Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco

Plant Physiology and Biochemistry 96 (2015) 388e400

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Research article

Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco Chen Meng b, 1, Song Zhang a, 1, Yong-Sheng Deng c, Guo-Dong Wang a, Fan-Ying Kong a, * a

State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Daizong Street, Tai'an, Shandong 271018, China Key Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, School of Life Sciences, Shandong University, Jinan 250100, China c Key Laboratory of Cotton Breeding and Cultivation in Huang-Huai-Hai Plain, Ministry of Agricultural, Cotton Research Centre, Shandong Academy of Agricultural Science, Jinan 250100, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 June 2015 Received in revised form 14 August 2015 Accepted 24 August 2015 Available online 29 August 2015

Flavonoids are secondary metabolites found in plants with a wide range of biological functions, such as stress protection. This study investigated the functions of a tomato (Solanum lycopersicum) flavanone 3hydroxylase-like protein gene SlF3HL by using transgenic tobacco. The expression of the gene was upregulated under chilling (4
C), heat (42
C), salt (NaCl) and oxidative (H2O2) stresses. The transgenic plants that displayed high SlF3HL mRNA and protein levels showed higher flavonoid content than the WT plants. Moreover, the expression of three flavonoid biosynthesis-related structural genes, namely, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) was also higher in the transgenic plants than in the WT plants. Under chilling stress, the transgenic plants showed not only faster seed germination, better survival and growth, but also lower malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and H2O2 and O$ 2 levels compared with WT plants. These results suggested that SlF3HL stimulated flavonoid biosynthesis in response to chilling stress. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Chilling stress Flavonoids Flavanone 3-hydroxylase-like protein Reactive oxygen species Tomato Transgenic tobacco

1. Introduction Chilling stress is a major environmental factor that limits plant growth, productivity and geographical distribution. Chillingsensitive plants, including important vegetable crops (e.g. sweet pepper, tomato and cucumber), suffer from chilling injury at

Abbreviations: 4CL, 4-coumarate-CoA ligase; ANS, anthocyanidin synthase; APX, ascorbate peroxidase; C4H, cinnamate4-hydroxylase; CHI, chalcone isomerase; CHS, chalcone synthase; DAB, 3,30 -diaminobenzidine; DFR, dihydroflavonol 4reductase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid 30 -hydroxylase; FLS, flavonol synthase; GPX, guaiacol peroxidase; H2O2, hydrogen peroxide; HPLC, high performance liquid chromatography; K, kaempferol; MDA, malondialdehyde; NAR, naringenin; NBT, nitroblue tetrazolium; O$ 2 , superoxide radical; PAL, phenylalanine ammonia lyase; PFD, photon flux density; Q, quercetin; qRT-PCR, quantitative realtime polymerase chain reaction; REC, relative electronic conductance; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SOD, superoxide dismutase; WT, wild type. * Corresponding author. State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, 61 Dai Zong Street, Tai'an, Shandong 271018, China. E-mail address: [email protected] (F.-Y. Kong). 1 Both these authors contributed equally to this work. http://dx.doi.org/10.1016/j.plaphy.2015.08.019 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.

temperatures below 10
Ce12
C. Therefore, the response and adaptation mechanisms of plants to chilling stress should be understood. Chilling stress limits enzyme activities in the Calvin cycle, which consequently reduces the utilization of absorbed light energy for CO2 assimilation and induces the overproduction of reactive oxygen species (ROS). ROS accumulation causes DNA and RNA damage, protein oxidation, membrane lipid peroxidation and, ultimately, cell death. Plants have evolved enzymatic and nonenzymatic antioxidant defence systems to scavenge ROS. The enzymatic system includes ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX). The non-enzymatic system includes several plant secondary metabolites, such as ascorbic acid (AsA), glutathione (GSH) and flavonoids, which play important roles in protecting plants from environmentally induced oxidative stress, and also pollen tube germination, seed dormancy, and auxin transport (Brown et al., 2001; Debeaujon et al., 2000; Hichri et al., 2011). Several studies have focused on the protective effects of flavonoids on plants exposed to abiotic stresses, such as low and high temperatures, high light, drought, salt, low nutrient availability and ultraviolet (UV) damage (Castellarin et al., 2007; Hern
andez et al., 2009; Park et al., 2007). Flavonoids possess antioxidant

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properties in vitro. For example, quercetin chelates transition metals, such as Fe2þ, to prevent the involvement of such metals in ROS-generating Fenton reaction. The observation that salt-tolerant species often accumulate more flavonoids than the salt-sensitive species suggests a link between flavonoids and salt stress tolerance (Liu et al., 2012). It has long been recognized that flavonoids accumulation was induced rapidly by abiotic stresses. For example, chilling stress increases flavonoid accumulation in grape, maize, red orange and apple (Christie et al., 1994; Mori et al., 2005; Piero et al., 2005; Ubi et al., 2006). In Arabidopsis, chilling stress significantly up-regulates chalcone synthase (CHS), chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H) to induce flavonoid accumulation (Zhang et al., 2010). In fact, flavonoid pathwayrelated genes are induced by stress treatment in various plants, such as potato, birch and rice; this finding suggests that these genes
et al., 2009; play important roles in plant stress tolerance (Andre Lenka et al., 2011; Liu et al., 2013; Morales et al., 2010). The overexpression of key flavonoid genes in transgenic plants induces flavonoid accumulation, which is often accompanied by increased antioxidant ability and stress tolerance (Mitsunami et al., 2014; Lukaszewicz et al., 2004; Reddy et al., 2007). By contrast, the down-regulation of flavonoid genes in mutants reduces antioxidant ability and stress tolerance (Oosten et al., 2013). Therefore, the antioxidant capacity of flavonoids could influence their protective effects against stress. Flavonoid biosynthesis is mainly controlled by structural genes in the highly conserved and well characterized phenylpropanoid pathway and regulatory genes. The structural genes encode multiple enzymes, such as phenylalanine ammonia lyase (PAL), cinnamate4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), CHS, CHI, F3H, flavonoid 3’-hydroxylase (F30 H), flavonol synthase (FLS), and dihydroflavonol 4-reductase (DFR, Fig. 1). F3H catalyzes the transition of flavanones to dihydroflavonols, which serve as intermediates for the biosynthesis of flavan 3-ols, flavonols, and

