Characterization of Brassica napus Flavonol ... - ACS Publications

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Characterization of Brassica napus Flavonol Synthase Involved in Flavonol Biosynthesis in Brassica napus L. Tien Thanh Vu,†,∥ Chan Young Jeong,†,‡,∥ Hoai Nguyen Nguyen,†,‡ Dongho Lee,† Sang A. Lee,† Ji Hye Kim,† Suk-Whan Hong,*,§ and Hojoung Lee*,†,‡ †

Downloaded by UNIV OF NEW SOUTH WALES on September 7, 2015 | http://pubs.acs.org Publication Date (Web): August 26, 2015 | doi: 10.1021/acs.jafc.5b02994

Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-713, Republic of Korea ‡ Institute of Life Science and Natural Resources, Korea University, Seoul 136-713, Republic of Korea § Department of Molecular Biotechnology, College of Agriculture and Life Sciences, Bioenergy Research Center, Chonnam National University, Gwangju, Republic of Korea S Supporting Information *

ABSTRACT: Recently, Brassica napus has become a very important crop for plant oil production. Flavonols, an uncolored flavonoid subclass, have a high antioxidative effect and are known to have antiproliferative, antiangiogenic, and neuropharmacological properties. In B. napus, some flavonoid structural genes have been identified, such as, BnF3H-1, BnCHS, and BnC4H-1. However, no studies on FLS genes in B. napus have been conducted. Thus, in this study, we cloned and characterized the function of BnFLS gene B. napus. By overexpression of the BnFLS gene, flavonol (kaempferol and quercetin) levels were recovered in the Arabidopsis atf ls1-ko mutant. In addition, we found that the higher endogenous flavonol levels of BnFLS-ox in vitro shoots correlated with slightly higher ROS scavenging activities. Thus, our results indicate that the BnFLS gene encodes for a BnFLS enzyme that can be manipulated to specifically increase flavonol accumulation in oilseed plants and other species such as Arabidopsis. KEYWORDS: anthocyanin, flavonols, BnFLS, Brassica napus, flavonoids, oilseed

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INTRODUCTION

dipping has been reported as a simple transformation method for B. napus.5 In plants, secondary metabolites can be classified on the basis of their biosynthesis pathway into three main groups: terpenes, phenolics, and nitrogen-containing compounds.6 In phenolics, flavonoids belong to a well-known subgroup that includes a C6−C3−C6 carbon framework in the chemical structure. Until the end of the last century, more than 6000 flavonoid compounds were identified and the number continues to increase.7 These substances have various functions in vascular plants, such as attraction of pollinating agents because of pigmentation of floral organs,8 protection from UV light,9 regulation of auxin transport,10 and defense against insects, fungi, and pathogens.11 Depending on the number and position of the hydroxyl group in the chemical structure, these compounds have an antioxidative effect.12 Flavonols, an uncolored flavonoid subclass, have a high antioxidative effect and are known to have antiproliferative, antiangiogenic, and neuropharmacological properties.13 Among flavonols, kaempferol has an antiproliferative effect on cancer cells in humans, which was widely studied in the past decade.14 In A. thaliana, the flavonoid biosynthesis pathway has been well-characterized.15,16 From phenylalanine, flavonoids are biosynthesized using some common key enzymes such as

Brassica napus L. belongs to the family Brassicaceae, which contains some well-known species such as B. rapa, B. oleracea, and the model plant Arabidopsis thaliana. B. napus L. is commonly known as rapeseed, oilseed, or canola. It is an important economic crop and a primary oil source (approximately 15% of global oil production).1 Total production of oilseed has increased from 36 775 505 tons in 2003 to 72 532 995 tons in 2013 worldwide (Food and Agriculture Organization of the United Nations, 2014). Recently, oilseed has become a very important crop and is considered as the world’s second and third major crop for animal feed and humans, respectively. In addition, canola oil contributes to a great extent to various industries that deal with biofuels and cleaners. Therefore, its cultivation area stretches throughout the world, especially in Europe, Canada, and China, with 93, 80, and 75 million hectares harvested in 2013, respectively (Food and Agriculture Organization of the United Nations, 2014). To increase seed yield, pathogen resistance, or abiotic stress tolerance, genomic breeding has been conducted to select new and better cultivars. In addition, transgenic oilseed plants were generated in many studies to increase the levels of secondary metabolites, which may play significant roles in human health. For instance, several transcription factor genes related to the anthocyanin biosynthesis pathway, such as Arabidopsis MYB75 (AtPAP1),2 were overexpressed in B. napus to increase anthocyanin accumulation. Materials commonly used for transformation are hypocotyls3 or cotyledons.4 However, floral © XXXX American Chemical Society

