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Naringenin inhibits seed germination and seedling root growth through a salicylic acid-independent mechanism in Arabidopsis thaliana
Plant Physiology and Biochemistry 61 (2012) 24e28
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Naringenin inhibits seed germination and seedling root growth through a salicylic acid-independent mechanism in Arabidopsis thaliana Iker Hernández*, Sergi Munné-Bosch Departament de Biologia Vegetal, Edifici Margalef, Facultat de Biologia, Universitat de Barcleona, Avda. Diagonal 643, Margalef Bldg, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2012 Accepted 4 September 2012 Available online 18 September 2012
Flavonoids fulfill an enormous range of biological functions in plants. In seeds, these compounds play several roles; for instance proanthocyanidins protect them from moisture, pathogen attacks, mechanical stress, UV radiation, etc., and flavonols have been suggested to protect the embryo from oxidative stress. The present study aimed at determining the role of flavonoids in Arabidopsis thaliana (L.) seed germination, and the involvement of salicylic acid (SA) and auxin (indole-3-acetic acid), two phytohormones with the same biosynthetic origin as flavonoids, the shikimate pathway, in such a putative role. We show that naringenin, a flavanone, strongly inhibits the germination of A. thaliana seeds in a dose-dependent and SA-independent manner. Altered auxin levels do not affect seed germination in Arabidopsis, but impaired auxin transport does, although to a minor extent. Naringenin and N-1-naphthylphthalamic acid (NPA) impair auxin transport through the same mechanisms, so the inhibition of germination by naringenin might involve impaired auxin transport among other mechanisms. From the present study it is concluded that naringenin inhibits the germination of Arabidopsis seeds in a dose-dependent and SAindependent manner, and the results also suggest that such effects are exerted, at least to some extent, through impaired auxin transport, although additional mechanisms seem to operate as well. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Auxin Flavonoids Germination Naringenin Salicylic acid
1. Introduction Seed germination and seedling establishment are two closely related processes with enormous importance in many aspects of plant biology, from plant ecology to crop productivity [1]. In seeds, flavonoids protect the seed from pathogen attacks and UV radiation, prevent water uptake, and protect the embryo against oxidative stress, among many other functions (for review, see Ref. [2]). Although seed germination is mainly driven by the opposite action of abscisic acid (ABA) and gibberellins (see Ref. [3] for a recent review), other phytohormones such as ethylene, salicylic acid (SA), auxins and jasmonic acid (JA) are also involved (reviewed by Ref. [4]). Among the hormones that have been reported to influence germination, SA and the auxin indole-3-acetic acid (IAA) share a common biosynthetic origin with flavonoids. The shikimate pathway uses trioses phosphate from the primary metabolism to
Abbreviations: ABA, abscisic acid; ACC, aminocyclopropane-1-carboxylic acid; IAA, indole-3-acetic acid; JA, jasmonic acid; NPA, N-1-naphthylphthalamic acid; SA, salicylic acid. * Corresponding author. Tel.: þ34 934033718; fax: þ34 934112842. E-mail addresses: [email protected] (I. Hernández), [email protected] (S. Munné-Bosch). 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.09.003
produce chorismate in plastids. SA is synthesized from isochorismate, which derives from chorismate directly [5]. Chorismate can also yield arogenate, the immediate precursor of phenylalanine and tyrosine. Phenylalanine is the precursor of most plant polyphenols, including flavonoids [6]. SA can be also synthesized from the phenylalanine-derived benzoic acid [5]. Alternatively, chorismate can be transformed, through several precursors, into tryptophan: one ebut not the onlye IAA precursor [7]. Flavonoids, at least some, have been reported to bind transmembrane auxin efflux carriers thereby impairing their functionality and thus dampening polar auxin transport (see Ref. [8] for review). Although several reports point toward a role of auxins during seed germination [9e12], little is known about such a role, and the possible mechanism(s) underlying it. On the other hand, it is extensively reported that SA inhibits seed germination in a dosedependent manner in many species [13e15], although the opposite effect has been also reported under salt stress conditions (see, for instance, Ref. [16]). In the present study we aimed at determining the effect of flavonoids in the process of seed germination in the model species Arabidopsis thaliana (L.). We show that naringenin, a flavonoid that is precursor of most flavonoids, in particular of flavonols, in Arabidopsis, strongly inhibits seed germination of seeds. To determine the possible involvement of SA and IAA, two phytohormones
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biosynthetically-related to flavonoids, in the effects of exogenous naringenin, we used a combined approach of mutant lines and exogenous applications to conclude that the germination inhibition caused by naringenin is independent from SA, and that impaired auxin transport impairs germination, although to a minor extent compared to exogenous naringenin.
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2. Results 2.1. Germination parameters Wild type (WT) Arabidopsis seedlings showed a normal germination response, reaching 50% germination (G50) by day 1.13 and a maximum germination percentage (Gmax) of 98% (Figs. 1 and 2). Exogenous IAA did not affect significantly the G50 or the Gmax, neither at 0.1 mM nor at 0.5 mM (1.07 and 0.97 days; and 99 and 98%, respectively; Figs. 1 and 2). Naringenin inhibited germination: at 0.2 mM WT seeds showed a G50 of 2.24 days and a Gmax of 74% (Fig. 2). At 1 mM, naringenin strongly inhibited seed germination: the first seeds, a 2%, germinated by day 2, reached 50% germination by day 8.63, and showed a Gmax of 33% (Fig. 2). Exogenous SA (1 mM) also impaired the germination of WT seeds: the first seeds (25%) germinated after 2 days, and reached a Gmax of 44.74% (Fig. 2). In the presence of 1 mM SA WT seeds do not (and are not predicted to) reach 50% germination (hence this parameter is absent in Fig. 2A). One mM naringenin strongly delayed the germination in cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seeds, showing G50 values of 12.81, 5.12, 8.51 and 7.96 days, respectively (Fig. 2A). A lower dose of naringenin (0.2 mM) also increased the G50 values of cyp79B2 cyp79B3 and sid2-1 seeds (to 2.45 and 4.01 days, respectively), but not those of eds5-1 or NahG seeds (1.94 and 1.85 days, respectively) (Fig. 2A). In the presence of 1 mM SA cyp79B2 cyp79B3, eds5-1 and sid2-1 seeds show G50 values of 8.85, 14.40 and 6.65 days, respectively (Fig. 2A). Transgenic NahG seeds, though, showed significantly lower G50 values (1.91 days), which are closer to those in plain MS medium (Fig. 2A).
