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

• Published in: Phytochemistry (Oxford) 2012-02, Vol.74, p.69-78
• Main Authors: Kim, Sun Ha, Ahn, Young Ock, Ahn, Mi-Jeong, Lee, Haeng-Soon, Kwak, Sang-Soo
• Format: Article
• Language: English
• Subjects: Abscisic acid; Abscisic Acid - metabolism; Alkyl and Aryl Transferases - metabolism; antioxidant activity; Antioxidants; Antioxidants - metabolism; beta -Carotene; beta Carotene - metabolism; biochemical pathways; biosynthesis; Biphenyl Compounds - metabolism; Carotenoid; Carotenoids; Carotenoids - metabolism; Cells, Cultured; Cloning, Molecular; complementary DNA; Crops; cultured cells; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; DNA, Complementary; Down-Regulation; Enzymes; Food sources; foods; free radical scavengers; Gene expression; gene expression regulation; Gene Expression Regulation, Plant; Gene Silencing; genes; Genes, Plant; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Hydroxylase; Hydroxylation; Immunological tolerance; industrial crops; Intramolecular Lyases - metabolism; Ipomoea batatas; Ipomoea batatas - enzymology; Ipomoea batatas - genetics; Ipomoea batatas - metabolism; lycopene; Metabolic engineering; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Phytoene synthase; Picrates - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Roots; Plant Tubers - enzymology; Plants, Genetically Modified; reactive oxygen species; Reactive Oxygen Species - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA Interference; Roots; salt stress; salt tolerance; Salt-Tolerance - genetics; Salts; Stress; stress tolerance; Stress, Physiological; sweet potatoes; Sweetpotato; transcription (genetics); Transgenic plants; Zeaxanthin; β-Carotene; β-Carotene hydroxylase
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2011.11.003

This study reports the metabolic engineering of carotenoid biosynthesis in cultured cells of sweet potato by down-regulation of β-carotene hydroxylase. Transgenic cells exhibited increases in carotenoid content, antioxidant activity and salt stress tolerance. [Display omitted] ► Sweetpotato IbCHY-β RNAi transgenic calli increased β-carotene and total carotenoids. ► Transgenic cell lines also showed enhanced antioxidant capacity under salt stress. ► Interestingly, the transgenic calli accumulated more ABA content. ► It is suggested that enhanced levels of carotenoids contribute to stress tolerance in transgenic plants. Sweetpotato (Ipomoea batatas Lam.) is an important industrial crop and source of food that contains useful components, including antioxidants such as carotenoids. β-Carotene hydroxylase (CHY-β) is a key regulatory enzyme in the beta–beta-branch of carotenoid biosynthesis and it catalyzes hydroxylation into both β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin. To increase the β-carotene content of sweetpotato through the inhibition of further hydroxylation of β-carotene, the effects of silencing CHY-β in the carotenoid biosynthetic pathway were evaluated. A partial cDNA encoding CHY-β was cloned from the storage roots of orange-fleshed sweetpotato (cv. Shinhwangmi) to generate an RNA interference-IbCHY-β 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-β gene expression in transgenic cultured cells. The expression level of phytoene synthase and lycopene β-cyclase increased, whereas the expression of other genes showed no detectable change. Down-regulation of IbCHY-β 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 117μg/g dry weight, of which β-carotene measured 34.43μg/g dry weight. In addition, IbCHY-β-silenced calli showed elevated β-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-β 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′-diaminobe