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Mol Biol Rep (2014) 41:8137–8148 DOI 10.1007/s11033-014-3714-4 Down-regulation of sweetpotato lycopene b-cyclase gene enhances tolerance to abiotic stress in transgenic calli Sun Ha Kim • Jae Cheol Jeong • Seyeon Park • Ji-Yeong Bae • Mi-Jeong Ahn • Haeng-Soon Lee Sang-Soo Kwak • Received: 18 July 2014 / Accepted: 28 August 2014 / Published online: 12 September 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Lycopene b-cyclase (LCY-b) is a key enzyme involved in the synthesis of a- and b-branch carotenoids such as a-carotene and b-carotene through the cyclization of lycopene. IbLCY-b had a length of 1,506 bp and approximately 80 % nucleotide sequence identity with that of tomato LCY-b. IbLCY-b was strongly expressed in leaves, and expression was enhanced by salt-stress and osmotic-stress conditions. To characterize the LCY-b gene (IbLCY-b) of sweetpotato (Ipomoea batatas), it was isolated and transformed into calli of white-fleshed sweetpotato using an IbLCY-b-RNAi vector. Transgenic IbLCY-bRNAi calli had yellow to orange color and higher antioxidant activity compared to that of white, nontransgenic (NT) calli. Transgenic cells had significantly higher contents of total carotenoids, although lycopene was not detected in transgenic or NT cells. All transgenic calli had strongly activated expression of carotenoid biosynthetic genes such as b-carotene hydroxylases (CHY-b), cytochrome P450 monooxygenases (P450), and carotenoid S. H. Kim J. C. Jeong S. Park H.-S. Lee (&) S.-S. Kwak (&) Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahak-ro 125, Daejeon 305-806, Korea e-mail: [email protected] S.-S. Kwak e-mail: [email protected] J.-Y. Bae M.-J. Ahn College of Pharmacy and Research Institute of Life Sciences, Gyeongsang National University, Jinjudae-ro 501, Jinju 660-751, Korea cleavage dioxigenase 1 (CCD1). Transgenic cells exhibited less salt-induced oxidative-stress damage compared to that of NT cells, and also had greater tolerance for polyethylene glycol (PEG)-mediated drought compared to that of NT cells, due to the higher water content and reduced malondialdehyde (MDA) content. The abscisic acid content was also higher in transgenic cells. These results show that a study of IbLCY-b can facilitate understanding of the carotenoid biosynthetic pathway in sweetpotato. IbLCY-b could be useful for developing transgenic sweetpotato enriched with nutritional carotenoids and with greater tolerance to abiotic stresses. Keywords Sweetpotato Carotenoid RNAi Lycopene b-cyclase Metabolic engineering Salt stress Drought stress Abbreviations ABA Abscisic acid CHY-b b-Carotene hydroxylases CRTISO Carotenoid isomerase CCD1 Carotenoid cleavage dioxigenase 1 DPPH 2, 2-Diphenyl-1-picrylhydrazyl HPLC High-performance liquid chromatography PSY Phytoene synthase PDS Phytoene desaturase LCY-b Lycopene b-cyclase LCY-e Lycopene e-cyclase MDA Malondialdehyde NCED 9-Cis-epoxycarotenoid dioxygenase NT Nontransgenic P450 Cytochrome P450 monooxygenases Ym Yulmi ZDS f-Carotene desaturase ZEP Zeaxanthin epoxidase 123 8138 Mol Biol Rep (2014) 41:8137–8148 Introduction Carotenoids are a subfamily of essential isoprenoid pigments synthesized by plants, algae, bacteria, and fungi. Carotenoids have important functions in photosynthesis and protecting plant cells against excess light energy and photooxidative damage [1–3]. Carotenoids are metabolites of phytoene, which is synthesized by phytoene synthase (PSY) from geranylgeranyl pyrophosphates (GGPP) that originate in the isoprenoid pathway [2]. Phytoene desaturase (PDS) and f-carotene desaturase (ZDS) convert phytoene to lycopene via phytofluene and f-carotene. Lycopene is the substrate for two competing lycopene cyclases, lycopene e-cyclase (LCY-e) and lycopene betacyclase (LCY-b). LCY-e introduces a single epsilon-ring into lycopene to produce d-carotene, and LCY-b introduces one or two beta-rings at either end of lycopene to produce b-carotene. Two carotenoid biosynthetic pathways proceed from lycopene, the a-branch (from a-carotene to lutein) and the b-branch (from b-carotene to neoxanthin), which play distinct and