Simple functional analysis of key genes involved in astaxanthin biosynthesis using Arabidopsis cultured cells

Abstract

Although several biotechnological researchers have reported the production of useful carotenoids in transgenic plants, the acquirement of genetically stable transgenic plants and subsequent examinations for effects of transgenes on the plants require a lot of time and efforts. Here, we show a simple and efficient approach to evaluate key genes involved in carotenoid biosynthesis using Arabidopsis thaliana suspension-cultured cell line T87. A plasmid for the expression of six key-gene candidates for astaxanthin production was constructed and introduced into A. thaliana T87 cells via Agrobacterium-mediated transformation. Five among twenty transgenic cell lines were isolated as lines resistant to a breaching herbicide norflurazon, and the expression of all the six transgenes in the lines was confirmed by quantitative transcriptional analysis. These five transgenic cell lines were shown to accumulate 4.3 to 21.9 μg g−1 fresh weight of astaxanthin, in addition to other ketocarotenoids containing adonirubin, canthaxanthin, echinenone and 3′-hydroxyechinenone. The amount of the ketocarotenoids was estimated to be 36% to 53% of the total carotenoids. The total carotenoid amount was also increased to 4.7 to 13.9 times that of control cells. We further employed microarray analysis to evaluate effects of the transgenes on endogenous gene expression in the transgenic cells. Transcriptional levels of many endogenous carotenogenic genes, e.g., genes for β-carotene hydroxylase, neoxanthin cleavage enzymes, abscisic aldehyde oxidase, 1-deoxy-D-xylulose 5-phosphate synthase, endoplasmic reticulum-targeted and mitochondria-targeted geranyl-geranyl diphosphate synthase were significantly elevated by the expression of the six genes for astaxanthin biosynthesis. These data suggest that transgenic cells adjust their whole cellular isoprenoid-metabolic system to direct the high-level production of the carotenoids including the ketocarotenoids in the plastids.