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

The cross talk among hydrogen peroxide (H2O2), 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 H2O2 and exogenous H2O2 addition significantly increased the anthocyanin concentration and UVR8 transcription, while these increases were severely inhibited by addition of dimethylthiourea (DMTU, a chemical trap for H2O2). It seems to suggest that H2O2 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 H2O2-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 H2O2 and NO are involved in UV-B-induced anthocyanin accumulation, and there is a crosstalk between them as well as a classical UVR8 pathway.

Scientific RepoRts | 6:29164 | DOI: 10.1038/srep29164 activation of expression of a target gene, or stabilization of the transcription factor complex 15 . The physical interactions of WDR and bHLH proteins with more specific MYB proteins determine the regulation of anthocyanin accumulation [13][14] . The MYB superfamily consists of more than 100 members in higher plants 11 , 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 genes 16 .
The induction of anthocyanin accumulation in vegetative tissues is often considered to be a response of plants to biotic or abiotic stress conditions, such as nutrient (nitrogen and phosphorus) deficiency, wounding, pathogen infection, water stress, and ultraviolet (UV) light 17,18 . 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 genes [19][20][21][22][23] . UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor that specifically mediates photomorphogenic responses to UV-B in plants 24 . 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 differentiation and functions in plant acclimation, and 4) survival under solar UV 25,26 . It is also found that the expression of UVR8 is up-regulated in plants grown under salt or osmotic stress 27 . Furthermore, the ectopic expression of UVR8 causes pleiotropic effects on plant growth, such as reduced plant organ size and root growth, and increased accumulation of flavonoids 28 .
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 signaling [29][30][31] . Recently, some studies [32][33][34] found that constitutively active UVR8 variants elevated levels of anthocyanins but UVR8 deficient 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 ROS 35 , among which H 2 O 2 is a major species generated in plants 36 . Because H 2 O 2 is relatively stable and diffusible through membrane, it is generally thought to serve as a signal molecule under various forms of abiotic stress 37 . It has been reported that H 2 O 2 could regulate the biosynthesis of anthocyanins 38,39 , and mediate the regulatory effect of UVR8 in stomatal closure 40 . In addition, nitric oxide (NO) is regarded as a downstream gaseous molecule of H 2 O 2 to mediate UV-B-induced stomatal closure 40,41 . Based on all these reports, we hypothesize that NO, H 2 O 2 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 compounds [42][43][44] . Consumption of radish sprouts has been confirmed to reduce the risk of cancer and oxidative stress both in vivo and in vitro [45][46][47] . 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 confirmed the involvement of the UVR8 pathway in UV-B-induced anthocynin accumulation. Moreover, our results demonstrated that H 2 O 2 -dependent production of NO interacted with UVR8 to regulate the biosynthesis of anthocyanins.

UVR8 was involved in the biosynthesis of anthocyanins.
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 significantly increased the concentration of anthocyanins, while the highest anthocyanin concentration (2 times greater than that under white light) was observed after 24 h UV-B irradiation (Fig. 1A). Similarly, the expression of UVR8 was significantly 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 conditions 17 . 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 significant 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 effects 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. Similarly, cadmium, chilling and salt treatments as well as UV-B irradiation all significantly increased the H 2 O 2 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 H 2 O 2 concentration, though no significant difference was noted between control and the treatment with 0.2 mM H 2 O 2 (Fig. 4B). The expression levels of UVR8 were also up-regulated by H 2 O 2 ranging from 0.5 to 10 mM, while no positive correlation between them was observed (Fig. 4C). Therefore, 0.5 mM H 2 O 2 was selected as the appropriate treatment concentration in the following experiments.

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 significantly increased by addition of 0.5 mM SNP, and decreased afterward with further increasing of SNP (Fig. S1B).

