Regulation of the carotenoid biosynthetic pathway in petals of California poppy (Eschscholzia californica) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand

Abstract

Carotenoids are essential plant pigments. They function in a wide range of processes including light harvesting in the photosynthetic apparatus, photoprotection against light damage, and pigmentation in flowers and fruits to attract pollinators and seed-dispersal herbivores. Carotenogenesis has been studied extensively in the last century in both photosynthetic and non-photosynthetic tissues of many plant species. Although most of the enzymes and their metabolites of the pathway have been identified, little is still known about how carotenoid production is regulated. Previous studies have proposed that regulation of the carotenoid pathway is through metabolite feedback occurring at both transcriptional and post transcriptional levels. This thesis examines the evidence for carotenogenesis gene transcription being feedback regulated by changes in carotenoid metabolites in petals of California poppy (Eschscholzia californica), and if so, by which metabolite(s). Virus-induced gene silencing (VIGS) was used to silence carotenoid biosynthetic genes in the petals of orange California poppy. High efficacy of silencing was achieved by first infiltrating and then drenching the California poppy seedlings with the Agrobacterium tumefaciens strain GV3101 containing the VIGS vectors. The VIGS vectors included portions of carotenoid gene fragments isolated from California poppy. qRT-PCR confirmed that transcript abundance of the targeted carotenogenesis genes EcaPDS, EcaZDS, EcaLCYb, EcaCHYb and EcaZEP was significantly reduced in the flower petals. Reduced transcript abundance of all genes apart from EcaLCYb altered flower colour. HPLC analyses revealed that the colour altered flower petals with knocked-down expression of each targeted gene resulted in a reduction of total carotenoid content and an altered profile of carotenoids. This manifested as an accumulation of higher amounts of intermediates including phytofluene, ζ-carotene, β-carotene and zeaxanthin, some of which are not usually seen in the flowers, and a reduction of the end products such as retro-carotene-triol and eschscholtzxanthin. However, these alterations in carotenoid profiles were not associated with any dramatic changes in transcript abundance of the non-TRV-targeted endogenous genes in the pathway. Therefore, little evidence was found for metabolite feedback regulation of transcriptional activity in the carotenoid biosynthetic pathway from this study. Other possible mechanisms for controlling carotenogenesis are discussed.