Introduction

Ultraviolet (UV) radiation has received increasing attention due to the thinning of the earth’s stratospheric ozone layer. While UV-C radiation (200–280 nm) is completely absorbed by atmospheric gases, a significant amount of UV-B (280–320 nm) reaches the earth’s surface (Jansen et al. 1998; Zhao et al. 2007). UV-B radiation causes a multitude of physiological and biochemical changes in plants, such as inhibition of photosynthesis, induction of stress responses, and damage to DNA and other molecules (Jansen et al. 1998; Frohnmeyer and Staiger 2003; Zhao et al. 2007).

Plants have developed numerous biochemical mechanisms to cope with environmental change (Zhao et al. 2007). In general, plants respond differently to irradiation with low or high doses of UV-B, either by stimulating protection mechanisms or by activating repair mechanisms to cope with the different types of stresses (Frohnmeyer and Staiger 2003). The most common protective mechanism against potentially damaging irradiation is the biosynthesis of UV-absorbing compounds (Hahlbrock and Scheel 1989; Frohnmeyer and Staiger 2003; Cakırlar et al. 2008). The accumulation of certain phenylpropanoid compounds, such as flavonoids and anthocyanins, plays an important role in mitigating UV-B-induced damage (Lois and Buchanan 1994; Bharti and Khurana 1997; Cakırlar et al. 2008). Genetic analysis of Arabidopsis mutants with altered UV-B responses further supports the importance of chemical sunscreens; constitutive accumulation of flavonoids and other phenolics results in resistance of mutants to UV-B (Bieza and Lois 2001; Zhao et al. 2007).

It is well documented that the responses to low UV-B are in part due to transcriptome changes (Frohnmeyer and Staiger 2003). Some genes have been reported to regulate the UV-B response, including Myb transcription factor 4 (MYB4), UV-light-insensitive 3 (Uli3), SENSITIVE TO ABA AND DROUGHT 2 (SAD2) and UV resistance locus 8 (UVR8) (Jin et al. 2000; Kliebenstein et al. 2002; Frohnmeyer and Staiger 2003; Zhao et al. 2007). Although recent studies have unraveled some of the key players in the UV-B response (Frohnmeyer and Staiger 2003; Zhao et al. 2007), molecular mechanisms underlying the regulation of the UV-B response remain to be determined further.

The plant hormone ethylene has been shown to be involved in modulating the plant responses to environmental stimuli from biotic and abiotic stresses, such as wounding, hypoxia, ozone, chilling or freezing (Wang et al. 2007). The Arabidopsis Ethylene-Insensitive 2 (EIN2) gene is a central component of the ethylene signaling pathway, and it is also a bifunctional transducer of ethylene and stress responses (Alonso et al. 1999). It was shown that EIN2 gene plays important roles in mediating ozone stress, high salt, oxidative stress, lead and disease resistances (Alonso et al. 1999; Thomma et al. 1999; Cao et al. 2006, 2009; Wang et al. 2007). However, it is unclear whether it also plays a role in the regulation of the UV-B response in Arabidopsis.

In this study, we showed that the ein2-1 mutation led to constitutive activation of CHALCONE SYNTHASE (CHS) and CINNAMATE 4-HYDROXYLASE (C4H) genes and increases in the biosynthesis of UV-absorbing compounds and, consequently, enhanced UV-B tolerance. These results suggest that EIN2 gene mediates the UV-B response, at least in part, through modulation of expression of CHS and C4H genes.

Materials and methods

Plant material and growth conditions

Wild-type Arabidopsis (Arabidopsis thaliana) ecotype Columbia Col-0 and the ein2-1 mutant (Guzmán and Ecker 1990) were obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA). Seeds were surface sterilized and plated on Murashige and Skoog (MS) media containing 0.8% agar and 1% sucrose. Plates were stored for 3 days in the dark at 4°C and then placed in a growth chamber set at 22°C, 100 μmol/m2 s light intensity and standard long-day conditions (16 h light/8 h dark).

Treatments and UV-B tolerance test

Twelve-day-old wild-type and ein2-1 seedlings were exposed to UV-B radiation (1.0 mW/cm2) for 0 (control), 2, 2.25, 2.5, 2.75 or 3 h, and then incubated for 3 days in a continuous-light growth chamber. To exclude UV-C radiation below 280 nm, the UV lamps were covered with cellulose acetate sheets. At the end of this incubation, plants were photographed and the percentage of plants with bleached cotyledons was calculated.

Spectrophotometric analysis

Twelve-day-old wild-type and ein2-1 seedlings were exposed to UV-B (1.0 mW/cm2) for 0 or 4.5 h, and then sampled for analysis of UV-absorbing pigments. Pigment content was determined according to the method described by Cakırlar et al. (2008). Briefly, 12-day-old seedlings (150 mg) were homogenized in 6 ml of medium containing methanol:HCl:H2O (79:1:20), centrifuged at 10,000g for 15 min and absorption for flavonoids and anthocyanins at 300 and 535 nm, respectively, was read using a UV–visible spectrophotometer (UV-1201 spectrophotometer).