complementary roles in photoprotection mechanisms (Fig. 1). Carotenoid synthesis pathways have been targeted for transgenic studies to modify expression of key genes such as PSY, PDS, and LCY-b [2, 3]. Lycopene is a natural carotenoid that imparts red color to fruits and vegetables such as tomato (Solanum lycopersicum), rosehip (Rosa canina), watermelon (Citrullus lanatus), and pink grapefruit (Citrus paradisi) [4]. Tomato fruits are considered an important source of lycopene, representing approximately 87 % of the dietary lycopene intake. Lycopene is a powerful antioxidant and has a protective role against cancer and heart disease [5]. However, lycopene biosynthesis and regulation are still poorly understood. Efforts to engineer carotenoid biosynthesis pathways in tomato have aimed to increase the levels of b-carotene, lycopene, and xanthophylls. Lycopene levels can be elevated by up-regulation of carotenogenic genes upstream of lycopene, such as CrtB and DXS, and down-regulation of lycopene beta-cyclases [6, 7]. Tomato mutant studies have provided more insight into lycopene biosynthesis. Mutants with reduced lycopene contents (yellow-fruited tomato) have reduced expression of PSY and a deletion mutation in the carotenoid isomerase gene (CRTISO). The delta mutant has high expression levels of LYCE and high levels of b-carotene accumulation. For other described mutants with increased levels of lycopene accumulation, the molecular mechanisms are more complicated. Ronen et al. analyzed an oldgold (og) mutant and found that higher lycopene accumulation was due to a deletion mutation in LYC-B [6]. Pepper capsanthin-capsorubin synthase (CCS), which shares high identity with LCY-b at the amino acid level, also has LCY-b activity and 123 Fig. 1 The carotenoid biosynthetic pathway in plants. IPI isopentenyl diphosphate isomerase, GGPS geranylgeranyl pyrophosphate, PSY phytoene synthase, PDS phytoene desaturase, ZDS f-carotene desaturase, CRTISO carotenoid isomerase, LCY-b lycopene b-cyclase, LCY-e lycopene e-cyclase, CHY-b b-carotene hydroxylase, P450 cytochrome P450 monooxygenases, ZEP zeaxanthin epoxidase, NSY neoxanthin synthase, NCED 9-cis-epoxycarotenoid dioxygenase, CCD1 carotenoid cleavage dioxigenase, ABA abscisic acid is strongly induced during fruit coloration [8]. Two LCY-b genes (LCY-B1 and LCY-B2) have been isolated from tomato, papaya, and citrus [6, 9–11]. It was reported that the enzymes encoded by LCY-B1 and LCY-B2 could generate b-carotene from lycopene in E. coli cells [6, 9, 10]. However, the roles of LCY-B1 and LCY-B2 in the production of a-carotene are still unclear. Fruit-specific overexpression of the native lycopene b-cyclase resulted in increased b-carotene accumulation without a related decrease in total carotenoids [7]. These reports suggest that differences in LCY-b gene expression lead to different levels of carotenoid accumulation. Mol Biol Rep (2014) 41:8137–8148 Sweetpotato [Ipomoea batatas (L.) Lam] is a nutritious crop that contains high levels of antioxidants such as carotenoids, anthocyanins, and vitamin C. Its wide adaptability on marginal lands in tropical climates can be exploited to secure the food supply. The United States Department of Agriculture (USDA) reported that sweetpotato yields two to three times more carbohydrate that of field corn, and has similar potential as an alternative source of bioenergy as some varieties of sugarcane [12]. In our previous studies, the metabolic engineering of carotenoids by silencing IbCHY-b or IbLCY-e resulted in higher levels of b-carotene and total carotenoids in sweetpotato culture cells [13, 14]. Carotenoid biosynthesis in sweetpotato is still poorly understood, so this study characterized the sweetpotato carotenoid biosynthetic pathway further. The sweetpotato LCY-b (IbLCY-b) gene was isolated and characterized in transgenic calli of white-fleshed sweetpotato using an IbLCY-b-RNAi vector. The results are discussed in terms of carotenoid regulation and abiotic stress responses. 