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anthocyanidins (Holton and Cornish, 1995). It is classified as a 2oxoglutarate-dependent dioxygenase (2-ODD) whose activity requires the presence of 2-oxoglutarate, ferrous iron (Fe2þ), molecular oxygen, and ascorbate. Since it was first cloned in Antirrhinum majus (Martin et al., 1991), F3H have been isolated and characterized in more than 50 plant species including Perilla frutescenes, Zea mays, Daucus carota, Ginkgo biloba, and Persea americana. In the present study, we isolated a tomato (Solanum lycopersicum) F3H-like protein gene (SlF3HL). This protein is a member of the 2-ODD superfamily characterized by a 2OG-FeII-Oxy domain. It shares approximately 67% amino acid identity with Arabidopsis downy mildew resistant 6 (DMR6), which encodes a 2-ODD superfamily member with unknown functions. The expression of DMR6 is associated with stress. DMR6 mutation enhances the expression of a subset of defence-associated genes, suggesting its roles in plant defence (Damme et al., 2008). The expression of SlF3HL is also stress associated. In particular, SlF3HL overexpression enhances the chilling tolerance of transgenic tobacco plants. 2. Materials and methods 2.1. Plant material, growth conditions and treatments Tomato seeds (S. lycopersicum cv. Zhongshu 4, provided by the Chinese Academy of Agricultural Sciences) were allowed to germinate on Murashige and Skoog (MS) medium at 25
C for 2 weeks. Sprouted seedlings were transplanted into sterilized soil with nutrient solution and grown at 25
C/20
C (day/night) with a 16/8 h photoperiod, 500 mmol m2 s1e600 mmol m2 s1 photon flux density (PFD) and 50%e60% relative humidity in a greenhouse. Two-month-old plants were used for subsequent abiotic stress assays in a growth chamber (GXZ-260C). For chilling treatment, the plants were exposed to 4
C in the illuminated incubation chamber with a PFD of approximately 200 mmol m2 s1 for 0, 3, 6, 9, 12 and 24 h. For heat treatment, the whole plants in pots were placed in the illuminated incubation chamber at 42
C for 0, 3, 6, 12 and 24 h. For oxidative treatment, the plant leaves were sprayed with 20 mM L1 H2O2. Salt stress was induced by completely immersing the plant roots in 200 mM L1 NaCl solution for 0, 3, 6, 12, 24, 48 and 72 h. For hormonal treatments, plant leaves were sprayed with 100 mM L1 abscisic acid (ABA) and 100 mM L1 jasmonic acid (JA) for 0, 1, 3, 6, 12 and 24 h. The treated plant leaves were harvested at an appropriate time, frozen in liquid nitrogen and then stored at 80
C. Tobacco seeds (Nicotiana tabacum cv. NC89) of the wild-type (WT) and transgenic plants were surface-sterilized and sown on MS medium for germination. The seedlings were transferred into fresh soil at the four-leaf stage and maintained under the same greenhouse conditions. When the sixth leaf was fully expanded (at about 2 months), the WT and transgenic tobacco plants were used for chilling stress treatment (for investigation of MDA content and REC, the plants were treated for 24 h. For investigation of other physiological parameters, the plants were treated for 12 h). The whole plants were exposed to low temperatures (4
C) in an illuminated incubation chamber (GXZ-260C) at a PFD of 200 mmol m2 s1. All the treated samples were immediately frozen in liquid nitrogen and then stored at 80
C for later use. 2.2. RNA gel blot analyses

Fig. 1. The flavonoid branch of the phenylpropanoid pathway.

Total RNA was isolated from tomato leaves using the total RNA isolation system (Tiangen, Beijing). The total RNA (20 mg) was separated in a 1.2% agarose formaldehyde gel, transferred to a nylon membrane and then fixed on the membrane by cross-linking with UV light. Pre-hybridization was performed at 65
C for 12 h. The 30

