The Paxillus involutus isolates MAJ and NAU, obtained from the Büsgen Institute: Institute of Forest Botany and Tree Physiology (Göttingen University, Germany), were grown on 2% modified Melin Norkrans (MMN) agar medium (g⋅L^-1): KH₂PO₄ 0.5, (NH₄)₂SO₄ 0.25, MgSO₄⋅7H₂O 0.15, CaCl₂⋅2H₂O 0.05, NaCl 0.025, FeCl₃⋅6H₂O 0.01, thiamine HCl 0.0001, glucose 10, malt extract 3, pH 5.2 (Gafur et al., 2004; Li J. et al., 2012). Prior to the colonization, the fungi were pre-grown on the agar culture medium for 1 week in petri dishes (diameter 90 mm) and kept in darkness at 23°C.

Plantlets of Populus × canescens (a hybrid of Populus tremula × Populus alba) were propagated by micropropagation as described by Leple et al. (1992). Regenerated P. × canescens plants were grown for 3–4 weeks on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962). Uniform plants with sufficient roots were used for ectomycorrhization. The colonization of P. × canescens with Paxillus involutus strains MAJ and NAU was followed the procedures described by Gafur et al. (2004). In brief, rooted plantlets from sterile culture were placed on the MMN agar medium in the presence or absence of EM mycelium. After fungal inoculation, the petri dishes were sealed with Parafilm and covered with aluminum foil to keep the roots in darkness. During the period of incubation, the temperature in the climate chamber was maintained at 23°C with a light period of 16 h (6:00 AM–22:00 PM). Photosynthetic active radiation (PAR) of 200 μmol m^-2 s^-1 was supplied by cool white fluorescent lamps. After 1 month of inoculation, EM and NM root tips for anatomical investigations were embedded, stained, and photographed as described previously (Gafur et al., 2004). EM and NM plants with similar height and growth performance were used for CdCl₂ treatment.

Liquid culture of P. involutus was grown as previously described (Ott et al., 2002; Langenfeld-Heyser et al., 2007; Li J. et al., 2012). In brief, mycelium from the agar plate was homogenized, transferred into 100 mL of liquid medium (pH 4.8) in flasks, and incubated on a rotary shaker in darkness (150 rpm, 23°C). P. involutus in submerged culture grew in the form of compact spherical masses of mycelium (pellets). For Cd²⁺ shock treatment, sterile filtered CdCl₂ solutions were added to achieve final concentrations of 50 μM. After ST (24 h) or LT (7 days) treatment, axenic cultures of MAJ and NAU were used for steady flux measurements of Cd²⁺, H⁺, and Ca²⁺.

Ectomycorrhizal and non-mycorrhizal plants were carefully removed from MMN agar medium. Rooted plantlets were cultivated in individual pots containing hydroponic MS nutrient solution (MS medium without agar and sucrose) (Murashige and Skoog, 1962). Plants were covered with plastic bags to reduce the rapid water loss in a growth room. NM and EM plantlets were subjected to 50 μM CdCl₂ for a short-term (ST) exposure, 24 h or a long-term (LT) exposure for 7 days. The required amount of CdCl₂ was added to the MS nutrient solution. Control plants were treated in the same manner without the addition of CdCl₂. The plants were maintained at 23°C with a light period of 16 h (6:00 AM–22:00 PM) and PAR was 200 μmol m^-2 s^-1. Plants were continuously aerated by passing air to hydroponic MS nutrient solution, which was regularly renewed. Steady fluxes of Cd²⁺, Ca²⁺ and H⁺ in NM and EM roots were examined after 24 h and 7 days of CdCl₂ treatment. In addition, ST-induced alterations of Cd²⁺ and Ca²⁺ fluxes were also examined in non-inoculated and P. involutus-inoculated roots after 7 days of co-culture.