8139 Table 1 Primer sequences for genes involved in carotenoid biosynthesis in sweetpotato Target name (accession no.) Sequence Direction GGPS TAAGACGGAGAGCGTGGAGA FOR (HQ828090) TGGGATGACCTTATCACCAT REV PSY (HQ828092) AAGTTCTTCGACGAGGCTGA GCAGTTTCTTTGGCTTGCTT FOR REV PDS GTACAAAACCGTGCCAGGAT FOR (HQ828091) TCCTTTAAAATCGCCTGTGC REV ZDS ATAGCATGGAAGGAGCAACG FOR (HQ828088) AGCTCATCAGATACAGCAGCAG REV CHY-b CAAGAGAAGGACCGTTCGAG FOR (HQ828095) GACGAACATGTAGGCCATCC REV LCY-e ACCAGTTGGAGGATCATTGC FOR (HQ828096) CACCCATACCAGGACTTCGT REV LCY-b ATGGTGTGACGATTCAAGCA FOR (HQ828094) GCCAATCCATGAAAACCATC REV P450 CAGCCAGGGAAATGGTGAGT FOR (JX177357) CAGCAAGTGTGCAAAAGGAG REV Materials and methods ZEP TGGTACTTGGATCACCGACA FOR Plant materials (BJ828089) NCED GCTGCCTGCAAAACTTTCAT GGGAAGATCCCGGAGTGTAT REV FOR (BJ563195) GGACCAATCTATGCGTCTCC REV CCD1 CGGTGGAGAAACTCACGATT FOR (KF30658) TCCCGATATCTTCGGCATAG REV ARF CTTTGCCAGAAGGAGATGC FOR (JX177359.1) TCTTGTCCTGACCACCAACA REV Sweetpotato (Ipomoea batatas L. Lam. cv. Yulmi) plants were kindly provided by the Bioenergy Crop Research Center, National Institute of Crop Science, Rural Development Administration, Korea. Nonembryogenic sweetpotato calli induced from shoot apical meristems were used as experimental material, following the method reported by Kim et al. [14]. Construction of RNAi vector To clone a partial IbLCY-b cDNA from sweetpotato, primers for LCY-b were synthesized based on the sequence of IbLCYb (accession no. JX393306). A partial IbLCY-b fragment was amplified from cDNA isolated from the SR of sweetpotato (cv. Shinhwangmi) by RT-PCR using advantage 2 pfu DNA polymerase mix (Clontech, Japan). The LCY-b primer sets were as follows: 50 -TAGATATGAAGGATATTCAGG AAAG-30 and 50 -AGTAGAATATCCATACCAAAAC AGA-30 . Then, a PCR product was cloned into the pGEM-T Easy vector (Promega, USA) and sequenced. For construction of the IbLCY-b-RNAi vector, IbLCY-b primers were designed from the sequence of a partial IbLCY-b fragment with restriction enzyme sites [(EcoRI and XhoI, underlined)]: 50 -GAATTCGTTAAATTTCATCAAGCCAAGG TT-30 ; 50 -CTCGAGGACGTTGATACCTAAGTGCCTT AA-30 . PCR was performed in a 20 lL reaction containing 1 lg of cDNA template and 0.1 lM of LCY-b primers under the following conditions: an initial denaturation step at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 20 s, annealing at 64 °C for 30 s, polymerization at 72 °C for 30 s and a final extension at 72 °C for 10 min. PCR product was cloned into the pGEM-T Easy vector. The pGEM::IbLCY-b vector was digested with EcoRI and XhoI, and ligated into the pENTR11 vector, which was already digested with the same enzymes. The pENTR11-IbLCY-b clones were subjected to site-specific recombination catalyzed by the LR Clonase enzyme mix (Invitrogen, USA) into a destination vector, (i.e. plant RNAi expression vector pH7GWIWG2(I) containing the cauliflower mosaic virus 35S promoter). The resulting expression construct was used to transform E. coli DH5a strains. Transformants were selected on LB agar plates containing 50 mg/L spectinomycin/streptomycin. Transformation into sweetpotato callus Sweetpotato calli (Ym) were transformed with Agrobacterium. tumefaciens strain EHA105 harboring the IbLCY-bRNAi expression vector according to the method described by Kim et al. [14]. The EHA105 strain of A. tumefaciens was 123 8140 transformed with IbLCY-b-RNAi construct using the freeze– thaw method [15]. The transformed calli were subcultured on fresh medium at 3 week intervals for selection. RNA isolation and qRT-PCR analysis Wild-type callus (Ym, NT) and transgenic callus were ground in liquid nitrogen with a mortar and pestle, and total RNA was extracted with the Easy-Spin total RNA extraction kit (iNtRON, Korea), according to the manufacturer’s instructions. For quantitative expression analysis of genes involved in carotenoid biosynthesis, first-strand cDNA was generated from total RNA (2 lg) using MMLV reverse transcriptase (Promega, USA), according to the manufacturer’s instructions. Quantitative RT-PCR was performed in a fluorometric thermal cycler (DNA Engine Opticon 2, MJ Research, Waltham, MA, USA) using Evergreen fluorescent dye, according to the manufacturer’s instructions. The expression levels of genes related to carotenoid biosynthesis were analyzed by quantitative