Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha.

Phytochemistry xxx (2015) xxx–xxx

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Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha Akito Kageyama a, Kimitsune Ishizaki b, Takayuki Kohchi c, Hideyuki Matsuura a, Kosaku Takahashi a,⇑ a

Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Graduate School of Science, Kobe University, Kobe 657-8501, Japan c Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan b

a r t i c l e

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Article history: Received 21 January 2015 Received in revised form 11 May 2015 Accepted 16 May 2015 Available online xxxx Keywords: Common liverwort Marchantiophyta Marchantia polymorpha Abscisic acid Bisbibenzyl Secondary metabolite UV-C irradiation

a b s t r a c t Environmental stresses are effective triggers for the biosynthesis of various secondary metabolites in plants, and phytohormones such as jasmonic acid and abscisic acid are known to mediate such responses in flowering plants. However, the detailed mechanism underlying the regulation of secondary metabolism in bryophytes remains unclear. In this study, the induction mechanism of secondary metabolites in the model liverwort Marchantia polymorpha was investigated. Abscisic acid (ABA) and ultraviolet irradiation (UV-C) were found to induce the biosynthesis of isoriccardin C, marchantin C, and riccardin F, which are categorized as bisbibenzyls, characteristic metabolites of liverworts. UV-C led to the significant accumulation of ABA. Overexpression of MpABI1, which encodes protein phosphatase 2C (PP2C) as a negative regulator of ABA signaling, suppressed accumulation of bisbibenzyls in response to ABA and UV-C irradiation and conferred susceptibility to UV-C irradiation. These data show that ABA plays a significant role in the induction of bisbibenzyl biosynthesis, which might confer tolerance against UV-C irradiation in M. polymorpha. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The study of plant secondary metabolism is one of the major fields in plant physiology research. Secondary metabolites in plants are involved not only in defense, but also a wide variety of physiological processes such as growth, development, signaling, senescence, and stress adaptation. Several secondary metabolites are known to accumulate in plants subjected to environmental stresses, and they can play significant roles in adapting to adverse environmental conditions (Nascimento and Fett-Neto, 2010). In flowering plants, the relationships between stresses and secondary metabolism have been studied extensively. For example, the synthesis of antimicrobial compounds is induced by pathogenic infection and wounding, herbivore attack triggers insect antifeedant production, and UV irradiation causes plants to produce photoprotective compounds that are recognized as UV-absorbing substances, such as phenolic compounds (Nascimento and Fett-Neto, 2010). In flowering plants, several phytohormones are known to play pivotal roles in stress responses. Jasmonic acid (JA) regulates various physiological processes, especially responses against ⇑ Corresponding author. Tel.: +81 11 706 3349; fax: +81 11 706 2505. E-mail address: [email protected] (K. Takahashi).

wounding, and has been shown to stimulate the production of many secondary metabolites, such as alkaloids, terpenoids, and flavonoids (Wasternack and Hause, 2013), as a defense response. Abscisic acid (ABA), the other major plant hormone, mediates various responses against environmental stresses, such as drought and high osmotic stresses (Hauser et al., 2011). Under drought or high osmotic conditions, ABA-mediated signaling rapidly alters the osmotic potential by the accumulation of compatible solutes in the stomatal guard cells, causing them to shrink and the stomata to close. In contrast to JA, ABA is not a ubiquitous signal molecule inducing synthesis of secondary metabolites in flowering plants. Bryophytes, including Marchantiophyta (liverworts), Bryophyta (mosses), and Anthocerotophyta (hornworts), are taxonomically positioned between algae and vascular plants (Asakawa, 1982; Asakawa et al., 2013a,b; Qiu et al., 2006; Bowman et al., 2007) and constitute an early diverging lineage of land plants. Therefore, bryophytes occupy a key evolutionary position and may help us to understand the molecular basis of the key innovations that allowed green plants to evolve from aquatic ancestors and adapt to a terrestrial environment (Bowman et al., 2007). Like flowering plants, bryophytes are known to produce various secondary metabolites, and several have been utilized as medicinal plants (Asakawa, 1982; Asakawa et al., 2013a,b). Nonetheless, there are few studies of the relationships among environmental

http://dx.doi.org/10.1016/j.phytochem.2015.05.009 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kageyama, A., et al. Abscisic acid induces biosynthesis of bisbibenzyls and tolerance to UV-C in the liverwort Marchantia polymorpha. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.05.009

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A. Kageyama et al. / Phytochemistry xxx (2015) xxx–xxx

stresses, plant hormones, and secondary metabolism in bryophytes. In the model moss Physcomitrella patens, UV-B irradiation is known to induce expression of the genes encoding chalcone synthases, which are biosynthetic enzymes leading to flavonoids, and flavonoid synthesis (Wolf et al., 2010). Production of momilactones A and B, which function as allelochemicals, are also induced by UVC irradiation, elicitors, and JA in the moss Hypnum plumaeforme (Kato-Noguchi, 2011). The role of ABA in stress adaptation has also been studied in bryophytes. In P. patens, ABA recruits the mechanism for adaptation to environmental stresses such as drought, osmotic stress, high salinity, and cold (Takezawa et al., 2011). In liverworts, the presence of ABA was reported in Marchantia polymorpha (Li et al., 1994). Furthermore, a negative regulator of ABA signaling, MpABI1, which encodes a protein of the carboxy-terminal protein phosphatase 2C (PP2C) domain, is known to participate in the ABA signaling system in M. polymorpha (Tougane et al., 2010). Thus, ABA has been suggested to play a central role in stress responses in bryophytes. Interestingly, it has been proposed that the family of PP2C, which act as negative regulators of ABA signaling, was acquired in land plants to suppress tolerance mechanisms for desiccation, possibly facilitating plants propagation on land (Komatsu et al., 2013). Given that ABA is a major plant hormone for stress adaptation in bryophytes, it can be hypothesized that ABA and environmental stresses could induce secondary metabolism as a stress adaptation in bryophytes. To test this hypothesis, the liverwort M. polymorpha was studied as a model system. M. polymorpha is being established as an experimental model organism because of its critical evolutionary position (Bowman et al., 2007) and the availability of molecular genetic tools, including transformation techniques (Ishizaki et al., 2008; Kubota et al., 2013) and gene-targeting strategies (Ishizaki et al., 2013). In this study, it was demonstrated that ABA plays important roles in the induction of biosynthesis of secondary metabolites and, moreover, in UV-C tolerance in the model liverwort M. polymorpha. 2. Results 2.1. Identification of compounds induced by treatment with ABA The effect of ABA treatment on secondary metabolism was examined in M. polymorpha grown for 30 days on 0M51C agar medium with or without 10 lM ABA supplementation. Plants were extracted with ethanol, and the resulting extract was analyzed by a reversed phase HPLC (Fig. 1a). As a result of ABA treatment, the intensities of peaks 1, 2, and 3 increased. Next, identification of the compounds induced by ABA treatment in M. polymorpha was attempted. To do this plants (400 g) were first soaked in ethanol. Then, several steps of chromatography afforded compounds 1 (11.0 mg, peak 1), 2 (1.8 mg, peak 2),

and 3 (1.1 mg, peak 3). UV spectral analysis of these isolated compounds showed a maximum absorbance at approximately 280 nm with similar UV absorbing profiles, suggesting that compounds 1, 2, and 3 have similar structures. MS spectral data demonstrated that the molecular formulae of compounds 1, 2, and 3 were C28H24O4 (found m/z, 424.16679; calculated, 424.16746), C28H24O4 (found m/z, 424.16566; calculated, 424.16746), and C29H26O4 (found m/z, 438.18382; calculated, 438.18311), respectively. The 1H NMR spectra of these compounds showed that most proton signals were derived from aromatic protons (dH 6.5– 8.0 ppm, Figs. S1–S3). Moreover, the characteristic olefin proton signal of bisbibenzyls, which was shifted to approximately dH 5.5 ppm, was observed in the 1H NMR spectra of these compounds. Through detailed investigations of MS and NMR spectroscopic data (Table S1), compounds 1, 2, and 3 were determined as isoriccardin C (1), marchantin C (2), and riccardin F (3), respectively (Asakawa et al., 1987; Speicher and Holz, 2010; Yoshida et al., 1996) (Fig. 1b). These compounds were categorized as bisbibenzyls, which are characteristic secondary metabolites of liverworts. Bisbibenzyls have a wide variety of biological activities such as antibacterial and anticancer activities (Asakawa et al., 2013a,b; Harrowven and Kostiuk, 2012), and their pharmacological actions have recently attracted attention. UPLC MS/MS analysis of 1, 2, and 3 showed that their concentrations were 2.5-, 1.7-, and 2.3-fold greater, respectively, in plants treated with 10 lM ABA, relative to untreated plants (Fig. 2). 2.2. Dose-dependent effect of ABA on accumulation of bisbibenzyls As 10 lM ABA induced the synthesis of bisbibenzyls in M. polymorpha, it was next investigated as to whether bisbibenzyls accumulated in an ABA-dose-dependent manner in M. polymorpha grown for 30 days (Fig. 2). The accumulation of 1 was significantly induced by treatment with 1 lM ABA. The increase of 1 and 2 was dose-dependent at ABA concentrations of 1 to 100 lM. The concentrations of 1 and 2 in plants treated with 100 lM ABA were 3.0- and 4.0-fold higher, respectively, than in untreated plants. In contrast, the amount of 3 was highest in the 10-lM ABA treatment, decreasing thereafter to approximately 80% of its maximum level in the 100-lM treatment. At a concentration of 100 lM, ABA might induce the expression of an additional metabolic enzyme, leading to a decline in the internal concentration of 3. Taken together, these results indicate that the concentrations of bisbibenzyls 1–3 increased depending on ABA concentration. 2.3. Elevation of ABA concentration in M. polymorpha subjected to UV-C treatment In flowering plants and the model moss P. patens, the accumulation of UV-absorbing compounds was triggered by UV irradiation

Fig. 1. Identification of bisbibenzyls induced by ABA in M. polymorpha. (a) HPLC analysis of ethanol extract of M. polymorpha grown on 0M51C agar medium with or without 10 lM ABA treatment for 30 days. Upper chromatogram; ABA treatment, lower chromatogram; control. Peaks 1, 2, and 3 are derived from isoriccardin C (1), marchantin C (2), and riccardin F (3), respectively. HPLC conditions are described in Experimental. (b) The structures of isoriccardin C (1), marchantin C (2), and riccardin F (3).



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bisbibenzyls in liverworts may be induced for protection against UV-C stress. The initial steps of the presumed biosynthetic pathway of bisbibenzyls have been proposed to overlap with the phenylpropanoid pathway (Friederich et al., 1999). Although the biosynthetic pathway of bisbibenzyls has not been confirmed, up-regulation of the phenylpropanoid pathway by UV-C might result in accumulation of bisbibenzyls. As opposed to UV-C, UV-B irradiation did not cause accumulation of bisbibenzyls in this study (data not shown). This result is consistent with the fact that flavonoids, which are also biosynthesized through the phenylpropanoid pathway, are not increased by UV-B irradiation in M. polymorpha (Markham et al., 1998). Fig. 2. Accumulation of bisbibenzyls in an ABA-dose-dependent manner. Plants were grown on 0M51C agar medium supplemented with ABA at 22 °C for 30 days. ABA concentration ranged from 1 to 100 lM. Data represent means ± s.d. of five separate experiments. Amounts of bisbibenzyls were analyzed by UPLC-MS/MS. The asterisks represent significant differences between the plants treated with ABA and the control plants (Student’s t-test, **p < 0.01, *p < 0.05).

2.5. Reduced response to ABA and UV-C in transgenic M. polymorpha overexpressing MpABI1

In plants, phenolic compounds such as flavonoids can accumulate by UV irradiation treatment as a defense system against UV stress (Roda et al., 2003; Emiliani et al., 2009). The data here showed that UV-C markedly elevated ABA levels and that ABA stimulated the biosynthesis of bisbibenzyls. Analysis of the concentration of bisbibenzyls 1–3 in M. polymorpha subjected to UVC irradiation established that the concentration of bisbibenzyls 1–3 in treated plants was significantly higher than in untreated plants (Fig. 4). This result suggests that ABA is involved in the mechanism of UV-C stress adaptation; i.e., ABA accumulation induced by UV-C triggers the biosynthesis of bisbibenzyls in M. polymorpha. These data suggest that the biosynthesis of

Molecular characterization of MpABI1, which encodes PP2C, has suggested that PP2C can act as a negative regulator in ABA signal transduction in M. polymorpha (Tougane et al., 2010). Overexpression of MpABI1 is presumed to result in an ABA-insensitive phenotype in M. polymorpha. To investigate the participation of ABA signal transduction in the biosynthesis of bisbibenzyls, transgenics of M. polymorpha overexpressing MpABI1 were generated. The size of these MpABI1 overexpressors was nearly the same as that of the empty vector control (Fig. 5) in the absence of ABA. When grown on agar medium supplemented with 10 lM ABA, the MpABI1 overexpressor showed enhanced growth relative to control plants (Fig. 5). The growth inhibitory activity of ABA was canceled by MpABI1 overexpression, suggesting that PP2C functions as a negative regulator of ABA in M. polymorpha. These data provide the first evidence that PP2C-mediated signal transduction is functional in a liverwort, M. polymorpha; previous analyses of the functions of MpABI1 have mainly been performed heterologously using P. patens (Tougane et al., 2010). Next, the influence of MpABI1 overexpression on the tolerance against UV-C damage was examined (Fig. 6). It was observed that UV-C irradiation resulted in severe damage to the MpABI1 overexpressor. In the MpABI1 overexpressor, the color of the thalli was changed significantly and internal fluid steeped out from the thalli, whereas in control plants, the color of the thalli had only turned slightly brownish and the UV-C damage was not as serious. The superficial tissues of the thalli appeared to suffer under UV-C irradiation in the MpABI1 overexpressor. These results indicate that MpABI1 overexpression reduced the response to ABA and conferred susceptibility to UV-C stress in M. polymorpha. Moreover, ABA showed an improved tolerance to UV-C in wild type plants and the vector controls (Fig. 6). These results indicate that ABA is essential for mediating stress adaptation to UV-C in M. polymorpha.

Fig. 3. Accumulation of ABA after UV irradiation. Marchantia polymorpha was grown on a 0M51C agar medium for 20 days and subjected to UV-B (280-320 nm) or UV-C (predominantly 254 nm) for 30 min each day for 5 days. ABA concentration was analyzed by UPLC-MS/MS. Data represent means ± s.d. of five separate experiments. The asterisk represents significant differences between the plants subjected to UV-C and the control plants (Student’s t-test, *p < 0.05).

Fig. 4. Accumulation of bisbibenzyls induced by UV-C irradiation. Marchantia polymorpha was grown on a 0M51C agar medium for 30 days and then subjected to intermittent UV-C irradiation for 30 min each day for 5 days. Data represent means ± s.d. of five separate experiments. The asterisks represent significant differences between the plants subjected to UV-C and the control plants (Student’s t-test, **p < 0.01).

(Wolf et al., 2010). As ABA was shown in the present study to elevate the concentration of bisbibenzyls, which have the capability to absorb UV, it was hypothesized that ABA is involved in UV-stress adaptation. Therefore, the accumulation levels of ABA in M. polymorpha subjected to UV irradiation were analyzed using UPLC-MS/MS. Under the experimental conditions of the present study, the ABA concentration in M. polymorpha subjected to UV-C irradiation was approximately 20 times larger than that in control plants (Fig. 3). This result demonstrated that UV-C has potent stimulatory effects on the elevation of endogenous ABA in M. polymorpha. In contrast, UV-B failed to increase ABA concentration (Fig. 3), showing that ABA is not involved in the response to UV-B in M. polymorpha. 2.4. Induction of accumulation of bisbibenzyls by UV-C irradiation


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Fig. 5. Effects of ABA on the phenotype of transgenic M. polymorpha overexpressing MpABI1. (a) Phenotypic observation of the transgenic M. polymorpha overexpressing MpABI1 (OX) and the empty vector control (VC). Each strain was grown on 0M51C agar medium at 22 °C for 20 days under white fluorescent light with or without 10 lM ABA. (b) Fresh weight of OX and VC. Growth conditions were as described above. Data represent means ± s.d. of five separate experiments. The asterisks represent significant differences between the ABA treated plants and the control plants (Student’s t-test, **p < 0.01).

were treated with ABA, the amounts of bisbibenzyls 1 and 2 in the MpABI1 overexpressor were significantly less than those in control plants. The amount of 3 also tended to decrease in the MpABI1 overexpressor, relative to that of control plants. Accordingly, ABA signaling was also found to play a significant role in the production of bisbibenzyls. When plants were subjected to UV-C irradiation, no significant differences in the amounts of 1 and 3 were found between the MpABI1 overexpressor and control plants. There was a tendency to decrease the amount of bisbibenzyls in the UV-C treated MpABI1 overexpressor. Although the effect of UV-C on the biosynthesis of bisbibenzyls was large, the inhibitory effect of MpABI1 overexpression on ABA signaling did not fully suppress the production of bisbibenzyls in response to UV-C. Fig. 6. Influence of UV-C irradiation on transgenic M. polymorpha overexpressing MpABI1. WT, VC, and OX represent wild-type, vector control, and MpABI1 overexpressor plants, respectively. Each strain was grown on 0M51C agar medium with or without 10 lM ABA at 22 °C for 20 days. For UV-C treatment, plants were exposed to UV-C light (predominantly 254 nm) for 30 min per day for 5 consecutive days. The scale bar represents 1 cm.

2.6. Quantification of bisbibenzyls in transgenics with MpABI1 overexpression Phenotypic analysis of M. polymorpha transgenics overexpressing MpABI1 indicated that the PP2C, which is encoded in MpABI1, functions as a negative regulator of ABA signaling in M. polymorpha. To investigate whether ABA signaling mediated by MpABI1 is involved in the regulation of biosynthesis of bisbibenzyls, the concentrations of bisbibenzyls 1–3 were analyzed in the MpABI1 overexpressor and control plants (Fig. 7). When plants were grown on agar medium without ABA, the amounts of bisbibenzyls 1–3 in the MpABI1 overexpressor were slightly increased relative to those in control plants. Given that ABA signaling was inhibited in the MpABI1 overexpressors, alteration of the ABA stimulus might influence the concentration of bisbibenzyls only slightly. When plants

3. Discussion Bisbibenzyls are characteristic secondary metabolites in liverworts and have various biological activities such as antimicrobial, anticancer, and antioxidant activities (Asakawa, 1982; Asakawa et al., 2013a,b; Harrowven and Kostiuk, 2012). When M. polymorpha was treated with ABA, the concentrations of three bisbibenzyls, isoriccardin C (1), marchantin C (2), and riccardin F (3), were increased (Fig. 1a). Accumulation of the bisbibenzyls 1–3 increased in a dose-dependent manner of ABA ranging from 1 to 10 lM (Fig. 2). These data show that ABA induces the biosynthesis of bisbibenzyls in M. polymorpha. To our knowledge, this is the first evidence that ABA stimulates secondary metabolism in liverworts. ABA has recently been shown to be involved in the induction of secondary metabolites in several flowering plants, such as Artemisia annua and Arabidopsis thaliana (Loreti et al., 2008; Zhang et al., 2013). The effect of ABA on the activation of secondary metabolism varies among species; therefore, ABA does not ubiquitously activate the synthesis of secondary metabolites in flowering plants. In the basal land plant (the liverwort), the study herein established that overexpression of MpABI1 suppressed synthesis



Fig. 7. Effects of ABA and UV-C on accumulation of bisbibenzyls in transgenic M. polymorpha overexpressing MpABI1. For ABA treatment, the transgenic M. polymorpha overexpressing MpABI1 (OX) and the empty vector control (VC) were grown on 0M51C agar medium supplemented with 10 lM ABA at 22 °C for 30 days. For UV treatment, each strain was exposed to UV-C light (predominantly 254 nm) for 30 min per day for 5 consecutive days. Data represent means ± s.d. of five independent experiments. The asterisks represent significant differences between OX and VC (Student’s t-test, **p < 0.01, *p < 0.05).

of bisbibenzyls in M. polymorpha in response to ABA (Fig. 7). This result is consistent with the ABA-stimulated production of bisbibenzyls in M. polymorpha (Fig. 2). Given that JA was not detected in M. polymorpha (Yamamoto et al., 2015), ABA signaling has a significant impact on the induction of secondary metabolism in M. polymorpha. UV has been shown to be one of the most effective stimulants inducing the production of secondary metabolites in flowering plants. In particular, the synthesis of phenolic compounds such as flavonoids, which are able to absorb UV and eliminate reactive oxygen species (ROS) produced by UV irradiation, is considered to be one of the most important defense systems against UV stress in land plants (Emiliani et al., 2009). Our study showed that M. polymorpha accumulated bisbibenzyls in response to UV-C irradiation (Fig. 4). In a manner similar to phenolic compounds in flowering plants, the function of the accumulation of bisbibenzyls in response to UV-C in M. polymorpha is to protect the plant from UV-C damage. Indeed, the production of bisbibenzyls might be one of the critical stress responses against UV-C in liverworts. As phenylpropanoids have been proposed to participate in the biosynthesis of bisbibenzyls, it is likely that the accumulation of phenolic compounds, which absorb UV, is a common mechanism allowing adaptation to UV stress in land plants (Emiliani et al., 2009). However, MpABI1 overexpression was not sufficient for depressing the synthesis of bisbibenzyls. It is possible that physiological responses, including the ABA-independent signal transduction stimulated by UV-C, cancel out the negative effect of MpABI1 overexpression on ABA signaling, such that the amounts of bisbibenzyls were not decreased entirely. Another reason for the insufficient suppression of the production of bisbibenzyls by MpABI1

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overexpression is that UV-C irradiation activates an unknown ABA signaling pathway bypassing the PP2C. In contrast to UV-C, UV-B did not induce the biosynthesis of bisbibenzyls in M. polymorpha, a result largely consistent with the observation that UV-B does not significantly increase flavonoids (Markham et al., 1998). As UV-C is more damaging to plants than UV-B, it is considered to be a more stimulating environmental cue to induce bisbibenzyls in M. polymorpha. Recently, UV-B irradiation was shown to stimulate the expression of chalcone synthase genes, which might be involved in the biosynthesis of flavonoids in P. patens (Wolf et al., 2010). The phenylpropanoid pathway is considered to have been significant in the defense of the first land plants from environmental stresses (Emiliani et al., 2009). Because phenylpropanoids have been proposed to participate in the biosynthesis of bisbibenzyls, it is likely that the accumulation of phenolic compounds, which absorb UV, is a common mechanism for adapting to UV stress in land plants. ABA is an important signaling molecule in adapting to stresses such as drought, high salinity, and osmotic stress. The framework of ABA signal transduction has been elucidated in A. thaliana (Hauser et al., 2011). PP2C, which is a negative regulator of ABA signaling, is also functional in the model moss P. patens and the model liverwort M. polymorpha (Tougane et al., 2010). The study herein showed that, under our experimental conditions, UV-C yielded significant damage to the transgenic M. polymorpha overexpressing MpABI1 but not to control plants (Fig. 6). Moreover, ABA treatment enhanced UV-C tolerance in wild type plants and the MpABI1 overexpressors (Fig. 6). These results indicate that ABA is a crucial signaling molecule for UV-C tolerance in M. polymorpha. Because the detailed mechanism of ABA functioning against UV-C stress remains unknown, further study is required to uncover the details of ABA signaling in the adaptation to UV-C stress in M. polymorpha. The ABA signaling system elucidated in A. thaliana is conserved in land plants. The role of ABA in adaptation to drought appears to be conserved in land plants, and ABA has acquired additional functions, such as stomatal closure and seed dormancy, during the course of evolution from bryophytes to flowering plants; ABA functions have changed during plant evolution. This study showed that ABA functions as a signaling molecule for UV-C tolerance in M. polymorpha, most likely via induction of secondary metabolites. These ABA functions, which were found in M. polymorpha, have never been shown in flowering plants. Given that M. polymorpha is the model liverwort as a basal land plant, these results suggest two possible hypotheses. One possibility is that ABA signaling for UV tolerance and secondary metabolite induction may have evolved once and then been lost in the vascular plant lineage: vascular plants might have evolved mechanisms other than ABA signaling for UV tolerance and induction of secondary metabolism (e.g., JA-mediated production of phenolic compounds against UV stress). Alternatively, liverworts may have acquired the ABA signaling system for UV tolerance and secondary metabolism independently of vascular plants. Identifying the correct scenario is critical for understanding the evolution of land plants because UV tolerance and the biosynthesis of secondary metabolite play important roles in the successful colonization of plants in the terrestrial environment. Our hypotheses might be addressed by research of ABA functions in plants intermediate between liverworts and vascular plants during plant evolution.

4. Concluding remarks This study showed that ABA induces the biosynthesis of bisbibenzyls, which is suggested to confer tolerance against UV-C irradiation in M. polymorpha. Overexpression of MpABI1, a negative


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regulator of ABA signaling, suppressed ABA responses and the production of bisbibenzyls and, moreover, reduced UV-C tolerance in M. polymorpha. Therefore, it is concluded that ABA is significant for UV-C stress adaptation in M. polymorpha. Given that liverworts represent the most basal lineage in extant land plants, ABA signal transduction, which induces UV tolerance and secondary metabolism, might be a key mechanism protecting early land plants from environmental stresses.