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partial cDNA of SlF3HL labeled with [a-32P]-dATP by random primed labeling (Prime-a-Gene-Labeling System, Promega) was used as the gene-specific probe. After 24 h of hybridization, filters were successively washed in 2  SSC (1  SSC comprises 0.15 M L1 NaCl and 0.015 M L1 sodium citrate, pH 7) with 0.2% SDS and in 0.2  SSC with 0.2% SDS at 42
C. Autoradiography was performed at room temperature. 2.3. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis Total RNA extraction was performed as previously described. For the reverse-transcription, 2 mg total RNA was denatured at 70
C for 5 min. Then 1 mL AMV reverse transcriptase (Fermentas) was added, mixed briefly, and incubated at 42
C for 1 h. The reaction was terminated at 70
C for 10 min. qRT-PCR was performed on a Bio-Rad CFX96TM Real-time PCR System using SYBR Real Master Mix (Tiangen, Beijing) with the following PCR thermal cycle conditions: denaturation at 95
C for 30 s, followed by 40 cycles of 95
C for 5 s, 58
C for 10 s, and 68
C for 10 s. EF-1a (GenBank accession number X144491) was used as the reference. Template-free, negative, and single primer controls were established before the examination. The results were represented by three biological replicates (each with three technical replicates) for each sample, and a standard curve method was used for statistical analysis. Primers used for qRT-PCR were: EF-1a forward 50 GGAACTTGAGAAGGAGCCTAAG-30 , and reverse 50 -CAACACCAACAGCAACAGTCT-30 ; SlF3HL forward 50 -CAGGCAGTAAGTAACGGTAAGTAC-30 , and reverse 50 - GAGATCCATCTTC TGTCAGCAG-30 ; NtPAL forward 50 -ATTGAGGTCATCCGTTCTGC-30 , and reverse 50 ACCGTGTAACGCCTTGTTTC-30 ; Nt4CL forward 50 -TCATTGACGAGGATGACGAG-30 , and reverse 50 -TGGGATGGTTGAGAAGAAGG-30 ; NtCHS forward 50 -TTGTTCGAGCTTGTCTCTGC-30 , and reverse 50 AGCCCAGGAACATCTTTGAG-30 ; NtCHI forward 50 -GTCAGGCCATTGAAAAGCTC-30 , and reverse 50 -CTAATCGTCAATGCCCCAAC-30 ; NtFLS forward 50 -GTCCACAACGTTGCATGGTG-30 , and reverse 50 CACAACTTCTCGCAGCCTC-30 ; NtDFR forward 50 -AACCAACAGTC AGGGGAATG-30 , and reverse 50 -TTGGACATCGACAGTTCCAG-30 . 2.4. Antibody preparation, total protein extraction and western blot analysis The coding region of SlF3HL was subcloned into the pET-30a (þ) vector between the BamHI and SacI sites. The expression and purification of the recombinant SlF3HL protein were processed using a NieNTA agarose system in accordance with the manufacturer's instructions (Qiagen, Hilden, Germany). The purified recombinant protein was used to immunize white mice. The obtained antiserum was purified in accordance with the antibody purification protocol of Amersham Biosciences (Piscataway, NJ). The secondary antibody was a peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). The primary and secondary antibodies were used at 1:500 and 1:5000 dilutions, respectively. Proteins were extracted from the tomato leaves with an extraction buffer (100 mM L1 Hepes, pH 7.5, 5 mM L1 ethylene diamine tetraacetic acid, 5 mM L1 ethylene glycol tetraacetic acid, 10 mM L1 dithiothreitol, 10 mM L1 Na3VO4, 10 mM L1 NaF, 1 mM L1 phenylmethanesulfonyl fluoride, 5 mg mL1 leupeptin, 5 mg mL1 aprotinin, 5% glycerol, and 50 mM L1 b-glycerophosphate). After centrifugation at 15,000 g for 30 min at 4
C, the supernatants were transferred into clean tubes, immediately frozen with liquid nitrogen, and then stored at 80
C. For western blot, 20 mg of total plant proteins separated by sodium dodecyl sulfate poly acryl amide gel electrophoresis (SDSPAGE) were electrophoretically transferred to polyvinylidene

fluoride membranes (Millipore, Billerica, MA), and then detected with antibody preparations. The protein content was determined with a dye-binding assay. 2.5. Isolation and sequencing of SlF3HL Total RNA was extracted from tomato leaves under chilling stress by using a simple Total RNA Kit (Tiangen). DNA-free RNA (2 mg) was reverse-transcribed in accordance with the protocol of the RT reagent Kit with gDNA Eraser (Takara). Putative full-length SlF3HL was amplified from the cDNA by using specific primers, which were designed based on the sequence (GenBank accession number NM001246911), SlF3HL F: 50 -CATAGATTCCATGGAAACC-30 and SlF3HL R: 50 - AACTTACTTCCGGGACAT-30 . A 1060 bp fragment was isolated, gel-purified, cloned into a pMD18-T vector (TaKaRa, Japan) and then sequenced. All primers were synthesized from Shanghai Bioasia Bioengineering Co., Ltd., Shanghai, China. Nucleotide and putative amino acid sequences were analyzed with DNAMAN version 5.2 (LynnonBiosoft, USA). 2.6. Plasmid construction and Agrobacterium-mediated transformation of tobacco plants The full-length cDNA of tomato SlF3HL was cloned into BamHI and SalI restriction sites of the binary vector pBI121, replacing the GUS gene, behind the cauliflower mosaic virus 35S (CaMV 35S) promoter. The recombinant plasmid was introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR and sequencing. Tobacco transformation was performed by co-cultivation of leaf discs for 15 min with A. tumefaciens strain LBA4404. Transformed leaf discs were selected on MS agar supplemented with 6-benzylaminopurine (3 mg L1), naphthalene acetic acid (0.2 mg L1), cefotaxime (250 mg L1), and kanamycin (100 mg L1). Regenerated shoots were rooted on MS basal medium containing kanamycin (100 mg L1). The profusely rooted plantlets were transferred into pots and maintained in greenhouse. The integration of the transgene into different transgenic lines was confirmed by qRT-PCR and western analysis. 2.7. Determination of contents of total flavonoids The total flavonoids was analyzed as described by Wang et al. (2012) with minor revision. 0.3 g fresh leaves was ground to powder in cold mortar, transferred to tubes for extraction by 3 mL of 80% (v/v) methanol for 2 h, ultrasonicated for 30 min, and then centrifuged at 9000 rpm for 10 min. For total flavonoid measurement, 1.5 mL of ethanol (95%), 0.1 mL of aluminum calorimetric (10%), 0.1 mL of potassium acetate (1 M L1), 2.8 mL of distilled water, and 0.5 mL of flavonoid extract were mixed. The reaction mixture was incubated at 25
C for 30 min, and the absorbance of the extract at 510 nm was measured to quantify the total flavonoid content. 2.8. Determination of contents of quercetin-3-glucoside, chlorogenic acids (CGA), naringenin (NAR), kaempferol (K) and quercetin (Q) Quercetin-3-glucoside, chlorogenic acid (CGA), naringenin (NAR), kaempferol (K) and quercetin (Q) were quantified by high performance liquid chromatography (HPLC) using a Shimadzu Prominence HPLC system (Shimadzu, Japan). Fresh leaves (0.3 g) were immersed in liquid N2, ground to powder, transferred to tubes for extraction with 3 mL of 80% (v/v) methanol for 2 h, ultrasonicated for 30 min, and then centrifuged at 9000 rpm for 10 min.

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The supernatant was filtered through a 0.45 mm membrane filter before HPLC analysis. For HPLC analysis, 10 mL of the filtrate was injected into a C-18 column (Shimadzu LC-20A, Japan, 150 mm  4.6 mm, 5.0 mm) which was maintained at 25
C. A linear HPLC gradient was employed with solvent A (0.1% formic acid, pH 3.6) and solvent B (methanol). The ratio of solvent A:B was 30:70, the temperature of the column was maintained at 35
C, and the flow rate was 1 mL min1. Quercetin-3-glucoside (1 mg mL1), CGA (1 mg mL1), NAR (1 mg mL1), K (1 mg mL1) and Q (1 mg mL1) (Sigma, St. Louis, USA) dissolved in methanol were used as standard samples.

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addition of 17 mM L1 p-aminobenzene sulfonic acid and 7 mM L1

a-naphthylamine. The mixture was then incubated at 25
C for 20 min and then centrifuged at 1500 g for 5 min. Finally, ethyl ether was added to the mixture. The water phase was used to determine the absorbance at 530 nm. For measurement of H2O2 concentration, leaf samples (0.5 g) were homogenized with 3 mL cold phosphate buffer (50 mM L1, pH 6.8). The homogenate was centrifuged at 6000 g for 15 min. After centrifugation, 3 mL of supernatant and 1 mL of 0.1% titanium sulfate in 20% (v/v) H2SO4 were added into a new tube, mixed, and then centrifuged again. The intensity of the yellow color of the supernatant was measured at 410 nm.

2.9. Total chlorophyll content and fresh weight measurement 2.12. Measurement of the activity of antioxidant enzymes Chlorophyll was analyzed by using UV spectrophotometry described as Kong et al. (2014). The total chlorophyll was extracted from the young seedlings incubated for 5 d at 4
C. The whole plants were homogenized in 5 mL of 95% ethanol for 3 d and the homogenate was centrifuged at 3500 g for 5 min. The supernatant was retained and the absorbance was recorded at 663 and 646 nm by UV spectrophotometry. The fresh weight of the seedlings was determined after 5 d of cultivation under the temperature stresses. 2.10. Measurement of malondialdehyde (MDA) and the relative electric conductivity (REC) MDA contents were evaluated as described by Cui and Wang (2006). Leaf tissue (0.5 g) was homogenized in 5 mL of 10% (w/v) trichloroacetic acid (TCA) with a mortar. After centrifugation at 10,000 g for 10 min, 2 mL of supernatant with 2 mL of 0.6% thiobarbituric acid (TBA, 0.6% in 10% TCA) was mixed, heated at 100
C for 15 min, and then quickly cooled and centrifuged at 5000 g for 10 min. The control contained 2 mL TCA instead of MDA extract. Absorbance was determined at 450, 532, and 600 nm. The MDA content was computed using a standard curve relating the MDA concentrations to absorbance. Electrolyte leakage was examined as described by Cao et al. (2007). Ten leaf discs (0.8 cm2 each) from each line were placed into a cuvette containing 10 mL of distilled water, vacuumed for 30 min, and then shaked for 30 min to measure the initial electric conductivity (S1). Subsequently, the cuvette with leaf discs was heated in boiling water for 30 min and cooled to room temperature to determine the final electric conductivity (S2). The distilled water was used as the blank and the electronic conductivity (S0) was measured. The relative electrical conductivity (REC) was evaluated as: REC (%) ¼ (S1S0)/(S2S0)  100%. 2.11. Analysis of the O$ 2 and H2O2 For NBT staining for O$ 2 , detached leaves were infiltrated with 10 mL NBT solution (0.5 mg mL1 NBT supplied in 10 mM L1 potassium phosphate buffer, pH 7.8) overnight in the dark. The following day, these stained samples were decolorized by boiling in acetic acid:glycerol:ethanol (1:1:4 [v/v/v]) solution. After cooling, the samples were cleared with chlorhydrate (10 mL of water added to 25 g chlorhydrate) and photographed. For NBT staining for H2O2, detached leaves were infiltrated with 10 mL DAB solution (1 mg mL1 DAB, pH 3.8) overnight in the dark. The following process was the same with NBT staining. To measure the concentration of O$ 2 , fresh leaves (0.5 g) without midrib were thoroughly ground in an ice bath with 3 mL cold phosphate buffer (50 mM L1, pH 7.8). The homogenate was centrifuged at 5000 g for 10 min at 4
C. The supernatant with phosphate buffer (pH 7.8) and 10 mM L1 hydroxylammonium chloride was incubated at 25
C for 20 min, followed by the

Leaf tissue (0.5 g) was ground in liquid Nitrogen, suspended in the 5 mL homogenization buffer containing 50 mM L1 Hepes (pH 7.0), 1 mM L1 AsA and 1% (v/v) Triton X-100. After centrifugation 13,000 g for 10 min at 4
C, supernatants were used to determine enzyme activity and protein concentration. APX activity was measured by monitoring the decrease in absorbance at 290 nm. The assay mixture (1 mL) contained 50 mM L1 Hepes (pH 7.0), 0.1 mM L1 ethylenediaminete-traacetic acid, 0.2 mM L1 H2O2, 0.5 mM L1 AsA and enzyme extract. The reaction was initiated by adding H2O2, one unit of enzyme was the amount of APX catalyzing the oxidation of 1 mM L1 AsA per minute. For measurement of SOD activity, the reaction mixture (1 mL) contained enzyme extract, 13 mM L1 L-methionine, 75 mM L1 NBT, 10 mM L1 EDTA-Na2 and 2 mM L1 riboflavin. The mixtures were reacted under low light (100 mM m2 s1) for 25 min and the absorbance was measured at 560 nm. Total CAT activity was assayed by measuring the rate of decomposition of H2O2 at 240 nm. GPX activity was determined in a reaction mixture (3 mL) containing 10 mM L1 potassium phosphate, pH 7.0, 10 mM L1 H2O2 and 20 mM L1 guaiacol and 0.01 mL enzyme extract. The reaction was started by adding H2O2 and guaiacol. The activity was determined by monitoring the increase in absorbance at 470 nm, as a result of guaiacol oxidation. The amount of enzyme producing 1 mM min1 of oxidized guaiacol was defined as 1U. 2.13. Statistical analysis Data points represent the mean ± standard deviation (SD) of three replications. Statistical significance of differences in the measured parameters between the wild type (WT) and transgenic plants was tested by Students t-text in Excel (select tools/data analysis/t-test: two sample assuming unequal variances). Significant differences in comparison with the control are indicated by *P < 0.05 and **P < 0.01. 3. Results 3.1. Characterization of the tomato cDNA A full-length cDNA related to chilling stress was isolated from tomato. This cDNA contains a 1014 bp open reading frame that encodes a 338-amino-acid protein with a calculated molecular weight of approximately 38.5 kDa and an isoelectric point of 5.57. Genomic DNA analysis showed that this gene contained four exons and three introns (Fig. 2A). NCBI protein blast revealed that the isolated cDNA belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODDs) superfamily which contains four enzymes of the flavonoid biosynthetic pathway: flavanone 3-hydroxylase (F3H), flavone synthase I (FNSI), anthocyanidin synthase (ANS) and flavonol synthase (FLS; Wellmann et al., 2004). A phylogenic tree of

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Fig. 2. Analyses of the SlF3HL sequence and phylogenetic tree of known 2-ODDs. (A) Sequence organization of SlF3HL (positions 1e4998 bp). UTRs, exons and introns are indicated in gray, black and white, respectively. (B) Phylogenetic tree of some known 2-ODDs (F3Hs, FNSIs, ANSs and FLSs). GbF3H for Ginkgo biloba (AAU93347); CsF3H for Citrus sinensis (BAA36553.1); GhF3H Gossypium hirsutum (ABM64799.1); LnF3H Ipomoea nil (BAA21897.1); PhF3H Petunia hybrid (AAC49929.1); VvF3H Vitis vinifera (P41090.1); AtF3H Arabidopsis thaliana (Q9S818); GmF3H Glycine max (AAT94365.1); MtF3H Medicago truncatula (FJ529406.1); MsF3H Medicago sativa (CAA55628.1); MdF3H Malus domestica (AAX89397.1); HvF3H Hordeum vulgare (CAA41146.1); ZmF3H Zea mays (AAA91227.1); AaFNSI Angelica archangelica (ABG78793.1); AcFNSI Aethusa cynapium (ABG78791.1); CmFNSI Conium maculatum (ABG78795.1); OsANS Oryza sativa (CAA69252.1); AtANS Arabidopsis thaliana (Q96323); OsFLS Oryza sativa (XP_467968.1); and AtFLS Arabidopsis thaliana (Q96330).

F3H, FNSI, ANS and FLS from various species was built using DNAman. The tree showed that the isolated cDNA showed more similarities to F3H proteins than to FNSI, ANS or FLS proteins (Fig. 2B). A sequence alignment analysis of F3Hs from different sources was carried out (Fig. 3). A high similarity among F3H proteins was observed from residues 25 to 350, with varying lengths and compositions in the N-terminal and C-terminal regions. The isolated polypeptide showed 71% similarity to other F3Hs. The 2OG-FeIIOxy domain, which is present in other F3H proteins, was also found in this polypeptide. Five similar motifs for F3Hs were labeled by “d”. Several amino acid residues (labeled by *) were strictly conserved. These amino acid residues include Pro142, Pro145 and Pro198 in motifs 2 and 3 for polypeptide folding; His70 in motif 1, His212 and Asp214 in motif 4 and His269 in motif 5 for Fe2þ ligation; and Arg279 and Ser281 in motif 5 for 2-oxoglutarate binding. Nevertheless, the predicted polypeptide SlF3HL differed from the typical F3H. For example, it lacked the provisional F3H N-terminal domain and several amino acid residues conserved in F3Hs were replaced in the predicted polypeptide. Moreover, it shared approximately 67% amino acid identity to Arabidopsis DMR6, which also contained the Fe (II)-dependent 2OG oxygenase domain but lacked the N-terminal domain. Thus the isolated polypeptide was annotated as a tomato (S. lycopersicum) flavonone 3hydroxylase-like protein (SlF3HL).

3.2. SlF3HL expression is stress associated Considering that SlF3HL was isolated from a tomato cDNA library that represents the expression patterns of genes affected by chilling, we first studied the expression of SlF3HL in response to chilling through RNA gel blot, qRT-PCR and western blot (Fig. 4). The transcript level of SlF3HL was obviously induced by chilling treatment. It reached the maximum level at 12 h, and slightly decreased after 12 h (Fig. 4A and B). As shown in Fig. 4C, protein signals were found at about 38 kDa and also gradually increased in strength after chilling treatment. The response of SlF3HL to chilling indicates that SlF3HL may play important roles at low temperatures once it was induced. The expression level of SlF3HL under heat (42
C), oxidative (20 mM L1 H2O2) and salt (200 mM L1 NaCl) stresses was detected through qRT-PCR analysis at different time points (Fig. 5A to C). SlF3HL expression initially increased and then decreased under all of the tested stresses. Under heat stress, the transcript level of SlF3HL increased by approximately 2.2-fold after 3 h of treatment and then gradually decreased thereafter (Fig. 5A). The expression of SlF3HL increased during the first 9 h and then decreased under 20 mM L1 H2O2 treatment (Fig. 5B). Under salt stress, the transcript level of SlF3HL increased, peaked at 6 h and then decreased (Fig. 5C). These results suggested that SlF3HL was involved in the response to various stresses.

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Fig. 3. Sequence alignment of the deduced SlF3HL protein with other plant F3Hs and AtDMR6. The positions of the five conserved motifs are labeled with bold lines. ‘*’ indicates the conserved amino acid residues in SlF3HL and other F3Hs for ligating Fe2þ and participating in 2-oxoglutarate binding; ‘o’ indicates the replaced amino acid residues in SlF3HL that were conserved in F3Hs.

