A comparative study of phenolic acids associated with cell walls and cytoplasmic extracts of host and non-host roots for AM fungi

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1 New Phytol. (1996), 133, A comparative study of phenolic acids associated with cell walls and cytoplasmic extracts of host and non-host roots for AM fungi BY GERALD NAGAHASHI*, GLORIA D. ABNEY AND LAND IS W. DONER U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Philadelphia, PA, 19118, USA (Received 27 July 1995 ; accepted 10 January 1996) SUMMARY Carrots (Daucus carota L.) are a ubiquitous host for arbusctilar myeorrhizal fungi whereas sugar beets (Beta vulgaris L.) are a non-host. Root cultures were used to compare the constitutive phenolic compounds associated with the cell wall or present in the cytoplasm of the host and non-host. Phenolic acids were released from purified cell walls by alkaline hydrolysis and were separated and identified by HPLC, TLC and u.v. absorption spectra analyses. Two phenolic acids unique to carrot root cell walls were identified as ^-hydroxybenzoic acid (p-hba) and vaniuic acid. Sugar beet root cell walls had ferulic acid as major constituent and contained several unique phenyl propanoids which were not identified. Caffeic acid was found only in the cytoplasm of carrot roots and was present in the conjugated form (chtorogenic acid). The sugar beet cytoplasm also contained several unidentified hydroxycmnaniic acid-type phenolics w'hich were not found in carrot roots. Key words: Cell-wall phenolics, cytoplasmic phenolics, AM host and non-host roots. INTRODUCTION wall as a likely source. In this study, the cell Vi'all was purified and separated from the cellular contents Considerable effort has heen put into the study of (cytoplasm). Phenolics were released from the cell oimpounds in root exudates and whole-root extracts wall and from cytoplasmic conjugates by alkaline which stimulate the growth of hyphae of arbuscular hydrolysis. The goal of the research was to determine mycorrhizal (AM) fungi (Elias & Safir, f987; Becard whether there were phenolic compounds uniquely & Piche, 1989; Chahot et al. 1992). The role of associated with host roots and not with non-host fiavonoids in host-microhe interactions is well roots, and to determine whether these compounds documented (Phillips, 1992) and recent efforts have were located in the cytoplasm or associated with the shown a stimulation or inhibition of germ-tube cell wall, growth by fiavonols and phenols (Becard, Douds & Pfeffer, 1992; Chabot et al. 1992; Phillips, 1992). p^i^^rl^ls AND METHODS Since germinating hyphae of G. margarita spores appear to be attracted to and to infect sites on the ^^^^ cultures primary root where secondary roots have initiated (Becard & Fortin 1988), it is likely that cell-wall Clones of Ri T-DNA-transformed roots of carrot constituents released during secondary root growth and sugar beet were obtained from Becard & Fortin will act as attractants or stimulants of fungal hypha! (1988) and were routinely propagated on minimal growth In the search for these plant chemical (M) medium m Petri dishes (Becard & Fortm, 1988). signals no studv has concentrated on the plant cell Root explants were propagated at specific stages as ' ' described by Becard & Piche (1992). Roots were * To whom correspondence should be addressed., harvested by rapidly dissolving the gellaii in 25 mm Mention of brand or firm names does not constitute an ^^.j^, ](,uffer containing 10 mm EDTA at ph IS endorsement by the U.S. Department of Agriculture over others ^ j^^^^^ ^, ^; of a similar nature not mentioned. '^ «

2 282 G. Nagahashi, G. D. Abney and L. W. Doner nm nm Caffeic acid \ p-hba / ". / " \ Ferulic acid \ Figure 1. HPLC chromatogram of a set of standards separated on a C18 reverse-phase column and eluted with a hnear gradient of acetonitrile in ph 30 water. Retention times were min (p-hba), min (caffeic acid), 1427 min (Ji-coumaric acid), and min (ferulic acid). Chlorogenic acid, m-coumaric, and vanillic acid had retention times of nain, min, and min, respectively (data not shown). Suhcellular fractionation procedures Harvested roots were pulverized in liquid nitrogen, suspended in homogenization medium containing 10 mm sodium metabisulphite in 50 mm HEPES- MES buffer at ph 7-7, and placed in a Parr nitrogen bomb at kg cm~' for 15 mm at 4 C. The contents of the bomb were extruded to atmospheric pressure to release cellular contents as described previously (Nagahashi & Seibles, 1986). The crude homogenate was separated into crude cell wall and cytoplasmic fractions by trapping the walls on cheesecloth and washing the cellular contents through the sieve. The cytoplasmic fraction was centrifuged at 0^ for 3 min to pellet any small cell wall fragments which passed through the filter. The cytoplasmic fraction, which contained subcellular membranes and cytosolic components, was decanted and used for further treatments. The isolated cell walls were purified by sonicating, washing in cold water, and trapping on cheesecloth as described (Nagahashi & Seibles, 1986), Vascular cell walls were separated from cortical cell walls by centrifuging the cell-wall fraction through a 50/60 % (w/w) sucrose gradient at 900 rpm for 5 min at room temperature (Nagahashi, Abney & Uknalis, 1994), Alkaline hydrolysis and butanol extraction The purified cell-wal! and cytoplasmic fractions were diluted 1:1 with an equal volume of 2 N sodium hydroxide and hydrolysed in the dark under a constant stream of nitrogen. After constant stirring at room temperature (25 C) for 15 h, the cell walls were removed by filtering through a sintered glass filter. Both the cell-wall hydrolysate and cytoplasmic fractions were then titrated to ph 1-5 with concentrated HCl and the phenols were extracted with two volumes of 1-butanol to one volume of hydrolysate. The extracts were roto-evaporated (Buchi Rotavapor RElll) to dryness, re-dissolved in methanol, and again evaporated to dryness. The methanol step was repeated several times to reniove the butanol. In the initial experiments, the cytoplasmic fraction was not alkahne-hydrolysed before extraction with alcohol, Chromatographic analyses High-performance liquid chromatography was performed with a Spectra-Physics 8800 ternary pump and SP 4270 integrator. The spectra Focus (forward optical-scanning detector) detection system was used, which allows for a rapid wavelength scan every

3 Phenolics in host and non-host roots for AM fungi Ferulic acid 1 p-hba V- 2 VanN'Ifc acid y Figure 2. HPLC separation of phenolics which were released by alkahne hydrolysis of carrot root eel! walls. Column and elution were identical to Figure 1. See text for details of the cell-wall extract preparation. second and is analogous to a photodiode array detector. A 5/xM spherical C18 column was eluted for 25 min with a linear acetonitrile gradient (0-55 % acetonitrile) in water adjusted to ph 3-0 with 85% phosphoric acid (Graham, 1991). This was followed by a step increase to % acetonitrile and held for 2 min before returning to % -svater at ph 3-0. Some chromatograms were run with the same pump, column and gradient, and an SP 4290 integrator, although detection was done with an HP 1047A RI detector. TLC was performed with Baker SiHPF-silica gel plates with a solvent system composed of toluene and acetic acid (8:2 (v/v)). Phenolics were detected by viewing untreated plates at 254 nm or 366 nm, treating plates with iodine vapour, or staining plates with the Folio-Ciocalteu phenol spray (Sigma). Further confirmation of the identified phenolics was made by paper chromatography and TLC with K2 cellulose plates, followed by a chromatic spray (Ibrahim & Towers, 1960). RESULTS Alkaline treatment was necessary to hydrolyse phenolic esters in the cell walls, and the extraction of phenolic compounds was performed at ph 1-5 in alcohol. The HPLC column used and the solvent system employed to elute the colutnn were described earlier (Graham, 1991). A set of standards was chromatographed; the retention times observed (Fig. 1) were very similar to previously reported values