Received: June 18, 2015 Revised: August 9, 2015 Accepted: August 12, 2015

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DOI: 10.1021/acs.jafc.5b02994 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Agrobacterium-mediated transformation of B. napus was conducted according to a previously reported protocol23 with some modifications. A single colony of Agrobacterium was inoculated into 25 mL of LB containing 25 μg/mL of rifampicin and 50 μg/mL of kanamycin and cultured at 30 °C with shaking (200 rpm) for 48 h. Agrobacterium cells were spun down and resuspended in MS liquid medium supplemented with 2% sucrose to achieve OD650 = 0.5 for generating the infection medium. Cotyledons were cut from 4-day-old seedlings grown in the dark and submerged in the infection medium for 10 min. The infected explants were then regenerated in various types of media, namely, cocultivation medium (2 days), callus induction medium (2 days), shoot initiation medium (2 weeks), shoot outgrowth medium (3 weeks), selection medium (3 weeks), and root initiation medium (3 weeks). Subsequently, the T1 transgenic plants were transferred to soil and grown in green house to collect seeds. These in vitro transgenic plants were also cultured in amplification medium for 3 weeks before further use. Detailed information on all the above-mentioned media is listed in Supporting Table 2. Measurement of Anthocyanin Levels. Anthocyanin levels were measured according to a previously described method with some modifications.24 The samples were ground to a fine powder in liquid nitrogen and extracted with 250 μL of methanol and 1% (v/v) HCl at 4 °C overnight. Then, 250 μL of distilled water and 250 μL of chloroform were added to the extracted samples, and the mixtures were vortexed and centrifuged for 5 min at 9 000g to separate the anthocyanin-containing phase. The aqueous phase was collected, and OD was measured at 535 and 650 nm. Anthocyanin levels were calculated as OD535 − OD650/fresh weight (grams). High-Performance Liquid Chromatography. Three-week old shoots on amplification medium were analyzed by high-performance liquid chromatography (HPLC) using Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA). The flavonol was analyzed on an YMCTriart C18+ column (4.6 mm × 250 mm, 5 μm) (YMC, Kyoto, Japan) using a mobile phase consisting of 100% methanol and 0.1% formic acid for elution. The flow rate was 0.6 mL/min and injection volume was 10 μL. The temperature was set at 4.5 °C for autosampler and 30 °C for column oven. The UV detector was set at 360 nm. Quercetin and kaempferol were selected as the standard samples. Antioxidant Activity Measured Using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay. Radical scavenging activity was measured using DPPH assay, as reported previously with some modifications.25 About 250 mg of the samples were ground in liquid nitrogen and extracted with 1 mL of 70% (v/v) acetone by sonication for 10 min. The samples were then separated to collect the supernatant by a centrifuge and extracted twice more. The supernatants were combined, dried by evaporation, and 1 mL of ethanol was added for resuspension and centrifuged. Then, 100 μL of the supernatant was mixed with 100 μL of DPPH solution (200 μM in ethanol) and maintained for 20 min at room temperature. Radical scavenging activity was shown as the decrease in absorbance at 517 nm because of DPPH radicals from the blank test by using 100% ethanol. 3,3′-Diaminobenzidine (DAB) Staining. In situ detection of hydrogen peroxide was performed by staining with DAB (SigmaAldrich), according to an adaptation of a previously reported method.26 Briefly, the samples were immersed in 1 mg/mL of DAB solution containing Tween 20 (0.05% v/v) and 10 mM sodium phosphate buffer (pH 7) and gently shaken for 4−6 h. The staining reaction was terminated and fixed in 3:1:1 ethanol/glycerol/acetic acid (bleaching solution) placed in a water bath at 95 °C for 15 min. The leaves were reimmersed in the bleaching solution until the chlorophyll was completely depleted and then visualized under white light and photographed. DBPA Staining. Flavonol accumulation in root tips was observed using DPBA staining, as described previously 27 with some modifications. Briefly, seedlings were stained for 5−15 min by using saturated (0.25%, w/v) DPBA with 0.02% (v/v) Triton X-100 and were then washed three times in distilled water for 5 min. The samples were visualized using a confocal laser scanning microscope (LSM 700, Carl-Zeiss).

