Effect of Cu2+ and pH on intracellular calcium content and lipid peroxidation in winter wheat roots


  • M. E. Riazanova Institute of Plant Physiology and Genetics of NAS of Ukraine
  • T. I. Makoveychuk Institute of Plant Physiology and Genetics of NAS of Ukraine
  • V. V. Schwartau Institute of Plant Physiology and Genetics of NAS of Ukraine
Keywords: copper toxicity, [Ca2 ]cyt, pH, malondialdehyde, Fluo-3 AM, Ca-channels

Abstract

The study investigates the effect of copper ions and pH of external solution on intracellular calcium homeostasis and lipid peroxidation in winter wheat roots. Experiment was carried out with winter wheat. Sterile seeds were germinated in Petri dishes on the filter paper soaked with acetic buffer (pH 4.7 and 6.2) at 20 °Cin the dark for 48 hours. Copper was added as CuSO4. It’s concentrations varied from 0 to 50 µM. The Ca2+-fluorescent dye Fluo-3/AM ester was loaded on 60 hour. Root fluorescence with Fluo-3 loading was detected using X-Cite Series 120 Q unit attached to microscope Olympus BX53 with camera Olympus DP72. Imaging of root cells was achieved after exciting with 488 nm laser and collection of emission signals above 512 nm. Preliminary analysis of the images was performed using software LabSens; brightness (fluorescence intensity) analysis was carried out by means of ImageJ. Peroxidation of lipids was determined according to Kumar and Knowles method. It was found that pH of solution had effect on release of calcium from intracellular stores. Low pH provokes an increase of [Ca2+]cyt which may be reaction of roots to acidic medium. Copper induces increase in non-selective permeability of plasma membrane and leads to its faster depolarization. This probably initiates Ca-dependent depolarization channels which are responsible for the influx of calcium from apoplast into the cell. Changing of the membrane permeability may occur due to interaction between Cu2+ ions and Ca-binding sites on plasma membrane or may be due to binding of copper with sulfhydryl groups and increasing of POL. Copper may also damage lipid bilayer and change the activity of some non-selective channels and transporters. Reactive oxygen species which are formed under some types of stress factors, especially the effect of heavy metals, can be activators of Ca-channels. Cu2+ ions rise MDA content and promote the oxidative stress. Low medium pH also induces its development. Oxidative stress apparently enhances activity of hyperpolarization of Ca-channels and leads to increase of [Ca2+]cyt. These Ca-channels may also be stimulated by calcium, which influxes into the cell, so Ca-signal can be self-enhanced one. Prolonged retention of high calcium concentration may be the evidence of copper toxicity to root cells and the consequence of deactivation of Ca-ATPases which promote Ca efflux from cell. The increase of [Ca2+]cyt may elicit changes in some metabolic processes.

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Published
2015-06-28
Section
Articles