4 284 G. Nagahashi, G. D. Abney and L. W. Doner Table 1. Rf values of phenolic standards compared with compounds associated with cell walls and cytoplasm of carrot roots and sugar beet roots Carrot Sugar beets Standards Cell Wall Cytoplasm Cell Wall Cytoplasm Vanillic acid Ferulic acid ^-HBA Caffeic acid TLC was performed on SiHPF-silica gel plates with a toluene; acetic acid (8:2 (v/v)) solvent system. 260 nm nm Fetulic acid E o 0 Figure 3. HPLC separation of phenolics which were released by alkaline hydrolysis of sugar beet root cell walls. Conditions -u'ere idetitical to Figure 2. (Graham, 1991). The HPLC chromatogram of a carrot cell-wall extract showed a prominent peak at 10-9 min and a minor peak at 12-4 min when monitored at 260 nm (Fig. 2). At 330 nm, a prominent peak at min and two small peaks at 18-2 min and 21-8 min -were observed. Rf values (Table 1) and absorption spectra analyses confirmed the presence of ^-HBA (10-9 min), vanillic acid

5 Phenolics in host and non-host roots for AM fungi Chlorogenic acid ^ Figure 4. HPLC separation of alcohol soluble compounds which were extracted from carrot root cytoplasm without pretreatment in alkali. See text for details. (12-4 min), and ferulic acid (15-15 min) in the cell wall of carrots. The unidentified compounds at 18-2 min and 21-8 min appear to be bydroxycinnamic acid derivatives. They both had two absorbance peaks c. 290 nm and 330 nm, with slightly greater absorbance at 330 nm. By contrast, the sugar heet cell walls had a considerably different phenolic content from the host cell walls. Ferulic acid was present (Fig. 3) but neither >-HBA nor vanillic acid were detected. F^our phenolics with retention times of 15-4, 17-1, 18-0 and 18-8 min, respectively, were unique to the non-host cell walls. These have not yet been identified, hut are likely to be hydroxycinnamic acid derivatives or coumarin derivatives, as indicated by their corresponding absorbance spectra (Fig. 3). The unidentified compound with a retention time of 154 min was not m-couinaric acid, since the absorption spectrum was very dissimilar to the m-coumaric standard. The unknown at 18-0 min in sugar beet cell walls (Fig. 3) was different from the unidentified compound in carrot cell walls with a similar retention time of 18-2 min but with dissimilar absorption spectra.

6 286 G. Nagakashi, G. D. Abney and L. W. Doner 260 nm Figure S. HPLC separation of compounds which were alkaline-hydrolysed from carrot-root cytoplasmic conjugates before extraction in butanol. See text for details. In our initial experiment with the cytoplasmic fraction of carrot roots, the crude fraction was not alkaline-h5'drolysed before butanol extraction. After HPLC separation, a very complicated chromatogram was observed (Fig. 4). The corresponding absorbance spectra for several of the peaks indicated the presence of many phenylpropanoid-type derivatives. Because most phenolic acids do not occur in the free form but are esterified to sugars, aliphatic and aromatic alcohols, or hydroxy acids, an alkaline hydrolysis was performed before acid extraction in butanol. The resulting chromatogram was considerably simplified (Fig. 5). The increase in free ferulic acid in the alkaline-hydrolysed extract indicated that several different feruloyl esters were hydrolysed. The disappearance of chlorogenic acid (retention time of 11-3 min) in the untreated control (Fig. 4) and the corresponding appearance of caffeic acid (Fig. 5) after alkaline treatment confirmed the hydrolysis of the ester linkages. One unidentified phenolic was present before and after alkaline hydrolysis and was very hydrophilic in nature (retention time of 8 min). Alkaline hydrolysis of the sugar beet cj^toplasmic fraction yielded ferulic acid as a major constituent (Fig. 6). The peak at 11-3 min had a similar retention time to ^-HBA but the absorbance maximum at 274 nm was unlike that of the ^-HBA standard (absorbance maximum at 254 nm). One phenyipropanoid standard (m-coumaric acid) had an absorbance maximum at 274 nm but its retention time on the column used in this study was min. Two minor peaks at 18-8 min and 19-5 min have not been identified yet. The compound with a retention

