Identification of a Factor That Complementarily Inhibits Somatic Embryogenesis With Vanillyl Benzyl Ether in Japanese Larch

By Umehara, Mikihisa Ogita, Shinjiro; Sasamoto, Hamako; Koshino, Hiroyuki; Et al

Abstract In Japanese larch (Larix leptolepis Gordon), a well- developed suspensor forms during somatic embryogenesis. The suspensor is the essential tissue for development of the embryo proper. In high-cell-density culture, the embryogenic cells proliferate, but no somatic embryos form because suspensor development is suppressed. Previously, we identified vanillyl benzyl ether (VBE) as a novel factor suppressing suspensor development from the high-cell-density conditioned medium (HCM), but the inhibitory effect of VBE was weaker than that of HCM added. Therefore, this study attempted to identify another inhibitory factor in the culture medium. Induction of somatic embryos was performed in a medium containing both VBE and a fraction of each chromatogram extracted from the culture medium. Results of the bioassay showed that a fraction had strong inhibitory activity with VBE, but weak activity without it. By physicochemical analyses of the fraction, 4- [(phenylmethoxy)methyl]phenol was identified as an inhibitory factor of larch somatic embryogenesis.

Keywords Inhibitory factor * Larix leptolepis * Somatic embryo * Vanillyl benzyl ether * 4-[(Phenylmethoxy)methyl] phenol

Introduction

In seed plants, a fertilized cell divides transversely and asymmetrically to form both a terminal cell, which gives rise to the embryo proper, and a basal cell, which often divides rapidly to form the suspensor [Meinke 1991; Yeung and Meinke 1993]. The events of fertilization and the subsequent development of zygotic embryos occur deep . within both the endosperm and the maternal cells [West and Harada 1993]. Because the physical inaccessibility of zygotic embryos renders biochemical and molecular ; analyses of zygotic embryogenesis difficult, developmental mechanisms of the embryo proper have been often investigated using somatic embryogenesis system in angiosperms [Borkird et al. 1988; Wurtele et al. 1993; Zimmerman 1993]. The development of somatic embryos closely resembles that of zygotic embryos, both morphologically and temporally [Zimmerman 1993]. In addition, the programs of gene expression and the accumulation of storage proteins appear to be similar, spatially and temporally, in somatic and zygotic embryos [Kiyosue 1992; Kiyosue et al. 1993].

In somatic embryogenesis of angiosperms, suspensors usually fail to develop during the culture [Yeung and Meinke 1993]. However, the somatic embryos of some conifers including Japanese larch (Larix leptolepis Gordon) consist of an embryo proper and a well-developed suspensor [Bonga et al. 1995; Ciavatta et al. 2001; Smertenko et al. 2003]. The characteristic makes it easy to study the interaction between the embryo proper and the suspensor. In cultures with high cell densities, somatic embryogenesis of Japanese larch is strongly inhibited although embryogenie cells (EC) actively proliferate [Ogita et al. 2000]. In this case, the cause of the inhibitory effect is not inhibition of cell proliferation but suppression of the suspensor development caused by inhibitory factors accumulated in high-cell-density-conditioned medium (HCM) [Umehara et al. 2004a]. The suspensor roles are generally thought to be the attachment of the embryo proper to mother tissue and the supply of nutrients and growth regulators to the embryo proper [Schwartz et al. 1997]. In Japanese larch, the suspensor is crucial for normal development of somatic embryos because the somatic embryo proper cannot develop further when the suspensor is excised [Umehara et al. 2004b]. Therefore, suppression of suspensor development indirectly might affect development of the embryo proper during somatic embryogenesis of Japanese larch.

Recently, one of the factors inhibiting somatic embryogenesis was purified from HCM of Japanese larch and identified as vanillyl benzyl ether (VBE) through physicochemical analysis [Umehara et al. 2005a]. In HCM, VBE was accumulated at concentrations as high at 10~5 M. Exogenously applied VBE particularly suppressed the suspensor development. Nevertheless, the inhibitory effect of VBE alone was weaker than that with HCM added. Consequently, we attempted to identify another inhibitory factor hidden in HCM, which was the main aim of the present study.

