Cloning and characterization of an Orange gene that increases carotenoid accumulation and salt stress tolerance in transgenic sweetpotato cultures.

The Orange (Or) gene is responsible for the accumulation of carotenoids in plants. We isolated the Or gene (IbOr) from storage roots of orange-fleshed sweetpotato (Ipomoea batatas L. Lam. cv. Sinhwangmi), and analyzed its function in transgenic sweetpotato calli. The IbOr gene has an open reading frame in the 942 bp cDNA, which encodes a 313-amino acid protein containing a cysteine-rich zinc finger domain. IbOr was strongly expressed in storage roots of orange-fleshed sweetpotato cultivars; it also was expressed in leaves, stems, and roots of cultivars with alternatively colored storage roots. IbOr transcription increased in response to abiotic stress, with gene expression reaching maximum at 2 h after treatment. Two different overexpression vectors of IbOr (IbOr-Wt and IbOr-Ins, which contained seven extra amino acids) were transformed into calli of white-fleshed sweetpotato [cv. Yulmi (Ym)] using Agrobacterium. The transgenic calli were easily selected because they developed a fine orange color. The expression levels of the IbOr transgene and genes involved in carotenoid biosynthesis in IbOr-Wt and IbOr-Ins transgenic calli were similar, and both transformants displayed higher expression levels than those in Ym calli. The contents of ß-carotene, lutein, and total carotenoids in IbOr-Ins transgenic lines were approximately 10, 6, and 14 times higher than those in Ym calli, respectively. The transgenic IbOr calli exhibited increased antioxidant activity and increased tolerance to salt stress. Our work shows that the IbOr gene may be useful for the biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

Down-regulation of ß-carotene hydroxylase increases ß-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato.

Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. ß-Carotene hydroxylase (CHY-ß) is a key regulatory enzyme in the beta-beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both ß-carotene to ß-cryptoxanthin and ß-cryptoxanthin to zeaxanthin. To increase the ß-carotene content of sweetpotato through the inhibition of further hydroxylation of ß-carotene, the effects of silencing CHY-ß in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-ß was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-ß construct. This construct was introduced into cultured cells of white-fleshed sweetpotato (cv. Yulmi). Reverse transcription-polymerase chain reaction analysis confirmed the successful suppression of IbCHY-ß gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene ß-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-ß gene expression changed the composition and levels of carotenoids between non-transgenic (NT) and transgenic cells. In transgenic line #7, the total carotenoid content reached a maximum of 117 µg/g dry weight, of which ß-carotene measured 34.43 µg/g dry weight. In addition, IbCHY-ß-silenced calli showed elevated ß-cryptoxanthin and zeaxanthin contents as well as high transcript level P450 gene. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) in transgenic cells was more than twice that in NT cells. RNA-IbCHY-ß calli increased abscisic acid (ABA) content, which was accompanied by enhanced tolerance to salt stress. In addition, the production of reactive oxygen species measured by 3,3'-diaminobenzidine (DAB) staining was significantly decreased in transgenic cultured cells under salt stress. Taken together, the present results indicate that down-regulation of IbCHY-ß increased ß-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity.