Shimuzu, 1989; Rathore, Hotary & Robinson, 1989/j; Harold & Caldwell, 1990). Instead the. 1990; Collings, White & Overall, 1992; Hush, current pattern may ...
Neiv Phytol. (1995), 129, 433-438
Electrical currents associated with arbuscular mycorrhizal interactions MORRIS", H. M. A. C. FONSECA', B. REID", N. A. R. GOW'^t AND M. J. DAFTi BY R. L. L. B E R B A R A ' * , B. M .
^ Biological Sciences Department, University of Dufidee, Dundee, DDl 4HN, Scotland, UK ^Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen, AB9 IAS, Scotland, UK (Received 9 June 1994; accepted 18 November 1994) SUMMARY Electrical currents associiited with the plant-fungus interaction between roots and an arbuscular mycorrhizal (AM) fungus Gigaspora mtirgarita were measured with a one-dimensional vibrating probe. Uninfected roots of clover (Trifolium repens L. cv. New Zealand White) and carrot (Daucus carota L. cv. Nantes) had an inwardly directed positive electrical current at the apical meristen-iatic and elongation zones and an outward flow at the mature non-growing regions. The current profile of endomycorrhizal colonized roots did not differ markedly in magnitude or pattern compared with non-colonized roots of an ecjuivalent stage of development. .^ sn-iall current was measured circulating around and through single mycorrhizal azygospores ot G. mtirgarita. CJerm tubes emerged within the zone of outward current and an inward current was found at the hyphal tip. When roots were colonized by the fungus an inward current was induced at the point of hyphal contact with the host root. This current declined over a period of 6 d. Because hyphal penetration of plant roots by AM fungi does not rupture the plant host membrane we propose that the host current is not the result of wounding but is induced by fungal elicitors at the infection court. Key words: Arbuscular mycorrhiza, electrical currents, plant-fungus interaction, root gern-iination, fungal spore.
: iNTRODUCTroN Vibrating, voltage-sensitive microelectrodes (Jaffe &: Nuccitelli, 1974; Schelfey, 1986; Nuccitelli, 1990) have facilitated precise non-invasive n-ieasurement of endogenous electrical fields and currents around plant roots (Weisenseel, Dorn & Jaffe, 1979; Behrens, Weisenseel & Sievers, 1982; Bjorkman & Leopold, l9H7a,b; Miller, Shand & Gow, 1988; Miller & Gow, I989rt,/); lwabuchi, Yano & Shimuzu, 1989; Rathore, Hotary & Robinson, 1990; Collings, White & Overall, 1992; Hush, Newman & Overall, 1992). Vibrating microelectrodes have also been used to detect electrical currents associated with the growth and differentiation of many other eukaryotic organisms (CJOW-, 1989rt; Nuccitelli, 1990; Raven, 1991) including fungi (Armbruster & Weisenseel, 1983; Kropf e/ al.,
1983 ; Kropf et al., 1984; Horwitz et al., 1984; Gow, ^^^^^ McGillivray & Gow, 1986; Schreurs & Harold, 1988; Youatt, Goyv & Gooday, 1988; Gow, 1989«; De Silva e/rt/., 1992). The electrical currents of fungi are carried mainly by protons (McGillivray & Gow, 1987; Takeucbi f/rt/. 1988 ; Gow, 1 989fl, De Silva et al., 1992). The direction and magnitude of flow of these currents do not correlate with growth or with hyphal extension rate and so they are not thought to play a direct role in these processes (Gow-, 1989/j; Harold & Caldwell, 1990). Instead the current pattern may reflect spatial differences in sites of nutrient trai-isport into fungal cells (De Sih'a c/rt/., 1992). In plants roots electrical currents are also carried mainly by protons and are the consequence of asymmetries in the distribution of ion transport proteins in the plasn-ia men-ibrane of cells in the root cortex (Weisenseel et al., 1979; Miller & Gow, 1989a; Miller et al., 1991). The physiological role of * Present address: Universidade Federal Rural do Rio de the currents or roots is not clear It has been ^2k^-9T^^l ' ' """'"• ^"•""='''™' ''"«""' "J- suggested that they may be inx-olved with the To whom c'orrespondence should be .sent. acquisition of nutrients (Miller et al., 1991 ; Raven,
434
R. L. L. Berbara and others
