NaCl dependent production of coniferin in Alluaudiopsis marnieriana suspension cultured cells

Abstract

A stable salt-tolerant cell-suspension culture of Alluaudiopsis marnieriana was established, and intracellular compounds that accumulated under salt-stress conditions were investigated. HPLC/MS, and NMR analyses indicated that enhanced accumulation of coniferin was found during the growth phase in medium containing 150 mM NaCl. Coniferin or its derivatives may play an important role in salt-tolerance mechanisms in this plant.

Salinity is known to affect many physiological and metabolic processes, and causes reductions in plant growth and productivity (Jia et al. 2015). However, some plant species, especially halophytes, have evolved a variety of mechanisms to deal with salt stress (Yamada et al. 2002a, b, 2003). Analysis of the mechanisms that enable halophytes to survive under high levels of NaCl will be useful in enhancing agricultural productivity in salinized regions.

Alluaudiopsis marnieriana is a member of the Didiereaceae, a family of arboreous fleshy plants that grow in dry salty soils in southwestern districts of Madagascar (Rowley 1992). Although there is little information about their mechanisms for salt tolerance, it can be postulated that this plant species has unique mechanisms for stress resistance. In this paper, stable cell-suspension cultures of A. marnieriana were established and the compounds that accumulated under salt-stress conditions were investigated to examine salt tolerance mechanisms in this species at the cellular level.

Leaves of A. marnieriana were surface-sterilized, cut into pieces, and placed on Murashige and Skoog’s agar plates (Murashige and Skoog 1962) supplemented with 10⁻⁶ M 2,4-dichlorophenoxyacetic acid, 10⁻⁶ M benzylaminopurine, 20 g l⁻¹ sucrose, and 0.8% agar at pH 5.6 to generate callus material. The plates were incubated at 25°C under 16 h light/8 h dark conditions. After one month of cultivation, yellowish-white calli were generated. The calli were transferred to Erlenmeyer flasks (300 ml) containing 50 ml of the liquid Murashige and Skoog’s medium and shaken at 80 rpm in the dark at 25°C. After growth was initiated, the suspension-cultured cells were reinoculated in the liquid Murashige and Skoog’s medium. Inoculation was repeated every two weeks for one year, and stable suspension-cultured cells were obtained.

Figure 1 shows a microscopic observation of the suspension-cultured cells. On the 8th day after the start of culture, the wet and dry weights of the cells were 12.2±0.00 g and 0.45±0.09 g, respectively, under 0 mM NaCl condition, and the water content was 96.3±0.07% whereas under 150 mM NaCl condition, the wet and dry weights were 6.2±0.00 g and 0.25±0.02 g, respectively, and the water content was 95.0±0.03%. Which formed small clusters consisting of many ellipsoidal cells in the absence of NaCl (Figure 1A). In contrast, increased spherical cells were found when the cells were cultured in the presence of 150 mM NaCl (Figure 1B). Enhancement of cell division under salt conditions may affect cell morphology. Similar cells were observed in medium containing 500 mM sorbitol [9.11% (w/v)] (date not shown). It can be postulated that these events were dependant on osmotic stress.

Figure 1. Microscopic observation of stable suspension-cultured cells of A. marnieriana grown for 8 days in NaCl-free MS medium (A), or in medium containing 150 mM NaCl (B). Each culture was precultured in each medium. Bars indicate 50 µm.

We focused on the soluble fraction, considering the possibility of compatible solutes peculiar to this plant. The intracellular compounds that accumulated in the suspension-cultured cells under salt-stressed conditions were investigated using high-performance liquid chromatography (HPLC, LaChrom Elite, Hitachi High-Technologies co., Tokyo, Japan; see Figure 2). Cells that grew in medium containing 0 or 150 mM NaCl for 8 days were filtered onto Whatman No. 1 filter papers and freeze-dried for 3 days using a freeze-dry system (Model Free Zone 18; LABCONCO, MO, USA). Distilled water (500 µl) was added to 10 mg dry cells, which were incubated at 4°C for 24 h. Each sample was then centrifuged at 20,000×g for 30 min and the upper phase (50 µl) used for HPLC analyses. Figure 2A and 2B show the chromatogram of each cell extract cultured without NaCl (A) or with 150 mM NaCl [0.877% (v/v)] (B). The five major peaks in each chromatogram were numbered on the basis of retention time, and the spectra were monitored using diode array detector (DAD) five peaks in Figure 2A and 2B were compared, and significant differences were found in peak no. 5. Absorption spectrum (200–350 nm) of peak no. 5 recorded online during HPLC/DAD analyses showed a characteristic spectrum with absorption maxima at 210, 258, and 296 nm (Figure 2C).

Figure 2. HPLC chromatograms of cell extracts from cells cultured without NaCl (A) or with 150 mM NaCl (B). Conditions for HPLC analysis are as follows: column, Nomura Chemicals Develosil RPAQUEOUS-AR-5 (250 mm×4.6 mm i.d.); column temperature, 30°C; solvent, 0.20% CH₃CN aq. (20 min); flow rate, 1 ml/min; monitored wavelength, 258 nm. The spectrum of compound 5 was monitored by DAD (C).