ABA and JA are plant phytohormones that regulate plant responses to environmental stresses. As shown in Fig. 5D and E, SlF3HL exhibited similar responses to ABA and JA. Both ABA and JA induced the gene but to different extent: ABA increased the transcript level, peaking at 3 h, by approximately 5.6-fold; JA peaking at 12 h, by approximately 2.1-fold. So the effect of ABA was stronger than that of JA. We also examined the expression patterns of SlF3HL in various organs. As shown in Fig. 5F, SlF3HL was constitutively expressed in the examined organs. However, the transcript level of SlF3HL was approximately 8.5-fold higher in the leaves than in the roots.

mRNA levels were approximately 39, 39, 48, 88, 183, 24, 35, 40 and 134 times higher in the transgenic lines S1, S3, S5, S6, S7, S8, S13, S19 and S26 than in the WT, respectively (Fig. 6A). Among these lines, S6, S7, and S26 were selected for western blot analysis with an antibody against SlF3HL. The protein level of SlF3HL showed similar changes to its mRNA level (Fig. 6B). Thus, S6, S7 and S26 were selected for the subsequent physiological measurements. Two-month-old transgenic and WT plants were used. The transcript level of SlF3HL was measured in three biological replicates (each with three technical replicates) and was normalized to EF-1a expression. Error bars represent the standard deviation. For (B), part of the Coomassie-stained total protein SDS-PAGE gel was selected as the loading control (LC).

3.3. Identification of transgenic plants Transgenic plants infected with A. tumefaciens LBA4404 carrying the SlF3HL gene (driven with 35S promoter) were detected by PCR after the first screening with 50 mg mL1 kanamycin (data not shown). A total of 29 individual kanamycin-resistant transgenic lines (T0) were obtained from tissue culture. The progeny obtained from T0 were named T1. Nine T1 lines (S1, S3, S5, S6, S7, S8, S13, S19 and S26) were selected for qRT-PCR analysis The relative SlF3HL

3.4. SlF3HL overexpression promoted flavonoid accumulation As shown in Fig. 7A, chilling stress caused flavonoid accumulation in both transgenic and WT plants. The transgenic plants accumulated more flavonoid than WT under normal condition (25
C) and after 12 h chilling treatment. In particular, the total flavonoid contents were 20.8%, 34.2% and 30.9% higher in

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transgenic plants S6, S7 and S26 than in WT under normal condition. Meanwhile, these contents were 31.4%, 56.8% and 46.2% higher in S6, S7 and S26 than in WT after 12 h chilling treatment. According to HPLC analysis, there was also increased accumulation of quercetin-3-glucoside in leaves of transgenic lines, relative to WT (Fig. 7B). Chilling stress also increased accumulation of a phenylpropanoid compound, chlorogenic acid (CGA), whereas no significant differences between the transgenic and WT plants were detected (Fig. 7C). Moreover, both the substrate and products of F3H, NAR, K and Q were higher in the transgenic plants than in the WT plants (Fig. 7D to F). To investigate the mechanisms underlying flavonoid accumulation in the transgenic plants, the expression profiles of some flavonoid synthesis-related genes were determined by qRT-PCR (Fig. 8). All of the genes, except for NtCHS in WT, were induced express by chilling stress. NtCHS, NtCHI, and NtFLS (labeled by red (in the web version) in Fig. 1) showed relatively higher expression levels in the transgenic plants than in WT, whereas NtPAL, Nt4CL and NtDFR showed little difference between the transgenic plants and WT (Fig. 8).

Fig. 4. Responses of SlF3HL to chilling stress in tomato. (A) RNA gel blot analysis of SlF3HL expression in leaves treated at 4
C. rRNA, ethidium bromide-stained rRNA, shown as a loading control. (B) qRT-PCR analysis of SlF3HL response to chilling stress. The transcript level of SlF3HL was measured in three biological replicates (each with three technical replicates) and was normalized to EF-1a expression. Error bars represent the standard deviation. (C) Western blot analysis of the SlF3HL protein levels in the leaves treated at 4
C. LC, loading control, part of the Coomassie-stained total protein SDSePAGE gel.

3.5. SlF3HL overexpression enhanced seed germination under chilling stress The effects of chilling stress on seed germination in the WT and transgenic plants are shown in Fig. 9. Germination percentage was scored every 2 d from 2 d to 10 d after sowing at 25
C. Most seeds both from WT and transgenic plants germinated 10 days after sowing at 25
C (Fig. 9A to C). When the seeds were treated at 4
C

Fig. 5. qRT-PCR analysis of the expression levels of SlF3HL under heat (A), 20 mM L1 H2O2 (B), 200 mM L1 NaCl (C) stresses, 100 mM L1 ABA (D), 100 mM L1 JA (E), and in different organs (F) in tomato. Two-month-old tomato plants were used. The transcript level of SlF3HL was measured in three biological replicates (each with three technical replicates) and was normalized to EF-1a expression. Error bars represent the standard deviation.

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395

to 25
C, the germination percentage of the WT seeds was approximately 51%, whereas those of S6, S7 and S26 were approximately 73%, 85% and 78%, respectively. The germination of the WT and transgenic seeds treated at 4
C was delayed by 4 d and 2 d as compared with that of the seeds treated at 25
C, respectively. These results indicate that the transgenic seeds germinated better than the WT seeds under chilling stress. 3.6. SlF3HL overexpression enhanced chilling stress tolerance

Fig. 6. Identification of transgenic plants by qRT-PCR (A) and western blot (B).

for 7 d, and then transferred to 25
C, the germination percentage decreased in both WT and transgenic plants. However, the decrease in germination percentage was more prominent in the WT seeds than in the transgenic seeds (Fig. 9D to F). At 10 d after the transfer