RT-PCR using the gene-specific primers listed in Table 1. The inter-experimental quality control comparisons of repeated samples were assessed using Ct values among the three replications. Liner data were normalized to the mean Ct of ARF as reference gene, and the relative expression ratio was using the 2D-Dct methods. Analysis of carotenoid contents Carotenoids were extracted from calli and analyzed by HPLC, according to a method previously described by Lim et al. [16] and following the method reported by Kim et al. [14]. The carotenoid content was measured by HPLC analysis with a minimum of five different calli. All contents were expressed as the mean (the average value of content per gram DW) ± SD (standard deviation) of two independent determinations. Analysis of radical-scavenging activity The DPPH radical-scavenging activity of the cells was measured as previously described [17], with some modification by Kim et al. [14]. The absorbance of the resulting solution was measured spectrophotometrically at 517 nm against a blank (MeOH). L-ascorbic acid (AsA, 0.015–0.125 mM) was used as the standard for the calibration curve, and the DPPH radical-scavenging activities were expressed as mole AsA equivalents per gram of tested samples. Mol Biol Rep (2014) 41:8137–8148 Fig. 2 Characterization and structural analysis of IbLCY-b. a Amino c acid sequences were deduced from LCY-b genes of 11 plants. b Phylogenetic analysis of IbLCY-b with other plant LCY-b. The evolutionary history was inferred using the neighbor-joining method and evolutionary analyses were conducted in MEGA 6. c Distribution of the IbLCY-b gene in various sweetpotato tissues. FR fibrous root, SR storage root. d Expression patterns of the IbLCY-b gene under various abiotic stress conditions in sweetpotato leaves. Different letters indicate statistically significant differences between the means (P \ 0.05) for time 0. Data are the average of three replicates. Statistical analyses were carried out using a one-way ANOVA with the least-significant difference post hoc test (*P \ 0.05; **P \ 0.01) contents, each callus was placed in a 1 mg/ml solution of DAB-HCl (pH 3.8) for 5 h at 25 °C under continuous light, as described previously [18]. Oxidized DABs were visualized in cells as dark-polymerization products from the reaction of DAB with H2O2. PEG-mediated drought stress To produce drought stress conditions, plates infused with PEG 6,000 (polyethylene glycol) were used according to a modified version of the method reported in Verslues et al. [19]. PEGinfused plates were prepared by dissolving solid PEG in a sterilized solution of half-strength MS medium with 6 mM MES buffer (pH 5.7), followed by overlaying of the PEG solution onto agar-solidified half-strength MS medium plates containing 7 % phytoagar. PEG water potential values were calculated from the van’t Hoff equation. The agar medium and PEG solution were equilibrated for at least 12 h before the excess PEG solution was removed. The addition of high sucrose concentrations can induce an osmotic response and thus we used half-strength MS medium without sucrose. Analysis of water content The degree of dehydration in tissues was assessed by measuring the water content (WC) of transgenic calli after PEG treatment. WC was measured using the following formula: WC (%) = (Fresh weight - Dry weight)/Fresh weight [20]. Analysis of lipid peroxidation Lipid peroxidation was measured using a modified thiobarbituric acid method [21], whereby the specific absorbance of extracts was recorded at A532. Nonspecific absorbance was measured at A600 and then subtracted from the A532 readings. The concentration of MDA was calculated as a measure of lipid peroxidation. DAB staining assay Analysis of ABA content Transgenic and nontransgenic calli were treated with MS1D liquid medium containing 150 and 200 mM NaCl for 24 h and then harvested. To measure cellular H2O2 123 The ABA content of sweetpotato callus was measured using a Phytodetek ABA enzyme immunoassay test kit Mol Biol Rep (2014) 41:8137–8148 8141 123 8142 Fig. 2 continued Mol Biol Rep (2014) 41:8137–8148 B C (Agdia, USA), as described elsewhere [22], with slight modifications. Color was detected with the Bio-Rad i-Mark Microplate Reader at a wavelength of 405 nm (Bio-Rad, USA). Three independent experiments were performed. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA). The subsequent multiple comparisons were examined based on the least-significant difference test. All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 12). Statistical significance was set at P [ 0.05 and P [ 0.01. Results IbLCY-b isolation and sequence analysis We isolated the IbLCY-b gene (accession no. JX393306) from leaves of sweetpotato (cv. Shinhwangmi). IbLCY-b had a length of 1,506 bp, which encoded 502 amino acid residues. The IbLCY-b nucleotide sequence showed 123 D 96 % homology with LCY-b of Ipomoea sp. Kenyan (accession no. BAI47577). IbLCY-b showed 81, 81, and 80 % sequence identity with LCY-b of Solanum tuberosum (accession no. XP006364433), S. lycopersicum (accession no. NP001234226.1), and Nicotiana tabacum (accession no. Q43578.1), respectively. The alignment of deduced amino acid sequence showed 98, 87, and 86 % sequence identity with LCY-b of Ipomoea sp. Kenyan, S. tuberosum chloroplastic-like LCY-b, and S. lycopersicum chloroplastic-like LCY-b, respectively (Fig. 2a). IbLCY-b has conserved motifs related to dinucleotide-binding motif in a specific protein region, which could be involved in the enzyme-substrate interaction, in membrane association, and in catalysis, such as the cyclase motif I, II, and a charged region in LCYbs. A domain named b-cyclase motif is fully conserved in all plant LCY-b; this domain is essential for lycopene b-cyclase activity [1, 23] (Fig. 2a). A phylogenetic tree was generated for IbLCY-b and the other nine plant LCY-bs (Fig. 2b). The phylogenic tree showed that IbLCY-b is more closely related to S. lycopersicum and N. tabacum LCY-b, especially the chloroplast-specific LCY-b. Mol Biol Rep (2014) 41:8137–8148 Fig. 3 Characterization of transgenic IbLCY-b-RNAi sweetpotato calli. a Construction of the CaMV 35S promoter::IbLCY-b-RNAi vector. b Phenotypes of transgenic sweetpotato calli overexpressing IbLCY-b-RNAi. c Expression of IbLCY-b in transgenic sweetpotato calli. Different letters indicate statistically significant differences between the means (P \ 0.05) for nontransgenic. d 2, 2-diphenyl-1picrylhydrazyl (DPPH) radicalscavenging activity in transgenic IbLCY-b-RNAi sweetpotato calli. Data are the means of three replicates. Statistical analyses were carried out using a one-way analysis of variance with the leastsignificant difference post hoc test (*P \ 0.05; **P \ 0.01) 8143 A B C Tissue-specific IbLCY-b expression and response to abiotic stress IbLCY-b expression was investigated in leaf, stem, fibrous root (FR), and storage root (SR) of sweetpotato (Fig. 2c). IbLCY-b was strongly expressed in leaf tissue and was weakly expressed in FRs. The expression of IbLCY-b under abiotic stresses such as salt-stress (NaCl), osmotic-stress (PEG polyethylene glycol), H2O2, and cold was investigated in leaf tissues. At 24 h after treatment (Fig. 2d), the IbLCY-b transcript level was significantly higher in response to NaCl and PEG treatments, whereas the transcript level was lower in response to cold temperature (4 °C). These results suggest that IbLCY-b might be involved in tolerance to osmotic or drought stress. Biochemical characterization of transgenic IbLCY-bRNAi sweetpotato calli The function of IbLCY-b in lycopene accumulation in sweetpotato was investigated by suppressing LCY-b. Transgenic sweetpotato calli expressing a 430 bp partial LCY-b construct fused with the RNAi (IbLCY-b-RNAi) under the control of the CaMV 35S promoter were prepared by Agrobacterium-mediated transformation (Fig. 3a). Forty-one transgenic cell lines were obtained and subsequently confirmed by genomic PCR analysis (data not shown). The colors of transgenic IbLCY-b-RNAi calli were yellow to orange, compared with white for nontransgenic D (NT) calli (Fig. 3b). Three independent transgenic lines with low IbLCY-b expression levels (Fig. 3c) were selected for further characterization. The expression of IbLCY-b transcripts was dramatically reduced in all