ammonia-lyase, chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase, flavonol synthase (FLS), dihydroflavonol reductase (DFR), and leucoanthocyanidin dioxygenase.16 Flavonols, anthocyanins, and proanthocyanidins share the same pathway before separating at the point of dihydroflavonol. Then, dihydroflavonols are biosynthesized to anthocyanins or flavonols by DFR or FLS, respectively. In Arabidopsis, AtMYB11, AtMYB12, and AtMYB111 activate the expressions of CHS, CHI, F3H, and FLS1 without having a bHLH partner.17 In grapevine, VvMYBF1 can regulate VvFLS1 expression independent of the bHLH transcription factor.18 However, in maize, ZmFLS1 expression is controlled by a complex of MYB-bHLH transcription factors.19 Altogether, these studies indicated that, in some plants, flavonol biosynthesis is regulated by MYB transcription factors with or without the collaboration of the bHLH transcription factor. In B. napus, some flavonoid structural genes have been identified, such as BnDFR (GenBank DQ767950), BnF3H-1 (GenBank DQ288239), BnCHS (GenBank DQ767948), and BnC4H-1 (GenBank DQ485130). However, to the best of our knowledge, no studies on FLS genes in B. napus have been conducted. Therefore, we attempted to identify the B. napus FLS (BnFLS) gene, whose cDNA sequences were retrieved from expressed sequence tag (EST) data.20 Transgenic B. napus plants that overexpress BnFLS (BnFLS-ox) show significantly higher flavonol accumulation than wild-type plants. In this study, we examined whether the cloned putative BnFLS is a real FLS gene of B. napus. To address this question, we overexpressed the BnFLS gene in Arabidopsis atf ls1-ko mutant and found that this gene can complement the lack of the AtFLS1 gene in terms of flavonol accumulation. By overexpression of the BnFLS gene, flavonol (kaempferol and quercetin) levels were recovered in the atf ls1-ko mutant, while anthocyanin content was lower in this line (BnFLS-ox/atf ls1ko) than in Arabidopsis wild-type plants. In addition, we found that the higher endogenous flavonol levels of BnFLS-ox in vitro shoots correlated with slightly higher ROS scavenging activities. Thus, our results indicate that the BnFLS gene encodes for a BnFLS enzyme that can be manipulated to specifically increase flavonol accumulation in oilseed plants and other species such as Arabidopsis.

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MATERIALS AND METHODS

Plant Materials and Growth Conditions. In this study, B. napus L. Hanla was used for all the experiments. Seeds were sterilized and germinated on Murashige and Skoog (MS) medium supplemented with 2% (w/v) sucrose and 0.4% (w/v) Phytagel (pH 5.8), and the following growth conditions were used: 23 °C and 16-h light/8-h dark cycle. For inducible abiotic-stress treatments, 5-day-old seedlings were submerged in MS medium supplemented with the indicated concentrations of sucrose, abscisic acid (ABA), gibberellic acid (GA3), NaCl, and mannitol for 24 h before use. RNA Extraction and RT-PCR. Total RNA was extracted from 3week-old leaves of B. napus growing in amplification media, as described by Sánchez and Carbajosa.21 First-strand cDNA was synthesized using M-MLV Reverse Transcriptase, according to the manufacturer’s instructions (iNtRON Biotechnology, Inc., Republic of Korea). PCR was performed using the cDNA and specific primers (Supporting Table 1). BnFLS Cloning and Transformation. Full-length BnFLS cDNA was isolated from B. napus Hanla by using RT-PCR and cloned using the pCR8/GW/TOPO TA Cloning Kit (Invitrogen). BnFLS was then subcloned into pEarleyGate202 vector22 to generate the construct 35Spro:FLAG:BnFLS. B


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Figure 1. Wild-type Brassica napus seedlings and flavonoid accumulation. (A) 1- to 7-day-old B. napus L. Hanla seedlings (from left to right). (DAS: day after seeding). (B) Anthocyanin accumulation in various tissues (root, hypocotyls, and cotyledons) of 5-, 6-, and 7-day-old seedlings. Wild-type seedlings were grown under normal conditions for 5, 6, and 7 days and used for anthocyanin measurement. (C) Flavonol accumulation in B. napus seedlings. Wild-type seedlings were grown under normal conditions for 5, 6, and 7 days and used for flavonol detection. The experiment was repeated three times and Duncan’s tests (p < 0.05) were performed. The letters above the columns indicate significant differences. Bars reflect standard errors. Statistical Analyses. Statistical analyses were performed using Duncan’s tests at 95% confidence level.

