Journal of Experimental Botany, Vol. 53, No. 371, pp. 1143-1154,
May 2002
© 2002 Oxford University Press
Original Papers |
Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis
1Institute of Experimental Phytopathology, Slovak Academy of Sciences, SK-90028-Ivánka pri Dunaji, Slovakia
2Mori-machi Laboratory of Plant Physiology, 443-5, Enden, Shizuoka, 437-0221, Japan
3Institut für Botanik, Technische Universität, Schnittspahnstr. 3, D-64287 Darmstadt, Germany
4University of California, Department of Land, Air, Water Resources, 151 Hoagland Hall, Davis, CA 95616-8627, USA
Received 21 June 2001; Accepted 17 January 2002
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Abstract |
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Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles. The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays. To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (Em) in cross-sections were mapped. The measured cells were identified by injected Lucifer Yellow. Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to Em values of about -170 mV, which suggest high plasma membrane proton pump activities. Rapidly dividing cells of cambia or small tumour parenchyma cells had low Em. The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential. The lowest values were recorded in stem pith cells. Cell K+ concentrations largely matched the respective Em. The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements. The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells. TTPs were inhibited by addition of NaN3, CN- plus SHAM or N2 gas in the xylem perfusion solution and by external N2 flushing. The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.
Key words: Agrobacterium tumefaciens-induced tumours, rays, respiration-dependent tumour energization, trans-tumour electropotential difference, xylem parenchyma.
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Introduction |
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Agrobacterium tumefaciens-induced plant tumours (crown galls) are structured by well differentiated concentric vascular bundles. They consist of xylem surrounded by phloem, and are functionally connected to the host bundles. The increased auxin production and accumulation, caused by the integration and expression of the bacterial T-DNA within the higher plant genome (Zambryski et al., 1989), leads to remarkable ethylene production. Ethylene induces typical structural changes in the stem/tumour interface, namely a higher number of vessels of significantly smaller diameter and multiseriate unlignified rays compared with the xylem of control stems. These findings are summarized in the ‘gall constriction hypothesis’, which describes the water flow to the shoot, giving priority to the gall at the expense of the host shoot. Assimilates are similarly attracted by the tumour at the expense of root development and ion absorption (Ullrich and Aloni, 2000; Mistrik et al., 2000). In general, rays are regarded as being necessary for the distribution of assimilates and nutrients in a lateral direction between the sieve elements and xylem across the cambial cell layer (Van Bel, 1990). In rapidly growing plant tumours, xylem parenchyma cells (XP) and the unusually multiplied multiseriate and unlignified rays were supposed to have particularly high membrane pump activities (Aloni et al., 1995).
Xylem parenchyma cells in roots and shoots have been shown to be highly active in ion release into the xylem vessels or in absorbing ions out of the vessels in an energy-dependent process (Läuchli et al., 1971; De Boer et al., 1985). The existence of back-to-back electrogenic H+ pump activity across the boundary membrane of the organ surface and across the xylem parenchyma symplast/ xylem interface has been demonstrated (Okamoto et al., 1978, 1984; De Boer et al., 1983). Strong evidence for the operation of XP pumps was derived from the electrical potential difference between different tissues of Vigna hypocotyls in whole plants and from data with a sophisticated xylem perfusion system in hypocotyl segments. Accordingly, XP cells must be well equipped with PM H+-ATPases. These findings were in agreement with the cytochemical localization of PM H+-ATPase hydrolytic activity in barley roots (Winter-Sluiter et al., 1977). The histochemical lead precipitation method revealed a substantial density in the stele in contrast to the cortex of wheat roots (Eleftheriou and Lazarou, 1997). Recently, voltage-dependent XP-located K+ channels, outward directed (SKOR), inward directed (KIRC and AKT1) and non-specific ion channels, have been identified (Wegner and Raschke, 1994; Roberts and Tester, 1995; Lagarde et al., 1996; De Boer and Wegner, 1997; Gaymard et al., 1998; De Boer, 1999). Some channels responded to water stress, and ABA decreased the outward-directed K+ current (Roberts, 1998; Gaymard et al., 1998; Roberts and Snowman, 2000). The addition of auxin to the perfusion solution significantly hyperpolarized XP and enhanced K+ absorption from the vessels (De Boer et al., 1985). In A. tumefaciens-induced tumours a high transformation rate of up to 100% with the auxin genes-containing T-DNA (Rezmer et al., 1999) is consistent with a strong auxin accumulation of up to 500 times that of control tissues (Kado, 1984). Therefore, the objective of the present study was to know whether T-DNA-transformed tumour cells, high in auxin content, differ in electrical properties from non-transformed stem tissue. More hyperpolarized cells would substantially contribute to the driving force for nutrient acquisition from the host shoot into the rapidly growing tumour.