Reverse transcription (RT)-PCR and real-time PCR analysis

Twelve-day-old wild-type seedlings exposed to UV-B (1.0 mW/cm2) for indicated times were sampled for RT-PCR and real-time PCR analysis. For RT-PCR, seedlings were homogenized with a mortar and pestle in liquid nitrogen. Total RNA was extracted using Trizol Reagent (Invitrogen, California). Semiquantitative RT-PCR was performed as previously described (Cao et al. 2009). For real-time PCR, total RNA extraction was performed as described above. cDNA was synthesized from total RNA by SuperScript II RNase H2 reverse transcriptase (Invitrogen) using Radom Hexamer Primer (Promega). Quantitative real-time PCR was performed according to the instructions provided for the Bio-Rad iCycler iQ system (Bio-Rad laboratories) with platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The fold change of transcripts was calculated based on an efficiency calibrated model (Yuan et al. 2006) and compared with the transcript level under normal condition. Statistical differences between samples were evaluated by Student’s t test using delta Ct values (Yuan et al. 2006). In each experiment, the mean of three biological replicates is used to generate means and statistical significance. Three experiments on independently grown plant material were performed to confirm that the results are reproducible. ACTIN11 (ACTIN) was used to normalize the reactions.

The following primers were designed for gene-specific transcript amplifications:

  • ACTIN11 (ACTIN, AT3g12110): forward, 5′-GATTTGGCATCACACTTTCTACAATG-3′, and reverse, 5′-GTTCCACCACTGAGCACAATG-3′;

  • EIN2 (AT5G03280): forward, 5′-GAAGACGAATCAATAGTGCGG-3′ and reverse, 5′-TGCGGAATGAAGGAGGAC-3′;

  • C4H (AT2G30490): forward, 5′-CCGGGATTATATTGGCATTG-3′ and reverse, 5′-CTGAATTGTCCACCTTTCTC-3′;

  • CHS (AT5G13930): forward, 5′-AGAGAAGATGAGGGCGACACG-3′ and reverse, 5′-GCCACACCATCCTTAGCTGAC-3′;

  • MYB4 (AT4G38620): forward, 5′-CAAGGGCATGGAAAGTCAAC-3′ and reverse, 5′-AAGTTTTGTACAGTTACGCCTAA-3′.

Statistical analysis

Data are the mean ± SD of three independent replicates. The analyses of variance were computed on statistically significant differences (P < 0.05) determined based on the appropriate F tests. The mean differences were compared utilizing Duncan’s multiple range test.

Results and discussion

The Arabidopsis EIN2 gene has been proposed to be a bifunctional transducer of ethylene and stress responses. To test whether EIN2 is mediated by UV-B, the expression pattern of EIN2 was analyzed in response to UV-B exposure. As shown in Fig. 1, the transcript level of EIN2 was significantly reduced after 1 h UV-B exposure and was not detected after 6 h UV-B exposure, which suggests the possible involvement of EIN2 in the regulation of the UV-B response.

Fig. 1
figure 1

Repress of EIN2 gene by UV-B exposure. The expression of EIN2 gene was analyzed by RT-PCR in 12-day-old wild-type seedlings treated with UV-B for 0 (control), 1, 3, and 6 h, respectively. ACTIN was used as a loading control

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To further investigate the role of EIN2 in regulating the response of plants to UV-B exposure, we evaluated the growth of wild-type and ein2-1 seedlings in response to UV-B exposure. Under normal growth condition, there was no significant (P > 0.05) difference between the wild type and the ein2-1 mutant (Fig. 2a). However, when wild-type and ein2-1 seedlings were exposed to UV-B for 2.5 h, more than 80% of the wild-type seedlings were bleached and died, whereas more than 60% of ein2-1 seedlings survived this treatment (Fig. 2b). Enhanced tolerance of ein2-1 seedlings to UV-B exposure is further supported by the fact that survival rates of ein2-1 seedlings were significantly (P < 0.05) higher than those of wild type in response to UV-B exposure for 2, 2.25, 2.5, 2.75 or 3 h (Fig. 2c). All these results suggest that ein2-1 seedlings were more tolerant to UV-B exposure than wild-type seedlings.