treatments, but no difference was observed between GA3 treatment results and control conditions (Figure 2A,B). In addition, flavonol levels in the root tips increased in response to sucrose, GA3, NaCl, and mannitol treatments, but no difference was observed between ABA treatment results and control conditions (Figure 2C). These results suggest that flavonoid levels increased in response to salt and drought stress conditions in young B. napus seedlings. Since flavonoids serve as antioxidants, we wanted to check whether stress treatment resulted in increased radical scavenging activity due to enhanced accumulation of flavonoids in B. napus seedlings. To measure radical scavenging activity, the DPPH assay was performed using 5-day-old wild-type seedlings grown under stress conditions. As shown in Figure 3, radical scavenging activity was slightly increased in response to NaCl and mannitol treatments, while sucrose, ABA, and GA3 treatments showed no effects (Figure 3). On the basis of these results, we can conclude that the increase in flavonoid levels is positively correlated with higher radical scavenging activity in response to salt and drought stress conditions. Isolation of BnFLS Gene from Wild-Type B. napus Seedlings. Overexpression of AtPAP1 transcription factor has been reported to result in the enhancement of anthocyanin levels in transgenic B. napus plants.2 Thus, in this study, we aimed to clone a B. napus FLS gene to test whether we can specifically increase flavonol levels in B. napus by manipulation of BnFLS, because flavonols can be used as antioxidants for food and medical purposes. For example, flavonols, including quercetin and kaempferol, are known to have antiproliferative effects on human cancer cells.14 cDNA fragments of BnFLS

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RESULTS Flavonoid Accumulation in Wild-Type B. napus Seedlings. B. napus Hanla seeds were sown on MS medium for germination and grown for 7 days (Figure 1A). To investigate flavonoid accumulation in the oilseed plants, we first detected anthocyanin and flavonol levels in 5-, 6- and 7-day-old wild-type seedlings (Figure 1). As demonstrated in Figure 1B, the anthocyanin levels were not different between 5- and 6-dayold seedlings. Besides, while roots and cotyledons of the 5-, 6and 7-day-old seedlings showed similar anthocyanin levels, hypocotyls of the 7-day-old seedlings accumulated a lesser amount of anthocyanin than younger seedlings (Figure 1B). Flavonol accumulation in the root tips of the seedlings was observed using DPBA staining. Root-tip tissues displayed green and yellow fluorescence because of kaempferol and quercetin accumulation, respectively. Flavonol levels in the root tips of 4and 5-day-old seedlings showed no differences, while this accumulation decreased in 6-day-old seedlings and increased in 7-day-old seedlings (Figure 1C). Because anthocyanin accumulation was higher in hypocotyls of 5- and 6-day-old seedlings than in those of 7-day-old seedlings and flavonol accumulation was higher in root tips of 5-day-old seedlings than in those of 6day-old seedlings, we chose 5-day-old seedlings for stress treatments. Five-day old seedlings were exposed to several stress conditions for 24 h and then used for measuring anthocyanin and flavonol accumulation. Anthocyanin levels increased in response to sucrose, ABA, NaCl, and mannitol C


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Figure 2. Flavonoid accumulation in Brassica napus seedlings in response to stress conditions. (A) Anthocyanin accumulation in 5-day-old seedlings in response to indicated stress conditions. (B) Anthocyanin accumulation in 5-day-old seedlings in response to sucrose, ABA, GA3, NaCl, and D


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mannitol. (C) Flavonol accumulation in the root tips of 5-day-old seedlings. The 5-day-old seedlings were grown under normal conditions and used for indicated treatments; control [MS liquid medium supplemented with 20 g/L of sucrose (2%)], ABA, sucrose, GA3, NaCl, or mannitol with indicated concentrations in the figure. After 24 h, these seedlings were used for anthocyanin measurement and flavonol detection. The experiment was repeated three times and Duncan’s tests (p < 0.05) were performed. The letters above the columns indicate significant differences. The letters (“ab” or “bc”) above the columns indicate no significant difference with a, b, or c, respectively. Bars reflect standard errors.