The flow of assimilates from source leaves into and across the tumour was elucidated in detail (Malsy et al., 1992). By using carboxyfluorescein, Lucifer Yellow CH movement and GFP-labelled Potato Virus X it could be demonstrated that, in spite of high acid cell wall invertase activity, the sieve element content is symplastically unloaded into tumour parenchyma cells (Pradel et al., 1996, 1999). The high cell wall invertase activity could recently be localized in the bundle-free, well-aerated aerenchyma (A Weilmünster and CI Ullrich, unpublished results).
The present experiments were designed to understand nutrient transfer from host vessels into different tumour cell types. Individual voltage measurements were performed in cross-sections of tumour and stem tissue. The cell type concerned was identified upon injection of the fluorescent Lucifer Yellow CH. To obtain information about cell membrane energetics in the intact, but otherwise inaccessible system, the xylem perfusion trans-tissue-electropotential method was employed (Okamoto et al., 1978). This method allows cell activities of XP and rays of the stem/tumour contact zone to be detected. It reveals cellular proton pump activities within the stele, causing voltage changes that are measurable between the xylem perfusion solution at any cut surface of the stem and the external tumour surface.
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Materials and methods |
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Tumour induction
Ricinus communis L. var. gibsonii cv. Carmencita (Walz Samen, Stuttgart, Germany) and cowpea, Vigna unguiculata L. cv. Otsubu (Asahi Noen, Sofue, Aichi, Japan), were grown in standard potting soil (LD 80) in growth chambers under universal white fluorescent lamps (L 58 W/25; Osram, München, Germany). They were exposed to 14 h light (200 µE m-2 s-1) at 27 °C and 10 h darkness at 21 °C with 60–80% relative humidity.
Ten days after sowing and after the appearance of the first leaves, the plant hypocotyls were wounded with a razor blade 10 mm below the cotyledons and inoculated with a bacterial pellet. The wild-type strain C58 of Agrobacterium tumefaciens, obtained from the Max-Planck-Institut für Züchtungsforschung (Köln, Germany), was grown for 24 h in YEB medium (1 g yeast extract, 5 g beef extract, 5 g peptone, 5 g sucrose l-1 and 2 mM MgSO4, pH 7.8). For inoculation the bacteria were harvested by centrifugation for 10 min at 3600 g.
Electrophysiology
Tissue cross-sections of 1 mm thickness were mounted in a horizontal Plexiglas chamber, perfused with the experimental solution, containing 0.5 mM KCl and 1 mM CaSO4. After 7 h of preincubation in the experimental solution voltage changes were recorded by means of glass microelectrodes connected to a high-impedance electrometer amplifier (L/M-1, List, Darmstadt, Germany). Further details have been described earlier (Ullrich and Guern, 1990).
After voltage measurements the cell type was identified by Lucifer Yellow CH (LYCH) injection. For this the tip of the glass micropipette was filled with 10 µl of 10 mg ml-1 LYCH solution (Sigma) and filled with 3 M KCl. The dye was iontophoretically injected by current pulses of -30 nA of up to 30 s duration, generated by the electrometer. LYCH fluorescence was detected under the epifluorescence microscope (Aristoplan, Leica, Bensheim, Germany; filter block I3, excitation BP between 450 and 490 nm and emission LP 515 nm).
Intracellular K+ concentration was determined with K+-selective microelectrodes in 1 mm tissue sections, after 7 h preincubation in 1x solution (containing 1 mM KCl, 1 mM NaH2PO4, 1 mM Ca(NO3)2 and 0.25 mM MgSO4, pH 5.6) and measured in 0.5 mM CaSO4 solution. K+-selective microelectrodes were filled with 7 µl K+ ionophore I cocktail B (No. 60398; Fluka, Buchs, Switzerland) stabilized with 1.2 mg PVC dissolved in 25 µl tetrahydrofurane. The K+-selective and the Em micropipettes and the reference electrode were filled with 0.5 mM LiCl as electrolyte and connected to a high-impedance differential electrometer amplifier (L/MPR 20, List). The K+ electrodes were calibrated before and after each experiment. The K+ concentration of whole tissues was determined by flame photometry (Eppendorf, Hamburg, Germany) after extraction with boiling distilled water for 30 min.
