Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts

Abstract

The cross talk among hydrogen peroxide (H₂ O₂ ), nitric oxide (NO) and UV RESISTANCE LOCUS8 (UVR8) in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated. The results showed that UV-B irradiation significantly increased the anthocyanin accumulation and the expression of UVR8, and a similar trend appeared in radish sprouts subjected to cadmium, chilling and salt stresses regardless of light source. However, these responses disappeared under dark exposure. These results suggest that abiotic stress-induced anthocyanin accumulation and UVR8 expression were light-dependent. Moreover, abiotic stresses all enhanced the production of H₂ O₂ and exogenous H₂ O₂ addition significantly increased the anthocyanin concentration and UVR8 transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for H₂ O₂ ). It seems to suggest that H₂ O₂ played an important role in the anthocyanin biosynthesis. Furthermore, addition of 0.5 mM sodium nitroprusside (SNP, a NO-releasing compound) substantially induced the anthocyanin accumulation, and H₂ O₂ -induced anthocyanin accumulation and UVR8 expression were significantly suppressed by co-treatment with 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO, a NO scavenger), which was parallel with the expression of anthocyanin biosynthesis-related transcription factors and structural genes. All these results demonstrate that both H₂ O₂ and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a classical UVR8 pathway.

Hydrogen peroxide, nitric oxide interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts

A

QiWu, Nana Su, Xiaoyan Zhang, Yuanyuan Liu, Jin Cui & Yongchao Liang

The cross talk among hydrogen peroxide (HO in UV-B-induced anthocyanin accumulation in the hypocotyls of radish sprouts was investigated. The expression of , and a similar trend appeared in radish sprouts subjected to cadmium, chilling and salt stresses regardless of light source. However, these responses disappeared under dark exposure. These results suggest that abiotic stress-induced anthocyanin accumulation and expression were light-dependent. Moreover, abiotic stresses all enhanced the production of HO and exogenous HO

transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for HO). It seems to suggest that HO played an important role in the anthocyanin biosynthesis. Furthermore,

anthocyanin accumulation, and HO-induced anthocyanin accumulation and expression were

transcription factors and structural genes. All these results demonstrate that both HO and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a

Anthocyanins are plant secondary metabolites synthesized through the avonoid pathway, generating the characteristic reddish, bluish, and purple hues, which contribute to ower pigmentation, attraction of pollinators and seed dispersers1. In addition, they are also important as antioxidant molecules to protect plant cells against damage by reactive oxygen species (ROS)2, allelopathy, or UV irradiation3, and the production of anthocyanins in autumn leaves reduces the risk of photo-oxidative damage and delays leaf senescence4.On the other hand, possessing valuable nutritional antioxidant activities, anthocyanins are recognized as compounds with anti-inammatory and anticancer eect, and potential health-benets, such as prevention of cardiovascular diseases and obesity5,6.

The biosynthesis of anthocyanins is through phenylpropanoid pathway, which is one of the best studied examples of secondary metabolism in higher plants, and most genes responsible for anthocyanin biosynthesis have been cloned and analyzed710. The anthocyanins are synthesized from phenylalanine which is subsequently catalyzed by PAL, CHS, CHI, F3H, DFR, ANS, LDOX and UFGT11, and the accumulation of anthocyanins is induced by the expression of these genes which are regulated by a ternary transcriptional complex (MBW complex) containing an R2R3-MYB-type transcription factor, a bHLH transcription factor, and a WD40 repeat (WDR) protein1214.

The MBW complex is highly organized, and each subunit plays a specic function such as binding to DNA,

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activation of expression of a target gene, or stabilization of the transcription factor complex15. The physical interactions of WDR and bHLH proteins with more specic MYB proteins determine the regulation of anthocyanin accumulation1314. The MYB superfamily consists of more than 100 members in higher plants11, among which both anthocyanin pigment 1 (PAP1) and anthocyanin pigment 2 (PAP2) are redundant R2R3-MYB transcription factors and positive regulate the expression of anthocyanin biosynthesis-related genes16.