To determine whether or not SlF3HL increases chilling tolerance in transgenic tobacco, we observed the growth performance of young seedlings (16-day-old) at 25
C or 4
C for 7 d (Fig. 10A and B). Without chilling treatment, the young seedlings of both WT and transgenic plants grew well and exhibited no significant differences. However, the growth of all seedlings was suppressed after 4
C treatment for 7 d. The transgenic seedlings showed better survival and growth than the WT seedlings. To illustrate, the cotyledons of the transgenic plants remained green, whereas those of the WT plants turned yellow. Consequently, the chlorophyll content in all seedlings decreased at 4
C. Nevertheless, the transgenic plants still showed higher chlorophyll content (approximately 57.8% that of the untreated WT plants on average) than the WT plants (approximately 39.1%, Fig. 10C). The fresh weight of the WT plants was 0.0325 ± 0.00642 g, which was lighter than those of S6 (0.0450 ± 0.00244 g), S7 (0.0524 ± 0.00725 g) and S26

Fig. 7. Variations in total flavonoids (A), quercetin-3-glucoside (B), CGA (C), NAR (D), K (E) and Q (F) content in WT and transgenic plants under normal and chilling condition. Error bars represent the SDs of triplicate biological replicates. * and ** indicate significant differences in comparison with the control at P < 0.05 and P < 0.01, respectively.

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Fig. 8. qRT-PCR analysis of the expression of NtPAL (A), Nt4CL (B), NtCHS (C), NtCHI (D), NtFLS (E), and NtDFR (F) in the investigated plants grown under normal condition and chilling stress. The transcript levels were measured in three biological replicates (each with three technical replicates) and was normalized to EF-1a expression. Error bars represent the standard deviation.

(0.0486 ± 0.00642 g, Fig. 10D). These results suggested that young seedlings with SlF3HL overexpression were more tolerant to chilling treatment than those without. The growth performance of the WT and transgenic plants (2month-old) at 25
C and 4
C was also observed (Fig. 11A). Although no significant difference in phenotype was observed between the WT and transgenic plants, MDA accumulation and REC, which indicate membrane damage and chilling injury, increased more obviously in the WT plants than in the transgenic plants. This result suggested that SlF3HL overexpression alleviated membrane damage under chilling stress (Fig. 11 B and C). 3.7. Overexpression of SlF3HL alleviated ROS accumulation under chilling stress The contents of H2O2 and O$ 2 in the leaves of WT and transgenic plants were analyzed by diaminobenzidine (DAB) and nitroblue terazolium (NBT) staining, respectively. As shown in Fig. 12A, slight DAB staining was detected both in WT and transgenic plants prior to treatment. After chilling treatment for 12 h, dark-brown DAB polymer products were detected in all plants, but the staining intensity was significantly lower in the transgenic plants than in the WT plants. Similarly, NBT staining showed that the blue NBT polymer products due to O$ 2 accumulation also increased in all plants after chilling treatment. However, this increase was more obvious in the WT plants than in the transgenic plants (Fig. 12B). The contents of H2O2 and O$ 2 were also examined, and the results

of this examination (Fig. 12C and D). These results indicate that SlF3HL overexpression alleviates the accumulation of H2O2 and O$ 2 under chilling stress in transgenic tobacco plants. No significant differences in APX, SOD, CAT and GPX activities between the WT and transgenic plants were observed before chilling treatment (Fig. 13). When the plants were treated at 4
C for 12 h, APX and CAT activities increased, whereas GPX activity restored to its original level. No significant differences in APX, SOD, CAT and GPX activities were observed between the WT and transgenic plants after chilling treatment. These results suggest that SlF3HL overexpression did not influence the activities of APX, SOD, CAT and GPX in the transgenic tobacco plants. Plants were treated at 4
C for 0 h and 12 h before measurement. Data of three independent experiments are represented as mean ± SD. 4. Discussion Flavonoid accumulate rapidly and exert protective effects on plants under chilling stress. The flavonoid biosynthetic pathway has been well characterized, and flavonoid biosynthesis-related genes, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3hydroxylase (F3H), have been cloned from various plants. However, only a few of these genes have been characterized for their catalytic activity in vivo. In this study, we identified a tomato 2ODD protein, SlF3HL, and investigated its roles in flavonoid

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Fig. 9. Germination of WT and transgenic seeds under normal condition and chilling stress. Germination of WT and transgenic seeds at 25
C for 7 d (A), 14 d (B) and germination percentage every 2 d from 2 d to 10 d (C). Germination of WT and transgenic seeds treated at 4
C for 7 d, then transferred to 25
C for 7 d (D), 14 d (E) and germination percentage every 2 d from R2 to R10 days (F). For (C) and (F), each column represents an average of three replicates, whereas bars indicate SDs. * and ** indicate significant differences in comparison with the control at P < 0.05 and P < 0.01, respectively.

accumulation and plant chilling tolerance. SlF3HL stimulates flavonoid biosynthesis under chilling stress although no catalytic function was confirmed in the current manuscript. It has long been recognized that chilling stress induces flavonoid accumulation. However, the regulatory mechanisms underlying this phenomenon remain elusive. The results of the present study showed that both chilling stress and SlF3HL overexpression induced flavonoid accumulation in tobacco (Fig. 7A and B). This result implies that SlF3HL plays a role in flavonoid biosynthesis or in the regulation of this biosynthetic pathway in response to chilling stress. By qRT-PCR, a possible reason for the higher flavonoid accumulation in the transgenic plants than the WT plants was found to due to the higher expression levels of three structural genes: CHS, CHI, and flavonol synthase (FLS) in the former than in the latter (Fig. 8). In fact, F3H gene was found to be coordinately expressed with CHS and CHI in Arabidopsis seedlings, whereas dihydroflavonol reductase (DHR) expression is controlled by distinct regulatory mechanisms (Pelletier and Shirley, 1996). As a key enzyme in the flavonoid biosynthetic pathway, F3H catalyzes the transition of flavanones to dihydroflavonols (Fig. 1; Holton and Cornish, 1995). In are mutant that defect in F3H in tomato: the

