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The ethylene signaling pathway has a negative impact on sucrose-induced anthocyanin accumulation in

J Plant Res (2011) 124:193–200 DOI 10.1007/s10265-010-0354-1


The ethylene signaling pathway has a negative impact on sucrose-induced anthocyanin accumulation in Arabidopsis
Yerim Kwon ? Jee Eun Oh ? Hana Noh Suk-Whan Hong ? Seong Hee Bhoo ? Hojoung Lee

Received: 23 December 2009 / Accepted: 15 April 2010 / Published online: 1 June 2010 ? The Botanical Society of Japan and Springer 2010

Abstract In an attempt to understand the complex regulatory mechanisms underlying sucrose-induced ?avonoid biosynthesis, we examined several Arabidopsis mutants with altered anthocyanin accumulation. We determined that disruption of ethylene signaling results in a dramatic increase in sucrose-induced anthocyanin accumulation. Furthermore, we investigated why the ein2-1 (ethylene insensitive) Arabidopsis mutant accumulates higher levels of anthocyanin in response to sucrose than wild-type Arabidopsis. An increased level of PAP1 transcript in the ein2-1 mutant appears to be the main factor responsible for the increased accumulation of anthocyanin in response to sucrose. Therefore, our results indicate that the ethylene signaling pathway plays a negative role in sucrose-induced anthocyanin accumulation. We believe that the explanation

for this observation may be related to the initiation of the senescence program in plants. Keywords Anthocyanin ? Arabidopsis thaliana ? EIN2 ? PAP1 ? Sucrose

Introduction Flavonoids are products of plant secondary metabolism that confer multiple advantages to the plant throughout its life cycle. Flavonoids are a functionally diverse group of molecules that participate in processes such as active oxygen scavenging, UV absorption, plant defense, and signaling between autotrophs and heterotrophs (Tahara 2007). Many studies have focused on elucidating the mechanisms by which ?avonoids affect cell function, and it has been established that these compounds possess conventional hydrogen-donating activity. Moreover, recent studies reveal that ?avonoids may also modulate the regulation of enzymes, receptors, signaling cascades, and gene expression (Williams et al. 2004; Sarkar and Li 2004). Anthocyanins are a group of ?avonoids known to possess anti-in?ammatory and anti-diabetic properties (Wang et al. 1999; Cignarella et al. 1996; Winkel-Shirley 2001). Anthocyanin synthesis is known to be increased by various environmental stresses, such as chilling, freezing, desiccation, wounding, and pathogenic infection (Christie et al. 1994; McKown et al. 1996, Ferreres et al. 1997; Farrant 2000). The anthocyanin biosynthetic pathway has been investigated in various plant systems (Holton and Cornish 1995; Bharti and Khurana 1997). The transcription factors reported to be involved in this process clearly indicate that developmental, environmental, and metabolic signal transduction pathways regulate the production of

Y. Kwon and J. E. Oh contributed equally.

Electronic supplementary material The online version of this article (doi:10.1007/s10265-010-0354-1) contains supplementary material, which is available to authorized users.
Y. Kwon ? J. E. Oh ? H. Lee (&) College of Life Sciences and Biotechnology, Korea University, 1, 5-ka Anam-dong, Sungbuk-ku, Seoul 136-713, Republic of Korea e-mail: lhojoung@korea.ac.kr H. Noh ? S.-W. Hong Division of Applied Plant Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea S. H. Bhoo Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Yong-In 446-701, Republic of Korea



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?avonoids (Mathews et al. 2003; Broun 2004; Matsui et al. 2004). In addition to serving as a source of energy and as structural components in plant growth and development, sugars act as signaling molecules that trigger various plant responses (Jang et al. 1997). It is well established that sucrose is a strong inducer of ?avonoid biosynthesis (Solfanelli et al. 2006), and is known to induce the accumulation of anthocyanin in a variety of plant species (Solfanelli et al. 2006; Nagira and Ozeki 2004; Nagira et al. 2006). Chalcone synthase catalyzes the ?rst step of the phenylpropanoid pathway that leads to ?avonoid production For example, chalcone synthase (CHS) genes catalyzes the ?rst step of the phenylpropanoid pathway that leads to ?avonoid production and were shown to contain Suc boxes in their 50 ?anking regions in Petunia hybrid and Arabidopsis thaliana (Tsukaya et al. 1991). To date, numerous lines of evidence have shown that the signaling pathways linking sucrose to anthocyanin accumulation involve various molecular components. A study by Vitrac et al. (2000) showed that several components common to multiple signal transduction pathways, such as Ca2?, calmodulin, and protein kinases/phosphatases, are involved in the induction of anthocyanin biosynthesis by sugar. However, it appears that plant sugar responses are complex, because they are modulated by additional signaling pathways, such as light, auxin, gibberellin, cytokinin, and ethylene pathways (Dijkwel et al. 1997; DeWald et al. 1994; Perata et al. 1997; Nemeth et al. 1998; Zhou et al. 1998). The molecular mechanism underlying the induction of anthocyanin accumulation in Arabidopsis in response to sucrose remains unknown. Therefore, we focused on further exploration of established studies showing that ethylene in?uences sucrose signaling with respect to seedling development (Rolland et al. 2006). The authors revealed a negative role for ethylene in the sucrose signaling pathway by demonstrating that sugar affected the stability of the transcription factor EIN3 (Yanagisawa et al. 2003). In the current study, we examined whether ethylene signaling mutants accumulate more anthocyanin than wild-type plants in response to sucrose. Furthermore, we investigated a possible molecular target of the cross-talk between ethylene and sucrose signaling pathways.

50 mM sucrose. Seeds were allowed to germinate and grow in a growth chamber at 22 ± 1°C with 16 h of light (100 lmol m-2 s-1 illumination)/8 h of darkness (72% relative humidity) before use. The CHS-GUS transgenic line was generated by fusing the promoter region (*1 kb from ATG) to the GUS reporter gene (vector pBI121). Measurement of anthocyanin content Determination of anthocyanin content was carried out by the spectrophotometric method described previously (Bariola et al. 1999). Brie?y, tissues were ground to a ?ne powder in liquid nitrogen and dissolved in 5 ml of extraction buffer [80% (v/v) methanol and 1% (v/v) HCl]. The supernatant was collected after centrifugation at 13,000g for 30 min and combined with 2 ml of chloroform. The absorbance of the aqueous/methanol phase was measured at A530 and A657, and the resulting values were normalized against the fresh weight of each sample to represent the anthocyanin content. Northern blot analysis and qRT-PCR Plants were grown on standard MS medium (0.6% agar, 50 mM sucrose) in a growth chamber for 14 days. The seedlings were then treated with ABA, or sucrose, alone or in combination, for the indicated period of time. The method used to extract total RNA from whole seedlings (*0.1 g) was described previously (Lee et al. 2002). Total RNA (20 lg in each lane) was separated by electrophoresis on formaldehyde-agarose gels [1.5% (w/v) agarose, 38% TM (w/v) formaldehyde], and transferred to a Hybond -XL membrane (Amersham Biosciences, NY, USA). Prehybridization was performed at 42°C for 1 h in prehybridization buffer (109 SSC, 509 Denhardt’s solution, 20% (w/v) SDS, 10 mg/ml denatured salmon sperm DNA). Probes labeled by the 32P-random-priming method were separately hybridized onto the blot at 42°C for 16 h. The blot was then washed twice at 45°C in 19 SSC and 0.1% SDS for 30 min, and then washed again at 45°C in 0.19 SSC and 0.1% SDS for 30 min. The membrane was then exposed and the ?lm was developed. Speci?ed DNA probes were obtained by PCR using the following primer pairs: PAP1 (50 -ATG GAG GGT TCG TCC AAA G-30 and 50 -CAA GGT GCT CCC CTT TTC TGT T-30 ), CHS (50 -ATG GTG ATG GCT GGT GCT TC-30 and 50 -TTA GAG AGG AAC GCT GTG CA-30 ). Probes were radiolabeled using a random primer kit (Amersham Bioscience). For qRT-PCR, total RNA was isolated from whole seedlings by TRI Reagent (MRC, Inc.). In brief, 1 lg of total RNA was subjected to cDNA synthesis using Oligo dT and Reverse Transcriptase (Promega). Quantitative Real-time PCR was performed with 10 ng of cDNA using

Materials and methods Plant materials, growth conditions, and stress treatment Arabidopsis thaliana Col-0, axr4, ein2, ein3, ein4, ein5, ein7, and eto1 were obtained from ABRC (Arabidopsis Bilolgical Resource Center) and seeded on MS (Murashige and Skoog salt base) agar medium supplemented with


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29 iQ SYBR Green Supermix by iCycler iQ (Bio-Rad). The relative expression level of the PAP1 transcript was calculated using the method described by Vandesompele et al. (2002). As an internal control to normalize the data, actin7 was used and relative expression level of the PAP1 transcript was calculated by calibrating with threshold cycles for ampli?cation of PAP1 and Actin7. The relative transcript level of PAP1 was detected by Gene Expression Analysis program for iCycler iQ Real-Time PCR Detection System (Vandesompele et al. 2002). Each experiment was repeated at least three times to con?rm statistical signi?cance. Quantitative real time PCR primers are as follows. PAP1-F: 50 -attcctacaacaccggcact-30 , PAP1-R: 50 -tgtccc ccttttctgttgtc-30 , actin7-F: 50 -GGA CCT GAC TCA TCG TAC TC-30 , actin7-R: 50 -TAC AGT GTC TGG ATC GGA GG-30 . ROS (H2O2) and GUS staining For the detection of H2O2, 7-day-old seedlings were examined according to the method described by ThordalChristensen et al. (1997), with some modi?cations. First, the seedlings were incubated with PBT buffer (50 mM potassium phosphate buffer) ?ve times before incubation for 10 min in 1 ml PBT buffer supplemented with 0.3 mg DAB (3,3-diamionbenzidine). Next, 1 ll 30% H2O2 was added to each tube for the color reaction. Each seedling was rinsed twice with PBT for 5 min before dehydration of the sample with 100% methanol. For histochemical GUS assays, 10-day-old Arabidopsis seedlings were treated or not with sucrose, ACC or AgNO3 as indicated in the ?gures. Whole seedlings were incubated at 37°C overnight before chlorophyll was removed with 70% ethanol at 70°C (Jefferson et al. 1987). Chlorophyll content measurement Chlorophyll content of leaf discs was determined as described previously (Tzvetkova-Chevolleau et al. 2007). All experiments were performed at least three times.

the increased expression of dihydro?avonol reductase (DFR) and anthocyanidin synthase (ANS) genes upon exposure to sucrose (Gollop et al. 2002). However, the mechanism underlying sucrose-mediated ?avonoid accumulation remains elusive. Our initial goal was to explore the regulatory mechanism of sucrose-induced anthocyanin accumulation. Because several phytohormones are known to regulate the expression of genes involved in ?avonoid biosynthesis (Jang et al. 1997; Pasqua et al. 2005; Loreti et al. 2008; Teng et al. 2005), we began by measuring the anthocyanin content in several mutants with altered hormone signaling. Anthocyanins provide defense against cellular damage caused by solar radiation and absorb light at frequencies in ¨ ndel et al. 2006). Therefore, expothe near-UV range (Pfu sure to high levels of sucrose may signal plant cells that their photosynthetic machinery is fully active and induce the biosynthesis of anthocyanin in an effort to protect the plant from the harmful effects of UV irradiation. As shown in Fig. 1, supplementation of the media with high levels of sucrose signi?cantly increased the amount of anthocyanins in wild-type plants. Five-day-old wild-type and various

Results and discussion The ein2-1 mutant accumulates high levels of anthocyanin in response to sucrose Sucrose is a potent inducer of ?avonoid accumulation, and plants grown on sucrose-containing medium accumulate high levels of anthocyanins (Baier et al. 2004; Ohto et al. 2001). The induction of anthocyanin biosynthesis by sucrose can be explained by the presence of sucrose boxes in the Arabidopsis CHS gene (Nagira and Ozeki 2004) and

Fig. 1 Anthocyanin accumulation in wild-type and axr4, ein2-1, eto1, and tt4 mutant seedlings in response to sucrose. a Anthocyanin content in wild-type and axr4, ein2-1, eto1, and tt4 mutant seedlings treated with sucrose. Five-day-old seedlings germinated in normal MS-agar medium (50 mM sucrose) were transferred to the designated medium supplemented with various concentrations of sucrose, allowed to grow for 3 days, and then collected for anthocyanin measurement. The data represent the mean values of three independent experiments (n = 50). b Anthocyanin content of wild-type (col0) and ein2, ein3, ein4, ein5, and ein7 mutant seedlings treated with sucrose. Seedlings were subjected to the same treatment as in a. The data represent the mean values of three independent experiments (n = 50)


196 Fig. 2 The ein2-1 mutant has a high level of anthocyanin. a Wild-type and ein2-1 mutant plants were germinated on MS medium with 0, 50, 100, 150 or 200 mM sucrose. Seedlings were photographed 21 days after germination. b Anthocyanin content of seedlings 10 days after germination. Data represent the mean values of three independent experiments (n = 50)

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mutant seedlings germinated in MS medium with 50 mM sucrose were transferred to test medium supplemented with 50, 100, 150, or 200 mM sucrose. Among them, the ethylene-insensitive mutant, ein2-1, exhibited a high level of anthocyanin accumulation in response to sucrose (Fig. 1a). In comparison to wild-type plants, ein2-1 mutants experienced an almost 200% increase in anthocyanin accumulation when grown in 200 mM sucrose medium. Other mutant plants, such as axr4 and eto1, did not accumulate increased amounts of anthocyanin in response to sucrose compared to wild-type. The tt4 mutant is a weak allele of chalcone synthase gene. We next examined whether other ethylene-insensitive mutants accumulated increased amounts of anthocyanin in response to high levels of sucrose. Hence, a series of ethylene-insensitive mutants, including ein3, ein4, ein5, and ein7, were assessed for increased anthocyanin accumulation in response to sucrose (Fig. 1b). To examine the germination phenotype in sucrose-containing medium, wild-type and ein2-1 plants were seeded directly onto MS medium with 0, 50, 100 or 200 mM sucrose. As shown in Fig. 2a, ein2-1 plants developed red pigmentation when subjected to concentrations of sucrose of at least 100 mM whereas the wild-type plants did not develop red pigmentation until the sucrose concentration reached at least 200 mM. Intriguingly, the ein2-1 mutant struggled to grow in 200 mM sucrose medium, although the reason for this is unknown. As demonstrated in Fig. 2b, anthocyanin levels were approximately eight-fold higher in the ein2-1 mutant than in the other genotypes tested when grown in high levels of sucrose (200 mM). However, we did not observe a signi?cant difference in the chlorophyll contents of wildtype and ein2-1 mutant plants grown in medium containing 50, 100, or 150 mM sucrose (Fig. 3). Disruption of the EIN2 is known to cause a substantial increase in the level of abscisic acid (ABA) (Wang et al. 2007). As reported previously, we also observed that, in comparison with wild-

Fig. 3 Chlorophyll contents of wild-type and ein2-1 mutant plants. Chlorophyll content of wild-type and ein2-1 seedlings grown for 18 days on MS-agar medium supplemented with 0, 50, 100 or 150 mM sucrose. Data represent the mean values of three independent experiments (n = 50)

type, the ein2-1 mutant was hyper-sensitive to 0.1 lM ABA during germination (Beaudoin et al. 2000; Ghassemian et al. 2000). Thus, it is plausible for us to reason that the ein2-1 mutant may have increased anthocyanin levels because it already contains more ABA than the wildtype. Mannitol treatment increased the level of anthocyanins more in the ein2-1 than the wild type, but its level was much lower in comparison to those of sucrose treatment (Fig. S1). Inhibition of ethylene biosynthesis enhanced anthocyanin accumulation in wild-type and ein2-1 mutant plants We next sought to determine why the disruption of ethylene signaling has such a strong effect on the biosynthesis of anthocyanins in the presence of high levels of sucrose (Fig. 1b). We hypothesized that disruption of ethylene signaling results in an increase in ethylene because the plant cells try to compensate for the impaired ethylene signaling response by synthesizing more ethylene. If elevated levels of ethylene are the cause of the increased


J Plant Res (2011) 124:193–200 Fig. 4 Anthocyanin contents and expression of CHS in wildtype and ein2-1 plants in response to sucrose or ACC. a Ten-day-old CHSpro-GUS (wild-type) seedlings germinated in normal MS-agar medium (50 mM sucrose) were transferred for incubation under the indicated conditions for 6 h, and then whole seedlings were incubated in GUS assay solution for 15 h before being photographed. b The anthocyanin content of seedlings shown in (a) 10 days after germination. Data represent the mean values of three independent experiments (n = 50)


levels of anthocyanins, the eto1 mutant should also accumulate a high level of anthocyanins in response to sucrose. However, our observations show that this is not the case (Fig. 2). In addition, treatment with the ethylene precursor, ACC, resulted in a reduction in anthocyanin accumulation in response to sucrose (Fig. 4). To examine whether disruption of ethylene reception affects anthocyanin accumulation in response to sucrose, we used the ethylene receptor blocker, silver nitrate (AgNO3) (Fig. 5). In agreement with our previous observation, AgNO3 signi?cantly increased the amount of anthocyanin accumulation in the ein2-1 mutant. A similar pattern of increased anthocyanin accumulation in the ein2-1 mutant was observed when ethylene signaling was blocked with AgNO3 treatment in the presence of sucrose (Fig. 5). These results demonstrate that ethylene inhibits anthocyanin accumulation in the ein2-1 mutant in response to sucrose. Although reactive oxygen species (ROS) act as second messenger molecules that are governed by various environmental stimuli in the regulation of gene expression and in signaling pathways, we did not detect a difference in the levels of ROS between wild-type and ein2-1 mutant plants in response to sucrose, ABA, or ABA plus sucrose (data not shown). The ein2-1 mutant has elevated levels of PAP1 transcript in response to elevated levels of sucrose To decipher the mechanism responsible for increased anthocyanin accumulation in the ein2-1 mutant, we examined the expression levels of genes known to be

involved in sucrose-mediated anthocyanin accumulation. The production of anthocyanin pigment (PAP1) gene encodes a transcription factor with a myb domain. Its transcript rapidly accumulates to high levels before anthocyanin starts to accumulate (Vom Endt et al. 2002). As shown in Fig. 6a, 50 mM sucrose induced the accumulation of PAP1 transcript after 15 h of treatment, and a more rapid induction of this gene was obtained following treatment with 100 mM sucrose. In both cases, the level of PAP1 transcript increased over the 24-h treatment period. CHS had a similar pattern of expression in response to sucrose (Fig. 6a). To establish whether the PAP1 transcript exhibited differential responses to sucrose in ein2-1 and wild-type plants, real-time PCR was performed (Fig. 6b). The level of PAP1 transcript was higher in the ein2-1 mutant than the wild-type upon exposure to 50, 100, 150, or 200 mM sucrose for 6 h. However, the difference in PAP1 transcript level was smallest between the mutant and the wild-type in the presence of 200 mM sucrose. To date, several transcription factors, including MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and a WD40 protein (Stracke et al. 2001), have been shown to control the biosynthesis of ?avonoids in a cooperative manner in many species. In addition, some MYB transcription factors, such as AtPAP1, AtPAP2, PhAN2, LeANT1, and GMYB10, can activate anthocyanin biosynthesis by themselves (Mathews et al. 2003; Quattrocchio et al. 1999; Borevitz et al. 2000; Elomaa et al. 2003). The PAP1 gene was ?rst identi?ed by Borevitz et al. (2000), who revealed that the mutant PAP1-Dominant (pap1-D) exhibited widespread activation of genes involved in



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Fig. 5 The expression of CHS in wild-type and ein2-1 plants in response to sucrose or AgNO3, or in the presence of a combination of sucrose and AgNO3. a Ten-day-old CHSpro-GUS (wild-type) and ein2-1/CHSpro-GUS seedlings germinated in normal MS-agar medium (50 mM sucrose) were transferred for incubation under the

indicated conditions for 6 h, and whole seedlings were then incubated in GUS assay solution for 15 h before they were photographed. b The anthocyanin content of the seedlings shown in a after 10 days of germination. Data represent the mean values of three independent experiments (n = 50)

senescence program would be triggered in the plant. Because photosynthetic pigments would no longer be required to protect the leaves during the senescence program, ethyleneinduced senescence would lead to the inhibition of anthocyanin biosynthesis by the down-regulation of PAP1 expression. However, this situation would be different in the petals or sepals, where coloration is part of the senescence process (Yoshida et al. 2008).
Acknowledgments This work was supported by a grant from the National Research Foundation (to Hojoung Lee, 2009; Grant #20090078046 & #2009-0065693) and in part by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (to Hojoung Lee; Grant #108066-3). Fig. 6 Northern analysis of PAP1 and CHS levels in wild-type and ein2-1 plants in response to sucrose. Wild-type and ein2-1 seedlings were grown on normal growth medium for 10 days before being subjected to sucrose treatment for the indicated period of time. Samples were then collected for northern blot analysis a or real-time PCR b. Transcript levels of PAP1 and CHS are shown. rRNA is shown as a loading control

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