H2O. After evaporation of the EtOAc extract, the extract was separated by a reversed phase HPLC (Capcell pak C18 UG80, 4.6 mm i.d. 250 mm, Shiseido, Tokyo, Japan; solvent, MeOH–H2O–AcOH (70:30:0.1, v/v); UV, 220 nm; flow, 1.0 ml/min). Further preparative TLC purification (SiO2 gel, 0.5 10 20 cm, Merck, Darmstadt, Germany) affords compounds 1 (11.0 mg; solvent, MeOH–CHCl3– AcOH, 5:95:0.1, v/v), 2 (1.8 mg; solvent, MeOH–CHCl3–AcOH, 8:92:0.1, v/v), and 3 (1.1 mg; solvent, MeOH–CHCl3–AcOH, 10:90:0.1, v/v).

5. Experimental 5.5. Identification of structures 1–3 5.1. Plant materials Male M. polymorpha, accession Takaragaike-1 (formally called ‘male E line’), and female M. polymorpha, accession Takaragaike-2 (formally called ‘female E line’), were asexually maintained and propagated through gemma growth as described previously (Okada et al., 2000; Takenaka et al., 2000). Takaragaike accessions have been selected for the on-going project by the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). F1 spores, which were produced by crossing Takaragaike-1 and Takaragaike-2, were employed for transformation. Formation of sexual organs was elicited by far-red irradiation as described previously (Chiyoda et al., 2008). Mature sporangia were collected 3– 4 weeks later after crossing, air-dried for approximately 10 days, and stored at 80 °C until use. Plants were propagated on 0M51C medium containing 1.4% agar medium under 50–60 lmol photons m2 s1 of continuous fluorescent light at 22 °C unless otherwise stated. 5.2. Abiotic stress treatments For UV-B irradiation, plants were exposed to UV-B light (280– 320 nm) from a distance of 15 cm using a UV-B lamp (15 W, Sankyo Denki, Tokyo, Japan) for 30 min per day for 5 consecutive days. For UV-C irradiation, plants were exposed to UV-C light (predominantly 254 nm) from a distance of 15 cm using a germicidal lamp (15 W, Hitachi, Tokyo, Japan) for 30 min per day for 5 consecutive days. 5.3. HPLC analysis of ABA-induced constituents in M. polymorpha M. polymorpha was grown on 0M51C agar medium with or without 10 lM ABA treatment for 30 days and then frozen in liq. N2. Samples (ca. 2 g) of frozen plants were crushed and extracted with EtOH (10 ml), with 2,4,6-trihydroxyacetophenone (0.2 lg) as an internal standard. After filtration and evaporation, the EtOH extract was dissolved in MeOH (1 ml) and analyzed by HPLC equipped with a reversed phase HPLC column (Capcell pak C18 UG80, 4.6 mm i.d. 250 mm, Shiseido, Tokyo, Japan; UV, 220 nm; flow, 1.0 ml/min) with mixed solvents of MeOH and H2O using a linear gradient mode, in which the gradient of MeOH with 0.05% AcOH to H2O with 0.05% AcOH was held at 20:80 from 0 min to 5 min and then increased linearly from 5 min to 25 min to 100:0. The gradient of 100:0 was maintained from 25 min to 30 min. 5.4. Isolation of compounds 1–3 M. polymorpha grown on 0M51C medium with ABA (10 lM) for 30 days (400 g) was frozen in liq. N2, crushed, and soaked in EtOH (1.2 l). After filtration and evaporation of the EtOH extract, it was dissolved in MeOH–H2O (9:1, v/v) and then partitioned with n-hexane. The resultant MeOH–H2O (9:1, v/v) was concentrated in vacuo, and the obtained residue was partitioned with EtOAc and