Fig. 2
figure 2

Tolerance of ein2-1 seedlings to UV-B exposure. Twelve-day-old wild-type (WT) and ein2-1 seedlings were exposed to UV-B for 0 h (control, a) or 2.5 h (b). After UV-B exposure, plants were transferred to a continuous-light growth chamber to recovery for 3 days and then photographed. c Survival rate of wild-type and ein2-1 seedlings exposed to UV-B for indicated times after 3-day recovery

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Plants utilize UV-B absorptive secondary metabolites from the phenylpropanoid biosynthetic pathway as sunscreens to avoid UV-B exposure (Kliebenstein et al. 2002). These compounds, especially colorless flavonoids (Chappell and Hahlbrock 1984) and hydroxycinnamic acids (Li et al. 1993; Landry et al. 1995; Ormrod et al. 1995), accumulate in plants in response to UV-B exposure. Several studies using Arabidopsis mutants deficient in flavonoids and hydroxycinnamic acids underscore the importance of chemical sunscreens in protecting against UV-B radiation (Li et al. 1993; Lois and Buchanan 1994; Landry et al. 1995; Bieza and Lois 2001). To determine whether enhanced tolerance of ein2-1 seedlings to UV-B exposure is associated with an increase in the accumulation of UV-absorbing compounds, we measured contents of flavonoids and anthocyanins in ein2-1 seedlings exposed to UV-B using absorption spectra analysis. As shown in Fig. 3a, under normal growth condition, there was no significant (P > 0.05) difference in absorbance at 300 nm (flavonoids) between wild-type and ein2-1 seedlings. After UV-B exposure for 4.5 h, however, a significant (P < 0.05) higher increase in absorbance at 300 nm was observed in ein2-1 seedlings than in wild-type seedlings. In contrast, a significant (P < 0.05) higher increase in absorbance at 535 nm (anthocyanins) was also detected in ein2-1 seedlings than in wild-type seedlings in the absence or presence of UV-B exposure (Fig. 3b). These results suggest that the ein2-1 mutation enhanced the accumulation of UV-absorbing compounds, flavonoids and anthocyanins.

Fig. 3
figure 3

Contents of UV-absorbing pigments at absorption spectrum values of 300 nm (a) and 535 nm (b) in 12-day-old wild-type (WT) and ein2-1 seedlings. Pigments were extracted from 12-day-old wild-type and ein2-1 seedlings exposed to UV-B for 0 h (control) and 4.5 h. Vertical bars represent SE for three independent replicates. Values with the same letter are not significantly different at P < 0.05, according to Duncan’s multiple range test

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CHS and C4H are two key enzymes involved in the biosynthesis pathway of UV-absorbing compounds (Kliebenstein et al. 2002). The constitutive increases in UV-absorbing pigments are often associated with the elevated transcript levels of CHS and C4H genes. Therefore, we analyzed the expression pattern of the two genes in wild-type and ein2-1 seedlings treated with or without UV-B. RT-PCR analysis showed that higher transcript levels of CHS and C4H genes were detected in ein2-1 plants than in wild-type plants in the absence or presence of UV-B exposure (Fig. 4a). To further confirm this result, we performed real-time PCR analysis. As shown in Fig. 4b, c, the transcript levels of CHS and C4H were significantly (P < 0.05) higher in ein2-1 plants than in wild-type plants in the absence or presence of UV-B exposure. These results suggest that the constitutive increase in UV-absorbing compounds in the ein2-1 mutant is associated with, at least in part, the constitutively increased transcript levels of CHS and C4H.

Fig. 4
figure 4

Effects of UV-B exposure on transcript levels of CHS and C4H genes in wild-type (WT) and ein2-1 seedlings. a RT-PCR analysis. Twelve-day-old wild-type and ein2-1 seedlings exposed to UV-B for 0 or 2 h were sampled for RT-PCR analysis. Three independent replicates were carried out. ACTIN was used as a loading control. b–d real-time PCR analysis. Twelve-day-old wild-type and ein2-1 seedlings exposed to UV-B for 0 or 2 h were sampled for real-time PCR analysis. Vertical bars represent SE for three independent replicates. Values with the same letter are not significantly different at P < 0.05, according to Duncan’s multiple range test

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It has been shown that MYB4 functions as a transcription repressor and downregulates C4H expression, and overexpression of MYB4 in the wild type repressed the expression of CHS and C4H genes (Jin et al. 2000). To test whether increases in the transcript levels of CHS and C4H are correlated to the downregulated expression of MYB4 in the ein2-1 mutant, we performed RT-PCR and real-time PCR analysis. However, there was no significant (P > 0.05) difference in the transcript level of MYB4 between ein2-1 and wild-type seedlings in the absence or presence of UV-B exposure (Fig. 4a–d), suggesting that EIN2 gene mediates the expression of CHS and C4H genes through a MYB4-independent pathway. Recently, UVR8 has been shown to act in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis (Kliebenstein et al. 2002). Therefore, it remains to further be determined how EIN2 gene mediates the expression of CHS and C4H genes.

In summary, the results presented here provide evidence that the ein2-1 mutation activates the expression of CHS and C4H genes, which in turn increases the levels of flavonoid and anthocyanin, and thereby leading to enhanced tolerance of ein2-1 seedlings to UV-B exposure.