Trans-tumour electropotentials (TTP) were determined (Okamoto et al., 1978). Stems of 8–10 cm length with attached tumours of various age were excised and mounted into a Perspex chamber (Fig. 1A
), fixed on a microscopic stage. The stems were sealed with instant glue in flanges of hard vinyl with a central hole fitting to the particular stem diameters between 3 mm and 15 mm diameter. Thus the stem base was separated from the main upper part to ensure that the perfusion stream is conducted only inside the stem xylem and to prevent electrical leakage along the stem and tumour surface. 20 min after gluing the stem to the flange, the lower cut end always being immersed in the experimental solution, the stem within the flange was mounted in the Perspex chamber and fixed with additional washers and O-rings. The chamber and thus only the external surface of the plant tissue was flushed with humidified air or purified, humidified N2 gas (5.0) through a separate gas inlet. A tygon tubing with 3% KCl in agar, connected to an Ag/AgCl electrode (EH-2FS, Wright, Guilford, CT, USA), which was filled with 3 M KCl, was positioned on the tumour surface through a hole in the main part of the chamber. The reference electrode was immersed in the perfusion exudate droplet at the top of the stem, which was continuously sucked off by a peristaltic pump. The exuding sap was not in contact with the tumour surface. Both electrodes were connected to the electrometer amplifier (L/M-1, List). The flow of the xylem perfusion solution of 1 ml h-1 (experiments in Figs 5–7, 9) and 20 ml h-1 (experiments in Fig. 8) was under slight hydrostatic pressure, which was applied over a tightly glued inlet into a small pre-chamber at the base of the stem (Fig. 1). The basic solution (1 mM KCl and 0.5 mM CaSO4) was rapidly exchanged with solutions containing fusicoccin (FC, 30 µM), abscisic acid (ABA, 100 µM, pH 5.6), glutamic acid (10 mM, pH 5.6), NaN3 (1 mM), or NaCN plus salicylhydroxamic acid (SHAM; 1 mM, pH 6) or was saturated by N2 gassing.
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(gm/gP) for the energy-dependent component (electrogenic H+ pump); ED=
(gm/gD) for the energy-independent component, diffusion potential (passive channels); (A, electrometer amplifier; E* based on measured data; el, electrode; gD, passive conductance; gm, membrane conductance; gP, pump conductance; PC, peripheral cells; R, chart recorder; T, stem tumour; TTP, trans-tumour electrical potentials; XP, xylem parenchyma cells).
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Histochemical staining
Hand-cut or vibratome tissue sections of about 50 µm thickness were prepared from tumour and host stem tissue. The xylem was stained with a 0.05% aqueous solution of toluidine blue for 0.5–3 min and immediately viewed in the microscope. Callose was stained with 0.1% aniline blue (in 1 M glycine at pH 9.4) for 1 min. The yellow-white fluorescence of the pit fields of the rays was viewed under UV-light of the epifluorescence microscope (Leica filter block A: excitation BP 340–380 nm; emission LP 430 nm). To detect endodermal suberin-containing Casparian strips around the stele of stems of Vigna unguiculata and Ricinus communis, tissue sections were stained with berberine-aniline blue (Brundrett et al., 1988). Sections were incubated in 0.1% berberine hemi-sulphate for 1 h, after rinsing they were transferred to 0.5% aniline blue solution for 30 min and after rinsing again in water they were transferred to the mounting medium, consisting of 0.1% FeCl3 in 50% glycerol on slides. Samples were viewed in the same way as those stained with aniline blue.
Mitochondrial and cytosolic reducing activity was visualized by staining the sections with 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT, Biomol, Hamburg, Germany). Samples were incubated for 2 h in a solution containing 5 mg MTT in 1 ml 0.15 M phosphate buffer at pH 7.5.