transgenic lines, but remained high in NT lines (Fig. 3c). All transgenic lines in this study also exhibited greater 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (Fig. 3d). To confirm the observed color changes in transgenic calli expressing IbLCY-b-RNAi, the carotenoid contents in transgenic callus lines were quantitatively analyzed using high-performance liquid chromatography (HPLC) (Fig. 4). The contents of total carotenoids in transgenic IbLCY-bRNAi calli were approximately 4.3–5.0 times higher than those in NT calli. The content of each carotenoid from abranch and b-branch pathways in the transgenic lines was higher than that in NT calli. In particular, a-carotene did not accumulate in NT calli, whereas the a-carotene content in all transgenic lines was approximately 1.1–2.7 lg/g dry weight (DW). The b-carotene contents in transgenic IbLCY-b-RNAi lines were approximately 1.4–1.8 times higher than those in NT calli. The contents of 13Z-b-carotene in transgenic calli were approximately 4.3–5.1 times higher than those in NT calli, whereas the contents of 9Zb-carotene in transgenic calli were approximately 4.8–7.8 times higher than those in NT calli. The contents of cryptoxanthin, zeaxanthin, and violaxanthin also significantly increased in transgenic calli. Lycopene was not detected in transgenic and NT calli, even though IbLCY-b was down-regulated in transgenic 123 8144 Mol Biol Rep (2014) 41:8137–8148 Fig. 4 Quantitative highperformance liquid chromatography (HPLC) analysis of total carotenoid contents and carotenoid compounds in transgenic IbLCY-b-RNAi sweetpotato calli. Each carotenoid was measured by HPLC using a minimum of five different calli. All levels are expressed as the mean (the average content in gram dry weight) ± standard deviation of two independent determinations calli. To understand why transgenic IbLCY-b-RNAi calli did not accumulate lycopene, the expression levels of genes involved in carotenoid biosynthesis in transgenic and NT calli were compared (Fig. 5). The results show that transcript levels of GGPS, PSY, CHY-b and cytochrome P450 monooxygenases (P450) in transgenic lines were significantly higher than those in NT cells. However, the transcript levels of PDS, ZDS, LCY-e, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED) were lower than those in NT lines. Carotenoid cleavage dioxigenase (CCD1) had high expression levels in transgenic lines #2 and #17. diaminobenzidine (DAB) staining, which turns to a dark brown color when oxidized by H2O2 (Fig. 6a). Transgenic IbLCY-b-RNAi calli retained a yellow color, indicating that oxidized species and DAB reactants were reduced. The NT calli (Ym) developed a dark brown color, indicating the accumulation of oxidized species and peroxides. Oxidized DAB contents in transgenic calli were significantly lower than those in NT cells, reflecting the oxidation damage in NT calli (Fig. 6b). Transgenic IbLCY-b-RNAi calli have greater tolerance to salt stress To evaluate the drought tolerance of transgenic IbLCY-bRNAi calli, transgenic and NT callus lines were treated with PEG for 72 h. Visible damage was apparent in NT calli, whereas transgenic calli retained essentially the same color (Fig. 7a). After 72 h of PEG treatment, the water content in transgenic calli was significantly higher than that in NT cells (Fig. 7b). Malondialdehyde (MDA) contents To assess the effects of IbLCY-b down-regulation on tolerance to oxidative stress induced by salt, 2 week-old calli were treated with 150 or 200 mM NaCl for 24 h. Saltinduced oxidative stress in calli was visualized with 30 , 30 - 123 Transgenic IbLCY-b-RNAi calli have greater tolerance to drought stress Mol Biol Rep (2014) 41:8137–8148 8145 Fig. 5 Expression patterns of genes involved in carotenoid biosynthesis in transgenic sweetpotato calli expressing IbLCY-b-RNAi. Different letters indicate statistically significant differences between the means (P \ 0.05) for nontransgenic. GGPS geranylgeranyl pyrophosphate, PSY phytoene synthase, PDS phytoene desaturase, ZDS fcarotene desaturase, LCY-b lycopene b-cyclase, LCY-e lycopene e-cyclase, CHY-b bcarotene hydroxylase, P450 cytochrome P450 