To determine whether Ca²⁺ ions compete with Cd²⁺ to penetrate across PM Ca²⁺-permeable channels, the effects of additional Ca²⁺ ions on Cd²⁺ electrodes was examined. Cd²⁺ calibrating solutions were added with 0, 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, or 2.0 mM Ca²⁺. Then Cd²⁺ microelectrodes were calibrated in Ca²⁺-supplemented solutions as described above. Moreover, the Nernst slope and intercept of the Cd²⁺ electrodes were calibrated in the measuring solution containing 0.1 mM KCl, 0.1 mM MgCl₂, and 0.05 mM CaCl₂.

Oscillations in membrane-transport activity are ubiquitous in plant response to salinity, temperature, osmotic, hypoxia, and pH stresses (Shabala et al., 2006). In our study, rhythmic (ultradian) flux oscillations in NM and EM P. × canescens roots were not noticeable as that observed in herbaceous species (Shabala et al., 1997, 2003, 2006; Shabala and Knowles, 2002). This finding is presumably due to a lower growth rate of woody roots compared with crop species (Li J. et al., 2012). The flux oscillations of the measured ions, e.g., H⁺, Ca²⁺, and Cd²⁺, were more like fluctuations as previously reported in poplar roots (e.g., Na⁺, K⁺, H⁺, and Ca²⁺; Li J. et al., 2012). In this study, H⁺, Ca²⁺, and Cd²⁺ fluxes were recorded for 6–8 min at each point, which is long enough to cover oscillatory periods of measured ions.

In this study, the effects of Ca²⁺, pH, H₂O₂, and PM transporter and channel inhibitors on Cd²⁺-altered ion flux profiles were examined in fungal mycelia and roots (NM and EM). Briefly,

Series 1: Ca²⁺ channel inhibitors. NM and EM roots were pre-treated with or without LaCl₃ (5 mM; Sun et al., 2010; Li et al., 2012b), GdCl₃ (500 μM, Demidchik et al., 2007, 2009; Sun et al., 2012), TEA (50 μM, White, 1998; Li J. et al., 2012), or verapamil (20 μM, Li et al., 2012a; He et al., 2015) for 24 h in the presence and absence of 50 μM CdCl₂. Fungal mycelia of the two P. involutus isolates, MAJ and NAU, were subjected to 0 or 5 mM LaCl₃ treatment for 24 h supplemented with or without 50 μM CdCl₂.

Series 2: Ca²⁺. After being subjected to Cd²⁺ stress (CdCl₂, 50 μM) for 24 h, NM and EM roots were then exposed to 25, 50, or 100 μM CaCl₂ for flux recordings in the presence of CdCl₂.

Cd²⁺ and Ca²⁺ fluxes in Series 1 and 2 were measured along root axes, 100–2,300 μm from the apex, at intervals of 200–300 μm. In P. involutus mycelia, Cd²⁺ and Ca²⁺ fluxes were continuously measured around the surface of pelleted hyphae over a recording period of 30 min.

Series 3: Hydrogen peroxide. NM and EM roots were sampled and immobilized in Cd²⁺ or Ca²⁺ measuring solutions for transient flux recordings in the apical region (100 μm from the root apex). The steady-state fluxes were continuously recorded for 10–20 min prior to the CdCl₂ shock. CdCl₂ stock (100 μM) was slowly added to the measuring solution until the final Cd²⁺ concentration reached 50 μM and transient kinetics of Cd²⁺ and Ca²⁺ were continuously for 20–30 min. Afterward, H₂O₂ (1.0 mM) was slowly added to the measuring solution and transient kinetics of Cd²⁺ and Ca²⁺ were restarted and continued for 20 min.

Series 4: ROS scavenger. NM and EM roots were pre-treated with or without 1, 3-Dimethyl-2-thiourea (DMTU, 5 mM, Chung et al., 2008; Sun et al., 2010) for 24 h in the presence and absence of 50 μM CdCl₂. Then Cd²⁺ and Ca²⁺ fluxes were measured along root axes, 100–2,300 μm from the apex, at intervals of 200–300 μm.

Series 5: External pH. NM and EM roots were pre-treated with 50 μM CdCl₂ for 24 h prior to flux measurements. Cd²⁺ and Ca²⁺ fluxes along root axes (100–2,300 μm from the apex) were recorded in Cd²⁺ or Ca²⁺ measuring solutions at pH 5.2, 6.2, or 7.2, respectively.