1). The putative BnFLS sequence was translated to the amino acid sequence, and alignment results showed that BnFLS is highly homologous to BrFLS1 and AtFLS1 (Figure 4). Because the flavonol accumulation in root tips was altered in response to different treatments as shown in Figure 2C, we then examined whether BnFLS can be induced by these conditions (Figure 5). As shown in Figure 5, BnFLS was highly expressed in response to mannitol treatment. However, expression of BnFLS was slightly decreased by sucrose, GA3, and NaCl treatments. Figure 3. Radical scavenging activity of wild-type Brassica napus seedlings in response to various signals. The 5-day-old seedlings were grown under normal conditions and exposed to various signals; control [MS liquid medium supplemented with 20 g/L of sucrose (2%)], 10 μM ABA, 60 g/L sucrose, 20 μM GA3, 200 mM NaCl, and 200 mM mannitol. After 24 h, these seedlings were subjected for DPPH assay to determine the radical scavenging activity. The experiment was repeated 3 times and Duncan’s tests (p < 0.05) were performed. The letters above the columns indicate significant differences. The letters (“ab”) above the column indicates no significant difference with a or b, respectively. Bars reflect standard errors. Figure 5. BnFLS expression in wild-type Brassica napus seedlings in response to various plant growth regulators and stress conditions. The 5-day-old seedlings were exposed to 6% sucrose, 10 μM ABA, 20 μM GA3, 200 mM NaCl, and 200 mM mannitol for 24 h; mRNA was then extracted to produce cDNA for qRT-PCR. The control medium was 2% sucrose. The BnACTIN was used for internal control. The primer information was shown in Supporting Table 1.

from Expressed Sequence Tag (EST) data20 were used to design a full-length specific primer pair for this gene. Total RNA was extracted from 5-day-old wild-type seedlings and used for synthesizing a first-strand cDNA library. PCR was performed using this primer set and the cDNA library. The PCR product was then cloned into TOPO vector (Invitrogen) and used to confirm the BnFLS sequence (Supporting Figure

Figure 4. Amino acid alignment among Brassica napus, B. rapa, and Arabidopsis thaliana FLS1. BnFLS cDNA sequence was translated to amino acid sequence and then aligned with Brassica rapa FLS1 (BrFLS1) and Arabidopsis thaliana FLS1 (AtFLS1). E


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Complementation Assay to Confirm That BnFLS Encodes a Functional FLS Enzyme by Using Arabidopsis atf ls1-ko Mutant. Although we found that the BnFLS sequence was highly homologous to BrFLS1 and AtFLS1, we needed to examine whether this gene encodes a functional FLS protein. To address this, we introduced BnFLS into Arabidopsis wild-type (Col-0) and atf ls1-ko mutant plants. Using the floraldipping transformation method,28 we obtained several transgenic plants on Col-0 (BnFLS/Col-0 #1; #2) and atf ls1-ko (BnFLS/atf ls1-ko #1; #2) backgrounds. RT-PCR results confirmed BnFLS overexpression in these transgenic plants (Figure 6A). As shown in Figure 6B, flavonol accumulation was

were confirmed using several methods (Figure 7). First, the putative transgenic plants were tested using instant strips that can detect the BAR gene product (Figure 7B). Real transgenic lines exhibited two bands instead of only one band, which is indicative of the wild-type as well as nontransgenic plants (Figure 7B). The putative transgenic plants were also examined using PCR with genomic DNA and PCR primers (Figure 7C and Supporting Table 1). In addition, RT-PCR was performed to show that these transgenic plants exhibited increased expression of BnFLS (Figure 7D). The expression levels of other genes involved in the flavonoid biosynthesis were similar in the wild type and transgenic BnFLS-ox plants (Supporting Figure 2). Accumulation of Anthocyanins and Flavonols in B. napus BnFLS-ox Plants. Transgenic BnFLS-ox plants (T1) were transferred onto amplification medium for further propagation (Supporting Table 2). After 3 weeks of growth on the amplification medium, around 5 new shoots were produced from the original shoot (Figure 8A). These shoots were separated and transferred to MS medium for further experiments. In vitro shoots of Brassica wild-type and BnFLS-ox (#1, #2, #3, and #4 lines) were transferred to MS medium supplemented with sucrose (2% or 6%) for growth and measurement of anthocyanin levels (Figure 8B). As shown in Figure 8B, anthocyanin levels of the wild-type and BnFLS-ox (#1, #2, #3, and #4) shoots were not significantly different in response to a low concentration of sucrose (2%). However, a higher concentration of sucrose (6%) increased anthocyanin accumulation in the wild-type samples, while there was no change in the BnFLS-ox shoots (Figure 8B). Next, we determined flavonol levels in the in vitro shoots by using HPLC (Figure 8C). The results showed that quercetin and kaempferol accumulation was higher in the BnFLS-ox line than in the wild-type, but sucrose treatment (6%) slightly decreased flavonol levels in comparison with control conditions (2%; Figure 8C). The transgenic shoots were transferred to MS medium and allowed to produce roots without adding any hormones in order to observe flavonol accumulation by using DPBA staining. As shown in Figure 8D, flavonol content in the BnFLS-ox (#1 #2, #3, and #4 lines) increased in comparison to the wild-type. Next, we checked whether the BnFLS-ox plants have increased antioxidative ability because of enhanced flavonol levels. Leaves of the BnFLS-ox and wild-type plants growing in the amplification medium were used for DAB staining to check reactive oxygen species (ROS) levels. An intense brown stain indicated that the samples had high levels of ROS (Figure 9). ROS levels were higher in wild-type leaves than in the BnFLSox leaves, which indicated that the BnFLS-ox plants may have higher scavenging activity than the wild-type.