The induction of anthocyanin accumulation in vegetative tissues is oen considered to be a response of plants to biotic or abiotic stress conditions, such as nutrient (nitrogen and phosphorus) deciency, wounding, pathogen infection, water stress, and ultraviolet (UV) light17,18. Among these various environmental stimuli, UV-B is reported to be a main factor in anthocyanin accumulation, which is concomitant with up-regulation of MYB and biosynthetic genes1923. UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor that specically mediates photo-morphogenic responses to UV-B in plants24. UVR8 is required for 1) a UV-B-stimulated compensatory increase in epidermal leaf cell size, 2) normal progression of endoreduplication in response to UV-B, 3) stomatal dierentiation and functions in plant acclimation, and 4) survival under solar UV25,26. It is also found that the expression of UVR8 is up-regulated in plants grown under salt or osmotic stress27. Furthermore, the ectopic expression of UVR8 causes pleiotropic eects on plant growth, such as reduced plant organ size and root growth, and increased accumulation of avonoids28.

UVR8 is a homodimer in its ground state, and UV-B exposure results in its instantaneous monomerization (the active form) followed by interaction with CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), a major factor in UV-B signaling2931. Recently, some studies3234 found that constitutively active UVR8 variants elevated levels of anthocyanins but UVR8 decient mutant had lower levels of anthocyanins, suggesting that UVR8 may be involved in regulation of the biosynthesis of anthocyanins. However, details in the primary mechanisms of the involvement of UVR8 in anthocyanin biosynthesis are still scant.

UV-B irradiation may induce the production of ROS35, among which H2O2 is a major species generated in plants36. Because H2O2 is relatively stable and diusible through membrane, it is generally thought to serve as a signal molecule under various forms of abiotic stress37. It has been reported that H2O2 could regulate the biosynthesis of anthocyanins38,39, and mediate the regulatory eect of UVR8 in stomatal closure40. In addition, nitric oxide (NO) is regarded as a downstream gaseous molecule of H2O2 to mediate UV-B-induced stomatal closure40,41. Based on all these reports, we hypothesize that NO, H2O2 and UVR8 may interact to regulate UV-B-induced anthocyanin biosynthesis.

In the present study, cherry radish sprouts were chosen as the materials, which contain substantial amounts of antioxidants, vitamin C and health-promoting compounds such as glucosinolates and phenolic compounds4244.

Consumption of radish sprouts has been confirmed to reduce the risk of cancer and oxidative stress both in vivo and in vitro4547. Furthermore, with red hypocotyls resulting from anthocyanin accumulation, cherry radish sprouts could provide visual evidence for the biosynthesis of anthocyanins, and consequently, the high concentration of anthocyanin accumulated in cherry radish sprouts could underpin their high antioxidant activity. Using radish sprouts, in the present paper we conrmed the involvement of the UVR8 pathway in UV-B-induced anthocynin accumulation. Moreover, our results demonstrated that H2O2-dependent production of NO interacted with UVR8 to regulate the biosynthesis of anthocyanins.

Results

To determine the involvement of UVR8 in the biosynthesis of anthocyanins, the concentration of anthocyanin and the transcript level of UVR8 in the hypocotyls of radish sprouts grown under dark, white light and UV-B were analyzed. As expected, compared with dark environment, white light signicantly increased the concentration of anthocyanins, while the highest anthocyanin concentration (2 times greater than that under white light) was observed aer 24h UV-B irradiation (Fig.1A). Similarly, the expression of UVR8 was signicantly induced by UV-B, which was 2-fold that under white light (Fig.1B).

Previous studies show that UVR8 is involved in salt or osmotic stress conditions17. To investigate whether there is a relationship between UVR8 and abiotic stresses in the biosynthesis of anthocyanins, we compared the anthocyanin accumulation and UVR8 expression under control condition and abiotic stress. The results showed that regardless of white light or UV-B irradiation, cadmium, chilling and NaCl treatments all induced signicant increases in anthocyanin accumulation and the transcript abundance of UVR8, compared with those under control (Fig.2).

However, it remains unknown whether abiotic stress-induced anthocyanin accumulation and UVR8 expression are light-dependent. To test this, we repeated the above experiment under a dark environment. In contrast to exposure to light, abiotic stresses under dark had no eects on the anthocyanin accumulation and the expression of UVR8 (Fig.3), suggesting that light was a necessary factor in stress-induced anthocyanin accumulation and UVR8 expression.