Series 6: PM H⁺-ATPase inhibitor. NM and EM roots were pre-treated with or without sodium orthovanadate (500 μM, Sun et al., 2010; Lu et al., 2013) for 24 h in the presence and absence of 50 μM CdCl₂. Then H⁺, Cd²⁺, and Ca²⁺ fluxes were measured along root axes, 100–2,300 μm from the apex, at intervals of 200–300 μm. P. involutus isolates, MAJ and NAU, were exposed to 0 or 500 μM sodium orthovanadate for 24 h prior to a 30-min of continuous recording of H⁺ flux.

An H₂O₂-sensititive microelectrode [tip diameter 2–3 μm, XY-DJ-502, Xuyue (Beijing) Science and Technology Co. Ltd., Beijing, China] was used to monitor H₂O₂ fluxes in EM and NM roots. H₂O₂ microelectrodes were prepared according to the method described by Twig et al. (2001). Before the measurement, H₂O₂ microelectrode was polarized at +0.60 V against an Ag/AgCl reference electrode. Thereafter, the microelectrodes were calibrated by the standard solution: 0.01, 0.1 and 1 mM H₂O₂. Roots sampled from control and CdCl₂ (50 μM,30 min)-treated EM and NM plants were immobilized in the measuring solution (0.1 mM NaCl, 0.1 mM MgCl₂, 0.1 mM CaCl₂ and 0.5 mM KCl, pH was adjusted to 5.2 with KOH and HCl) and equilibrated for 25 min. The fluxes were recorded 100 μm from the apex and conducted along the root axis until 2300 μm, at intervals of 200–300 μm, and then calculated.

Ionic fluxes were calculated using the program JCal V3.2.1, a free MS Excel spreadsheet, which was developed by the Yue Xu¹. The experimental data were subjected to SPSS (SPSS Statistics 17.0, 2008) for statistical tests and analyses. Unless otherwise stated, P < 0.05 was considered as significant.

H₂O₂-sensitive microprobes were used to detect the H₂O₂ response to Cd²⁺ exposure in NM and EM roots. In the absence of Cd²⁺, NM roots exhibited a stable H₂O₂ efflux (0.7–1.5 pmol cm^-2 s^-1) along the root axis; the mean flux rate increased 2.4-fold in response to Cd²⁺ treatment (50 μM CdCl₂, 30 min, Figure 6). Ectomycorrhization of poplar roots with P. involutus stains, MAJ and NAU, resulted in a significant increase of H₂O₂ efflux along the roots (Figure 6). However, upon CdCl₂ exposure EM roots displayed decreased H₂O₂ efflux in contrast to NM roots (Figure 6).

The woody Cd²⁺-hyperaccumulator P. × canescens exhibited a vigorous Cd²⁺ uptake after a 50 μM CdCl₂ shock (40 min), ST (24 h), and LT (7 days) treatment (Figures 1A and 2). The result is consistent with previous findings where P. × canescens roots exhibited a high Cd²⁺ uptake after 40 days of CdSO₄ exposure (50 μM, Ma Y. et al., 2014). Similarly, a high entry of Cd²⁺ was recorded in hyperaccumulating ecotypes of Sedum alfredii (Lu et al., 2010; Sun et al., 2013a) and Suaeda salsa under Cd²⁺ stress (Li et al., 2012a). An important result was that EM roots exhibited higher Cd²⁺ influx than NM roots irrespective of Cd²⁺ stress conditions, shock, ST, and LT (Figures 1A and 2). Substantial evidence indicates that Cd²⁺ can be enriched in ectomycorrhizal plants (Sell et al., 2005; Baum et al., 2006; Krpata et al., 2008, 2009; Sousa et al., 2012; Ma Y. et al., 2014). The enhanced Cd²⁺ uptake in EM roots is partly due to the capacity of the fungus to take up Cd²⁺ because CdCl₂ shock resulted in a net Cd²⁺ influx in the mycelia of the two P. involutus strains and the flux rate increased with the prolonged duration of CdCl₂ treatment from 24 h to 7 days (Figures 1A and 2). In liquid cultures, P. involutus cultures also showed high capacities for Cd²⁺ accumulation (Ott et al., 2002). P. involutus could bind Cd²⁺ onto the cell walls or accumulate the metal in the vacuolar compartment (Blaudez et al., 2000; Ott et al., 2002). Moreover, the ectomycorrhizal fungus appears to detoxify high concentrations of Cd²⁺ by (i) the chelation of metal ions in the cytosol with thiol-containing compounds, e.g., glutathione, phytochelatins, or metallothioneins (Courbot et al., 2004; Jacob et al., 2004), and (ii) activation of antioxidative defense system (Jacob et al., 2001; Ott et al., 2002). Our pharmacological data revealed that Cd²⁺ entered the fungal hyphae mainly through PM Ca²⁺ channels because the influx was suppressed by LaCl₃, a Ca²⁺ channel blocker (Supplementary Figure S8B). Therefore, Cd²⁺ enriched by ectomycorrhizal hyphae is thought to be transferred to the host roots, probably through the apoplastic space during the period of Cd²⁺ stress.