7 Phenolics in host and non-host roots for AM fungi Z Ferulic acid.o < Figure 6. HPLC separation of compounds which were alkaline-hydrolysed from cytoplasmic conjugates of sugar beet roots before extraction in butanol. See text for details. time of 18-8 min was also present m the cell-wall extract of sugar beet roots (cf. Figs 3, 6). DISCUSSION If the cell walls are a source of signals that are involved in recognition events, stimulation of hyphal growth, and possibly appressorium forniation, then the cell walls of a host plant should show inherent differences from those of a non-host plant. This study showed that the carrot cell wall has at least four phenolic compounds (including ^-HBA and vanitlic acid) which are not present in sugar beet cell walls. Furthermore, the sugar beet eel! wall has at least four compounds (retention times of 15'4, 17-1, 18'0 and 18'8 min) which are not present in carrot root cell walls. To complete the comparative study, the cytoplasmic fractions from sugar beets and carrots were also compared. Caffeic acid, and an unidentified peak with retention time of 8 min were unique to the carrot cytoplasm. Five compounds with retention times of 11-3, 14-3, 17-1, 18-8, 19-5 min were only found in the sugar beet cytoplasm. The results reported here are significant and relevant to future research on host-am fungi

8 288 G. Nagahashi, G. D. Abney and L. W. Doner interactions. Carrot roots contain unique phenolics both in the ceil wall and in the cytoplasm. Similarly, sugar beet roots contain unique phenolic compounds extraceilularly and intracellularly. In our subsequent paper (Douds et al., 1996), the efficacy of the identified phenolics on the growth of germinated AM fungal spores is tested. This approach would be useful in determining what makes a root a host or non-host for mycorrhizal association. REFERENCES Becard G, Douds DD, Pfeffer PE Extensive hyphal growth of vesicular arbuscular mycorrhizal fungi in the presence of CO^ and fiavonols. Applied Environmental Microbiology 58: Becard G, Fiche \ Establishment of VA mycorihizae in root organ culture: review and proposed methodology. In: Norris JR, Read DJ, Varma AK, eds. Methods in Microbiology, Vol. 24, London: Academic Press, Becard G, Piche Y Fungal growth stimulation by CO, and root exudates m vesicular-arbuscular inycorrhizal symbiosis. Applied Environmental Microbiology 55: , Becard G, Fortin JA Early events of vesicular-arbuscular mycorrhizal formation on Ri T-DN.A transfornned roots. New Phytologist 108: 211. Chabot S, Bel Rhlid R, Chenevert R, Pjche Y Hyphal growth promotion in vitro of the VA mycorrhizal fungus, Gigaspora margavita Becker & Hall, by the activity of activity of structurally specific flavonoid compounds under CO^-enriched conditions. New Phytologist 122: 461^67. Douds DD, Nagahashi G, Abney GD The differential effects of cell wall-associated phenolics, cell walls, and cytoplasmic phenolics of host and non-host roots on the growth of two species of AM fungi. New Phytologist, this volume, Elias KS, Safir GR H^'phal elongation of Glomus fasciculatum in response to root exudates. Applied Environmental Microbiology Graham T A rapid, high resolution high performance liquid chromatography profiling procedure for plant and microbial aroinatic secondary metabolites. Plant Physiology 95; Ibrahim RK, Towers GHN The identification, by chromatography, of plant phenolic acids. Archives of Biochemistry of Biophysics 81: , Nagahashi G, Abney GD, Uknalis J Separation of vascular cell walls from cortical celt w-alls of plant roots. Protoplasma 178: Nagahashi G, Doner L, Ahney GD, Tsao A The low temperature, rapid dissolution of gellan away from root cultures, biotechnology Techniques 7: Nagahashi G, Seibles TS Purification of plant cell w alls: isoelecttic focusing of CaCl,, extracted enzymes. Protoplasma 134: , Phillips DA Flavonoids: plant signals to soil microbes. In: Stafford HA, Ibrahim RK, eds. Recent Advances in Phytochemistry, Vol. 26. New York : Plenum Press

Appressorium formation by AM fungi on isolated cell walls of carrot roots

New Phytol. (1997), 136, 299-304 Appressorium formation by AM fungi on isolated cell walls of carrot roots BY G. NAGAHASHI* AND D. D. DOUDS, JR USDA, Agricultural Research Service, Eastern Regional Research