HO was involved in UV-B-induced biosynthesis of anthocyanins. To identify the role of H2O2 under dierent stresses and the relationship between H2O2 and the anthocyanin accumulation, the H2O2 concentrations under stresses and the eects of H2O2 on anthocyanin accumulation and UVR8 expression were tested. Similarly, cadmium, chilling and salt treatments as well as UV-B irradiation all signicantly increased the H2O2

concentration, with the maximum observed under UV-B, which is about 20 times as great as that of control (Fig.4A). The accumulation of anthocyanins was positively correlated with the H2O2 concentration, though no signicant dierence was noted between control and the treatment with 0.2mM H2O2 (Fig.4B). The expression levels of UVR8 were also up-regulated by H2O2 ranging from 0.5 to 10mM, while no positive correlation between them was observed (Fig.4C). Therefore, 0.5mM H2O2 was selected as the appropriate treatment concentration in the following experiments.

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Figure 1. The concentration of anthocyanins (A) and the expression of UVR8 (B) in the hypocotyls of radish sprouts under dark (D), white light (W) and UV-B. Aer 36h dark incubation, the radish sprouts were then exposed to dark, white light or UV-B for another 36h when the samples were collected and detected. The data are means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

Figure 2. The concentration of anthocyanins (A,C) and the expression of UVR8 (B,D) in the hypocotyls of radish sprouts under dierent treatments. Aer 24h dark culture, sprouts were subjected to cadmium (Cd), chilling, NaCl or nothing (Con) for 12h still under dark, then these sprouts were transferred to white lightor UV-B for 24h. Values are the means standard error of three independent experiments with at least three replicates for each. Measurements in the same column followed by dierent letters are signicantly dierent at P< 0.05 level by Duncans test.

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Figure 3. The concentration of anthocyanins (A) and the expression of UVR8 (B) in the hypocotyls of radish sprouts under dierent treatments. Aer 24h dark culture, sprouts were subjected to nothing (Con), cadmium (Cd), chilling or NaCl for 12h still under dark, then these sprouts were transferred to white light, UV-B orstill dark for 24h. The data represent means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

Addition of 0.5mM H2O2 signicantly enhanced the pigmentation in the hypocotyls of radish sprouts, regardless of exposure to white light or UV-B, while addition of DMTU (dimethylthiourea, a chemical trap for H2O2)

eectively inhibited UV-B-induced anthocyanin accumulation (Fig.5A,B). The concentration of H2O2 and the level of UVR8 both showed similar trends with the accumulation of anthocyanins (Fig.5C,D).

NO was involved in UV-B-induced biosynthesis of anthocyanins. To verify the involvement of NO in activating anthocyanin biosynthesis, the endogenous NO levels and anthocyanin concentrations in response to various concentrations of exogenous SNP (sodium nitroprusside, a NO-releasing compound) were determined. As shown in Fig. S1, the endogenous levels of NO almost increased linearly with increasing SNP concentrations (Fig. S1A). The anthocyanin accumulation was signicantly increased by addition of 0.5mM SNP, and decreased aerward with further increasing of SNP (Fig. S1B).

The relationship underlying NO, HO Similar to the results above, addition of H2O2, SNP or their combination all considerably enhanced the anthocyanin bio-synthesis, while in comparison with SNP, co-treatment with DMTU slightly oset this increase, though it was not statistically signicant. Moreover, H2O2-induced increase of anthocyanins was dramatically suppressed by co-treatment with PTIO (Fig.6A). The NO level and UVR8 expression were both consistent with this trend, except that the combination of DMTU and SNP signicantly inhibited SNP-induced increase of UVR8 expression (Fig.6B,C).

Considering that H2O2 induces the generation of NO which is involved in many plant physiologic responses, we examined the eect of H2O2 on the enzymes and genes responsible for NO generation. As reported previously48,49, H2O2 signicantly enhanced the activities of NOS and NR, and up-regulated the expression of NOA1 and NR by 5 and 1.2-fold, respectively (Fig.7).