The relationship underlying NO, H 2 O 2 and UVR8 in anthocyanin accumulation. Similar to
the results above, addition of H 2 O 2 , SNP or their combination all considerably enhanced the anthocyanin biosynthesis, while in comparison with SNP, co-treatment with DMTU slightly offset this increase, though it was not statistically significant. Moreover, H 2 O 2 -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 significantly inhibited SNP-induced increase of UVR8 expression (Fig. 6B,C).
Considering that H 2 O 2 induces the generation of NO which is involved in many plant physiologic responses, we examined the effect of H 2 O 2 on the enzymes and genes responsible for NO generation. As reported previously 48,49 , H 2 O 2 significantly 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 biosynthesis were further determined by qRT-PCR. The relative expressions of COP1, PAP1 and PAP2 displayed similar trends. Compared with the control, H 2 O 2 , SNP and their combination all significantly up-regulated the transcript levels, while DMTU and PTIO had no effects on the transcription of COP1, PAP1 and PAP2, with the exception of the considerable decrease of COP1 under DMTU treatment. Compared with H 2 O 2 , co-treatment with PTIO significantly 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, H 2 O 2 , SNP and their combination significantly

Discussion
Since its initial discovery in 2002 37 , UVR8 has been the focus of recent attention due to numerous reports of its regulatory effects on UV-B signaling pathway and stress responses in plants 50,51 . 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 understood 52 . In this study, we explored the cross talk among H 2 O 2 , NO and UVR8 in regulating anthocyanin accumulation in the hypocotyls of radish sprouts.
Similar to previous reports in peach, strawberry and lettuce 53-55 , 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 COP1 40,52 , 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 intensity 57 , low temperature 58 , and nutrient deficiency 59 . In this study, we also observed that the anthocyanin accumulation was significantly increased by three typical abiotic stresses, cadmium, chilling and salt, regardless After 24 h dark incubation, the radish sprouts were then subjected to different abiotic stresses for 12 h, and then they were exposed to white light for another 24 h. The anthocyanin accumulation (B) and the expression of UVR8 (C) in the hypocotyls of radish sprouts treated with increasing exogenous H 2 O 2 . After 24 h dark incubation, the radish sprouts were then subjected to H 2 O 2 with different concentrations for 12 h, and then they were exposed to white light for another 24 h. The data represent means ± SD of three independent experiments. Significance between experimental values was assessed by Duncan's 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 H 2 O 2 in abiotic stresses and anthocyanin accumulation, we speculated that H 2 O 2 may be a conjunctive point between anthocyanins and UVR8. As expected, compared with control, the concentration of H 2 O 2 under various abiotic stresses (Cd, chilling, NaCl and UV-B) was increased significantly (Fig. 4A), and exogenous addition of H 2 O 2 (0.5-10 mM) enhanced both the anthocyanin accumulation and the expression of UVR8 (Fig. 4B,C). Furthermore, UV-B-induced anthocyanin accumulation, H 2 O 2 concentration and UVR8 expression were all suppressed by addition of DMTU (an inhibitor of H 2 O 2 ), although DMTU did not affect these factors under white light (Fig. 5), which might be due to the low H 2 O 2 accumulation under normal growth condition. These results imply that UV-B irradiation induces more production of H 2 O 2 , 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 H 2 O 2 to regulate the stomatal closure 40,60 , which prompted us to further investigate the role of NO in anthocyanin accumulation and the relationship among NO, H 2 O 2 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 significantly increased the anthocyanin accumulation (Fig. S1B). Furthermore, we observed that H 2 O 2 -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 far 62,63 . 