Determination and Detection of Reactive Oxygen Species (ROS), Lipid Peroxidation, and Electrolyte Leakage in Plants

Abstract

Reactive oxygen species or intermediates are formed by the incomplete reduction of oxygen. Organisms living in aerobic environment generate various kinds of reactive oxygen species (ROS) molecules, such as superoxide (*O(2)(-)), hydrogen peroxide (H(2)O(2)), hydroxyl radical (OH(-)), singlet oxygen, and lipid hydroperoxides. ROS are highly reactive molecules and are extremely unstable, so detection of ROS relies on measuring the end products that are formed when they react with particular substances. The end products can be measured by changes in their fluorescence, color, or luminescence. ROS causes lipid peroxidation wherein the lipids in the cell membranes are damaged. Lipid peroxidation is usually quantified using a colorimetric assay. When ROS concentrations reach a certain threshold, it activates a programmed cell death response in the cells. This is quantified by measuring the amount of ion leakage. ROS such as superoxide and hydrogen peroxide have been detected traditionally by staining techniques. Superoxide anion is detected with nitroblue tetrazolium (NBT) and hydrogen peroxide by Diaminobenzidine tetrahydrochloride (DAB) staining. In this chapter, methods for determining total ROS and lipid peroxidation assay, histochemical staining techniques for superoxide and H(2)O(2) molecules are described.

An integrated method for removal of harmful cyanobacterial blooms in eutrophic lakes

“…entering the cells could cause lipid peroxidation (Imlay, 2003;Jambunathan, 2010) and phycocyanin, an essential pigment for light harvesting, to dissociate from the thylakoid membrane. This algal control method focuses on inactivating the photosynthetic apparatus but not destroying the whole cell, which is not consistent with some previous reports that only electron transport is inhibited by H 2 O 2 (Samuilov et al, 2001).…”

In vivo antioxidant assessment of two antimalarial plants–Allamamda cathartica and Bixa orellana

“…Lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) was measured using the methods in previous studies [19,20] . A volume of 0.1 mL of liver tissue homogenate and blood samples were treated separately with 2 mL of mixture [1:1:1 TBA-TCA-HCl reagent (thiobarbituric acid 0.37%), 0.25 N HCl, 10% TCA] and incubated for 45 min in a water bath (100 °C ), cooled and centrifuged at room temperature for 10 min at 1 000 r/min.…”

Phytotoxicity induced by Phragmites australis: an assessment of phenotypic and physiological parameters involved in germination process and growth of receptor plant

“…The MDA level was used as an index of LP and was expressed as nmol g (1 fresh weight (f. wt) (Jambunathan 2010). About 200 mg of control and treated plantlets were homogenized with 4 mL of 0.1% TCA, and then centrifuged at 15,000 rpm for 15 min.…”