Finally, the expression levels of transcription factors and structural genes responsible for anthocyanin bio-synthesis were further determined by qRT-PCR. The relative expressions of COP1, PAP1 and PAP2 displayed similar trends. Compared with the control, H2O2, SNP and their combination all signicantly up-regulated the transcript levels, while DMTU and PTIO had no eects on the transcription of COP1, PAP1 and PAP2, with the exception of the considerable decrease of COP1 under DMTU treatment. Compared with H2O2, co-treatment with PTIO signicantly down-regulated the transcripts of COP1, PAP1 and PAP2. Compared with SNP, combined treatment with DMTU and SNP decreased the transcription of COP1 and PAP1 (Fig. S3). The transcript levels of anthocyanin-biosynthesis-related structural genes (PAL, CHS, CHI, F3H, DFR, ANS, LDOX and UFGT) displayed the similar trends to the transcription factors (Fig.8). In detail, H2O2, SNP and their combination signicantly

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Figure 4. The H2O2 concentration in the hypocotyls of radish sprouts subjected to Cd, chilling, NaCl or UV-B (A). Aer 24h dark incubation, the radish sprouts were then subjected to dierent abiotic stresses for 12h, and then they were exposed to white light for another 24h. The anthocyanin accumulation (B) and the expression of UVR8 (C) in the hypocotyls of radish sprouts treated with increasing exogenous H2O2. Aer 24h dark incubation, the radish sprouts were then subjected to H2O2 with dierent concentrations for 12h, and then they were exposedto white light for another 24h. The data represent means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

up-regulated the transcript levels of these genes, while H2O2+ PTIO and SNP+ DMTU substantially suppressed their expressions.

Discussion

Since its initial discovery in 200237, UVR8 has been the focus of recent attention due to numerous reports of its regulatory eects on UV-B signaling pathway and stress responses in plants50,51. However, the exploration of UVR8 function in vivo is still at an early stage and information about UVR8 is virtually derived from studies with Arabidopsis. Therefore its physiological roles in other diverse species remain poorly understood52. In this study, we explored the cross talk among H2O2, NO and UVR8 in regulating anthocyanin accumulation in the hypocotyls of radish sprouts.

Similar to previous reports in peach, strawberry and lettuce5355, compared with white light and dark, UV-B considerably increased the anthocyanin accumulation in the hypocotyls of radish sprouts (Fig.1A). Though it has been found that UV-B exposure induces the conversion of UVR8 from a homodimer to a monomeric which will interact with transcription factor COP140,52, the UVR8 expression showed the consistent trend with the anthocyanin accumulation (Fig.1B), suggesting that as a UV-B photoreceptor, UVR8 is likely to be involved in mediating anthocyanin biosynthesis. This hypothesis is supported by a recent study by Mao et al.56 who found that PeUVR8 in Populus euphratica could increase the anthocyanin accumulation in the Arabidopsis uvr8 mutant.

Apart from UV-B, other abiotic stresses could also induce the biosynthesis of anthocyanins, such as high light intensity57, low temperature58, and nutrient deciency59. In this study, we also observed that the anthocyanin accumulation was signicantly increased by three typical abiotic stresses, cadmium, chilling and salt, regardless

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Figure 5. Eects of exogenous addition of H2O2 or DMTU on the pigmentation of hypocotyls (A), anthocyanin accumulation (B), H2O2 concentration (C) and UVR8 expression (D). Aer 36h dark incubation, the radish sprouts were then subjected to H2O2 or DMTU under white light or UV-B for 24h. The data represent means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

of supply with white light or UV-B (Fig.2A,C). Meanwhile, the expression of UVR8 showed a similar trend (Fig.2B,D), which was in line with the study of Fasano et al.27, suggesting that UVR8 was involved in abiotic stresses, and there was a potential relationship between anthocyanins and UVR8. By contrast, these increases were not observed under dark (Fig.3), illustrating that abiotic stress-induced anthocyanin accumulation and increased expression of UVR8 were light-dependent.

Taking into consideration the importance of H2O2 in abiotic stresses and anthocyanin accumulation, we speculated that H2O2 may be a conjunctive point between anthocyanins and UVR8. As expected, compared with control, the concentration of H2O2 under various abiotic stresses (Cd, chilling, NaCl and UV-B) was increased signicantly (Fig.4A), and exogenous addition of H2O2 (0.510mM) enhanced both the anthocyanin accumulation and the expression of UVR8 (Fig.4B,C). Furthermore, UV-B-induced anthocyanin accumulation, H2O2

concentration and UVR8 expression were all suppressed by addition of DMTU (an inhibitor of H2O2), although DMTU did not aect these factors under white light (Fig.5), which might be due to the low H2O2 accumulation under normal growth condition. These results imply that UV-B irradiation induces more production of H2O2,

which further up-regulates UV-B-induced UVR8 expression to modulate the biosynthesis of anthocyanins.

