Production of ketocarotenoids in transgenic carrot plants with an enhanced level of β-carotene

One of the drawbacks in inducing the synthesis of ketocarotenoids in transgenic plants is the accompanying decrease in beta-carotene level, especially in transgenic plants overexpressing only a beta-carotene ketolase. In an attempt to overcome this drawback, we isolated a beta-carotene ketolase (HpBkt) cDNA from Haematococcus pluvialis and generated transgenic carrot plants overexpressing HpBkt cDNA under the control of the ibAGP1 promoter and its transit peptide sequence. Semi-quantitative reverse transcriptase-PCR with total RNAs extracted from taproot tissues of five lines of the generated HpBkt-transgenic carrot plants revealed variable levels of the inserted HpBkt transcript, ranging from very low to high. All of the HpBkt-transgenic carrot plants tested produced astaxanthin, resulting in a color change from the light-orange coloration of wild-type taproots to a dark-orange-reddish coloration in the taproots of the HpBkt-transgenic plants. The astaxanthin content was not correlated with the HpBkt transcript level. Overexpression of HpBkt caused an increase in the transcript levels of the endogenous carotenogenic genes, including phytoene synthase 1 (PSY1), phytoene synthase 2 (PSY2), lycopene beta-cyclase 1 (LCYB1), and beta-carotene hydroxylase 1 (CHXB1), which resulted in elevated beta-carotene levels in the HpBkt-transgenic plants (range 1.3- to 2.5-fold) compared to the wild-type plants. These results demonstrate that HpBkt-overexpressing carrot plants under the control of the ibAGP1 promoter and its transit peptide are capable of both newly synthesizing ketocarotenoids and enhancing their beta-carotene level.