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Fig. 2. Time required to reach a 50% germination (G50; panel A), and maximum germination percentages (Gmax; panel B), in Arabidopsis thaliana seeds of different genotypes in MS medium, or MS medium supplemented with 0.1 mM indole-3-acetic acid (IAA), 0.5 mM IAA, 0.05 mM N-1-naphthylphthalamic acid (NPA), 0.1 mM NPA, 0.2 mM naringenin (N), 1 mM N, or 1 mM salicylic acid (SA). The values shown are the mean SEM of 6e7 replicates of 20e25 seeds each. The G50 value calculated for SAtreated Col 0 wild type (WT) seeds is omitted since it does not reach 50% germination in the 7 days and, according to the nonlinear regression (see Materials and methods), it is not expected to reach such percentage. ANOVA tests show that differences in Gmax and G50 among lines, treatments, and the interaction between both factors, are statistically significant (P < 0.01).
Fig. 1. Germination percentages of Arabidopsis thaliana Col 0 wild type seeds in MS medium and MS medium supplemented with 0.2 mM naringenin (N), 1 mM N, 0.1 mM indole-3-acetic acid (IAA), 0.5 mM IAA or 1 mM salicylic acid (SA). The values shown are the mean SEM of 6 or 7 replicates of 20e25 seeds each.
In WT seeds, the presence of 0.1 mM N-1-naphthyl phthalamic acid (NPA) increased G50 significantly from 1.13 to 1.74 days (Fig. 2A). This increment was also observed in cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seeds (1.94, 2.78, 2.74 and 2.32 days, respectively). Exogenous IAA hardly affected the G50 in any of the lines tested, which was around 1 day in all cases (Fig. 2A and B). All lines reached Gmax values over 90% in MS medium. IAA and NPA treatments hardly affected Gmax in any of the lines tested (Fig. 2B). 1 mM SA reduced the Gmax to 32, 13 and 43% in cyp79B2
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cyp79B3, eds5-1 and sid2-1 seeds, respectively, but this treatment hardly affected the Gmax in NahG seeds, which attained a 82% (Fig. 2B). 1 mM naringenin also decreased the Gmax in cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seeds (to 33, 30, 32 and 38%, respectively). And so did 0.2 mM naringenin (to 88, 90, 66, 91%, respectively), although in this case the decrease in eds5-1 and NahG lines was not statistically significant (Fig. 2B). 2.2. Effects of naringenin, IAA and SA on seedling main root growth After 8 days, the main root of the seedlings grown in control medium ranged between 39.4 and 30.6 mm (Fig. 3). This parameter responded equally in all lines to the different treatments (Fig. 3). 0.1 mM IAA increased seedling main root length between 10 and 32% (Fig. 3); 0.5 mM IAA, in contrast, reduced it between 6 and 22% (Fig. 3); 0.05 mM NPA strongly reduced it by ca. 80%; 0.1 mM NPA inhibited it by ca. 85%; and finally, 0.2 mM naringenin also reduced it between 67 and 79%. 1 mM SA- and 1 mM naringenin-treated seeds hardly germinated and their roots were shorter than 1 mm. 2.3. Levels of SA and IAA in seedlings The levels of SA in 8 days-old MS-grown cyp79B2 cyp79B3, eds51, sid2-1 and NahG seedlings were similar to those of the WT (29.5, 28.7, 21.6, 12.8 and 20.5 ng g1 FW, respectively; Table 1). The levels of IAA in MS-grown cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seedlings were similar to those of the WT as well (5.3, 7.0, 4.1, 6.2 and 7.4 ng g1 FW; Table 1). However, when seedlings grown in the different media are taken together, cyp79B2 cyp79B3 seedlings show lower IAA levels (P < 0.05) compared to the WT (3.08 vs. 5.92 ng g1 FW: values similar to those reported previously [7]) (see Table A).
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Table 1 Levels of salicylic acid (SA) and indole-3-acetic acid (IAA) in 8 days-old seedlings grown in MS medium. Values show the mean SEM (3 < n < 6). Every replicate is a pool of 15e25 seedlings. No statistically significant difference was found in SA and IAA levels among the WT and the transgenic or mutant lines (P > 0.05). Line
Compound SA (ng g1 FW)
WT cyp79B2 cyp79B3 eds5-1 sid2-1 NahG
20.5 29.5 28.7 21.6 12.5
8.3 3.1 15.6 5.6 2.2
IAA (ng g1 FW) 7.4 5.3 7.0 4.1 6.2
3.6 3.7 1.7 1.5 1.8
3. Discussion 3.1. Naringenin strongly inhibits Arabidopsis seed germination and seedling main root growth Results show that naringenin is a powerful dose-dependent inhibitor of Arabidopsis seed germination. This flavanone is even more effective than SA, a well-known inhibitor of seed germination [13e15], inhibiting the process. It has been previously reported that naringenin inhibits seed germination in soybean (Glycine max), but in this study it was observed a better inhibition of germination at 0.18 mM (50 mg L1) than at 0.32 mM (100 mg L1) [17], while in our case the effect of naringenin was clearly dose-dependent. Moreover, naringenin strongly inhibited seedling main root growth: seedlings developed in 0.2 mM naringenin showed roots at least 67% shorter than in MS medium, and seedlings developed in 1 mM naringenin showed roots smaller than 1 mm in all cases (Fig. 3). It has been recently reported that naringenin inhibits root growth in soybean [19]. In this report, the authors suggest that the effect of naringenin on root growth cessation is due to enhanced lignification. However, Bido et al. [18] reported on root growth inhibitions of 25e30% while, at the same naringenin concentration, we hereby report on an inhibition of 70e80% (Fig. 3). The differences between the results shown hereby and those reported in soybean [17,18] may be due to differences among species and experimental setup. 3.2. Naringenin impairs Arabidopsis seed germination through a SA-independent mechanism Given the constitutive degradation of SA in the NahG line, the presence of SA in the germination medium hardly affected the germination of these seeds, while WT, sid2-1 and eds5-1 seeds showed strongly impaired germination (Fig. 2A and B). 1 mM naringenin strongly impaired seed germination in all lines, including NahG, thereby showing that the effects of the flavanone over germination are independent from SA. Moreover, exogenous naringenin did not affect the levels of SA in young WT, sid2-1, or eds5-1 seedlings (data not shown), which supports such independence. 3.3. Naringenin might inhibit germination by impairing auxin transport
Fig. 3. Main root length in 8 days-old Arabidopsis thaliana seedlings of different genotypes germinated in MS medium, or MS medium supplemented with 0.1 mM indole-3-acetic acid (IAA), 0.5 mM IAA, 0.05 mM N-1-naphthylphthalamic acid (NPA), 0.1 mM NPA, 0.2 mM naringenin (N), 1 mM N, or 1 mM salicylic acid. WT stands for Col 0 wild type. The values shown are the mean SEM of 6e7 replicates of 5 seedlings each. ANOVA tests show that differences in Gmax and G50 among lines, treatments, and the interaction between both factors, are statistically significant (P < 0.01).
