The Modulation Of Ca²⁺-permeable Channels In Cd²⁺Enrichment In Paxillus Involutus-populus × Canescens Ectomycorrhizae

The presence of highly toxic cadmium(Cd2+)in the environment is a serious threat to human health as heavy metals can be enriched in plants and eventually enter the human body through the food chain.Nowadays it is of particular importance for phytoremediation of Cd2+ pollution in soils.Populus×canescens has been identified as a woody Cd2+-hyperaccumulator.Colonization with the ectomycorrhizal fungus Paxillus involutus enhances both cadmium uptake and tolerance in P.x canescens.The two P.involutus strains,MAJ and NAU,form compatible and incompetent ectomycorrhizae associations with P.x canescens roots.The colonization of P.x canescens roots with the competent strain MAJ results in enriched Cd2+ levels under cadmium stress.Whether the incompatible fungal isolate NAU also affects the Cd2+ entry into P.x canescens host plants needs to be clarified.Previous studies showed ectomycorrhizal formation induced the production of hydrogen peroxide(H2O2)and activated PM H+-ATPase activity of the host plants.It is possible that H2O2 stimulates the entry of Cd2+ through PM Ca2+-permeable channels(CaPCs)as the activity of these channels has been shown to be stimulated by H2O2.In addition,high external Cd2+ concentrations establish a large electrochemical gradient facilitating the rapid movement of Cd2+ ions through PM Cd2+-permeable channels.However,it is still unclear whether the CaPCs-mediated Cd2+ flow and H202 and PM H+-ATPase could mediate the entry of Cd2+ in mycorrhizal plants under cadmium stress.Using a Non-invasive Micro-test Technique,flux profiles of Cd2+,Ca2+,and H+were investigated in axenically grown cultures of two strains of P.involutus(MAJ and NAU),ectomycorrhizae formed by these fungi with the woody Cd2+-hyperaccumulator,P.x canescens,and non-mycorrhizal(NM)roots.The aim was to elucidate whether the Cd2+influx through Ca2+-permeable channels is stimulated byH2O2 and H+-ATPase in ectomycorrhizal roots since the ectomycorrhizas exhibit enhanced H2O2 production and upregulated H+-pumping activity.In addition to mature ectomycorrhizae,flux profiles of Cd2+and Ca2+were recorded in P.involutus-inoculated roots after seven days of co-culture.The aim was to determine whether flux profiles of mature EM associations resemble the pattern of those from host roots at early stages of fungal colonization.The main findings are as follows:1.P.× canescens exhibited a vigorous Cd2+ uptake after shock(50 ?M),short-term(50 ?M,24 h)and long-term(50 ?M,7 d)cadmium treatments.EM roots exhibited a higher Cd2+influx than NM roots irrespective of cadmium stress conditions.The mycelia of the two P.involutus strains exhibited a stable Cd2+influx and the flux rate increased with the prolonged duration of CdCl2 treatment(24 h?7 d).However,there were marked differences between the two strains in Cd2+ uptake given the shock treatment.Pure fungal mycelium of MAJ accumulated Cd2+with a higher rate than NAU.In accordance,MAJ roots exhibited a higher influx than NAU roots after the onset of CdC12 shock.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.x canescens.Thus,in MAJ-colonized roots the host cells might have been more accessible to Cd2+.2.Cadmium-induced alterations of Cd2+ and Ca2+ fluxes were also examined in non-inoculated and P.involutus-inoculated roots after 7 d of co-culture.NAU-and MAJ-colonized roots showed larger flux rates than non-inoculated roots after short-term cadmium stress,indicating that P.involutus enhanced the absorption of Cd2+at the early stages of colonization.Furthermore,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 cadmium treatments(shock,ST,and LT).These results suggest that the continuous flow of Cd2+and Ca2+ in EM roots detected by NMT microelectrodes was largely driven by the host rather than the fungal partner.3.The relationships between the total flux rates of Cd2+and Ca2+ in the presence of Cd2+(Eca2++cd2+)and Ca2+flux in the absence of Cd2+(?Ca2+(-Cd2+))were highly significant.These suggest that the entry of Cd2+ and Ca2+ is mainly through the same pathway in NM and EM roots,mostly likely through calcium-permeable channels in the PM.P.involutus colonization and Cd2+stress activated PM Ca2+-permeable channels in fungal hyphae and P.x canescens roots,enhanced the absorption of Cd2+The activated PM Ca2+-permeable channels were permeable to Cd2+and allowed the entry of Cd2+into host plants under Cd2+stress.A co-application of Cd2+and Ca2+suppressed the entry of Cd2+in P.