (Demmig-Adams and Adams, 1996). The role of the carotenoids that ac- cumulate to high levels in many fruits and flowers is thought to be to serve as visual attractants for insects and animals to aid in pollination and seed dispersal. Interest in carotenoid synthesis has also been stim- ulated by the various roles that carotenoids play in the human diet (DellaPenna, 1999; Hirschberg, 1999). Some β -ring-containing carotenoids are precursors of vitamin A in the human diet. Vitamin A deficiency is a significant cause of blindness and early mortal- ity, particularly in countries where rice, which is low in vitamin A, is the main staple in the diet. In ad- dition, diets rich in carotenoids, such as lycopene, that lack β rings and thus do not have provitamin A activity have other health-promoting effects (Mayne, 1996). The nutritional benefits of carotenoids have led to metabolic engineering of crops deficient in carotenoids for increased production of these com- pounds (Burkhardt et al., 1997; Shewmaker et al., 1999; Ye et al., 2000). The first committed step in the biosynthesis of carotenoids is the head-to-head condensation of two molecules of geranylgeranyl pyrophosphate to form the colorless intermediate phytoene. This reaction is catalyzed by the enzyme phytoene synthase (Harker and Hirschberg, 1998). In plants, four subsequent de-