1991), the transport of hormones across tissues, the regulation of gravitropic root bending (Behrens et al., 1982; Bjorkman & Leopold, 1987a, b; Iwabuchi et al., 1989; Collings et al., 1992), and growth and structural polarity of the root (Weisenseel et al., 1979; Toko et al., 1987; Miller & Gow, \9%9a,b\ Rathore et al., 1990; Nick & Furuya, 1992), The electrical current of plant roots is a labile and dynamic process and its magnitude and direction is dependent on a number of factors which include pH, root maturity, plant species, the presence of plant growth regulators and the nitrogeti status of the root (W^eisenseel et al., 1979; Miller & Gow, 1989a, 6; Rathore et al., 1990; Miller et al., 1991; Gow, Morris & Reid, 1992), The current direction can be inward or outward at the meristematic and elongation zone of plant roots; however, the majority of plant species thus far examined have an inward current flow at the actively growing region at the root apex and an outward How at the more mature areas of the root, distant from the tip (Miller & Gow, 19896, Gow et aL, 1992), Wound sites are invariably sites of large, inwardly directed ion currents (Miller et al., 1988; Hush & Overall, 1989; Hush et al., 1992). These proton circulations result in the establishment of substantial p H gradients in the rhizosphere (Weisenseel et al., 1979; Miller et al., 1991; Hush et al., 1992) whose significance is only now being explored. The endogenous electrochemical gradients generated in the rhizosphere may have an important influence on microorganisms that form pathogenic or symbiotic associations with plant roots. For example, zoospores of phytopathogens have been shown to exhibit electrotaxis in weak electrical fields of a magnitude comparable with those generated by roots (Miller et al., 1988; Gow et al., 1992; Morris, Reid & Gow, 1992; Gow, 1993; Morris & Gow, 1993). Zoospores may therefore use a combination of chemotaxis and electrotaxis to locate the root surface of a host plant. Since plant roots of species that are commonly influenced with mycorrhizal fungi are known to generate ion currents (see Gow et al., 1992 for a review), we have investigated the electrical currents of plant roots, infected and non-infected with a mycorrhizal fungus and the spores and hyphae of the arbuscular mycorrhizal fungus Gigaspora margarita. The possible significance of the electrophysiological interactions between the two partners in this plant-fungus interaction is discussed. MATERIALS AND METHODS
Production of root cultures and infection protocol Transgenic roots of clover {Trifolium repens L. cv. New Zealand White) and carrot {Daucus carota L. cv. Nantes) were used as a model system in these experiments. These were obtained by transforming plants with Arobacterium rhizogens LBA 9402
carryitig the Ri T-plasmid and binary vector pBin 19 as described by Kumar & Forrest (1990), Transgenic roots had been cultured over a 2 yr period and were chosen for these studies on the basis that they had maintained a stable phenotype over this period. Roots were grown in Petri dishes filled with a minimum salts medium (Becard & Fortin, 1988) overlaid with a dialysis membrane. Spores of Gigaspora margarita Becker & Hall were supplied by Dr M. Paula (Emrapa-CNPBS, Brazil), surfacesterilized as described by Becard & Piche (1992) and placed in Petri dishes with or without roots (one spore per Petri dish). Once colonization of a root system by the fungus was well established the dish was flooded with a low-salt artificial pond water solution (APW; 1-0 mM NaCl, 0-1 mM KCl and 0-1 mM CaClj, adjusted to pH 6-5) prior to electrophysiological examination with a vibrating microelectrode. This medium had a conductivity of 160//S cm-'. Measurement of ion currents The ion currents produced by colonized and noncolonized transformed roots, the tips of AM hyphae and by spores were measured with a one-dimensional vibrating microelectrode as described by Miller et al. (1988), A stainless steel microelectrode (Microprobe Inc, PO Box 87, Clarksburg, USA) was electroplated to form a platinum black ball of ~ 25 fim diam. at the electrode tip. The electrode was vibrated at 325 Hz with an amplitude of 30//m. The potential difference across the vibration distance was determined using a two-phase lock-in differential amplifier (Model 5208, EG & G Instruments, Bracknell, UK). The conductivity of the bathing medium was measured using a Kent Eil conductivity meter (Model MC-1, Mk 4, Kent Industrial Instruments Ltd,, Surrey, UK). Measurements were made adjacent to the fungus or root and at a reference position at least 1 cm from the test site. Current densities were calculated from electrical field measurements and a knowledge of the medium resistivity (Jaffe & Nuccitelli, 1974). Measurement of ion currents around roots The current flow associated with colonized and noncolonized roots was determined at 100//m ititervals from the growing tip, at a fixed distance of 50/^m from the root surface. At least six separate roots were measured. The current flow associated with the penetration point of colonized T. repens roots was determined 50 fim from the root surface, opposite the attachment and penetration point, and up to 200 /im either side of the penetration point. All measurements were carried out in APW at pH 6-5, Three separate, colonized roots were used to obtain values for mean current densities.