To follow the pathway of water movement in the xylem out of the host stem into the tumour vessels, only the base of excised host stems was placed into PbEDTA solution for 48 h (modified after Crowdy and Tanton, 1970). The lead was precipitated in the intact tumours as PbS by releasing H2S gas from FeS with 5 N HCl for 30 min in a glass chamber. 25 mM PbEDTA was prepared by dissolving 4.15 g Pb(NO3)2 and 4.65 g of Na2EDTA in water and mixing 1:1. The chelate precipitate was centrifuged, redissolved in water at pH 7.2 and made up to 500 ml. Hand-cut sections of the treated tumours were immediately viewed by light microscopy. Micrographs were reproduced from colour slides taken with an Orthomat E camera system (Leica) on Kodak Ektachrome Elite 100 or 200 ASA daylight or EPY 64T film.
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Results |
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Cell membrane potentials
Different tissue types were investigated with respect to their electrical membrane potentials (Em) in cross-sections. Independent of the different tissue types, values attained their maximum in 3–4-week-old tumours (Figs 2A
, B, 3A–C). Xylem parenchyma cells (XP) of the pathological xylem in the tumour/host stem interface (Fig. 4A, I), identified by Lucifer Yellow CH injection and viewed under fluorescent light (Fig. 4N), displayed the highest Em values, more negative than -170 mV (Fig. 2A). Em values of XP cells in bundles distal from the tumour (Fig. 4A, E) and those within the tumour bundles were not significantly different. However, Em values of XP from control stems of the same age as those with tumours were considerably lower: between -140 and -150 mV (Fig. 2A).
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Like Em of XP cells, that of rays, multiseriate or uniseriate (Fig. 4A
, C, F) was similarly high with -160 to -180 mV, in rays between the stem and tumour and in rays located distal to the tumour as well. However, Em of rays of control stems were, like those of control XP cells, about -130 to -140 mV (Fig. 2B). Ray and XP cells were characterized upon MTT staining by high mitochondrial reducing activity (Fig. 4B, F, I). Rays across the cambium were identified by strong callose staining, indicating the abundance of pit fields and, hence, of plasmodesmata (Fig. 4G). By contrast, the large non-transformed aerenchyma cells (Fig. 4B, H) had Em values of only about -100 mV (Fig. 2B), with an energy-independent diffusion potential of about -70 mV, as determined with N2 gassing (Fig. 9B).
Em of phloem parenchyma (PP) cells (Fig. 3A) was also high in the tumour/stem interface region where host and tumour bundles merge, and in a similar range as that of XP and that within the tumour. Em of phloem parenchyma cells distal from the tumour was lower and that of control stems was only about -120 mV. Correspondingly, carboxyfluorescein (CF), applied to the apical leaves, was restricted to the SE-CC complex in the control stem (Fig. 4D
). However, in plants with tumours, CF rapidly migrated out of the distal sieve elements into the PP cells, indicating a strong signal from the tumour towards the host stele (Fig. 4Dß). Em of sieve elements of the uninfected control stem and in the tumour did not differ and was in the range of -175±6 mV (n=12).
Interestingly, Em of rapidly dividing cells like those of the cambium was low, between -80 and -100 mV, irrespective of whether the cells were located at the stem/tumour interface, opposite the tumour or in control stems (Fig. 3B). Also Em of small parenchyma cells, which existed only in the tumour (Fig. 4H) was only slightly higher with -100 to -110 mV (Fig. 3B). Em of cortex cells of control stems (Fig. 4E) and those distal to the tumour (Fig. 4A) had a similarly low level of -100 mV as the tumour aerenchyma (Fig. 3C). Em of pith cells of stems with tumours and control stems (Fig. 4A, B, M) was the lowest with -40 to -60 mV (Fig. 3C).
Mapping of tissue K+ concentrations
High Em may indicate high proton pump activity or a pump state with low membrane permeability for K+ and may lead to higher ion absorption and accumulation. Therefore, intracellular K+ concentrations of the different cell types were measured, which was done with K+ selective electrodes. A comparison of these data with the K+ concentration of whole tissues (Table 1) suggests that the K+ electrode had been located in the vacuole of the respective cells. Therefore, results are assumed to reflect the vacuolar K+ concentration. Tumour sieve elements had the highest K+ concentration of about 220 mM, those in the stem slightly less with 170 mM. Small tumour parenchyma cells, in spite of relatively low Em, still had a considerable K+ concentration of 140 mM. Corresponding to their lower Em, stem cortex and tumour aerenchyma cells had only 110 and 93 mM K+ on average and stem pith cells only 65 mM K+. The lowest K+ concentration of about 30 mM was recorded in stem vessels (Table 1).