There were marked differences between the two strains in Cd²⁺ uptake given the shock treatment (Figure 1A). Pure fungal mycelium of MAJ accumulated Cd²⁺ with a higher rate than NAU (Figure 1A). In the P. involutus-ectomycorrhizal symbioses, the incompatible fungal isolate NAU is unable to induce a functional ectomycorrhizae while MAJ forms a typical Hartig net with the roots of P. × canescens (Gafur et al., 2004). Thus, in MAJ-colonized roots the host cells might have been more accessible to Cd²⁺. In accordance, MAJ roots exhibited a higher influx than NAU roots after the onset of CdCl₂ shock (Figure 1A). However, Cd²⁺ influx into NAU-colonized roots was similar to that of MAJ-colonized roots during ST or LT Cd²⁺ treatment (Figure 2). This was likely due to (i) similar capacities for Cd²⁺ uptake of MAJ and NAU hyphae during a 24-h or 7-days of Cd²⁺ exposure (Figure 2), or (ii) similar uptake capacity of the fungus-ensheathed inner root cells (Figure 2). The observed correlation between EM and NM roots showed that the continuous Cd²⁺ flow was mainly the consequence of host roots in the Cd²⁺-stressed ectomycorrhizal symbioses during a prolonged period of Cd²⁺ exposure (24 h to 7 days; Supplementary Figure S2).

Our data revealed that the entry of Cd²⁺ is likely mediated through PM Ca²⁺ channels in the fungal hyphae and poplar roots, and P. involutus-ectomycorrhizas facilitated the channel-mediated Cd²⁺ influx under Cd²⁺ stress. The experimental evidence for these conclusions is briefly listed below.

Collectively, under CdCl₂ stress Cd²⁺ ions could penetrate the PM Ca²⁺ channels in fungal hyphae and in P. × canescens roots. At present we cannot exclude the possibility that Cd²⁺ penetrated the PM through transporters for Cd²⁺ (Ma Y. et al., 2014; He et al., 2015) or other nutritional ions (Gussarsson et al., 1996; Cohen et al., 1998; Zhao et al., 2002; Cosio et al., 2004; Clemens, 2006), because (1) the four types of Ca²⁺ channel inhibitors applied here were not able to fully block the Cd²⁺ influx in NM and EM roots (Figure 7B, Supplementary Figures S5B, S6B and S7B), and (2) the total flux of Cd²⁺ and Ca²⁺ (Σ_Ca²⁺_+Cd²⁺, molar ratio of Cd²⁺ to Ca²⁺ is 1:1) under ST and LT Cd²⁺ stress was 10.9–27.7% higher than the flux rate of Ca²⁺ under non-Cd²⁺ conditions (Figure 4, Supplementary Figure S4). This implies that a small fraction of Cd²⁺ ions penetrated the PM through other channels and transporters.