As discussed before, naringenin inhibits seed germination and seedling root growth in a dose-dependent and SA-independent manner. The auxin efflux inhibitor NPA, at 0.1 mM (and in some cases, at 0.05 mM, although not statistically significant), impaired significantly the germination of WT, eds5-1, sid2-1 and NahG seeds, which suggests that impaired auxin transport alters seed germination (Fig. 2A). This inhibition, as in the case of naringenin, is SAindependent since NahG plants respond similarly to the WT and the
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SA-induction deficient lines sid2-1 and eds5-1 (Fig. 2A). Some particular flavonoids displace NPA from plasma membrane and microsome binding sites. These binding sites have been reported in several transmembrane IAA transporters of the ABC (ATP-binding cassette) family (for review, see Ref. [8]). Thus, although further research is needed to confirm this, flavonoids and NPA might inhibit germination by the same mechanism: impairing auxin transport. However, the inhibition caused by NPA was very mild compared to that observed in naringenin (and SA) treatments (Fig. 2A), which suggests that additional mechanisms operate in the inhibition of germination, aside of the inhibition of auxin transport. The treatment with 0.1 mM IAA resulted on increased main root length in all genotypes (Fig. 3), but did not affect the germination of any of them (Figs. 1 and 2). It is well reported that the adequate auxin doses promote root growth (and root hair formation) (e.g. Refs. [19e21]), but it is also well-known that extremely high auxin levels have a negative impact on plant growth; for that feature synthetic auxins are widely used as herbicides (for review, see Ref. [22]). On the other hand, cyp79B2 cyp79B3 seeds showed WT-like germination and root growth under control conditions and also under the different treatments tested (Figs. 2 and 3). The facts that cyp79B2 cyp79B3 show WT-like germination and that IAA treatment does not affect this process in any of the genotypes tested hereby, and that NPA inhibits seed germination in all lines tested, suggests that auxin transport, and not whole plant auxin levels, are involved in the process of seed germination. 3.4. Considerations on the widely used SA-deficient lines sid2-1, eds5-1 and NahG It is noteworthy to highlight that WT, eds5-1, sid2-1 and NahG seedlings have the same SA basal levels (Table 1; Fig. A shows the confirmation of the identity and homozygosis of the eds5-1 and sid2-1 lines). In agreement, it has been previously reported that unchallenged young sid2-1 seedlings show WT SA levels [23], and that EDS5 shows low expression levels in unchallenged plants [24]. Thus, we suggest that the SA deficiency in these genotypes (sid2-1 and eds5-1) is only appreciable when SA accumulation is induced, while SA remains at basal levels otherwise in young seedlings. Moreover, some of the treatments applied in this study trigger the accumulation of SA in sid2-1 (and eds5-1) mutants (data not shown), which evidences that SID2 is not the only path for the biosynthesis of SA. A set of alternative SA biosynthetic pathways that use phenylalanine as a precursor is well documented, and the importance of these alternative pathways seems to vary depending on the process triggering SA biosynthesis (reviewed by Ref. [25]). The present work shows that naringenin strongly inhibits seed germination in a dose-dependent manner. This inhibition is exerted independently from SA, a well-known germination inhibitor that shares biosynthetic precursors with naringenin. Naringenin also shares biosynthetic precursors with IAA, and the flavanone, in turn, is a precursor of the flavonoids (mainly flavonols) that inhibit auxin efflux. Although the inhibition of seed germination by naringenin is independent from IAA concentrations, IAA transport might be involved in such inhibition. 4. Material and methods 4.1. Plant material and growth conditions For the experiments, the A. thaliana (L.) Col 0 WT was chosen, and all the mutants and transgenics were in Col 0 background. The sid2 and eds5 (also called sid1) mutants were identified by their inability to accumulate SA upon pathogen challenge [26,27]. SID2 encodes for the isochorismate synthase (isochorismate
27
hydroxymutase; EC 5.4.4.2), a key enzyme in the biosynthesis of SA [5]. EDS5 encodes for a transporter of the Multi-Drug and Toxic Extrusion (MATE) family with an as yet unknown function [24]. NahG transgenic plants constitutively express a bacterial SA hydroxylase (salicylate 1-monooxygenase; EC 1.14.13.1), and thus constitutively degrade SA [28]. The cyp79B2 cyp79B3 double KO is deficient in two cytochrome P450 monooxygenases, CYP79B2 and CYP79B3, involved in indole-3-acetic acid (IAA) biosynthesis from tryptophan. cyp79B2 cyp79B3 double KOs show ca. 65% WT IAA levels when grown at 26 C, but WT IAA levels when grown at 21 C [7]. WT and the homozygous mutant and transgenic plants were grown side by side under controlled conditions (16 h photoperiod of