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

Abstract

Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. beta-Carotene hydroxylase (CHY-beta) is a key regulatory enzyme in the beta-beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both beta-carotene to p-cryptoxanthin and beta-cryptoxanthin to zeaxanthin. To increase the beta-carotene content of sweetpotato through the inhibition of further hydroxylation of beta-carotene, the effects of silencing CHY-beta in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-beta was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-beta 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-beta gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene p-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-beta 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 mu g/g dry weight, of which beta-carotene measured 34.43 mu g/g dry weight. In addition, IOCHY-beta-silenced calli showed elevated p-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-beta 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-beta increased beta-carotene contents and total carotenoids in transgenic plant cells and enhanced their antioxidant capacity. (C) 2011 Elsevier Ltd. All rights reserved.