Plasma membrane Ca²⁺ channels in P. involutus hyphae maybe more permeable to Cd²⁺ compared to the channels in P. × canescens roots as the fungal mycelium displayed a typical higher Ca²⁺ influx than poplar roots under control and Cd²⁺-stress conditions (Figures 1B and 3). We cannot discriminate between the channels of the fungus and those of the plant in the ectomycorrhizal symbiosis, but the Cd²⁺ and Ca²⁺ fluxes in EM roots appear to mainly reflect the response of the host plants to Cd²⁺ stress because (1) EM roots exhibited a different pattern from the P. involutus mycelia in enhancing Ca²⁺ and Cd²⁺ uptake under hydroponic Cd²⁺ conditions. Cd²⁺-shocked MAJ and NAU fungal strains usually displayed a stable Cd²⁺ influx with the exception of an initial transient increase (Figure 1A). However, EM roots showed a declined Cd²⁺ influx over the duration of Cd²⁺ exposure, similar to the Cd²⁺ kinetics in NM roots (Figure 1A). Moreover, the Cd²⁺ influx in the mycelia of the two P. involutus strains increased with the prolonged CdCl₂ exposure from 24 h to 7 days (from 23.9 to 72.7 pmol cm^-2 s^-1; Figure 2). In contrast, the Cd²⁺ fluxes in EM roots were relatively stable under ST (43.7 ± 8.4 pmol cm^-2 s^-1) and LT treatments (35.9 ± 6.0 pmol cm^-2 s^-1; Figure 2). ST, LT, and Cd²⁺ shock increased the Ca²⁺ influx in P. involutus mycelia, while the Ca²⁺ influx in EM roots was declined by these Cd²⁺ treatments (Figures 1B and 3). (2) NMT data showed that ion fluxes in mature P. × canescens–P. involutus symbiotic associations bear a striking resemblance to the ST inoculated roots (Supplementary Figure S1). Similar findings have been previously reported in a salt stress study where P. × canescens roots were inoculated with P. involutus for 10 and 20 days (Ma X. et al., 2014). At early stages of fungal co-culture the Cd²⁺ and Ca²⁺ influx is mostly the result of host properties. Therefore, the Cd²⁺ and Ca²⁺ stimulation in P. involutus-ectomycorrhizal roots reflects the enhanced root uptake ability. (3) The correlation analyses revealed that Cd²⁺ and Ca²⁺ influxes in EM roots show a significant relationship with NM roots but not with fungal mycelia under various Cd²⁺ treatments (shock, ST, and LT; Supplementary Figures S2 and S3). Taken together, these data suggest that the continuous flow of Cd²⁺ and Ca²⁺ in EM roots detected by NMT microelectrodes was largely driven by the host and that the fungal partner enhanced fluxes leading to enriched Cd²⁺ and Ca²⁺ concentrations.

The observed patterns of Cd²⁺ and Ca²⁺ fluxes upon Cd²⁺ exposure could be explained by channel-mediated ion fluxes. NMT data show that the Ca²⁺ flux in EM roots was negatively correlated with the Ca²⁺ influx in fungal hyphae upon Cd²⁺ shock treatment (Supplementary Figure S3). This is presumably the result of Cd²⁺-Ca²⁺ competition across the Ca²⁺ channels in the root PM. After being exposed to Cd²⁺ shock, Ca²⁺ entry was enhanced in the hyphae (Figure 1B). However, the fungal hyphae which were enriched in Ca²⁺ ions, were unable to deliver Ca²⁺ to the root cells because the Cd²⁺ ions competitively inhibited the entry of Ca²⁺ through the PM channels. As a result, the high influx of Ca²⁺ through fungal hyphae led to an apparently greater Ca²⁺ efflux in Cd²⁺-exposed EM roots (Figures 1B and 8B).

Paxillus involutus colonization enhanced the uptake of Cd²⁺ under shock, ST, and LT stress, compared to NM roots (Figures 1A and 2). The increased entry of Cd²⁺ is likely due to the activation of PM Ca²⁺ channels in the ectomycorrhizas. The stimulated Ca²⁺ influx by P. involutus inoculation revealed the activation of PM Ca²⁺ channels since the ectomycorrhiza-enhanced entry of Ca²⁺ was suppressed by Ca²⁺ channel blockers (LaCl₃, GdCl₃, verapamil, or TEA; Figure 7A, Supplementary Figures S5A, S6A, and S7A). The activated PM Ca²⁺ channels allowed the entry of Cd²⁺ in addition to Ca²⁺ under Cd²⁺ stress (Figures 1A and 2).