ca. 80 mmol m2 s1, constant temperature of 21 C and relative humidity of ca. 70%). After selfing, their seeds were collected in parallel and employed for the experiments described hereby. Seeds were surface-sterilized with an excess of 70% ethanol containing 0.1% Triton X-100, followed by 4 washes with sterile water. Surface-sterilized seeds were germinated in 10% agarose vertical plates with half strength MS medium supplemented with 1 mM SA, 0.1 mM IAA, 0.5 mIAA, 0.05 mM NPA, 0.1 mM NPA, 0.2 mM naringenin, 1 mM naringenin or 1 mM SA. Sowed seeds were allowed to stratify for 3 days at 4 C in the darkness, and then transferred to a germination cabinet (Sanyo MLR-35OH, Sanyo Electric Co. Ltd., Moriguchi, Osaka, Japan) at 26 C with 70% relative humidity and a 16-h photoperiod of ca. 60 mmol m2 s1. Germination was scored every 24 h at the beginning of the light period during 7 days by scoring root tip protrusion with the aid of a magnifying glass. The eighth day pictures were taken, and plants were snap-frozen in liquid nitrogen. Root length was measured afterward from the pictures using the ImageJ software. The germination assays and root length measurements were repeated to verify the results. All the data shown hereby belong to the same repeat. 4.2. Determination of phytohormones Phytohormones were determined essentially as described [29] with minor modifications. Frozen plant material (ca. 50 mg) was grinded in a Retsch mill and extracted with 300 mL MeOH/iPrOH/ AcH (59/40/1; v/v/v) including 100 ppb of each deuterium-labeled standards, as described [29]. Phytohormones were determined by UPLC-ESI-MS/MS with the gradient and settings described [29]. 4.3. Statistical analyses Statistical analyses were performed with the PASW Statistics 17.0 software (IBM Corp., Somer, NY). Differences were considered statistically significant (*) when P < 0.05 and very significant (**) when P < 0.01, in ANOVA or Student’s t-tests. The time required to achieve a 50% germination percentage (G50) was calculated with the Prism 5 software (GraphPad Software Inc., La Jolla, CA). Acknowledgments The authors are very grateful to the Chromatography platform of the Serveis Cientifico-Tècnics of the University of Barcelona and Dr. Maren Müller for their assistance in the determination of phytohormones. We are also grateful to Dr. John Celenza (Boston University) for kindly providing us with homozygous cyp79B2 cyp79B3 double mutants, and Luis Mur for kindly providing us with SA-deficient mutants and transgenics. The authors are indebted to the Spanish Ministry of Economy and Competitivity (Project Nr. PIB2010BZ-00472 and Juan de la Cierva fellowship awarded to IH) and the Catalan Government for the ICREA Academia prize awarded to SMB.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2012.09.003. References [1] J.D. Bewley, M. Black, Physiology and Biochemistry of Seeds in Relation to Germination, vol I, Springer, Berlin-Heidelberg-New York, 1983. [2] L. Lepiniec, I. Debeaujon, J. Routaboul, A. Baudry, L. Pourcel, N. Nesi, M. Caboche, Genetics and biochemistry of seed flavonoids, Annu. Rev. Plant Biol. 57 (2006) 405e430. [3] K. Weitbrecht, K. Mueller, G. Leubner-Metzger, First off the mark: early seed germination, J. Exp. Bot. 62 (2011) 3289e3309. [4] A. Linkies, G. Leubner-Metzger, Beyond gibberellins and abscisic acid: how ethylene and jasmonates control seed germination, Plant Cell. Rep. 31 (2012) 253e270. [5] M.C. Wildermuth, J. Dewdney, G. Wu, F.M. Ausubel, Isochorismate synthase is required to synthesize salicylic acid for plant defence, Nature 414 (2001) 562e565. [6] T. Vogt, Phenylpropanoid biosynthesis, Mol. Plant 3 (2010) 2e20. [7] Y.D. Zhao, A.K. Hull, N.R. Gupta, K.A. Goss, J. Alonso, J.R. Ecker, J. Normanly, J. Chory, J.L. Celenza, Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3, Genes Dev. 16 (2002) 3100e3112. [8] W.A. Peer, A.S. Murphy, Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci. 12 (2007) 556e563. [9] M. Ogawa, A. Hanada, Y. Yamauchi, A. Kuwalhara, Y. Kamiya, S. Yamaguchi, Gibberellin biosynthesis and response during Arabidopsis seed germination, Plant Cell 15 (2003) 1591e1604. [10] D.A. Ni, L.J. Wang, C.H. Ding, Z.H. Xu, Auxin distribution and transport during embryogenesis and seed germination of Arabidopsis, Cell Res. 11 (2001) 273e278. [11] K. Bialek, J.D. Cohen, Free and conjugated indole-3-acetic-acid in developing bean-seeds, Plant Physiol. 91 (1989) 775e779. [12] P. Liu, T.A. Montgomery, N. Fahlgren, K.D. Kasschau, H. Nonogaki, J.C. Carrington, Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages, Plant J. 52 (2007) 133e146. [13] L.Q. Guan, J.G. Scandalios, Developmentally related responses of maize catalase genes to salicylic acid, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 5930e5934. [14] N. Nishimura, N. Kitahata, M. Seki, Y. Narusaka, M. Narusaka, T. Kuromori, T. Asami, K. Shinozaki, T. Hirayama, Analysis of ABA hypersensitive germination2 revealed the pivotal functions of PARN in stress response in Arabidopsis, Plant J. 44 (2005) 972e984.
