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
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Date
2012
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Massey University
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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.
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Keywords
Carotenoids, California poppy, Eschscholzia californica, Plant pigments, Gene regulation