Zinc and nickel disrupt tubular vacuole and mitochondrial networks, but only nickel disrupts microtubules in hyphal tip cells of two Paxillusinvolutus strains

Ectomycorrhizal fungi are able to ameliorate heavy metal stress to host trees inpolluted soils. Their sensitivity or tolerance to heavy metals is usually examined based on growth and proliferation on heavy metal amended media. However, there are no data on cellular effects of heavy metals and detoxification in live cells of these fungi. Organelle morphology has recently been recognized as an indication of cellular health and its changes can be used to assess cytotoxicity. The aim of this study was, therefore, to investigate shortterm effects of common heavy metal pollutants, Zn2+ and Ni2+ on the morphology ofvacuoles, mitochondria and microtubules in hyphal tip cells of two Paxillus involutusstrains.Vacuoles, mitochondria and microtubules were labeled with Oregon Green® 488carboxylic acid diacetate, 3,3'-dihexyloxacarbocyanine iodide ((DiOC6(3)) and anti-α-tubulin antibodies, respectively. They were treated with 0-1 mmol L-1 NiSO4 or 0-100mmol L-1 ZnSO4 or K2SO4 (SO42- control) and examined by fluorescence microscopy.Vacuoles and mitochondria in untreated hyphal tip cells of strain P2 which originatedfrom a heavy metal-rich soil were motile and tubular forming networks. Exposure to themetals caused tubular vacuole thickening and vesiculation as well as fragmentation oftubular mitochondria in living hyphal tip cells. The highest K2SO4 concentration also hadsevere effects on mitochondria. These effects were metal, concentration and exposure timedependent. NiSO4 caused these effects at a hundred fold lower concentration than ZnSO4and induced severe microtubule disruption. Vacuole tubularity recovered after removal ofthe metal solutions depending on the metal, concentration and exposure time.Mitochondrial tubularity recovered to pretreatment morphology in a shorter time and evenduring exposure to the highest metal concentration.Vacuoles of strain P46 which originated from a non-polluted soil were pleomorphic,but mainly spherical with occasional tubular interconnections. The vacuoles were toosensitive to UV light exposure required for fluorescence microscopy to investigate theirresponse to heavy metals. The mitochondrial network and microtubules resembled those ofstrain P2. The highest ZnSO4 and NiSO4 concentrations caused similar trends in response ofmitochondria and microtubules of the two strains. However, mitochondria of strain P46were less sensitive at lower metal concentrations. The highest K2SO4 concentration hadmore severe effects on mitochondria of strain P46 than in strain P2 from which tubularitywas not recovered.This investigation is the first to reveal that heavy metals affect organelle morphologyin two strains of an ectomycorrhizal fungus. Nickel effects on the organelles are likely toresult from microtubule disruption. Metal induced mitochondrial fragmentation is possiblyan apoptotic response and the recovery of tubular organelle networks suggests cellulardetoxification