[15] Z. Xie, Z. Zhang, S. Hanzlik, E. Cook, Q.J. Shen, Salicylic acid inhibits gibberellininduced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY gene, Plant Mol. Biol. 64 (2007) 293e303. [16] S. Lee, S. Kim, C. Park, Salicylic acid promotes seed germination under high salinity by modulating antioxidant activity in Arabidopsis, New Phytol. 188 (2010) 626e637. [17] F.T. Colpas, E.O. Ono, J.D. Rodrigues, J.R.D. Passos, Effects of some phenolic compounds on soybean seed germination and on seed-borne fungi, Braz. Arch. Biol. Technol. 46 (2003) 155e161. [18] G.S. Bido, M.L. Lucio Ferrarese, R. Marchiosi, O. Ferrarese-Filho, Naringenin inhibits the growth and stimulates the lignification of soybean root, Braz. Arch. Biol. Technol. 53 (2010) 533e542. [19] K.V. Thimann, Auxins and the growth of roots, Am. J. Bot. 23 (1936) 561e569. [20] M.L. Evans, H. Ishikawa, M.A. Estelle, Responses of Arabidopsis roots to auxin studied with high temporal resolution e comparison of wild-type and auxinresponse mutants, Planta 194 (1994) 215e222. [21] V.A. Grieneisen, J. Xu, A.F.M. Maree, P. Hogeweg, B. Scheres, Auxin transport is sufficient to generate a maximum and gradient guiding root growth, Nature 449 (2007) 1008e1013. [22] K. Grossmann, Mode of action of auxin herbicides: a new ending to a long, drawn out story, Trends Plant Sci. 5 (2000) 506e508. [23] S.C. Venugopal, R. Jeong, M.K. Mandal, S. Zhu, A.C. Chandra-Shekara, Y. Xia, M. Hersh, A.J. Stromberg, D. Navarre, A. Kachroo, P. Kachroo, Enhanced disease susceptibility 1 and salicylic acid act redundantly to regulate resistance genemediated signaling, PLoS Genet. 5 (2009) e1000545. [24] C. Nawrath, S. Heck, N. Parinthawong, J.P. Metraux, EDS5, an essential component of salicylic acid-dependent signaling for disease resistance in Arabidopsis, is a member of the MATE transporter family, Plant Cell 14 (2002) 275e286. [25] Z. Chen, Z. Zheng, J. Huang, Z. Lai, B. Fan, Biosynthesis of salicylic acid in plants, Plant Signal. Behav. 4 (2009) 493e496. [26] J. Glazebrook, E.E. Rogers, F.M. Ausubel, Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening, Genetics 143 (1996) 973e982. [27] C. Nawrath, J.P. Metraux, Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation, Plant Cell 11 (1999) 1393e1404. [28] T. Gaffney, L. Friederich, B. Vernooij, D. Negrotto, G. Nye, S. Uknes, E. Ward, H. Kessmann, J. Ryals, Requirement of salicylic acid for the induction of systemic acquired resistance, Science 261 (1993) 754e756. [29] M. Müller, S. Munne-Bosch, Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry, Plant Meth. 7 (2011) 37.
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Research article
Naringenin inhibits seed germination and seedling root growth through a salicylic acid-independent mechanism in Arabidopsis thaliana Iker Hernández*, Sergi Munné-Bosch Departament de Biologia Vegetal, Edifici Margalef, Facultat de Biologia, Universitat de Barcleona, Avda. Diagonal 643, Margalef Bldg, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 July 2012 Accepted 4 September 2012 Available online 18 September 2012
Flavonoids fulfill an enormous range of biological functions in plants. In seeds, these compounds play several roles; for instance proanthocyanidins protect them from moisture, pathogen attacks, mechanical stress, UV radiation, etc., and flavonols have been suggested to protect the embryo from oxidative stress. The present study aimed at determining the role of flavonoids in Arabidopsis thaliana (L.) seed germination, and the involvement of salicylic acid (SA) and auxin (indole-3-acetic acid), two phytohormones with the same biosynthetic origin as flavonoids, the shikimate pathway, in such a putative role. We show that naringenin, a flavanone, strongly inhibits the germination of A. thaliana seeds in a dose-dependent and SA-independent manner. Altered auxin levels do not affect seed germination in Arabidopsis, but impaired auxin transport does, although to a minor extent. Naringenin and N-1-naphthylphthalamic acid (NPA) impair auxin transport through the same mechanisms, so the inhibition of germination by naringenin might involve impaired auxin transport among other mechanisms. From the present study it is concluded that naringenin inhibits the germination of Arabidopsis seeds in a dose-dependent and SAindependent manner, and the results also suggest that such effects are exerted, at least to some extent, through impaired auxin transport, although additional mechanisms seem to operate as well. Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords: Auxin Flavonoids Germination Naringenin Salicylic acid
1. Introduction Seed germination and seedling establishment are two closely related processes with enormous importance in many aspects of plant biology, from plant ecology to crop productivity [1]. In seeds, flavonoids protect the seed from pathogen attacks and UV radiation, prevent water uptake, and protect the embryo against oxidative stress, among many other functions (for review, see Ref. [2]). Although seed germination is mainly driven by the opposite action of abscisic acid (ABA) and gibberellins (see Ref. [3] for a recent review), other phytohormones such as ethylene, salicylic acid (SA), auxins and jasmonic acid (JA) are also involved (reviewed by Ref. [4]). Among the hormones that have been reported to influence germination, SA and the auxin indole-3-acetic acid (IAA) share a common biosynthetic origin with flavonoids. The shikimate pathway uses trioses phosphate from the primary metabolism to
Abbreviations: ABA, abscisic acid; ACC, aminocyclopropane-1-carboxylic acid; IAA, indole-3-acetic acid; JA, jasmonic acid; NPA, N-1-naphthylphthalamic acid; SA, salicylic acid. * Corresponding author. Tel.: þ34 934033718; fax: þ34 934112842. E-mail addresses: [email protected] (I. Hernández), [email protected] (S. Munné-Bosch). 0981-9428/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2012.09.003
produce chorismate in plastids. SA is synthesized from isochorismate, which derives from chorismate directly [5]. Chorismate can also yield arogenate, the immediate precursor of phenylalanine and tyrosine. Phenylalanine is the precursor of most plant polyphenols, including flavonoids [6]. SA can be also synthesized from the phenylalanine-derived benzoic acid [5]. Alternatively, chorismate can be transformed, through several precursors, into tryptophan: one ebut not the onlye IAA precursor [7]. Flavonoids, at least some, have been reported to bind transmembrane auxin efflux carriers thereby impairing their functionality and thus dampening polar auxin transport (see Ref. [8] for review). Although several reports point toward a role of auxins during seed germination [9e12], little is known about such a role, and the possible mechanism(s) underlying it. On the other hand, it is extensively reported that SA inhibits seed germination in a dosedependent manner in many species [13e15], although the opposite effect has been also reported under salt stress conditions (see, for instance, Ref. [16]). In the present study we aimed at determining the effect of flavonoids in the process of seed germination in the model species Arabidopsis thaliana (L.). We show that naringenin, a flavonoid that is precursor of most flavonoids, in particular of flavonols, in Arabidopsis, strongly inhibits seed germination of seeds. To determine the possible involvement of SA and IAA, two phytohormones
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biosynthetically-related to flavonoids, in the effects of exogenous naringenin, we used a combined approach of mutant lines and exogenous applications to conclude that the germination inhibition caused by naringenin is independent from SA, and that impaired auxin transport impairs germination, although to a minor extent compared to exogenous naringenin.