×canescens roots and mycelium,and the restriction increased with the increasing fraction of Ca2+in the mixture(Ca2+:Cd2+=1:2,1:1,2:1).Additionally,four typical calcium-permeable channels inhibitors(LaC13,GdCl3,verapamil,and TEA)effectively inhibited Ca2+and Cd2+influxes in NM and EM roots Similarly,in the pure P.involutus mycelia,calcium-permeable channels inhibitors also effectively restricted influx of Ca2+and Cd2+or induced net efflux.These results suggest that the divalent cations,Cd2+and Ca2+,competitively permeated the plasma membrane through Ca2+-permeable channels.In addition,a small fraction of Cd2+ions may penetrate the PM through other channels and transporters that were permeable to Cd2+in P.x canescens roots and mycelium4.The inoculation with P.involutus and Cd2+stress caused the production of H2O2 in P.×canescens roots.In the absence of Cd2+,NM roots exhibited a stable H2O2 efflux and increased 2.4-fold in response to Cd2+treatment(50 ?M CdC12,30 min).Mycorrhization of poplar roots with P.involutus stains,MAJ and NAU,resulted in a significant increase of H2O2 efflux.However,upon CdC12 exposure mycorrhizal roots displayed decreased H2O2 efflux in contrast to NM roots.H2O2 induced by cadmium and fungal colonization mediates the influx of Cd2+and Ca2+ through Ca2+-permeable channels in P.x canescens roots and mycelium.Meanwhile,Cd2+and Ca2+influx in P.x canescens roots and mycelium were both suppressed by the ROS scavenger,DMTU,and Cd2+influx in NAU-roots was less restricted than in MAJ-roots by DMTU.These results suggest that Cd2+ and Ca2+ions enter NM and EM roots by the same pathway involving PM Ca2+-permeable channels that are activated by Cd2+-elicited H2O2.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+.5.The inoculation with P.involutus and Cd2+stress activated PM H'-ATPase of the host plants and mycelium.The colonization of P.× canescens with P.involutus caused a marked H+efflux,suggesting that the fungal colonization could activate the PM H+-ATPase in EMs since the H+efflux was significantly suppressed by sodium orthovanadate,an inhibitor of PM H+-ATPase.The activated PM H+-ATPase provides an electrochemical H+gradient for PM hyperpolarization.NMT profiles of P.x canescens roots(NM,MAJ,NAU)and mycelium showed the maximum influx of Ca2+and Cd2+ at pH 5.2.Meanwhile,H+,Ca2+ and Cd2+influxes were markedly suppressed by the application of sodium orthovanadate,and Cd2+ influx in NAU-roots was less restricted than in MAJ-roots by sodium orthvanadate.These results suggest that the activated PM H+-ATPase,on the one hand maintains a more suitable acidic environment to promote the Cd2+and Ca2+influx the PM,and on the other hand,provides an electrochemical H+gradient for PM hyperpolarization,thus increasing Cd2+influx via hyperpolarisation-activated Ca2+ channels(HACCs).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.In conclusion,we propose a signaling pathway that triggers Ca2+-channel-mediated Cd2+influx in NM P.x canescens roots and explains the pronounced Cd2+stimulation in ectomycorrhizal associations under cadmium stress.The Cd2+-elicited H2O2 and active H+-ATPase favored the Cd2+influx through Ca2+-permeable channels in NM roots and P.involutus-ectomycorrhiza,while these channels mediate Ca2+ influx in the absence of cadmium stress.In EMs,Cd2+enriched in hyphae is thought to be delivered to the host roots.Moreover,the colonization of P.x canescens roots with the fungal strains MAJ and NAU stimulates H2O2 production and increases H+-ATPase activity,and thus accelerates Cd2+entry through Ca2+-permeable channels,in particular through HACCs,under excessive cadmium.Cd2+ions competitively enter Ca2+-permeable channels,and thus diminish the entry of Ca2+,leading to a marked Cd2+enrichment in ectomycorrhizal roots under cadmium stress.