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Trans-tumour electrical potential difference
In cowpea (Vigna unguiculata) electropotentials measured between the stem surface and xylem exudate solution, as indicated in Fig. 1A, B, were slightly negative for control stems (Fig. 5A). Flushing the stem surface with N2 resulted in an apparent hyperpolarization of about 20–30 mV. Re-aeration immediately restored the original level (Fig. 5A). Such a potential difference results from back-to-back cell membrane potentials of tissues separated by conductivity barriers (Fig. 1B). While N2 was flushed around the stem surface, the xylem vessels, to which xylem parenchyma and ray cells are directly exposed, remained supplied with O2 by the perfusion solution (Fig. 1A). Hence, N2 only affected the outer cells and the activities of the inner cells became apparent (Figs 5–9). Since these are back-to-back values, the voltage differences of the interior were increasingly negative with decreasing values in the outer cell layers. Both cowpea and Ricinus stems with well-developed tumours exhibited anoxia-induced potential changes greater than 60 mV, which in cowpea only slowly recovered upon re-aeration (Fig. 5B, C). In cowpea, xylem and phloem tissue are separated from the cortex cells by the endodermal cylinder with Casparian strips in the cell walls (Fig. 4K, L), which cause the insulating effect. In Ricinus no such distinct berberine–aniline blue stainable material could be detected. Nevertheless, anoxia-induced long-lasting electrical differentiation between outer and inner tissues was similar to that known from cowpeas.
Stems with tumours were cut 3 cm below the tumour (Fig. 1). One hour after cutting off the stems and the onset of xylem perfusion, the TTP was most negative with about -50 mV and became constant at about -10 mV for more than 14 h more (Fig. 6A). Experiments were usually performed between 4 h and 7 h after excision. Anoxia-induced 
TTPs were largest in 3-week-old tumours (Fig. 6B), which means tumours of about 2 g FW (Fig. 6B, inset). Such electropotential changes were nearly twice those of control stems (Fig. 6B).
The measurement of TTPmax was dependent on the position of the recording surface electrode. TTPs were largest, i.e. 50 mV on average with the electrode at the tumour (Table 2). Values obtained from the stem surface opposite the tumour or above were the same as from control stems. Only below the tumour, where the xylem is considerably enlarged, were intermediate values of about 30 mV recorded (Table 2).
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Addition of the PM H+-ATPase activator fusicoccin (FC) to the xylem perfusion solution resulted in a more negative TTP (Fig. 7B
), presumably due to the multiseriate and tumour-dependent activated XP cells. By immunofluorescence microlocalization these XP cells were recently found to contain high concentrations of auxin and cytokinins (M Langhans, unpublished results). Anoxic gassing inhibited the outer membrane pumps and resulted in increasingly negative TTP values (Fig. 7B). While the anoxia effect was reversible, the FC effect persisted due to irreversible binding of the toxin to the plasma membrane (Fig. 7B).
Omitting K+ from the CaSO4 perfusion solution resulted in smaller anoxia effects but similar FC effects (Fig. 7C). Abscisic acid (ABA), known to decrease outward-directed K+ channel activity of XP membranes, slowly decreased the equilibrium voltage by 10–20 mV (Fig. 7D), while the anoxia effect was even slightly enhanced (Fig. 7D). Addition of 5 mM NaF to the xylem perfusion solution had no effect on TTP, neither in cowpea stem controls or cowpea tumours, nor in Ricinus tumours of various sizes, not even during xylem perfusion for up to 3.5 h (data not shown).
Addition of glutamic acid to the xylem perfusion solution (10 mM at pH 5.6) induced characteristic TTP changes due to H+/glutamic acid co-transport. After an apparent hyperpolarization an apparent depolarization was evident, indicating a transient XP depolarization with a subsequent repolarization overshoot, which is typical for acid amino acids (Pavlovkin et al., 1984). Anoxia caused a further strong apparent TTP hyperpolarization (Fig. 7E).
