Mi-Jeong Ahn

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 β-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.

Downregulation of the lycopene ϵ‐cyclase gene increases carotenoid synthesis via the β‐branch‐specific pathway and enhances salt‐stress tolerance in sweetpotato transgenic calli

Lycopene ε-cyclase (LCY-ε) is involved in the first step of the α-branch synthesis pathway of carotenoids from lycopene in plants. In this study, to enhance carotenoid synthesis via the β-branch-specific pathway [which yields β-carotene and abscisic acid (ABA)] in sweet potato, the expression of IbLCY-ε was downregulated by RNAi (RNA interference) technology. The RNAi-IbLCY-ε vector was constructed using a partial cDNA of sweet potato LCY-ε isolated from the storage root and introduced into cultured sweet potato cells by Agrobacterium-mediated transformation. Both semi-quantitative Reverse transcription polymerase chain reaction (RT-PCR) of carotenoid biosynthesis genes and high-performance liquid chromatography (HPLC) analysis of the metabolites in transgenic calli, in which the LCY- εgene was silenced, showed the activation of β-branch carotenoids and its related genes. In the transgenic calli, the β-carotene content was approximately 21-fold higher than in control calli, whereas the lutein content of the transgenic calli was reduced to levels undetectable by HPLC. Similarly, expression of the RNAi-IbLCY-ε transgene resulted in a twofold increase in ABA content compared to control calli. The transgenic calli showed significant tolerance of 200 mM NaCl. Furthermore, both the β-branch carotenoids content and the expression levels of various branch-specific genes were higher under salt stress than in control calli. These results suggest that, in sweet potato, downregulation of the ε-cyclization of lycopene increases carotenoid synthesis via the β-branch-specific pathway and may positively regulate cellular defenses against salt-mediated oxidative stress.

Enhanced accumulation of carotenoids in sweetpotato plants overexpressing IbOr-Ins gene in purple-fleshed sweetpotato cultivar

Sweetpotato [Ipomoea batatas (L.) Lam] is an important root crop that produces low molecular weight antioxidants such as carotenoids and anthocyanin. The sweetpotato orange (IbOr) protein is involved in the accumulation of carotenoids. To increase the levels of carotenoids in the storage roots of sweetpotato, we generated transgenic sweetpotato plants overexpressing IbOr-Ins under the control of the cauliflower mosaic virus (CaMV) 35S promoter in an anthocyanin-rich purple-fleshed cultivar (referred to as IbOr plants). IbOr plants exhibited increased carotenoid levels (up to 7-fold) in their storage roots compared to wild type (WT) plants, as revealed by HPLC analysis. The carotenoid contents of IbOr plants were positively correlated with IbOr transcript levels. The levels of zeaxanthin were ∼ 12 times elevated in IbOr plants, whereas β-carotene increased ∼ 1.75 times higher than those of WT. Quantitative RT-PCR analysis revealed that most carotenoid biosynthetic pathway genes were up-regulated in the IbOr plants, including PDS, ZDS, LCY-β, CHY-β, ZEP and Pftf, whereas LCY-ɛ was down-regulated. Interestingly, CCD1, CCD4 and NCED, which are related to the degradation of carotenoids, were also up-regulated in the IbOr plants. Anthocyanin contents and transcription levels of associated biosynthetic genes seemed to be altered in the IbOr plants. The yields of storage roots and aerial parts of IbOr plants and WT plants were not significantly different under field cultivation. Taken together, these results indicate that overexpression of IbOr-Ins can increase the carotenoid contents of sweetpotato storage roots.

Overexpression of the IbMYB1 gene in an orange‐fleshed sweet potato cultivar produces a dual‐pigmented transgenic sweet potato with improved antioxidant activity

The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity.

Overexpression of the sweet potato IbOr gene results in the increased accumulation of carotenoid and confers tolerance to environmental stresses in transgenic potato.

In a previous study, we have evidenced that the overexpression of the IbOr gene isolated from sweet potato conferred a tolerance activity against salinity and methyl viologen (MV) treatment in transgenic sweet potato calli along with an enhanced carotenoid content. In this study, to further examine the function of the IbOr gene in heterologous organism, we transformed the IbOr gene into potato under the direction of SWPA2 promoter, a strong inducible promoter upon treatment with various environmental stresses. Consistently with our previous study of sweet potato calli, the level of total carotenoid was elevated up to 2.7-fold (38.1 μg g(-1)DW) compared to the non-transgenic control, Atlantic cultivar. However, the composition of carotenoid was not influenced by the overexpression of the IbOr gene since only pre-existing carotenoids in the non-transgenic control including violaxanthin, lutien and β-carotene were elevated at a similar level of total carotenoids. In general, the transcript levels for most of carotenogenesis-related genes were elevated in transgenic tuber, whereas they remained at similar levels in transgenic leaf tissues compared to those of non-transgenic controls. The increased levels of carotenoid content in the leaf or tuber tissue of transgenic lines were correlated with the enhanced tolerance activity against salt- or MV-mediated oxidative stresses and DPPH radical-scavenging activity. Our preliminary results suggest that further investigation is required for the development of a crop tolerant to salinity and other environmental stresses through the overexpression of the IbOr gene.