A
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2. Results 2.1. Germination parameters Wild type (WT) Arabidopsis seedlings showed a normal germination response, reaching 50% germination (G50) by day 1.13 and a maximum germination percentage (Gmax) of 98% (Figs. 1 and 2). Exogenous IAA did not affect significantly the G50 or the Gmax, neither at 0.1 mM nor at 0.5 mM (1.07 and 0.97 days; and 99 and 98%, respectively; Figs. 1 and 2). Naringenin inhibited germination: at 0.2 mM WT seeds showed a G50 of 2.24 days and a Gmax of 74% (Fig. 2). At 1 mM, naringenin strongly inhibited seed germination: the first seeds, a 2%, germinated by day 2, reached 50% germination by day 8.63, and showed a Gmax of 33% (Fig. 2). Exogenous SA (1 mM) also impaired the germination of WT seeds: the first seeds (25%) germinated after 2 days, and reached a Gmax of 44.74% (Fig. 2). In the presence of 1 mM SA WT seeds do not (and are not predicted to) reach 50% germination (hence this parameter is absent in Fig. 2A). One mM naringenin strongly delayed the germination in cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seeds, showing G50 values of 12.81, 5.12, 8.51 and 7.96 days, respectively (Fig. 2A). A lower dose of naringenin (0.2 mM) also increased the G50 values of cyp79B2 cyp79B3 and sid2-1 seeds (to 2.45 and 4.01 days, respectively), but not those of eds5-1 or NahG seeds (1.94 and 1.85 days, respectively) (Fig. 2A). In the presence of 1 mM SA cyp79B2 cyp79B3, eds5-1 and sid2-1 seeds show G50 values of 8.85, 14.40 and 6.65 days, respectively (Fig. 2A). Transgenic NahG seeds, though, showed significantly lower G50 values (1.91 days), which are closer to those in plain MS medium (Fig. 2A).
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Fig. 2. Time required to reach a 50% germination (G50; panel A), and maximum germination percentages (Gmax; panel B), in Arabidopsis thaliana seeds of different genotypes in MS medium, or MS medium supplemented with 0.1 mM indole-3-acetic acid (IAA), 0.5 mM IAA, 0.05 mM N-1-naphthylphthalamic acid (NPA), 0.1 mM NPA, 0.2 mM naringenin (N), 1 mM N, or 1 mM salicylic acid (SA). The values shown are the mean SEM of 6e7 replicates of 20e25 seeds each. The G50 value calculated for SAtreated Col 0 wild type (WT) seeds is omitted since it does not reach 50% germination in the 7 days and, according to the nonlinear regression (see Materials and methods), it is not expected to reach such percentage. ANOVA tests show that differences in Gmax and G50 among lines, treatments, and the interaction between both factors, are statistically significant (P < 0.01).
Fig. 1. Germination percentages of Arabidopsis thaliana Col 0 wild type seeds in MS medium and MS medium supplemented with 0.2 mM naringenin (N), 1 mM N, 0.1 mM indole-3-acetic acid (IAA), 0.5 mM IAA or 1 mM salicylic acid (SA). The values shown are the mean SEM of 6 or 7 replicates of 20e25 seeds each.
In WT seeds, the presence of 0.1 mM N-1-naphthyl phthalamic acid (NPA) increased G50 significantly from 1.13 to 1.74 days (Fig. 2A). This increment was also observed in cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seeds (1.94, 2.78, 2.74 and 2.32 days, respectively). Exogenous IAA hardly affected the G50 in any of the lines tested, which was around 1 day in all cases (Fig. 2A and B). All lines reached Gmax values over 90% in MS medium. IAA and NPA treatments hardly affected Gmax in any of the lines tested (Fig. 2B). 1 mM SA reduced the Gmax to 32, 13 and 43% in cyp79B2
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cyp79B3, eds5-1 and sid2-1 seeds, respectively, but this treatment hardly affected the Gmax in NahG seeds, which attained a 82% (Fig. 2B). 1 mM naringenin also decreased the Gmax in cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seeds (to 33, 30, 32 and 38%, respectively). And so did 0.2 mM naringenin (to 88, 90, 66, 91%, respectively), although in this case the decrease in eds5-1 and NahG lines was not statistically significant (Fig. 2B). 2.2. Effects of naringenin, IAA and SA on seedling main root growth After 8 days, the main root of the seedlings grown in control medium ranged between 39.4 and 30.6 mm (Fig. 3). This parameter responded equally in all lines to the different treatments (Fig. 3). 0.1 mM IAA increased seedling main root length between 10 and 32% (Fig. 3); 0.5 mM IAA, in contrast, reduced it between 6 and 22% (Fig. 3); 0.05 mM NPA strongly reduced it by ca. 80%; 0.1 mM NPA inhibited it by ca. 85%; and finally, 0.2 mM naringenin also reduced it between 67 and 79%. 1 mM SA- and 1 mM naringenin-treated seeds hardly germinated and their roots were shorter than 1 mm. 2.3. Levels of SA and IAA in seedlings The levels of SA in 8 days-old MS-grown cyp79B2 cyp79B3, eds51, sid2-1 and NahG seedlings were similar to those of the WT (29.5, 28.7, 21.6, 12.8 and 20.5 ng g1 FW, respectively; Table 1). The levels of IAA in MS-grown cyp79B2 cyp79B3, eds5-1, sid2-1 and NahG seedlings were similar to those of the WT as well (5.3, 7.0, 4.1, 6.2 and 7.4 ng g1 FW; Table 1). However, when seedlings grown in the different media are taken together, cyp79B2 cyp79B3 seedlings show lower IAA levels (P < 0.05) compared to the WT (3.08 vs. 5.92 ng g1 FW: values similar to those reported previously [7]) (see Table A).