After being subjected to CdCl₂ exposure, NM roots displayed an increased H₂O₂ efflux along the root axis (Figure 6). It is well documented that Cd²⁺ induced accumulation of H₂O₂ in pine roots (Schützendübel et al., 2001), P × canescens roots (Schützendübel et al., 2002), and in suspension cultures of tobacco (Piqueras et al., 1999) and P. euphratica (Sun et al., 2013b; Han et al., 2016). H₂O₂ efflux was evident in EM roots irrespective of the presence or absence of Cd²⁺ treatments (Figure 6). Our results suggest that the Cd²⁺ influx through PM Ca²⁺ channels is stimulated by H₂O₂ in NM and EM roots. The experimental evidence and explanations are briefly listed here.

Taken together, these results suggest that Cd²⁺ and Ca²⁺ ions enter NM and EM roots by the same pathway involving PM Ca²⁺ channels that are activated by Cd²⁺-elicited H₂O₂.

The high Cd²⁺ influx in EM roots resulted from the pronounced activation of PM Ca²⁺ channels that were stimulated, at least in part, by the fungal-elicited H₂O₂. Compared to NM roots, MAJ- and NAU-ectomycorrhizal roots displayed a significant higher H₂O₂ efflux in the absence of Cd²⁺ stress (Figure 6), suggesting that the inoculation with P. involutus caused a strong production of H₂O₂ in EM roots. This finding agrees with Gafur et al. (2004) and Langenfeld-Heyser et al. (2007), who detected strong H₂O₂ accumulation in the outer hyphae mantle of compatible (MAJ) and incompatible (NAU) interactions. H₂O₂ production in the hyphae is suggested to regulate host’s root growth, defense against other invading microbes, and increasing plant-innate immunity (Salzer et al., 1999; Gafur et al., 2004). In our study, H₂O₂ produced in the ectomycorrhizae accelerated the influx of Ca²⁺ in the absence of Cd²⁺, whereas it increased entry of Cd²⁺ in the presence of high external Cd²⁺ (Figure 8). ROS scavenging by DMTU simultaneously decreased Ca²⁺ and Cd²⁺ influxes along the root axis of EM plants (Figure 9). These observations suggest that H₂O₂ produced in compatible (MAJ) and incompetent (NAU) ectomycorrhizal associations activated Ca²⁺ permeable channels, which allowed the entry of Cd²⁺ under Cd²⁺ stress.

We noticed that the H₂O₂ efflux in MAJ and NAU-ectomycorrhizal roots was lowered by Cd²⁺ stress (Figure 6). This reduction may have resulted from the activation of antioxidant enzymes and increased amounts of ROS scavengers produced as a defense response. It has been repeatedly shown that the antioxidant enzyme activities are activated under heavy metal stresses (Schützendübel et al., 2001, 2002; Rozpądek et al., 2014; Chen et al., 2015; Tan et al., 2015). The enhanced activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase play an important role in scavenging the Cd²⁺-elicited H₂O₂ in plants (Garg and Aggarwal, 2012; Anjum et al., 2015; Tan et al., 2015). To combat Cd²⁺-induced superoxide and H₂O₂, P. × canescens plants were found to rely mainly on antioxidant enzymes and the formation of the potential radical scavenging molecules, such as proline, sugar alcohols and soluble phenolics (He et al., 2011). However, the lowered H₂O₂ efflux in EM roots (Figure 6) did not reduce the Cd²⁺-elicited entry of Cd²⁺, because (i) the fungal-elicited H₂O₂ had already activated the Ca²⁺-channels before the Cd²⁺ addition, and/or (ii) the H₂O₂ level is still high enough to activate the channels under Cd²⁺ stress. The observation that stressed EM roots still contain high concentrations of H₂O₂ in the hyphae (Langenfeld-Heyser et al., 2007) supports these speculations.

