Using a Non-invasive Micro-test Technique, flux profiles of Cd²⁺, Ca²⁺, and H⁺ were investigated in axenically grown cultures of two strains of Paxillus involutus (MAJ and NAU), ectomycorrhizae formed by these fungi with the woody Cd²⁺-hyperaccumulator, Populus × canescens, and non-mycorrhizal (NM) roots. The influx of Cd²⁺ increased in fungal mycelia, NM and ectomycorrhizal (EM) roots upon a 40-min shock, after short-term (ST, 24 h), or long-term (LT, 7 days) exposure to a hydroponic environment of 50 μM CdCl₂. Cd²⁺ treatments (shock, ST, and LT) decreased Ca²⁺ influx in NM and EM roots but led to an enhanced influx of Ca²⁺ in axenically grown EM cultures of the two P. involutus isolates. The susceptibility of Cd²⁺ flux to typical Ca²⁺ channel blockers (LaCl₃, GdCl₃, verapamil, and TEA) in fungal mycelia and poplar roots indicated that the Cd²⁺ entry occurred mainly through Ca²⁺-permeable channels in the plasma membrane (PM). Cd²⁺ treatment resulted in H₂O₂ production. H₂O₂ exposure accelerated the entry of Cd²⁺ and Ca²⁺ in NM and EM roots. Cd²⁺ further stimulated H⁺ pumping activity benefiting NM and EM roots to maintain an acidic environment, which favored the entry of Cd²⁺ across the PM. A scavenger of reactive oxygen species, DMTU, and an inhibitor of PM H⁺-ATPase, orthovanadate, decreased Ca²⁺ and Cd²⁺ influx in NM and EM roots, suggesting that the entry of Cd²⁺ through Ca²⁺-permeable channels is stimulated by H₂O₂ and H⁺ pumps. Compared to NM roots, EM roots exhibited higher Cd²⁺-fluxes under shock, ST, and LT Cd²⁺ treatments. We conclude that ectomycorrhizal P. × canescens roots retained a pronounced H₂O₂ production and a high H⁺-pumping activity, which activated PM Ca²⁺ channels and thus facilitated a high influx of Cd²⁺ under Cd²⁺ stress.

Cadmium (Cd²⁺) pollution occurring in salt-affected soils has become an increasing environmental concern in the world. Fast-growing poplars have been widely utilized for phytoremediation of soil contaminating heavy metals (HMs). However, the woody Cd²⁺-hyperaccumulator, Populus × canescens, is relatively salt-sensitive and therefore cannot be directly used to remediate HMs from salt-affected soils. The aim of the present study was to testify whether colonization of P. × canescens with ectomycorrhizal (EM) fungi, a strategy known to enhance salt tolerance, provides an opportunity for affordable remediation of Cd²⁺-polluted saline soils. Ectomycorrhization with Paxillus involutus strains facilitated Cd²⁺ enrichment in P. × canescens upon CdCl₂ exposures (50 μM, 30 min to 24 h). The fungus-stimulated Cd²⁺ in roots was significantly restricted by inhibitors of plasmalemma H⁺-ATPases and Ca²⁺-permeable channels (CaPCs), but stimulated by an activator of plasmalemma H⁺-ATPases. NaCl (100 mM) lowered the transient and steady-state Cd²⁺ influx in roots and fungal mycelia. Noteworthy, P. involutus colonization partly reverted the salt suppression of Cd²⁺ uptake in poplar roots. EM fungus colonization upregulated transcription of plasmalemma H⁺-ATPases (PcHA4, 8, 11) and annexins (PcANN1, 2, 4), which might mediate Cd²⁺ conductance through CaPCs. EM roots retained relatively highly expressed PcHAs and PcANNs, thus facilitating Cd²⁺ enrichment under co-occurring stress of cadmium and salinity. We conclude that ectomycorrhization of woody hyperaccumulator species such as poplar could improve phytoremediation of Cd²⁺ in salt-affected areas.

Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca²⁺) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca²⁺ sensors, and can act as an intermediate in the Ca²⁺ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca²⁺/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca²⁺ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.