downstream flavonoids, kaempferol (K) and quercetin (Q), are lower in the mutant, whereas the substrate, naringenin (NAR), was higher (Maloney et al., 2014). However, in the transgenic plants both the F3H products (K and Q) and the substrate (NAR) were more accumulated (Fig. 7D to F). The reason for this is possible that SlF3HL has been shown to activate the flavonoid pathway: in addition to NtFLS, the expression of NtCHS and NtCHI, which are in the upstream of narigenin, was also upregulated by SlF3HL overexpression (Fig. 8). But still further research is needed to confirm this assertion. SlF3HL overexpression enhanced the chilling stress tolerance of transgenic tobacco, as indicated by the following lines of evidence. Firstly, SlF3HL expression was induced by all the tested stresses, including chilling, as a protective response of the plants against the stresses (Figs. 4 and 5). Actually, many flavonoid pathway-related genes, such as PAL, F3H, flavonoid 30 -hydroxylase (F30 H), dihydroflavonol 4-reductase (DFR), and anthocyanin synthase (ANS), have been found to response to various stresses, such as chilling, heat, salt, drought and UV-B irradiation, suggesting that they were involved in response to various stresses (Castellarin et al., 2007; Desikan et al., 2001; Han et al., 2010; Park et al., 2007). Secondly,

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Fig. 10. Growth performance of the young seedlings under 25
C (A), 4
C (B), chlorophyll content (C) and fresh weight (D). For (C) and (D), each column represents an average of three replicates, whereas bars indicate SDs. * indicate significant differences in comparison with the control at P < 0.05.

the transgenic plants showed faster seed germination, and better survival and growth after chilling treatment (Fig. 9 to 11). In fact, previous studies have already established that manipulating flavonoid metabolism in plants under stress has unpredicted effects. For instance, the overexpression of OsANS in rice increases

antioxidant activity (Reddy et al., 2007). Overexpression of CsF3H improved salt tolerance of transgenic tobacco plants (Mahajan and Yadev, 2014). In addition, the overexpression of key flavonoid genes in potato increases antioxidant ability in transgenic plants

Fig. 11. Growth performance (A), MDA content (B) and REC (C) of the grown plants (about 2-month-old). For (A), the top panel represents plants grown under normal condition. Bottom panel represents plants treated at 4
C for 24 h. For (B) and (C), plants were treated at 4
C for 24 h before measurement. Each column represents an average of three replicates, whereas bars indicate SDs (*, P < 0.05; **, P < 0.01).

Fig. 12. H2O2 and O$ 2 analysis in the WT and transgenic plants. (A) DAB staining for
H2O2. (B) NBT staining for O$ 2 . Top panel represents seedlings grown at 25 C. Bottom panel represents seedlings treated at 4
C for 12 h. (C) H2O2 contents. (D) O$ 2 contents. The experiment was repeated three times with similar results. The data are presented as the mean ± SD of three biological replicates (*, P < 0.05; **, P < 0.01).

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Fig. 13. APX (A), SOD (B), CAT (C) and GPX (D) activity in the investigated plants.

(Lukaszewicz et al., 2004). Transgenic plants that overexpress PAP1, which encodes an MYB transcription factor, exhibit strong resistance to Spodoptera litura (Mitsunami et al., 2014). Additional line of evidence that supports the roles of SlF3HL in chilling stress tolerance includes the less accumulation of ROS in the transgenic plants than in the WT plants. Chilling stress leads to metabolism disturbance and causes oxidative damage to proteins, lipids, and DNA by increasing the accumulation of reactive oxygen species (ROS; Desikan et al., 2001; Foyer and Noctor, 2005; Mittler et al., 2004). In the present study, the transgenic plants maintained lower H2O2 and O$ 2 levels than the WT plants, suggesting that SlF3HL can help reduce ROS accumulation (Fig. 12). In plants, stressinduced ROS accumulation induces lipid peroxidation, which leads to electrolyte leakage and finally membrane damage (Madhava-Rao and Sresty, 2000). Therefore, the reduction in H2O2 and O$ 2 levels in the transgenic tobacco plants can alleviate the damage induced by membrane lipid peroxidation. Our results confirmed this viewpoint. That is, the transgenic plants showed less accumulation of MDA (indicator of lipid peroxidation extent) and lower REC than the WT plants (Fig. 11B and C). This result implies that membrane damage was less serious in the transgenic plants than in the WT plants and further highlights the important roles of SlF3HL in chilling tolerance. Plants have evolved enzymatic and non-enzymatic antioxidant defense systems to regulate ROS accumulation. The enzymatic system, which comprises ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) scavenges ROS to protect plant cells. The non-enzymatic system, which includes several plant secondary metabolites such as ascorbic acid (AsA), glutathione (GSH) and flavonoids, also contributes to the cellular redox balance (Mittler et al., 2004). From our results, no significant differences in APX, SOD, CAT and GPX activities were detected between the transgenic and WT plants (Fig. 13). Therefore, the lower H2O2 and O$ 2 levels in the transgenic plants than in the WT plants were not due to the enzymatic antioxidant defense systems. As one of the most important antioxidants, flavonoids can modulate ROS accumulation and protect plants from oxidative damage because of their strong capacity to donate elec
ndez et al., 2008; trons or hydrogen atoms (Bais et al., 2006; Herna Pourcel et al., 2007; Taylor and Grotewold, 2005). Thus, the possible

reason for the lower ROS accumulation in the transgenic plants was the higher flavonoid accumulation in these plants. But this still need further study to exclude other antioxidants. In conclusion, the present results suggested that overexpression of SlF3HL increased flavonoid accumulation and reduced ROS accumulation, which protected the biological membrane system, thereby increased the chilling stress tolerance of transgenic tobacco. Author contributions C Meng and FY Kong conceived and designed the experiments; C Meng and S Zhang performed the experiments; YS Deng and GD Wang analyzed the data; QW Meng contributed reagents/materials/analysis tools; C Meng and FY Kong wrote the paper. Acknowledgments This research was supported by the State Key Basic Research and Development Plan of China (2009CB118505), the Natural Science Foundation of China (31171474, 31371553) and Project Funded by China Postdoctoral Science Foundation (2015M570604). References
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