Figure 6. Functional complementation analysis of the BnFLS. (A) The BnFLS gene was introduced into Arabidopsis Col-0 (BnFLS/Col-0) as well as the atf ls1-ko mutant seedlings (BnFLS/atf ls1-ko) to examine whether this gene can recover the genetic defects of the atf ls1-ko mutant. Seedlings of the wild-type (Col-0), BnFLS/Col-0, BnFLS/ atf ls1-ko, and atf ls1-ko grown on 1/2× MS medium were used for RNA extraction and RT-PCR using specific primers (Supporting Table S1). ACTIN2 was used as an internal control. (+) indicates the PCR product from bacterial plasmid containing the BnFLS gene. (B) Root tips of the wild-type (Col-0), BnFLS/Col-0, BnFLS/atf ls1-ko, and atf ls1-ko grown on 1/2× MS medium were used for the detection of flavonol by using DPBA staining. BnFLC: BnFLS transforms into Col0; BnFLf: BnFLS transforms into f ls1-ko. The 6-day-old seedlings grown under normal conditions were used for flavonol detection.

observed in root tips of 6-day-old seedlings by using DBPA staining; BnFLS/Col-0 #1 showed higher flavonol accumulation than Col-0, but BnFLS/Col-0 #2 showed decreased flavonol levels. In contrast, fluorescence of atf ls1-ko was less intense, showing lower flavonol accumulation. However, the higher flavonol accumulation of BnFLS/atf ls1-ko #1 and #2 than atfls1-ko confirmed that BnFLS serves as an FLS enzyme. Generation of Transgenic Seedlings That Ectopically Express BnFLS. After confirming the flavonol synthase function of BnFLS, we decided to generate B. napus transgenic plants that overexpress the gene. To do this, the cDNA of BnFLS was cloned into pEarleyGate202,22 in which the expression of this gene was driven by the cauliflower mosaic virus 35S promoter (CaMV 35Spro; Figure 7A). Next, the 35Spro:FLAG:BnFLS construct was transformed into wild-type B. napus Hanla cotyledon samples, according to a method described by Bhalla and Singh.23 The putative transgenic plants

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DISCUSSION Anthocyanins and flavonols are biosynthesized using the flavonoid biosynthesis pathway. In this pathway, dihydroflavonols can be catalyzed to produce anthocyanins or flavonols by DFR or FLS, respectively. In a DFR-defective mutant, accumulation of flavonols is higher than that in wild-type plants,29 whereas anthocyanin content is higher in the atfls1-ko mutant than in wild-type plants.30,31 Therefore, it is important for plants to decide which enzymes need to be activated on the basis of the conditions. In this study, we cloned a newly isolated B. napus FLS and characterized transgenic plants that overexpressed this gene aiming for a high production of F