Materials and Methods

Plant materials and cell cultures. The Japanese larch culture system was established previously [Ogita et al. 1997; Ogita et al. 1999]. Details of bioassay for identification of the inhibitory factor in larch somatic embryogenesis have been described previously [Umehara et al. 2004a]. EC were maintained by 3-wk subculture in a modified Campbell and Durzan’s medium (mCD medium) [Campbell and Durzan 1975] supplemented with 7 [mu]M 2,4-dichlorophenoxyacetic acid and 3 [mu]M 6-benzylaminopurine. To induce somatic embryogenesis, EC were transferred to 10 ml of phytohormone-free mCD medium at the cell density of 0.5 ml packed cell volume per liter in a 100-ml flask (This condition was used as control). Each fraction of octade-cylsilyl (ODS) chromatography and ODS high-performance liquid chromatography (HPLC) was dissolved in ethanol to produce 1,000-fold concentration; 10 [mu]l of each was dropped in a 100-ml flask. After the volatilization of ethanol, 10 ml of mCD medium with or without 1.0 x 10^sup -5^ M VBE was poured into the flask. The EC were suspended in the mCD medium. All cultures were incubated on a gyratory shaker (100 rpm) at 25[degrees]C under a 16-h photoperiod provided by fluorescent lamps (white light at 30 [mu]mol photons m^sup -2^s^sup -1^). The resultant somatic embryos were counted with a counting chamber under a microscope after 3 wk culture. The experiments were repeated at least twice with four replications.

Chemicals. The VBE was synthesized as described previously [Umehara et al. 2005a]. The synthesized compound was dissolved in dimethyl sulfoxide, and added to phytohormone-free mCD medium at a concentration of 1.0 x 10^sup -5^ M.

Purification of inhibitory factors. Details of the purification process have been described previously [Umehara et al. 2005a]. The strongest inhibitory factor, VBE, was purified by dialysis, extraction by ethyl acetate, ODS column chromatography and twice- repeated HPLC. In the first ODS HPLC, fractions were eluted with a 40-100% acetonitrile gradient with a flow rate of 1 ml min^sup -1^. The UV absorption of the eluate was monitored at 220 nm. The second ODS HPLC was performed by isocratic elution with a 40% acetonitrile with a flow rate of 1 ml min^sup -1^. The UV absorption of the eluate was monitored at 220 nm.

Thin-layer chromatography. An aliquot was spotted on a silica gel plate (60 F254, Art. 5715; Merck and Co., Inc., Munich, Germany) to investigate the purity of fraction with a mixture of Hexane and ethyl acetate (1:1, v/v) as the mobile phase. The pattern of the spot was examined by irradiation with UV light.

Mass spectrometry. A Bruker Daltonics (Billerica, MA, USA) APEX- II Fourier transformation ion cyclotron resonance (FT-ICR) mass spectrometer (equipped with a 7T passively shielded magnet and a nanoelectrospray ion source) was used for determining the molecular mass of the purified compound. The mass spectrometer was externally calibrated with sodium iodide cluster ions. The sample was introduced into the nanospray emitter as a chloroform/methanol ( 1/ 2 v/v) solution (modified with 10 mM ammonium acetate).

Figure 1. A purification scheme for inhibitory factors of somatic embryogenesis in Japanese larch. Values in respective steps reflect the relative values of inhibitory effects when the effect of HCM added was defined as 1. Asterisks show the purification step investigated in Figs. 2, 3, 4.

Figure 2. Purification of inhibitory factor(s) by ODS column chromatography and assay of the resulting fractions for their effect on somatic embryo formation. Each fraction of 10, 20, 40, and 100% EtOH was added to the mCD medium with (white box) or without (black box) 1.0 x 10^sup -5^ M VBE. After 3 wk culture, the somatic embryos in each culture were quantified. C shows control. Results are shown as the mean and SD (n=4).

Nuclear magnetic resonance (NMR) analysis. The respective ^sup 1^H and ^sup 13^C NMR spectra were obtained using a JEOL ECA600 spectrometer operated at 600 MHz and 150 MHz. Chemical shifts were recorded in parts per million (ppm) relative to the internal standard, trimethylsilane. The spinspin bond in this compound was confirmed at 8 MHz by heteronuclear multiple bond correlation (HMBC) spectra.

