Reactive oxygen species production and antioxidative defense system in pea root tissues treated with lead ions: mitochondrial and peroxisomal level

Abstract

Reactive oxygen species (ROS) production and enzymatic antioxidative system [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX), alternative oxidase (AOX)] in nonphotosynthesizing pea plant cells were investigated. From the roots of pea plants cultivated hydroponically on a Hoagland medium with the addition of 0.1 and 0.5 mM of Pb(NO3)2, the three following fractions were isolated by means of a Percoll gradient: cytosol, peroxisomal, and mitochondrial. Lead stress caused H2O2 production in these organelles. The mitochondria from pea cell roots were the main site of H2O2 production. Intensive stress caused by 0.5 mM of Pb(NO3)2 brought about a decrease of H2O2 concentration in mitochondria and peroxisomes after 3 days of the exposition, which was due to an increase of CAT activity. The isoenzymatic profile of antioxidative enzymes indicates mitochondrial and peroxisomal localization of MnSOD and cytoplasmic localization of CuSOD. APOX activity was estimated for all three fractions: cytosol, mitochondria, and peroxisomes. Simultaneously, we observed an increased expression of AOX genes on the basis of the amount of mRNA transcript and confirmed it immunologically on the level of synthesized AOX protein (36 kDa). This has been the first evidence of AOX genes expression of which is induced by the treatment of plants with lead ions and it increases along with the concentration of metal.