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Figure 7. Generation of the BnFLS overxepressing transgenic Brassica napus plants (BnFLS-ox). (A) Construction map of the 35Spro:FLAG:BnFLS plasmid for the generation of BnFLS-ox transgenic plants. Bar: Basta resistant gene; 35S: cauliflower mosaic virus 35S promoter; FLAG: FLAG epitope tag for target proteins; BnFLS: Brassica napus Flavonol Synthase gene. (B) Selection method of real BnFLS-ox transgenic plants; the wild-type or nontransgenic plants showed only one band when instant basta detection strips were used, while real transgenic lines exhibited one additional band indicated with red arrows. (C) Genotyping of the BnFLS-ox transgenic plants by using PCR with genomic DNA and specific primers (Supporting Table 1). (D) Three-week-old BnFLS-ox transgenic plants were grown under normal conditions and used for RT-PCR to check for BnFLS expression level in two different PCR cycles. BnACTIN was used for internal control (primer information is listed in Supporting Table 1). (+) indicates the PCR product from bacterial plasmid containing the BnFLS gene. WT: wild type.

flavonols. In particular, we intended to redirect the flavonoid biosynthesis pathway by using genetic manipulation to increase the production of specific flavonoids, such as quercetin or kaempferol, in the oilseed plants. Several genes that encode FLS enzymes have been isolated and characterized from various plant species, including A. thaliana,32 Ginkgo biloba,33 and Glycine max.34 Arabidopsis is known to contain 6 FLS genes, while several other flavonoid biosynthesis enzymes are encoded by only 1 gene.32,35 Flavonols such as kaempferol and quercetin exhibit variations in their accumulation levels and tissue specificities under different environmental circumstances, implying the involvement of various FLS genes.17,36,37 In the current study, anthocyanins accumulated more in the hypocotyls and cotyledons of the wild-type B. napus seedlings than in the roots (Figure 1B). This result was expected because it is well-established that anthocyanins have light-protection and light-filtration functions in plants. We observed that anthocyanin levels significantly decreased in 7-day-old seedlings, indicating the younger seedlings may be more active in producing anthocyanins. Young seedlings may be vulnerable to various types of biotic or abiotic agents, resulting in a high production of flavonoids to combat against these attacks like in other plant systems.38 We observed that the levels of both anthocyanins and flavonols were significantly enhanced in the wild-type in response to several stresses (Figure 2). Interestingly, ABA increased anthocyanin levels but did not affect flavonol accumulation (Figure 2). In contrast, GA3 did not alter anthocyanin levels but enhanced flavonol accumulation (Figure 2). These plant growth regulators are reported to control flavonoid biosynthesis by regulating the expression levels of certain transcription factor genes.39,40 For instance, GA3 inhibits the induction of DFR expression by sucrose but barely affects FLS expression, while ABA can increase DFR expression.41 Several studies have reported that anthocyanin

biosynthesis is promoted by UV-B light in Arabidopsis thaliana42 and B. rapa.43 In many cases, flavonoids serve antioxidants in stress tolerance. Flavonoids have been shown to function as excellent free radical scavengers because they are strongly reactive as hydrogen or electron donors.44 Previous studies proposed that the radical scavenging activity of flavonoids can be determined by the number and location of the phenolic OH groups in their structure.44 Overaccumulation of flavonols and anthocyanins was proposed to increase drought stress tolerance and antioxidant activity in A. thaliana.44 This led us to examine radical scavenging activity in B. napus plants after stress treatment. Radical scavenging activity was higher in response to salt and drought stress conditions, which appears to be correlated with the increase in flavonoid accumulation in response to various stresses (Figures 2 and 3). In B. napus, several structural genes for flavonoid biosynthesis, such as BnF3′H,45 BnIS,46 and transcription factor genes BnTT2,47 BnTT16,48 have been identified and studied, while no studies have been conducted on BnFLS. This prompted us to characterize BnFLS and generate BnFLS-ox transgenic plants to expand our understanding on the functions of FLS in B. napus. On the basis of gene expression data for B. napus,20 we found 2 cDNA fragments of BnFLS, which is a homologue of AtFLS1. Alignment results of BnFLS and AtFLS1 sequences helped us to deduce the full-length cDNA sequence of BnFLS (Supporting Figure 1). On the basis of this sequence, we designed a specific primer pair to amplify and clone BnFLS from B. napus cDNAs. The sequence of the cloned putative BnFLS was checked, and the open reading frame was confirmed to be 1011 bp (Supporting Figure 1). Then, BnFLS cDNA sequences were converted into amino acid sequences and aligned again with AtFLS1 and BrFLS1 amino acid sequences. This alignment indicated that BnFLS is highly homologous to BrFLS (98%) and AtFLS1 (91%) (Figure 4). The high homology between G