monooxygenases, ZEP zeaxanthin epoxidase, NCED 9-cis-epoxycarotenoid dioxygenase, CCD1 carotenoid cleavage dioxigenase 1. Data are the means of three replicates. Statistical analyses were carried out using a oneway analysis of variance with the least-significant difference post hoc test (*P \ 0.05; **P \ 0.01) were also significantly lower in transgenic lines compared to that for NT lines (Fig. 7c). We investigated the relationship between greater drought tolerance and the levels of abscisic acid (ABA), the drought-stress-related plant hormone. ABA levels were compared in both transgenic and NT calli, with the content in transgenic calli approximately 1.4 times higher than that in NT cells (1.98 pM/mL) (Fig. 8). Discussion As a key branch point in the carotenoid biosynthesis pathway, LCY-b introduces a ring at both ends of lycopene to form a-carotene and b-carotene in plant plastids. To characterize the sweetpotato LCY-b gene (IbLCY-b), we isolated it and transformed it into white-fleshed sweetpotato culture cells using an IbLCY-b-RNAi vector. IbLCY-b has a length of 1,506 bp and contains the conserved regions of plant LCY-bs, which was determined by comparing the amino acidic sequence of IbLCY-b with LCY-b sequences of ten other plants. The plant LCY-b conserved region, containing dinucleotide-binding domain, cyclase motifs (CM) I and II, and a charged region, is proposed to be essential for the association of LCY-b with membrane components and also for its catalytic activity [1, 8, 23, 24] (Fig. 2a). The chloroplast-specific LCY-b of S. lycopersicum and N. tabacum had three and four amino acid changes, respectively [23]. The CM I and II and the charged region were the same or with one amino acid change in S. lycopersicum and N. tabacum, respectively. These results indicate that IbLCY-b is closely correlated with the chloroplast-specific LCY-b family. 123 8146 Mol Biol Rep (2014) 41:8137–8148 A A B B Fig. 6 Transgenic sweetpotato calli expressing IbLCY-b have greater tolerance to salt-induced oxidative stress. a Visible damage of transgenic and nontransgenic Ym sweetpotato calli treated with a 30 , 30 -diaminobenzidine (DAB)-HCl solution after treatment with 150 and 200 mM NaCl for 24 h. b Analysis of oxidized DAB contents in transgenic IbLCY-b and nontransgenic Ym sweetpotato calli after treatment with 150 mM NaCl. Data are the means of three replicates. Statistical analyses were carried out using a one-way analysis of variance with the least-significant difference post hoc test (*P [ 0.05; **P [ 0.01) In our previous studies, we characterized the function of IbCHY-b and IbLCY-e in transgenic calli by down-regulating their expression using RNAi technology [13, 14]. For the current study of carotenoid biosynthesis, transgenic sweetpotato culture cells were chosen as a model system because transgenic calli can be easily prepared and compared with transgenic sweetpotato plants [13, 14]. We constructed an IbLCY-b-RNAi vector and introduced it into white-fleshed sweetpotato culture cells by Agrobacteriummediated transformation. Three independent transgenic lines with low expression levels of IbLCY-b (Fig. 3c) were selected for detailed characterization. All transgenic lines in this study displayed significantly higher levels of total and individual carotenoids than those in NT calli (Fig. 4). Lycopene did not accumulate in transgenic calli. We anticipated that lycopene would accumulate in transgenic calli due to down-regulation of IbLCY-b, even though sweetpotato does not accumulate lycopene in SRs. We suggest that sweetpotato converts lycopene to other metabolites, so lycopene levels do not become elevated. There are several possible reasons why transgenic calli do not accumulate lycopene. First, transgenic sweetpotato 123 C Fig. 7 Effect of drought (-0.5 MPa) stress on transgenic IbLCY-b RNAi calli. a Visible damage of sweetpotato calli after treatment with PEG for 72 h. b Water content in sweetpotato calli after treatment with PEG for 72 h. c Quantitative analysis of lipid peroxidation in sweetpotato calli after PEG treatment for 72 h. Malondialdehyde (MDA) is a measure of lipid peroxidation. Data