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/ribosomes 64 . In addition, it has been reported that NOA1-mediated NO production was involved in plant responses against salinity stress 65 and pathogens 66 . In plants the NO production can also be generated via nitrate reductase (NR), a critical enzyme responsible for nitrate assimilation 67,68 . In the present study, the enzyme activity and gene expression related to NO biosynthesis were all significantly enhanced by addition of H 2 O 2 (Fig. 7). All these results suggest that irradiation with UV-B enhanced the production of H 2 O 2 , which increased the level of NO to further magnify UV-B-induced expression of UVR8 to regulate the anthocyanin biosynthesis. This hypothesis was further verified by the expressions of transcription factors and structural genes responsible for anthocyanin biosynthesis under various treatments ( Fig. S3 and 8). The UVR8-dependent UV-B signaling pathway operates at low fluence rates to initiate classical UV-B photomorphogenic responses, such as the induction of flavonoid biosynthesis 69 . 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 monomers 70,71 , 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 fluence of UV-B could activate UVR8 73 . In this study, we reported that both H 2 O 2 and NO were involved in high-intensity-UV-B-induced anthocyanin accumulation. In detail, high fluence of UV-B or abiotic stress-induced production of H 2 O 2 could regulate the biosynthesis of anthocyanins through the following three pathways: 1) H 2 O 2 directly up-regulates the expression of UVR8, which is light-dependent; 2) H 2 O 2 increases the concentration of NO which magnifies the effects of UV-B on UVR8 transcription; and 3) H 2 O 2 influences 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 280 nm to 360 nm with its peak of 306 nm (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 306 nm, 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
Plant materials and treatments. 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 Hoagland's 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, Zhejiang, China) or UV-B lamps (Sankyo Denki, Shinagawa, Tokyo, Japan) which emit ultraviolet rays between 280 nm and 360 nm with its peak of 306 nm (Fig. S4). The light intensity of white light was set at 50 ± 5 μ mol·m −2 ·s −1 , while the UV-B dose was set at 10 W·m −2 measured with a pocket UV light meter (UV-340 A, Lutron, Taiwan). The temperature was 25 °C and the relative humidity (RH) was 65-75%.
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. Briefly, samples (0.5 g) were incubated in 5 mL 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 quan- 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 conserved 72 , 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, after 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   After 24 h dark incubation, the radish sprouts were then subjected to H 2 O 2 for 12 h, and then they were exposed to white light for another 24 h. The data represent means ± SD of three independent experiments. Significance between experimental values was assessed by Duncan's test (P < 0.05).
To determine NOS activity, the total protein was extracted using the buffer containing 100 mM 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 20 μ M FAD. NOS activity was then measured after centrifugation at 13 000 g for 20 min at 4 °C according to Gonzalez et al. 76    1 mM hydroxylamine. The homogenate was centrifuged at 6,000 g for 25 min. To determine H 2 O 2 concentration, the extracted solution was mixed with 0.1% titanium chloride in 20% (v/v) H 2 SO 4 . 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 H 2 O 2 .
Quantification of NO concentration. Nitric oxide concentration was determined using the method described by Hu et al. 78 and Zhou et al. 74 with slight modifications. Samples (1.0 g) were ground in a mortar and pestle in 8 ml of 50 mM cool acetic acid buffer (pH 3.6, containing 4% zinc diacetate). The homogenates were centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was collected. The pellet was washed by 1 ml of extraction buffer and centrifuged as before. The two supernatants were combined and 0.1 g of charcoal was added. After vortex and filtration, the filtrate was leached and collected. The mixture of 1.5 ml of filtrate 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 NaNO 2 .

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 Duncan's multiple range test (p < 0.05). All the statistical analyses were performed using SPSS 19.0 for Windows.

Figure 9. Model of H 2 O 2 and NO participating in UV-B-induced anthocyanin biosynthesis.
In a identified path, UVR8, the photoreceptor of UV-B, firstly 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), finally 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 H 2 O 2 which could also be induced by abiotic stress. H 2 O 2 may promote the biosynthesis of anthocyanins through three pathways: 1) H 2 O 2 interacts with UVR8, which is lightdependent; 2) H 2 O 2 firstly stimulates the production of NO that interacts with the UVR8; 3) H 2 O 2 up-regulates the expression of transcription factors directly, which is also light-dependent.