In addition to H₂O₂, PM H⁺-ATPase activated by Cd²⁺ and enhanced by fungal colonization also accelerated Cd²⁺ influx through PM Ca²⁺ channels in NM and EM roots. PM H⁺-ATPases pump protons into the external medium to maintain an electrochemical H⁺ gradient across the PM (Blumwald et al., 2000; Zhu, 2003). Krämer (2010) suggested that H⁺-ATPases play an important role in adaptation of plants to heavy metal stress. The finding that the net H⁺ efflux in fungal mycelia and EM roots was markedly reduced by a specific inhibitor of PM H⁺-ATPase (sodium orthovanadate) in the presence and absence of Cd²⁺ stress (Figure 11A, Supplementary Figure S9) supports that the vigorous H⁺ efflux is the consequence of H⁺-ATPase activity. Accordingly, the increased H⁺ efflux upon Cd²⁺ shock (NM, MAJ and NAU roots; Figure 1C), ST (MAJ and NAU roots; Figure 5) and LT stress (NM roots; Figure 5) indicates the activated H⁺-pumping activity. In NM and EM roots, Cd²⁺ exposure led to a marked upregulation of HA2.1 and AHA10.1, two important genes encoding PM H⁺-ATPases (Ma Y. et al., 2014). The activation of PM H⁺-ATPase by Cd²⁺ is likely associated with the Cd²⁺-elicited H₂O₂, since (i) H₂O₂ increased H⁺ pumping activity in P. euphratica callus cells (Sun et al., 2010), in roots of P. euphratica (Sun et al., 2010) and secretor and non-secretor mangrove species (Lu et al., 2013; Lang et al., 2014), and (ii) the expression of genes encoding PM H⁺-ATPase are stimulated by H₂O₂ in Cucumis sativus roots (Janicka-Russak and Kabala, 2012; Janicka-Russak et al., 2012).

The activated PM H⁺-ATPase enabled NM and EM roots to maintain an acidic environment, which favors the entry of Cd²⁺ across the PM (Figure 10A). Similarly, He et al. (2015) showed that pH 5.5 accelerates Cd²⁺ influx into poplar roots compared to pH 4.0 or pH 7.0. Moreover, the Cd²⁺ influx was markedly suppressed by the application of sodium vanadate, an inhibitor of PM H⁺-ATPase (Figure 11B). These results indicate that the PM H⁺-pumps play a crucial role in enhancing the entry of Cd²⁺ (Ma Y. et al., 2014). Accordingly, NMT profiles of NM and EM roots showed that the maximum influx of Ca²⁺ was observed at pH 5.2 (Figure 10B), and that Ca²⁺ influx was blocked by sodium vanadate (Figure 11C). Therefore, we infer that Cd²⁺ activated H⁺-pumping in the PM, which led to hyperpolarization of the PM and increased Cd²⁺ influx through hyperpolarization-activated Ca²⁺ channels (HACCs). However, at present we cannot exclude the possibility that Cd²⁺ ions also penetrated through depolarization-activated (DACCs) and voltage-independent Ca²⁺ channels (VICCs), because the inhibitor of PM H⁺-ATPase, sodium vanadate, could not fully block the Cd²⁺ influx in NM and EM roots (Figure 11B). It has been shown that NSCCs co-exist with HACCs in the root cell plasma membrane to mediate the entry of Ca²⁺, but the two Ca²⁺ influx routes differ in their voltage sensitivity (Demidchik et al., 2002).