represent the average of three replicates. Statistical significance of differences between the control and transgenic lines were determined by one-way analysis of variance with the least-significant difference post hoc test (*P [ 0.05; **P [ 0.01) culture cells did not have well differentiated chloroplasts. IbLCY-b had high homology with the tomato chloroplastspecific LCY-b, SlLcyb [11, 25]. In tomato, SlLcyb is preferentially expressed in green leaves, where the conversion of lycopene to b-carotene and a-carotene is usually complete [25]. The LCY genes are differentially expressed in photosynthetic and nonphotosynthetic organs; thus it is probably partially true that culture cells can lose the potential to accumulate lycopene in the cell independently from that induced by gene expression. Second, LCY-b is encoded by a single gene in several plants including Mol Biol Rep (2014) 41:8137–8148 8147 with greater abiotic stress tolerance through the metabolic engineering of carotenoid biosynthesis. Genomic information for sweetpotato is very limited compared to its agronomic importance. The biosynthetic pathway will become fully known after completing the whole-genome sequencing of sweetpotato in the near future. Transgenic sweetpotato plants expressing the IbLCY-bRNAi vector should be generated and characterized in terms of carotenoid content and abiotic stress tolerance. In summary, our results show that it should be possible to generate transgenic plants with increased tolerance to environmental stresses and enriched nutritional value by manipulating the genes involved in carotenoid biosynthesis. Fig. 8 Analysis of abscisic acid (ABA) content in transgenic IbLCYb-RNAi and nontransgenic Ym sweetpotato calli. Data represent the average of three replicates. Statistical significance of differences between the control and transgenic lines was determined by one-way analysis of variance with the least-significant difference post hoc test (*P [ 0.05; **P [ 0.01) Arabidopsis, rice, and maize. Tomato, pepper, papaya, and orange plants have organ-specific LCY-b function mediated by small gene families [1, 6, 9–11, 23, 26–31]. By contrast, tomato fruit-specific LCY-b can elevate b-carotene accumulation without a concomitant decrease in total carotenoids [7]. We can expect that sweetpotato also has multiple LCY-b genes to regulate lycopene accumulation. In spite of the observed LCY-b down-regulation, a-branch and bbranch carotenoids such as b-carotene and a-carotene, respectively, displayed high levels of accumulation in transgenic IbLCY-b-RNAi lines. Finally, CCD1 gene expression was significantly higher in transgenic lines. CCDs might be involved in oxidative cleavage of carotenoids such as lycopene, a-carotene, b-carotene, and zeaxanthin to generate apocarotenoids [32]. Bixa orellana CCD1 is related to carotenoid dioxygenases cleavage of lycopene [33]. In this study, transgenic lines with high levels of CCD1 expression can convert lycopene to cleavage metabolites mediated by CCD1. This scenario remains to be studied. Recent studies have demonstrated that increased carotenoid levels mediated by plant metabolic engineering improves the responses to abiotic stresses such as salt stress, drought stress, and oxidative stress [13, 14, 34, 35]. In the present study, DPPH radical activity and ABA content in transgenic lines was much higher than that in the NT line, indicating that the IbLCY-b-RNAi vector might contribute to abiotic stress tolerance on the whole-plant level. The antioxidant activity of carotenoids involves quenching of singlet oxygen and rapid interaction with peroxyl radicals (ROO), as opposed to that of unsaturated acyl chains [36–38]. ABA is a well-known plant hormone that responds to abiotic stress [39, 40]. This study demonstrates that it is possible to develop transgenic plants Acknowledgments This work was supported by ‘‘Cooperative Research Program for Agriculture Science & Technology Development (Project Nos. PJ009506, PJ009488 and PJ008097)’’ the Rural Development Administration, Republic of Korea, and the KRIBB Initiative Program. References 1. 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