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Figure 8. Flavonoid accumulation in wild type and the BnFLS-ox transgenic plants. (A) Growth phenotype of the wild type and BnFLS-ox #2 shoots growing in the amplification medium. (B) Anthocyanin levels in the wild type and BnFLS-ox shoots in response to 2 or 6% sucrose. (C) Flavonol (quercetin and kaempferol) levels in the wild type and BnFLS-ox #2 shoots in response to 2 or 6% sucrose. The 3-week-old transgenic plants grown under normal conditions were incubated in the liquid medium containing the indicated sucrose concentrations for 24 h. Then, these shoots were H


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used for anthocyanin and flavonol detection. The experiment was repeated three times and Duncan’s tests (p < 0.05) were performed. The letters above the columns indicate significant differences. The letters (“ab” or “bc”) above the columns indicate no significant difference with a, b, or c, respectively. Bars reflect standard errors. (D) Flavonol accumulation in the root tips of in the wild type and BnFLS-ox lines. After growing for 3 weeks in the amplification medium, the roots were used for DPBA staining.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02994. Materials details and characterization (PDF)

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AUTHOR INFORMATION


Corresponding Authors

*E-mail: [email protected]. Phone: +82-2-3290-3006. Fax: +82-2-3290-3508. *E-mail: [email protected]. Phone: +82-62-530-2180. Fax: +82-62-530-2180. Author Contributions ∥

Figure 9. DAB staining of BnFLS-ox transgenic plants. Leaves of 3week old wild type and BnFLS-ox (#1, #2, #3, #4 lines) shoots grown in the amplification medium were incubated in liquid medium supplemented with DAB reagent to visualize the ROS levels. Four separate leaves were examined.

T.T.V. and C.Y.J. contributed equally.

Funding

This study was supported by a grant from the National Research Foundation of Korea (to Hojoung Lee, 2014; grant NRF-2014R1A1A3050272) and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (to H.L., 2012; grant #2012-112068-3). Notes

The authors declare no competing financial interest.

BrFLS and BnFLS may be explained by the theory of origin of B. napus.49,50 In addition, BnFLS cDNA was transformed into the Arabidopsis fls1-ko mutant and was shown to complement the flavonol-lacking phenotype of this mutant (Figure 6B). Arabidopsis atfls1-ko showed a strong reduction in flavonol content (Figure 6B) and accumulated higher levels of anthocyanins and glycosylated dihydroflavonols than the wildtype.30,31,35 However, differences in substrate preference between the enzymes of different plant species or the expression of other flavonol synthase genes may be the reasons why flavonol accumulation in BnFLS/Col-0 #2 and BnFLS/ atfls1-ko #2 root tips were lower than that in Col-0. To characterize the functions of BnFLS, we generated BnFLS-ox plants and confirmed the expression level of BnFLS in these transgenic plants by using RT-PCR (Figure 7C). In addition, we measured anthocyanin and flavonol levels in BnFLS-ox transgenic and wild-type seedlings (Figure 8). Higher expression of BnFLS in BnFLS-ox shoots was related to higher flavonol content (Figure 8C). Anthocyanin levels in 3-week-old seedlings of the wild-type were significantly increased in response to a high sucrose concentration (6%; Figure 8B). However, this high sucrose concentration could not succeed in promoting anthocyanin biosynthesis in the BnFLS-ox plants. Because DFR and FLS catalyze the same substrate, overexpression of BnFLS led to a higher accumulation of flavonols and resulted in lower anthocyanin levels in these transgenic plants (Figure 8). According to our observations, BnFLS overexpression confers increased antioxidative capability to transgenic oilseed plants, because ROS levels were decreased in the BnFLS-ox plants (Figure 9). Since high levels of flavonols as well as anthocyanins were suggested to be correlated with increased drought stress tolerance in A. thaliana,44 it would be of great interest to examine BnFLS-ox plants in the future.

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ACKNOWLEDGMENTS We would like to thank to Prof. Prem Bhalla and Dr. Dany Heang (Plant Molecular Biology and Biotechnology Laboratory, The University of Melbourne) for advising us the Canola genetic transformation technique. We would like to thank Dr. Christoph Ringli (Institute of Plant Biology, University of Zürich) for donating the f ls1-3 mutant seeds.

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ABBREVIATIONS USED DPPH, 2,2-diphenyl-1-picrylhydrazyl; ROS, reactive oxygen species; DAB, 3,3′-Diaminobenzidine REFERENCES

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