Since the tumour cells did not respond to NaF, inhibitors of mitochondrial respiration were added to the xylem perfusion solution. NaCN plus SHAM, NaN3 and also N2 gassing resulted in apparent TTP depolarization by 27±11 mV (9) (Fig. 8A, C, E). In contrast, in the control stems these inhibitors had only a very small effect by 5±3 mV (9) or in many experiments even no effect (Fig. 8B, D, F). Flushing the tumours with N2 gas from outside apparently hyperpolarized TTP and additional xylem perfusion with the respiration inhibitors completely reversed the apparent TTP changes by additional inhibition of the interior electrogenic pumps (Fig. 8G–K).
These measurements are not easy to interpret because they are a complex sum of cell polarization with opposite signs (Fig. 1B). Therefore, the results were completed by simultaneous measurements of TTP (Fig. 9A) and intracellular voltage of the peripheral tumour cells (Fig. 4H) at the same tumour-stem system (Fig. 9B). With the electrode located in the tumour cells, external anoxia caused a rapid membrane depolarization and internal fusicoccin a considerable hyperpolarization, which was affected by anoxia as well. During simultaneous TTP measurements, external anoxia, by decreasing the outer membrane potential, induced hyperpolarization, thus revealing XP activities, i.e. polarized XP cells: FC, applied to the inner tissues with the xylem perfusion solution, induced hyperpolarization as well, in this case by hyperpolarizing the XP cells (Fig. 9A).
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Discussion |
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Agrobacterium tumefaciens-induced plant tumours were, until recently, regarded as an undifferentiated mass of cells, but are now known to be characterized by a dense net of vascular bundles (Ullrich and Aloni, 2000). In the present study, the flow of water through the host stem into even remote tumour vessels could be labelled by precipitating Pb as PbS from a soluble PbEDTA-chelate (Fig. 4J
). While Pb remains in the apoplast, nutrients have to be efficiently absorbed from the vessels by xylem parenchyma cells via an energy-requiring process. The high auxin (x500) and cytokinin (x1600) concentrations of such tumours induce ethylene emission, which dramatically changes the xylem structure at the host/tumour interface (Aloni et al., 1998; Wächter et al., 1999). A large xylem area develops smaller vessels, which are surrounded by multiseriate layers of unlignified rays (Fig. 4A, C). These parenchyma cells are supposed to be the essential site of considerable inorganic ion acquisition. A precondition for efficient ion transport is energized proton pumps at the plasma membrane, which in turn energize carriers and ion channels. In particular, auxin is supposed to induce not only structural changes but also enhanced proton pump activity and hence ion absorption from the xylem apoplast, as known from cowpea xylem perfusion experiments (De Boer et al., 1985). In Kalanchoë daigremontiana total tumour K+ concentration was found to amount up to five times the control values and inorganic phosphate concentration even to eight times that of the control (Marx and Ullrich-Eberius, 1988).
The Em measurements revealed high membrane potentials of XP, PP and rays (Figs 2, 3). In particular, the XP cells were highly activated by tumour-induced signals such as considerably increased concentrations of auxin and cytokinins, as recently detected by immunolocalization with monoclonal antibodies (M Langhans, unpublished results). In agreement with the energetics, the increased K+ concentration of tumour cells corresponded to higher Em values, except those in cambium cells (Table 1). Rather low membrane transport activity was observed for stem cortex cells, non-transformed tumour aerenchyma and pith cells and, apparently, the rapidly dividing cambial cells. Em values were highest at a tumour age of 3 weeks (Figs 2, 3). These maxima were preceded by the earlier accumulation maxima of the phytohormones auxin and zeatin riboside at 2 weeks after infection (D Veselov et al., unpublished results).