Results and Discussion

Figure 1 shows that inhibitory factors were purified from HCM by dialysis, extraction with ethyl acetate, ODS-column chromatography, and twice-repeated ODS HPLC. Physicochemical analyses identified the fraction that exhibited a strong inhibitory effect as VBE [Umehara et al. 2005a]. However, the relative inhibitory activity declined as fractions were purified (Fig. 1). The effects of VBE were approximately 0.6 at relative value when the inhibitory strength of HCM was determined as 1. The amounts of inhibitory factors might gradually reduce during the purification. Especially, it declined markedly during ODS column chromatography and twice-repeated ODS HPLC. For that reason, we expected that another inhibitory factor existed in other fractions of these purification steps. Figure 3. Purification of inhibitory factor(s) by the first ODS HPLC and bioassay of the resultant fractions for effects on somatic embryo formation. The inhibitory fraction recovered from ODS column chromatography, the 80% EtOH eluate, was subjected to ODS HPLC. (a) Absorbance of eluate at 220 nm. Successive 1-ml fractions were collected as indicated and eluted with a 40-100% acetonitrile gradient. (b) Fraction nos. 1-8 and 13-22 were added to mCD medium with (white box) or without (black box) 1.0 x 10^sup -5^ M VBE and assayed for their effects on somatic embryo formation. After 3 wk culture, the somatic embryos in each culture were quantified. C shows control. Results are shown as the mean and SD (n=4).

Figure 4. Rechromatography of inhibitory factor(s) by second HPLC and bioassay of the resultant fractions for effects on somatic embryo formation, (a) Absorbance of eluate at 280 nm. The inhibitory fraction recovered from the first HPLC, a mixture of fraction nos. 9- 12, was subjected to second HPLC and eluted with isocratic mode in 40% acetonitrile. (b) Fraction nos. 1, 3, 4, and 5 were added to mCD medium with (white box) or without (black box) 1.0 x 10^sup -5^ M VBE and assayed for their effects on somatic embryo formation. After 3 wk culture, the somatic embryos in each culture were quantified. C shows control. Results are shown as the mean and SD (n=4).

Table 1. ^sup 1^H NMR (600 MHz, CDCl^sub 3^) and ^sup 13^C NMR (150 MHz, CDCl^sub 3^) assignments of 4- [(phenylmethoxy)methyl]phenol

In ODS-column chromatography, VBE was eluted in 80% ethanol faction. Other fractions, 10%, 20%, 40%, and 100% ethanol fractions, were added to the fresh medium with or without 10^sup -5^ M VBE for the bioassay. Results indicate that the 100% ethanol fraction with VBE showed stronger inhibition of somatic embryo formation than the effect of VBE alone (Fig. 2). However, the inhibitory effect was weaker than that of HCM [Umehara et al. 2004a; Umehara et al. 2005a], and has been lost during further purification (data not shown).

In the first ODS HPLC, VBE was eluted in fraction numbers 9-12. Other fractions were added to the fresh medium with or without 10^sup -5^ M VBE for the bioassay. Other fractions with VBE showed no stronger inhibition than the effect of VBE alone (Fig. 3).

In the second ODS HPLC, VBE was eluted in fraction number 2. Other fractions, nos. 1, 3, 4, and 5 were added to the fresh medium with or without 10^sup -5^ M VBE for the bioassay. Results showed that fraction number 1-that with VBE-much more strongly inhibited somatic embryo formation than did VBE alone (Fig. 4). The inhibitory effect was equal to that of HCM [Umehara et al. 2004a; Umehara et al. 2005a]. The fraction was concentrated and physicochemically analyzed after confirmation of the purity by TLC.

Figure 5. Chemical structures of inhibitory factors in somatic embryogenesis of Japanese larch. (a) Vanillyl benzyl ether (VBE), (b) 4-[(phenylmethoxy) methyl] phenol (4PMP). Arrows show the PFG- HMBC correlations of 4PMP.

