Development of an Adonis aestivalis expressed sequence tag population as a resource for genes of the carotenoid pathway.

Expressed sequence tag (EST) libraries are a powerful tool for gene discovery in plants with genomes that are not fully sequenced. Adonis aestivalis (Ranunculaceae) is unusual among higher plants in that it accumulates large amounts of the valuable red ketocarotenoid astaxanthin in the flower petals, in addition to other carotenoids, and should therefore be a rich source of transcripts involved in carotenoid biosynthesis. To create a resource for gene discovery and increase our understanding of ketocarotenoid biosynthesis in plants, we sequenced 4189 cloned cDNAs from the floral tissues of A. aestivalis. The EST collection was annotated using sequence homology searches comparing the A. aestivalis ESTs with reference databases. ESTs involved in carotenoid biosynthesis were identified based on sequence homology to published sequences and by functional prediction using InterProScan. This study identified expressed transcripts for most of the known genes involved in the carotenoid pathway. Using an Escherichia coli functional expression system, two candidate transcripts from the A. aestivalis cDNA library were functionally characterized and identified as a novel beta-carotene 3-hydroxylase (CrtH2) and a beta-carotene monooxygenase. The expanded use of this EST collection combined with the E. coli expression system as presented here will greatly facilitate the cloning and characterization of candidate genes involved in carotenoid biosynthesis in plants.