The present data were obtained with intracellular microelectrodes from tissue slices of about 1 mm thickness. Therefore, it was important to know whether in the intact host stem/tumour system similar transport activities could be observed. To exclude possible slicing-induced changes in activity, trans-tumour electropotentials were obtained by measuring voltage differences between the xylem perfusion solution exuding on top of the cut stem and the tumour surface. Indeed, anoxia flushing of the gas space around the tumour depolarized the membranes of outer cells and apparently hyperpolarized cells of the interior. Actually, the latter maintained their activity due to the xylem perfusion with the aerated experimental solution. Since both tissue types are localized back-to-back (Fig. 1B), the Em of XP became apparent. By perfusing the stem vessels with the PM H+-ATPase activator fusicoccin, hyperpolarization of the inner cells became prominent and confirmed the existence of highly active xylem-located cells (Fig. 7B, C). Simultaneous measurements of the TTP and Em of peripheral cells with an intracellular microelectrode support this interpretation (Fig. 9). In these experiments the position of the intracellular Em electrode was apparently within the cytosol, according to gentle Lucifer Yellow injection after Em measurements (Fig. 4M, shown in a pith cell). Only after injection for a long-time and under higher current, the vacuole became fluorescent as well. Moreover, the distinct response of the TTP pattern to anoxia suggests the existence of insulating material within the tumour, which divides the apoplastic compartment surrounding each symplast into two parts: the xylem-side space and the peripheral-side space. Otherwise such a reaction in the TTP would be difficult to explain. In cowpea stems Casparian strips could be identified in the endodermis around the stele of the hypocotyl (Fig. 4K, L). In Ricinus hypocotyls the berberine–aniline blue test was negative. But the presence of a cell layer similar to a dense starch-sheath around the stele and transformed tumour tissue suggests a similar insulating function (Fig. 4H).
Animal and human tumours are known to maintain a high rate of glycolysis under aerobic conditions (Dang and Semenza, 1999). A similar metabolism was assumed for plant tumours (Beiderbeck, 1977). The strong reaction of the TTP to anoxia and the absence of any NaF effect, however, infer that the energy metabolism in the tumour tissue is mainly oxygen-respiration dependent. Both tissue compartments, the well-aerated peripheral and the inner one facing the xylem, were both equally sensitive to the inhibitors of mitochondrial respiration, CN- plus SHAM, NaN3 and N2 (Fig. 8). Hence the high membrane pump activity of XP and ray cells in the tumour/stem interface together with low permeability for K+ increases ion absorption out of the host xylem solution. By contrast, perfusing the xylem of the control stem with these inhibitors had only a minor or no effect (Fig. 8). Thus the hypocotyl-located XP proton pumps apparently have low or no activity and will energize ion absorption out of the xylem fluid to a much lesser extent.
Not only XP and ray cells in the tumour/stem interface, but also the stem XP and phloem parenchyma cells (PP) opposite to the tumour were hyperpolarized beyond the respective values of the control stem. A signal from the tumour must be released into the host stem. Auxin, cytokinins and also ABA concentrations were significantly increased in the tumours. ABA accumulated up to 45 times in tumours and was still 20 times more concentrated in the phloem sap opposite to the tumour than in the non-infected control stem (Mistrik et al., 2000). ABA was found to inhibit K+ release from XP into the vessels through the outward rectifying K+ channels SKOR (Roberts, 1998; Gaymard et al., 1998; Roberts and Snowman, 2000). This suggests that the high tumour ABA concentration contributes to the ion attraction to and retention within the tumour parenchyma cells instead of release into the vessels. Also the sieve elements opposite to the tumour in the host stem are clearly affected by such signals from the tumour. Carboxyfluorescein, as a label of assimilate flow basipetally transported from leaves to roots, was restricted to the sieve element–companion cell complex in the control stem. In stems with tumours CF was transported in the lateral direction towards the tumour (Fig. 4D, ß)
The present data infer that the ethylene-caused multiplication of XP and in particular of ray cells is functionally essential for nutrient acquisition from the host vessels for tumour growth. Moreover, the data show that the fundamental electrophysiological structure of intact stems, as described by the back-to-back H+-pump model (Okamoto et al., 1978; De Boer and Prins, 1985; De Boer et al., 1985), is still maintained and even reinforced in the tumour tissue. It remains to be investigated whether proton pumps, carriers and channels of XP, PP and rays in the tumour are different iso-proteins in transformed tissue or are just highly multiplied and activated due to the considerable increase in phytohormone concentration of auxin, cytokinins, ethylene, and abscisic acid (Aloni et al., 1998; Wächter et al., 1999; Mistrik et al., 2000).
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Acknowledgements |
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This work was supported by grants of the Deutsche Forschungsgemeinschaft (SFB 199 and GK 340) to CIU. AL gratefully acknowledges a US Senior Scientist Award by the Alexander von Humboldt-Foundation (Bonn, Germany) and RW the support in CLSM techniques by Professor AJE Van Bel and Dr M Knoblauch (University Giessen, Germany).
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Footnotes |
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5 To whom correspondence should be addressed. Fax: +49 6151 164630. E-mail: uleb{at}bio.tu\|[hyphen]\|darmstadt.de
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References |
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