Several previous studies showed that NO acts as downstream gaseous signal molecule of H2O2 to regulate the stomatal closure40,60, which prompted us to further investigate the role of NO in anthocyanin accumulation and the relationship among NO, H2O2 and UVR8. The endogenous NO concentration in radish sprouts showed an almost linear increase with the concentration of SNP (the donor of NO) (Fig. S1A), with 0.5 mM SNP signicantly increased the anthocyanin accumulation (Fig. S1B). Furthermore, we observed that H2O2-induced increases of anthocyanin accumulation, NO concentration and UVR8 expression were all considerably restrained by co-treatment with PTIO (a scavenger of NO) (Fig.6).

In animals, NO is synthesized through NO synthase (NOS)61 which has also been detected widely in plants, and inhibitors of mammalian NOS also have the ability to inhibit the generation of NO in plants, though no archetypal NOS-encoding gene(s) have been isolated in higher plants so far62,63. The nitric oxide associated1 (NOA1), reported to encode a protein with NOS activity, has been shown to be a GTPase playing a role in binding RNA/ribosomes64. In addition, it has been reported that NOA1-mediated NO production was involved in plant responses against salinity stress65 and pathogens66. In plants the NO production can also be generated via nitrate reductase (NR), a critical enzyme responsible for nitrate assimilation67,68. In the present study, the enzyme activity and gene expression related to NO biosynthesis were all signicantly enhanced by addition of H2O2 (Fig.7).

All these results suggest that irradiation with UV-B enhanced the production of H2O2, which increased the level of NO to further magnify UV-B-induced expression of UVR8 to regulate the anthocyanin biosynthesis. This hypothesis was further veried by the expressions of transcription factors and structural genes responsible for anthocyanin biosynthesis under various treatments (Fig. S3 and 8).

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Figure 6. The anthocyanin accumulation (A), endogenous NO level (B) and UVR8 expression (C) under dierent treatments. Aer 36h dark incubation, the radish sprouts were then subjected to H2O2, DMTU, SNP,

PTIO or their combination under white light for 24h. The data represent means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

The UVR8-dependent UV-B signaling pathway operates at low uence rates to initiate classical UV-B photomorphogenic responses, such as the induction of avonoid biosynthesis69. Therefore, the common pathway of low-intensity-UV-B-induced anthocyanin accumulation may be that UVR8 perceives the UV-B signal, and then transforms it into monomers70,71, which interact with the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)72 to activate gene expression involved in anthocyanin biosynthesis in plants. However, it was reported that both low and high uence of UV-B could activate UVR873. In this study, we reported that both H2O2 and NO were involved in high-intensity-UV-B-induced anthocyanin accumulation.

In detail, high uence of UV-B or abiotic stress-induced production of H2O2 could regulate the biosynthesis of anthocyanins through the following three pathways: 1) H2O2 directly up-regulates the expression of UVR8, which is light-dependent; 2) H2O2 increases the concentration of NO which magnies the eects of UV-B on UVR8 transcription; and 3) H2O2 inuences the redox which changes the expressions of anthocyanin biosynthesis-related transcription factors, and this process is also light-dependent (Fig.9). In this study, the UV-B lamps were used as the light source and the spectrum ranges from 280nm to 360nm with its peak of 306nm (Fig. S4), which is a related broad spectrum of light including UV-A. Therefore, the UV-B induced anthocyanin biosynthesis might be partly due to the broad spectrum of light. As the spectrum peak of UV-B lamps is 306nm, we estimated that in this study, the enhancement of anthocyanin was mainly induced by UV-B, which should be further investigated under a narrow band of UV-B. In addition, the emergence of LED UV-B would facilitate the related researches.

Materials and Methods

Seeds of radish (Raphanus sativus L cv. Yanghua) were soaked in deionized water for about 12 h, and then put on moist gauze to germinate for 24 h. Uniform-sized germinated seeds were selected and sown into cases containing1/4 strength Hoaglands solution and covered with gauze. These cases were covered with gauze and were maintained in an incubator in dark at 25 C for 24 or 36 h. Then the sprouts were treated grown in growth chambers equipped with LED white light (Safe Instrument Experimental Factory,

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Figure 7. Eects of H2O2 on the activities of NOS and NR and the expression levels of NOA1 and NR. Aer 24h dark incubation, the radish sprouts were then subjected to H2O2 for 12h, and then they were exposed to white light for another 24h. The data represent means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

Zhejiang, China) or UV-B lamps (Sankyo Denki, Shinagawa, Tokyo, Japan) which emit ultraviolet rays between 280nm and 360nm with its peak of 306nm (Fig. S4). The light intensity of white light was set at 505molm2s1, while the UV-B dose was set at 10 Wm2 measured with a pocket UV light meter (UV-340 A, Lutron, Taiwan). The temperature was 25C and the relative humidity (RH) was 6575%.