The structure of the unusual compound 5 was identified by purifying the extract of cells grown in the presence of 150 mM NaCl under the following conditions. Distilled water (5 l) was added to approximately 100 g of freeze-dried cells and extracted at 4°C for 24 h. The extract was prefiltered using Advantec No. 3 filter paper, and then filtered with a Millipore membrane filter (HVLP04700). Following filtration, the extract was applied to an 8 cm×3 cm column of Wakosil 25C18 (Wako Pure Chemical Industries, Ltd., Osaka, Japan). After washing with 10% aqueous acetonitrile, acetonitrile : water 10 : 90 [% (v/v)], the 15% aqueous acetonitrile fraction was collected, dried, and resuspended in 50 ml of distilled water. This fraction was applied to a 3 cm×3 cm column of Dowex Marathon WBA (Sigma-Aldrich, MO, USA) to remove the anionic compounds, and the aqueous wash was collected. The collected fraction was then used with DOWEX 50WX8-100 (Sigma-Aldrich), and the aqueous wash was collected, dried, and resuspended in 8 ml of distilled water to remove the cationic compounds. Finally, compound 5 in the partially purified sample was fractionated using HPLC under the following conditions: Develosil RPAQUEOUS-AR-5 (250 mm×4.6 mm i.d.; Nomura Chemical Co., Ltd., Aichi, Japan); column temperature, 30°C; solvent, 12.16% (v/v) aqueous acetonitrile (9 min); flow rate, 1 ml/min.

Using positive ion electrion-mass spectrometry (MS), the purified compound 5 produced a quasimolecular ion at m/z 365.12 [M+Na]⁺. The ¹H-nuclear magnetic resonance (NMR) spectrum (CD₃OD) showed characteristics of a coniferyl alcohol moiety: δ 3.87 (s, 3H), 4.2 (d, J=6.9, 2H), 6.28 (dt, J=19.5, 5.7, 1H), 6.54 (d, J=19.5, 1H), 6.94 (dd, J=8.3, 1.9, 1H), 7.07 (d, J=2.3, 1H), and 7.1, (d, J=8.3, 1H). The ¹³C NMR spectrum of compound 5 showed an oxygenated methylene carbon (δ 62.5) and five oxygenated methine carbons (δ 71.4, 75.0, 77.9, 78.3, and 102.8) (Daubresse et al. 1998). This suggested the presence of a hexosyl moiety in the molecule. Using enzymatic hydrolysis, the hexosyl moiety in compound 5 was determined to be a β-glucopyranoside. The heteronuclear multiple bond correlation observed between an anomeric proton at δ 4.88 (d, J=8.7, 1H) and the quaternary carbon signal at δ 147.7 indicated that glucose was attached at the C-4 position of coniferyl alcohol. Therefore, the structure of compound 5 was identified as coniferin (Greca et al. 1998).

The relationship between cell growth and coniferin accumulation in the suspension-cultured cells was investigated by culturing them in medium containing 0, 50, 150, and 250 mM NaCl. Coniferin contents were then determined. Growth curves are shown in Figure 3A and 3B, and growth was estimated using fresh weight (FW; Figure 3A) and dry weight (DW; Figure 3B). Reduced biomass of these cells can be thought to depend on cell death, as well as Arabidopsis thaliana suspension-cultured cells after the exponential growth phase (Kobae et al. 2006). The suspension-cultured cells were able to grow in the presence of 250 mM NaCl. Approximately 0.1–0.2 mg/g DW of coniferin was detected in cells cultured with 0 and 50 mM NaCl (Figure 3C). Eight days after inoculation, a maximum coniferin content of 0.9 mg/g DW was observed in cells cultured with 150 mM NaCl. The content gradually decreased after this time (Figure 3C). The coniferin content was less than 0.1 mg/g DW in the presence of 250 mM NaCl (Figure 3C).

Figure 3. Growth curves of suspension-cultured cells of A. marnieriana on the basis of FW (A) and DW (B). Time courses of coniferin content in the cells are shown in (C). Each culture was precultured in medium containing the concentration of NaCl to which cells are adapted; 0, 50, 150, 250 mM, and cell cultures were initiated by inoculation of 0.7–1.0 g FW per culture bottle. Cells were cultured in medium containing 0 (○), 50 (●), 150 (△), or 250 (▲) mM NaCl, and Each point represents the means of three replicates±s.d.

Coniferin, a coniferyl alcohol-β-D-glycoside, is an important precursor of the cell-wall constituent, lignin (Freudenberg 1965). It is widely spread in various plant species including gymnosperms (Terasawa et al. 1984a) and angiosperms (Terasawa et al. 1984b). However, there is little information regarding the relationship between coniferin production and salt stress in plants. However, it has recently been shown that coniferin accumulation is increased in Arabidopsis callus-suspended cells adapted to high salinity compared with control cells. (Chun et al. 2019). Sánchez-Aguayo et al. showed that tomato plants under salinity stress undergo xylem lignification (Sánchez-Aguayo et al. 2004). In the next step, it is necessary to investigate the relationship between salt stress and lignification in these cultured cells.

Coniferin, although incorporated at the coniferyl-alcohol level, is also known as an efficient precursor of podophyllotoxin, an anticancer agent. To enhance the production of podophyllotoxin, coniferin was added to cell cultures of Podophyllum hexandrum (Woerdenbag et al. 1990). This was because coniferin synthesis is the rate-determining step in the production of podophyllotoxin. The NaCl-dependent production of coniferin shown in this work will be useful in enhancing the production of coniferin and its derivatives.

In this paper, a salt-tolerant stable cell-suspension culture of A. marnieriana was established, and an intracellular compound that accumulated under salt-stress conditions was identified as coniferin by HPLC/MS, and NMR analyses. The discovery of NaCl-dependent coniferin synthesis will contribute to an elucidation of the salt tolerance mechanisms of members of the family Didiereaceae.

Abbreviations

MS

mass spectrometry

DAD

diode array detector

FW

fresh weight

DW

dry weight