Transgenic Alfalfa Plants Expressing the Sweetpotato Orange Gene Exhibit Enhanced Abiotic Stress Tolerance

Alfalfa (Medicago sativa L.), a perennial forage crop with high nutritional content, is widely distributed in various environments worldwide. We recently demonstrated that the sweetpotato Orange gene (IbOr) is involved in increasing carotenoid accumulation and enhancing resistance to multiple abiotic stresses. In this study, in an effort to improve the nutritional quality and environmental stress tolerance of alfalfa, we transferred the IbOr gene into alfalfa (cv. Xinjiang Daye) under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter through Agrobacterium tumefaciens-mediated transformation. Among the 11 transgenic alfalfa lines (referred to as SOR plants), three lines (SOR2, SOR3, and SOR8) selected based on their IbOr transcript levels were examined for their tolerance to methyl viologen (MV)-induced oxidative stress in a leaf disc assay. The SOR plants exhibited less damage in response to MV-mediated oxidative stress and salt stress than non-transgenic plants. The SOR plants also exhibited enhanced tolerance to drought stress, along with higher total carotenoid levels. The results suggest that SOR alfalfa plants would be useful as forage crops with improved nutritional value and increased tolerance to multiple abiotic stresses, which would enhance the development of sustainable agriculture on marginal lands.

Isolation and identification of phytochemical constituents from Scrophularia takesimensis

Phytochemical constituents were isolated from Scrophularia takesimensis (Scrophulariaceae) using repeated chromatography and prep-HPLC. Their structures were elucidated as stigmast-5-en-3-ol (1), αspinasterol 3-O-β-D-glucopyranoside (2), 5-hydroxypyrrolidin-2-one (3), trans-cinnamic acid (4), 4methoxycinnamic acid (5), 2-methoxycinnamic acid (6), and 5,7-dihydroxy-4′-methoxyflavone (7) by spectroscopic analysis. All compounds were isolated for the first time from S. takesimensis and compounds 2, 3, 6, and 7 were isolated for the first time from Scrophularia species. Among them, 5,7dihydroxy-4′-methoxyflavone (7) exhibited strong AR inhibitory activity, with an IC50 value of 4.96 μM. The content of 5,7-dihydroxy-4′-methoxyflavone (7) was measured in aerial parts of S. takesimensis, S. kakudensis and S. boreali-koreana (54.3, 25.4, and 15.0 μg/g, respectively) by HPLC.

Inner morphological and chemical differentiation of Boehmeria species

The present study was designed to establish quality control parameters for pharmacognostic evaluation and differentiation of eight locally derived Boehmeria species, B. gracilis, B. nivea, B. pannosa, B. platanifolia, B. quelpaertensis, B. spicata, B. splitgerbera, B. tricuspis, and two varieties named B. japonica var. longispica, B. nivea var. concolor, which have been utilized as the folk medicine, ‘Mo-Si-Pool’ in Korea. Although the outer morphological study of these species had been reported, there is no pharmacognostical description yet. Therefore, inner morphological evaluation on leaf midrib, petiole and stem of eight Boehmeria species and two varieties was accomplished along with preliminary phytochemical analysis by HPLC–DAD profiling. The microscopic data showed discriminative inner morphological characteristics such as collenchyma cell layer, thickness of cortex and xylem, frequency of druse and hairs, and number of vascular bundles. The HPLC profiles exhibited more than four characteristic peaks. The molecular ions of the four peaks (1–4) were tentatively identified by ESI–MS, and their structures were identified by NMR spectroscopy to be the flavonoids, rutin (1), isoquercetin (2) and kaempferol-3-O-rutinoside (3), and a phenanthroquinolizidine alkaloid, (−)-cryptopleurine (4). While compounds 1 and 2 were detected in all samples, compound 4 was determined only in B. japonica var. longispica, B. pannosa and B. quelpaertensis and B. splitgerbera. These findings provide the initial scientific criteria for proper identification and establishment of standards for use of Boehmeria species in traditional medicine.