Anthocyanin analysis. The total anthocyanin concentration in the radish hypocotyls was determined according to the method developed by Zhou et al.74, which involves measuring the absorbance (530) of extracts. Briey, samples (0.5g) were incubated in 5mL of 1% HCl in methanol at room temperature in the dark for 24 h. Tubes were shaken with a vortex mixer every 6 hours. The anthocyanins in the aqueous phase were then quantied spectrophotometrically (A530 0.25 A657) (UV-5200 spectrophotometer, Shanghai Metash Instruments

Co., Ltd, Shanghai, China).

Observation of the hypocotyls cross section. The hypocotyls of radish sprouts were transected with a blade and then observed under a light microscope (Model Stemi 2000-C; Carl Zeiss, Germany) and photographed on a color lm (Powershot A620, Canon Photo Film, Japan).

Total RNA was isolated from root tissues using Trizol extraction reagent (Invitrogen, Gaithersburg, MD, USA) and the RNA purity was veried by the ratio (>1.9) of 260/280 nm absorbance. DNA-free total RNA (8 l) from dierent treatments was used for rst-strand cDNA synthesis in a 20l reaction volume (Thermo Scientic, MD, Lithuania) according to the manufacturers instructions. Real-time quantitative PCR reactions were performed using a Mastercycler eprealplex real-time PCR

system (ABI 7500, MD, USA) with Bestar SybrGreen qPCR mastermix (DBI, Bioscience Inc., Germany) in a

20l reaction volume according to the user manual.

PCR primers targeting actin, UVR8, COP1, NOA1, NR, PAL, CHS, CHI, F3H, DFR, LDOX, ANS, UFGT, PAP1 and PAP2 were designed using Primer Express version 3.0 (Applied Biosystems). In order to design the UVR8

primer, the UVR8 mRNA of Arabidopsis was blasted against the radish data base (RadishBase), and a similar mRNA (Unigene ID: UN17090, Length: 1690) was obtained. The similarity is 90%. As UVR8 proteins occur widely among plant species and are well conserved72, the products from this prime were thought to be UVR8. All primers (Table S1) were synthesized by Genewiz Bio-engineering Ltd. Company (Suzhou, China). The relative expression levels were presented as values relative to that of corresponding control sample at the indicated time, aer normalization to actin transcript levels.

Determination the activities of NR and NOS. The activity of NR was determined as described by Sun et al.75. The hypocotyls (0.5g) were homogenized with a mortar and pestle on ice with 5ml of extract buer containing 50mM HEPES-KOH (pH 7.5), 5% glycerol (v/v), 10mM MgCl2, 1mM dithiothreitol (DTT), 1mM phenylmethylsulfonyl uoride (PMSF), and 10 M avin adenine dinucleotide (FAD). The extract was centrifuged at 13 000g for 20min at 4C.

The activity of NR was measured immediately by mixing 250l of supernatant with 250l prewarmed (25C) assay buer containing 50 mM HEPES-KOH (pH 7.5), 10 mM MgCl2, 1 mM DTT, 2 mM KNO3 and 200 M

NADH. The reaction was started by adding assay buer, incubated at 30C for 30min and then stopped by adding

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Figure 8. The transcript levels of anthocyanin biosynthesis-related genes PAL (A), CHS (B), CHI (C), F3H (D), DFR (E), ANS (F), LDOX (G) and UFGT (H) under dierent treatments. Aer 36h dark incubation, the radish sprouts were then subjected to H2O2, DMTU, SNP, PTIO or their combination under white light for 24h. The data represent means SD of three independent experiments. Signicance between experimental values was assessed by Duncans test (P<0.05).

50 l 0.5 M Zn-acetate. The nitrite produced was measured colorimetrically at 540 nm aer adding 1 ml of 1% sulfanilamide in 3M HCl plus 1ml of 0.02% N-(1-naphthyl) ethylenediamine in 0.2M HCl.