Ectomycorrhizal Populus × canescens show highly activated H⁺-pumping activity in the PM, which favors the Cd²⁺ influx through HACCs. Our NMT data showed that colonization of P. × canescens with P. involutus caused a marked H⁺ efflux (Figures 1C, 5, and 11A), suggesting that the fungal colonization could activate the PM H⁺-ATPase in ectomycorrhizas. This is consistent to our previous studies (Li J. et al., 2012; Ma X. et al., 2014). It has been documented that some host PM H⁺-ATPase isoforms show high activity in arbuscular mycorrhizal associations (Ramos et al., 2005; Rosewarne et al., 2007). Obviously, H⁺-pumping activity was activated by Cd²⁺ shock and ST exposure, as the H⁺ efflux in MAJ- and NAU-ectomycorrhizal roots were significantly higher than the NM roots (Figures 1C and 5). Increased abundance of HA2.1 and AHA10.1 encoding PM H⁺-ATPase in ectomycorrhizas compared to NM roots of P. × canescens were suggested to lead to higher activities of PM H⁺-ATPases (Ma Y. et al., 2014). The highly activated PM H⁺-ATPase, on the one hand maintains a more suitable acidic environment to promote the Cd²⁺ and Ca²⁺ influx across the PM (Figure 10) and on the other hand, provides an electrochemical H⁺ gradient for PM hyperpolarization, thus increasing Cd²⁺ influx via HACCs. Accordingly, the Cd²⁺-stimulated Cd²⁺ and Ca²⁺ in the P. involutus mycelia (Figures 1–3) was associated with the activated H⁺ pumps since Cd²⁺ treatment markedly upregulated the transcription of PM H⁺-ATPase 1 (Jacob et al., 2004).

Importantly, the H₂O₂ produced in the ectomycorrhizal associations may accelerate the Cd²⁺ through the PM H⁺-ATPase-mediated HACCs. Whole-cell patch clamp recordings of Arabidopsis guard cells showed that the PM hyperpolarization only activates Ca²⁺ currents in the presence of H₂O₂ (50 μM to 5 mM), and the Ca²⁺ current amplitudes increase with increasing H₂O₂ concentrations (Pei et al., 2000). Demidchik et al. (2007) showed that application of H₂O₂ (10 mM) to the external PM face of elongation zone epidermal protoplasts resulted in the appearance of a hyperpolarization-activated Ca²⁺ permeable conductance. In mature epidermal protoplasts, PM HACCs were activated only when H₂O₂ was present at the intracellular membrane face, and channel opening probability increased with intracellular H₂O₂ concentrations at hyperpolarized voltages (Demidchik et al., 2007). A massive presence of H₂O₂ was demonstrated in the outer hyphae mantle of P. involutus symbiosis (Gafur et al., 2004; Langenfeld-Heyser et al., 2007) and obviously could be released from the hyphae into the surrounding medium (Figure 6). Therefore, we suppose that in ectomycorrhizal P. × canescens, H₂O₂ elicited by fungal colonization stimulated Cd²⁺ influx through the HACCs that had been activated by P. involutus colonization. In addition, we found that Cd²⁺ influx in NAU-roots was less restricted than in MAJ-roots by DMTU and sodium orthvanadate (Figures 9B and 11B). The difference in the sensitivity to antagonists of H₂O₂ and PM H⁺-ATPase indicates the involvement of voltage-independent Ca²⁺ channels (VICCs) in the mediation of Cd²⁺ uptake in NAU-roots, in addition to the dominant Cd²⁺ entry through HACCs.

We noticed that LT stress in hydroponic conditions caused a pronounced shift of H⁺ efflux toward an influx in EM roots (Figure 5). LT-stressed P. involutus mycelia exhibited a trend similar to that in EM roots (Figure 5). These results imply that ectomycorrhization activated an H⁺/Cd²⁺ antiport to reduce excessive Cd²⁺ uptake and accumulation under prolonged stress conditions (Sun et al., 2013b). Similarly, we have previously shown that NaCl-treated P. euphratica roots retain an active PM Na⁺/H⁺ antiport to avoid the excessive buildup of Na⁺ when exposed to LT salinity (Sun et al., 2009a,b). Here, the rate of H⁺/Cd²⁺ antiport could not be determined, because our NMT data only show the net flux of the target element across the PM, instead of an unidirectional flux. In addition, EM roots were able to avoid the ROS burst in Cd²⁺ environments (Figure 6), probably because these roots were characterized by elevated H₂O₂ production (Gafur et al., 2004). Therefore, EM roots are likely to control the Cd²⁺ influx through the H₂O₂-activated PM Ca²⁺ channels, thus avoiding an excessive accumulation of the heavy metal ions under prolonged period of Cd²⁺ stress.