Electrical currents of myeorrhizae
435
Measurement of ion currents around spores and hyphae of G. margarita The current flow associated with the growing tips of eight hyphae of G. margarita was measured 20 //m from the hyphal surface. Hyphae were gro'wn from individual spores germinated oti the mitiimal medium of Becard & Fortin (1988). Each germinated spore was mounted on a silver wire loop prior to measuring with the vibrating microelectrode. The loop was of silver wire (50 //m diam.; Goodfellow Metals, Science Park, Cambridge, UK) and was mounted onto die base of a Petri dish usitig autoclave tape. The loop formed a platform ~ 5 tnni above the Petri dish surface. Spores were placed in the loop allowing free access to the spore circumference by the vibrating microelectrode. Current profiles produced by germinated and non-germinated spores were measured 20 //m frotn their surface in APW at pH 6-5. Cotitrol experiments tested for possible barrier artefacts as described by SchefTey (1986). Specimens were observed during vibrating electrode measurements with an Olympus CK2 inverted microscope at a magtiification of x 100. Error values, 'where cited, are statidard deviations. RESULTS
Electrical currents in colonized and non-colonized transgenie roots Non-colonized transgenie roots of T. repens and D, carota had electrical current patterns similar to those found previously for tion-transformed roots (Miller et aL, 1986; Miller & Gow, 1989«). The currents were itiwardly directed at the root cap and the detisity of this current iticreased basipetally towards the meristem, peaked at the zones of elongation then diminished in the region of root maturatioti and at the root hair zone. The current direction then
Outwatd -2-0 0.00 0-25
0-50
0-75
1-00
1-25
1-50
Distance from root tip (mm)
Figure 2. Magnitude and direction of ion current flow transgenie roots ot D. carota colonized (O) or noneolonized ( • ) with G. margarita. Values are means±s.D for at least six roots. reversed to an outward one in the mature root region (Figs 1, 2). The maxitnum current density measured at the apices of either T. repens or D. carota roots was 1-06 ±0-49 /lA cm"- and 1-46 ±0-38 //A cm"- {n = 6) respectively. Roots of T, repens atid D. earota colonized by G. margarita showed no marked differetice in their current profile compared with noti-colonized roots (Figs 1, 2). Colonized roots of T. repens and D. carota had meati apical current densities of 0-28 + 0-08/*Acm-' and 0-16 ±0-04/(A cm"" (maximum values of 1-65+ 0-09//A cm"" and L66 + 0-40/;A ctn~'^; n = 6) respectively. Electrical currents around spores and AM hvphae
The niagtiitude and directioti of etidogetious electrical currents measured around germinating spores atid the tips of hyphae of G. margarita growing in a tninimal salts solution containing no exogenous carbon or tiitrogen source are sliowti in Figure 3. No Inwatd currents were detected around dormant spores. Prior 1-5to germitiation, weak asymmetric curretits were I foutid circulatitig aroutid isolated spores with no 1-0 o obvious relationship between the architecture of the \T 0-5 spore and the sites of inward and outward current. The maximum outward and inward current densities 0-0 were 17 + 7 atid 35 + 6 (« = 6) nA cm"- respectively. -0-5Germinating hyphae emerged from areas of outward fiow and were associated with a tiew zone of itiward -10curretit at the point of hyphal etiiergence. A small inward current (barely detectable except at high -1-5sensitivity in tnedia of high resistivity) with a mean Outward ?-0 density of 2-0 + 0-1 nA cm"- {n = 8) was found at the 0-00 0-25 0-50 0-75 1-00 125 1-50 hyphal tip. The current direction opposite the Distance from root tip (mm) germination site was unstable, in some cases Figure 1. Magnitude atid direction of ion current flow reverting to a small (10 + 5 nA cm"'', n = 6) inward around traiisgenie roots of T. repens colonized (O), or noncolonized (0) with G. margarita. Values are means+ S.D flow. No measurable current flow was detected alotig the letigth of hyphae. Current measurements made for at least si.x roots.
N
1
k
436
R. L. L. Berbara and others
(a)
Electrical currents at the site of penetration
o