To determine NOS activity, the total protein was extracted using the buer containing 100mM HEPES-KOH (pH 7.5), 1 mM EDTA, 10% glycerol (v/v), 5 mM DTT, 0.5 mM PMSF, 0.1% Triton X-100 (v/v), 1% polyvinylpyrrolidone (PVP) and 20M FAD. NOS activity was then measured aer centrifugation at 13 000g for 20min at 4 C according to Gonzalez et al.76. Briey, NOS activity was detected in1 ml of reaction mixture containing 100mM phosphate buer(pH 7.0), 1mM L-Arg, 2mM MgCl2, 0.3mM CaCl2, 4M BH4, 1M FAD, 1M avin mononucleotide (FMN), 0.2 mM DTT, 0.2 mM NADPH, and 200 l of protein extract. The decrease in absorbance as a result of NADPH consumption was determined at 340nm for 5min. NOS activity was calculated using the extinction coefficient of NADPH (e=6.22mM1 cm1).

Detection of HO concentration. The H2O2 concentration was measured colorimetrically as described by Hung77. H2O2 was extracted by homogenizing hypocotyl tissue with phosphate buer (50mM, pH 6.5) containing

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Figure 9. Model of H2O2 and NO participating in UV-B-induced anthocyanin biosynthesis. In a identied path, UVR8, the photoreceptor of UV-B, rstly perceives the signal from low-intensity-UV-B irradiation, and then initiates the transcription factors (COP1, MBW complex) which further up-regulates the expression of anthocyanin biosynthesis-related structural genes (PAL, CHS, CHI, F3H, DFR, ANS), nally to enhance the accumulation of anthocyanins. Besides the signal, high-intensity-UVB could also act as an environmental stress which, to some extent, induces the production of H2O2 which could also be induced by abiotic stress. H2O2 may promote the biosynthesis of anthocyanins through three pathways: 1) H2O2 interacts with UVR8, which is light-dependent; 2) H2O2 rstly stimulates the production of NO that interacts with the UVR8; 3) H2O2 up-regulates the expression of transcription factors directly, which is also light-dependent.

1mM hydroxylamine. The homogenate was centrifuged at 6,000g for 25min. To determine H2O2 concentration, the extracted solution was mixed with 0.1% titanium chloride in 20% (v/v) H2SO4. The mixture was then centrifuged at 6,000 g for 25 min. The absorbance was measured at 410 nm. The NO concentration was calculated by comparing to against a standard curve of H2O2.

Nitric oxide concentration was determined using the method described by Hu et al.78 and Zhou et al.74 with slight modications. Samples (1.0 g) were ground in a mortar and pestle in 8 ml of 50 mM cool acetic acid buer (pH 3.6, containing 4% zinc diacetate). The homogenates were centrifuged at 10,000g for 15min at 4C. The supernatant was collected. The pellet was washed by 1ml of extraction buer and centrifuged as before. The two supernatants were combined and 0.1g of charcoal was added. Aer vortex and ltration, the ltrate was leached and collected. The mixture of 1.5 ml of ltrate with1.5 ml of the Greiss reagent was incubated at room temperature for 30 min. Absorbance was determined at 540 nm. The NO concentration was calculated against a standard curve of NaNO2.

Statistical analyses. Values presented are means standard deviation (SD) of three replicates. Data was subjected to analysis of variance (ANOVA), and mean values were compared by Duncans multiple range test (p< 0.05). All the statistical analyses were performed using SPSS 19.0 for Windows.

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Acknowledgements

This work was supported by the National Science Foundation of China (31171998) and (31572169).

Author Contributions

Q.W., N.S. and J.C. conceived the study, designed all the experiments. Q.W., N.S., X.Z. and Y.L. performed the experiments and interpreted experimental date. Q.W., N.S., Yongchao L. and J.C. participated in writing the manuscript. All the authors read and approved the nal manuscript.

Additional Information

Supplementary information accompanies this paper at http://www.nature.com/srep

Competing nancial interests: The authors declare no competing nancial interests.

How to cite this article: Wu, Q. et al. Hydrogen peroxide, nitric oxide and UV RESISTANCE LOCUS8 interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts. Sci. Rep. 6, 29164; doi: 10.1038/ srep29164 (2016).

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