The inhibitory fraction of the ODS HPLC procedure was collected from approximately 24 1 of HCM; approximately 2 mg of purified oil was obtained. The compound that had inhibitory activity gave deprotonated molecule ([M-H]^sup -^) at m/z 213.0923 (electrospray ionization FT-ICR mass spectrometry; negative ion mode; calculated for [C^sub 14^H^sub 13^O^sub 2^]^sup -^ 213.0921).

Table 1 shows spectral data results of ^sup 1^H and ^sup 13^C NMR analyses. These results indicate that this compound has a phenolic hydroxyl group, two methylene groups, and two benzene rings. These results suggest that the purified compound is 4-[(phenylmethoxy) methyl] phenol (4PMP), which has a chemical structure that resembles VBE (Fig. 5). The structure was confirmed by pulsed field gradient (PFG)-HMBC correlation to construct it.

It is expected that 4PMP accumulates at a high concentration of 10^sup -5^ M order in HCM because the peak area of 4PMP in HPLC is similar size to that of VBE, although the amount of 4PMP in HCM has not been investigated yet. Addition of authentic benzyl ether (nacalai tesque, Inc.) at 1.0 x 10^sup -5^ M strongly inhibited somatic embryogenesis of Japanese larch (unpublished results). Benzyl ether compounds such as VBE and 4PMP are inhibitory substances in somatic embryogenesis of Japanese larch; 4PMP has not been reported as a bioactive product. The biosynthetic pathway and action mechanism of 4PMP have remained unknown. Simple phenolic compounds are generally synthesized from phenylalanine via the phenyl-propanoid pathway. They are subsequently converted to compounds with more complex structures such as anthocyanins, lignins, and flavonoids, which play important roles in various developmental processes and defense responses [Dixon and Paiva 1995; Shaari and Waterman 1995]. VBE might also be synthesized via the phenylpropanoid pathway. Elimination or dilution of these compounds in the medium will be required to improve somatic embryogenesis in Japanese larch or some conifer tissue culture.

On the other hand, amounts of stimulatory factors in the medium might affect somatic embryogenesis. Phytosulfokine (PSK), which was well known as a plant peptidyl growth factor, was isolated from a conditioned medium of Asparagus mesophyll cells [Matsubayashi and Sakagami 1996]. PSK stimulated somatic embryo formation of carrot [Kobayashi et al. 1999], Japanese cedar [Igasaki et al. 2003], and Japanese larch [Umehara et al. 2005b], as well as proliferation of Asparagus suspension cells [Matsubayashi and Sakagami 1996]. The effects of PSK are considered to be both activation of cell division and cell differentiation. The dose-balance of stimulatory and inhibitory factors in the medium might contribute to somatic embryogenesis in plant cell cultures. In the future, we attempt to investigate the physiological traits of inhibitory and stimulatory factors through competitive experiments using VBE, 4PMP, and PSK.

Acknowledgment This work was supported in part by a Grant-in-Aid from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF00L01601).

Received: 13 August 2006/Accepted: 5 December 2006 / Published online: 11 March 2007 / Editor: R. J. Newton

(c) The Society for In Vitro Biology 2007

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M. Umehara (*) * H. Kamada

Gene Research Center, University of Tsukuba,

Tsukuba 305-8572, Japan

e-mail: umehara@farc.pref.fukuoka.jp

S. Ogita

Biotechnology Research Center, Toyama Prefectural University,

Kosugi 939-0398, Japan

H. Sasamoto

Graduate School of Environmental and Information Sciences,

Yokohama National University,

Hodogaya 240-8501, Japan

H. Koshino

Molecular Characterization Team, RIKEN,

Wako 351-0198, Japan

T. Nakamura

Biomolecular Characterization Team, RIKEN,

Wako 351-0198, Japan

T. Asami * S. Yoshida

Plant Function Laboratory, RIKEN,

Wako 351-0198, Japan

M. Umehara

Department of Biotechnology,

Fukuoka Agricultural Research Center,

Chikushino 818-8549, Japan

Copyright Society for In Vitro Biology May/Jun 2007

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