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Table 1 Levels of salicylic acid (SA) and indole-3-acetic acid (IAA) in 8 days-old seedlings grown in MS medium. Values show the mean SEM (3 < n < 6). Every replicate is a pool of 15e25 seedlings. No statistically significant difference was found in SA and IAA levels among the WT and the transgenic or mutant lines (P > 0.05). Line
Compound SA (ng g1 FW)
WT cyp79B2 cyp79B3 eds5-1 sid2-1 NahG
20.5 29.5 28.7 21.6 12.5
8.3 3.1 15.6 5.6 2.2
IAA (ng g1 FW) 7.4 5.3 7.0 4.1 6.2
3.6 3.7 1.7 1.5 1.8
3. Discussion 3.1. Naringenin strongly inhibits Arabidopsis seed germination and seedling main root growth Results show that naringenin is a powerful dose-dependent inhibitor of Arabidopsis seed germination. This flavanone is even more effective than SA, a well-known inhibitor of seed germination [13e15], inhibiting the process. It has been previously reported that naringenin inhibits seed germination in soybean (Glycine max), but in this study it was observed a better inhibition of germination at 0.18 mM (50 mg L1) than at 0.32 mM (100 mg L1) [17], while in our case the effect of naringenin was clearly dose-dependent. Moreover, naringenin strongly inhibited seedling main root growth: seedlings developed in 0.2 mM naringenin showed roots at least 67% shorter than in MS medium, and seedlings developed in 1 mM naringenin showed roots smaller than 1 mm in all cases (Fig. 3). It has been recently reported that naringenin inhibits root growth in soybean [19]. In this report, the authors suggest that the effect of naringenin on root growth cessation is due to enhanced lignification. However, Bido et al. [18] reported on root growth inhibitions of 25e30% while, at the same naringenin concentration, we hereby report on an inhibition of 70e80% (Fig. 3). The differences between the results shown hereby and those reported in soybean [17,18] may be due to differences among species and experimental setup. 3.2. Naringenin impairs Arabidopsis seed germination through a SA-independent mechanism Given the constitutive degradation of SA in the NahG line, the presence of SA in the germination medium hardly affected the germination of these seeds, while WT, sid2-1 and eds5-1 seeds showed strongly impaired germination (Fig. 2A and B). 1 mM naringenin strongly impaired seed germination in all lines, including NahG, thereby showing that the effects of the flavanone over germination are independent from SA. Moreover, exogenous naringenin did not affect the levels of SA in young WT, sid2-1, or eds5-1 seedlings (data not shown), which supports such independence. 3.3. Naringenin might inhibit germination by impairing auxin transport
Fig. 3. Main root length in 8 days-old Arabidopsis thaliana seedlings of different genotypes germinated in MS medium, or MS medium supplemented with 0.1 mM indole-3-acetic acid (IAA), 0.5 mM IAA, 0.05 mM N-1-naphthylphthalamic acid (NPA), 0.1 mM NPA, 0.2 mM naringenin (N), 1 mM N, or 1 mM salicylic acid. WT stands for Col 0 wild type. The values shown are the mean SEM of 6e7 replicates of 5 seedlings each. ANOVA tests show that differences in Gmax and G50 among lines, treatments, and the interaction between both factors, are statistically significant (P < 0.01).
As discussed before, naringenin inhibits seed germination and seedling root growth in a dose-dependent and SA-independent manner. The auxin efflux inhibitor NPA, at 0.1 mM (and in some cases, at 0.05 mM, although not statistically significant), impaired significantly the germination of WT, eds5-1, sid2-1 and NahG seeds, which suggests that impaired auxin transport alters seed germination (Fig. 2A). This inhibition, as in the case of naringenin, is SAindependent since NahG plants respond similarly to the WT and the
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SA-induction deficient lines sid2-1 and eds5-1 (Fig. 2A). Some particular flavonoids displace NPA from plasma membrane and microsome binding sites. These binding sites have been reported in several transmembrane IAA transporters of the ABC (ATP-binding cassette) family (for review, see Ref. [8]). Thus, although further research is needed to confirm this, flavonoids and NPA might inhibit germination by the same mechanism: impairing auxin transport. However, the inhibition caused by NPA was very mild compared to that observed in naringenin (and SA) treatments (Fig. 2A), which suggests that additional mechanisms operate in the inhibition of germination, aside of the inhibition of auxin transport. The treatment with 0.1 mM IAA resulted on increased main root length in all genotypes (Fig. 3), but did not affect the germination of any of them (Figs. 1 and 2). It is well reported that the adequate auxin doses promote root growth (and root hair formation) (e.g. Refs. [19e21]), but it is also well-known that extremely high auxin levels have a negative impact on plant growth; for that feature synthetic auxins are widely used as herbicides (for review, see Ref. [22]). On the other hand, cyp79B2 cyp79B3 seeds showed WT-like germination and root growth under control conditions and also under the different treatments tested (Figs. 2 and 3). The facts that cyp79B2 cyp79B3 show WT-like germination and that IAA treatment does not affect this process in any of the genotypes tested hereby, and that NPA inhibits seed germination in all lines tested, suggests that auxin transport, and not whole plant auxin levels, are involved in the process of seed germination. 3.4. Considerations on the widely used SA-deficient lines sid2-1, eds5-1 and NahG It is noteworthy to highlight that WT, eds5-1, sid2-1 and NahG seedlings have the same SA basal levels (Table 1; Fig. A shows the confirmation of the identity and homozygosis of the eds5-1 and sid2-1 lines). In agreement, it has been previously reported that unchallenged young sid2-1 seedlings show WT SA levels [23], and that EDS5 shows low expression levels in unchallenged plants [24]. Thus, we suggest that the SA deficiency in these genotypes (sid2-1 and eds5-1) is only appreciable when SA accumulation is induced, while SA remains at basal levels otherwise in young seedlings. Moreover, some of the treatments applied in this study trigger the accumulation of SA in sid2-1 (and eds5-1) mutants (data not shown), which evidences that SID2 is not the only path for the biosynthesis of SA. A set of alternative SA biosynthetic pathways that use phenylalanine as a precursor is well documented, and the importance of these alternative pathways seems to vary depending on the process triggering SA biosynthesis (reviewed by Ref. [25]). The present work shows that naringenin strongly inhibits seed germination in a dose-dependent manner. This inhibition is exerted independently from SA, a well-known germination inhibitor that shares biosynthetic precursors with naringenin. Naringenin also shares biosynthetic precursors with IAA, and the flavanone, in turn, is a precursor of the flavonoids (mainly flavonols) that inhibit auxin efflux. Although the inhibition of seed germination by naringenin is independent from IAA concentrations, IAA transport might be involved in such inhibition. 4. Material and methods 4.1. Plant material and growth conditions For the experiments, the A. thaliana (L.) Col 0 WT was chosen, and all the mutants and transgenics were in Col 0 background. The sid2 and eds5 (also called sid1) mutants were identified by their inability to accumulate SA upon pathogen challenge [26,27]. SID2 encodes for the isochorismate synthase (isochorismate
27
hydroxymutase; EC 5.4.4.2), a key enzyme in the biosynthesis of SA [5]. EDS5 encodes for a transporter of the Multi-Drug and Toxic Extrusion (MATE) family with an as yet unknown function [24]. NahG transgenic plants constitutively express a bacterial SA hydroxylase (salicylate 1-monooxygenase; EC 1.14.13.1), and thus constitutively degrade SA [28]. The cyp79B2 cyp79B3 double KO is deficient in two cytochrome P450 monooxygenases, CYP79B2 and CYP79B3, involved in indole-3-acetic acid (IAA) biosynthesis from tryptophan. cyp79B2 cyp79B3 double KOs show ca. 65% WT IAA levels when grown at 26 C, but WT IAA levels when grown at 21 C [7]. WT and the homozygous mutant and transgenic plants were grown side by side under controlled conditions (16 h photoperiod of ca. 80 mmol m2 s1, constant temperature of 21 C and relative humidity of ca. 70%). After selfing, their seeds were collected in parallel and employed for the experiments described hereby. Seeds were surface-sterilized with an excess of 70% ethanol containing 0.1% Triton X-100, followed by 4 washes with sterile water. Surface-sterilized seeds were germinated in 10% agarose vertical plates with half strength MS medium supplemented with 1 mM SA, 0.1 mM IAA, 0.5 mIAA, 0.05 mM NPA, 0.1 mM NPA, 0.2 mM naringenin, 1 mM naringenin or 1 mM SA. Sowed seeds were allowed to stratify for 3 days at 4 C in the darkness, and then transferred to a germination cabinet (Sanyo MLR-35OH, Sanyo Electric Co. Ltd., Moriguchi, Osaka, Japan) at 26 C with 70% relative humidity and a 16-h photoperiod of ca. 60 mmol m2 s1. Germination was scored every 24 h at the beginning of the light period during 7 days by scoring root tip protrusion with the aid of a magnifying glass. The eighth day pictures were taken, and plants were snap-frozen in liquid nitrogen. Root length was measured afterward from the pictures using the ImageJ software. The germination assays and root length measurements were repeated to verify the results. All the data shown hereby belong to the same repeat. 4.2. Determination of phytohormones Phytohormones were determined essentially as described [29] with minor modifications. Frozen plant material (ca. 50 mg) was grinded in a Retsch mill and extracted with 300 mL MeOH/iPrOH/ AcH (59/40/1; v/v/v) including 100 ppb of each deuterium-labeled standards, as described [29]. Phytohormones were determined by UPLC-ESI-MS/MS with the gradient and settings described [29]. 4.3. Statistical analyses Statistical analyses were performed with the PASW Statistics 17.0 software (IBM Corp., Somer, NY). Differences were considered statistically significant (*) when P < 0.05 and very significant (**) when P < 0.01, in ANOVA or Student’s t-tests. The time required to achieve a 50% germination percentage (G50) was calculated with the Prism 5 software (GraphPad Software Inc., La Jolla, CA). Acknowledgments The authors are very grateful to the Chromatography platform of the Serveis Cientifico-Tècnics of the University of Barcelona and Dr. Maren Müller for their assistance in the determination of phytohormones. We are also grateful to Dr. John Celenza (Boston University) for kindly providing us with homozygous cyp79B2 cyp79B3 double mutants, and Luis Mur for kindly providing us with SA-deficient mutants and transgenics. The authors are indebted to the Spanish Ministry of Economy and Competitivity (Project Nr. PIB2010BZ-00472 and Juan de la Cierva fellowship awarded to IH) and the Catalan Government for the ICREA Academia prize awarded to SMB.
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