The 1H NMR and 13C NMR spectra were recorded on a Bruker AMX-500 NMR spectrometer (Bruker, Karlsruhe, Germany). 1H NMR chemical shifts were referenced to the residual CDCl3 solvent peak at d 7.24 ppm, and 13C NMR chemical shifts were referenced to the CDCl3 solvent peak at d 77.0 ppm. HRFDMS were recorded on a JEOL JMS T100GCV mass spectrometer (Jeol, Tokyo, Japan). The NMR spectroscopic data of 1–3 are shown in Figs. S1–S3 and Table S1. 5.6. UPLC-MS/MS analysis of ABA and bisbibenzyls in M. polymorpha Plant tissue (ca. 1.0 g) was frozen with liq. N2, crushed, and soaked overnight in EtOH (15 ml). UPLC separation was performed using a Waters ACQUITY ethylene-bridged (BEH) C18 column (2.1 mm i.d. 100 mm) and the Waters Micromass Quanttro Premier Tandem Quadrupole Mass Spectrometer (Waters, Milford, MA, USA). The endogenous ABA concentration was analyzed according to the method of Kobayashi et al. (2010). For analysis of bisbibenzyls, the analytes were eluted from the column with mixed solvents of MeOH–H2O–AcOH (20:80:0.05) (solvent A) and MeOH–AcOH (100:0.05) (solvent B) using a linear gradient mode, when the solvent gradient was held at 10:90 solvent A:solvent B from 0 min to 0.2 min, linearly increased from 0.2 min to 2.0 min to 70:30, and remained at 70:30 from 2.0 min to 3.0 min. Then, from 3.0 min to 4.0 min, the gradient changed linearly from 70:30 to 100:0 and then was held at 100:0 from 4.0 min to 5.0 min. Flow rate was 0.25 ml/min. The ionization settings of isoriccardin C (1), marchantin C (2), riccardin F (3), and quercetin-30 -OCD3 (internal standard) are shown in Table S2. 5.7. Generation of transgenic M. polymorpha overexpressing MpABI1 Total RNA was extracted from M. polymorpha and reverse transcription (M-MLV reverse transcriptase, Invitrogen, Carlsbad, CA, USA) was carried out according to the manufacturer’s instructions to yield cDNA. PCR was carried out with the cDNA using a forward primer (MpABI1-F1, 50 -AGGGACTATGGAGACGCTGGTCACTTCCGA30 ), a reverse primer (MpABI1-R1, 50 -AGACAAACTACGCCGAAGA TCCCGTCGTTAA-30 ) and KOD FX DNA polymerase (Toyobo, Osaka, Japan). Then, the resultant PCR product was ligated into cloning vector pBluescript SK II (+) (Stratagene, La Jolla, CA, USA). After the sequence of this construct was verified, PCR was carried out using primers for seamless cloning: a forward primer (MpABI1F2, 50 -AAAGCAGGCTCCACCATGGAGACGCTGGTC-30 ) and a reverse primer (MpABI1-R2, 50 -AGCTGGGTCTAGATACTACGCCGAAGATCC30 ). Then, seamless cloning was carried out to insert the PCR product into pENTR4 (Invitrogen, Carlsbad, CA, USA) using Seamless Cloning and Assembly Enzyme Mix (Invitrogen, Carlsbad, CA, USA). After sequence verification, an LR reaction was carried out to transfer MpABI1 to the overexpression vector pMpGWB102 using LR Clonase II Enzyme Mix (Invitrogen, USA). Agrobacterium-mediated transformation of M. polymorpha was conducted as described previously (Ishizaki et al., 2008).



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Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

The Elegant Simplicity of the Liverwort Marchantia polymorpha.

Correction: A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha.

TALEN-mediated genome-editing approaches in the liverwort Marchantia polymorpha yield high efficiencies for targeted mutagenesis.

DRP3 and ELM1 are required for mitochondrial fission in the liverwort Marchantia polymorpha.

AgarTrap: a simplified Agrobacterium-mediated transformation method for sporelings of the liverwort Marchantia polymorpha L.

Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.

Vacuolar ion channels in the liverwort Marchantia polymorpha: influence of ion channel inhibitors.

Development of Gateway Binary Vector Series with Four Different Selection Markers for the Liverwort Marchantia polymorpha.

Phytochrome Signaling Is Mediated by PHYTOCHROME INTERACTING FACTOR in the Liverwort Marchantia polymorpha.

Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorpha L.

Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha.

Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha.

RNA sequencing analysis of the gametophyte transcriptome from the liverwort, Marchantia polymorpha.

Phototropin encoded by a single-copy gene mediates chloroplast photorelocation movements in the liverwort Marchantia polymorpha.

Gene organization and newly identified groups of genes of the chloroplast genome from a liverwort, Marchantia polymorpha.

Transfer RNA genes in the mitochondrial genome from a liverwort, Marchantia polymorpha: the absence of chloroplast-like tRNAs.

Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

Phytochrome-mediated regulation of cell division and growth during regeneration and sporeling development in the liverwort Marchantia polymorpha.

Essential role of the E3 ubiquitin ligase nopperabo1 in schizogenous intercellular space formation in the liverwort Marchantia polymorpha.

Carotenoid analysis of a liverwort Marchantia polymorpha and functional identification of its lycopene β- and ε-cyclase genes.

Efficient and Inducible Use of Artificial MicroRNAs in Marchantia polymorpha.

Abscisic acid biosynthesis in roots : I. The identification of potential abscisic acid precursors, and other carotenoids.

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture.