Constitutive and induced polyphenol oxidases of chickpea infected by Fusarium oxysporum f. sp ciceri: Purification and their physiological significance

Abstract

Constitutive and induced polyphenol oxidases (PPOs) were purified and characterized from chickpea ICCV10 genotype, treated with salicylic acid. PPO-I was purified to 124-fold with 1.3% yield, while PPO-II was purified to 142-fold with a yield of 1.2%. The molecular mass of each isozyme was estimated to be 81 and 64 kDa for PPO-I and PPO-II, respectively, by SDS-PAGE. The optimum pH and temperature for both isozymes was pH 7.0 and 25 degrees C, respectively. Using catechol as a substrate, K-m for PPO-I and PPO-II were 5 mM and 2.5 mM, respectively, while V-max for PPO-I and PPO-II were 250 U and 500 U, respectively. In addition, induced PPO-II was found to possess laccase activity. Salicylic acid (1-10 mu M) had no effect on PPO-I activity, whereas PPO-II/laccase activity increased by 20%. Differential inhibition of both the enzymes was observed by kojic acid, CTAB and sodium azide. Among the various PPO inhibitors tested, L-ascorbic acid, citric acid, L-cysteine, and sodium bisulphite were found to be potent inhibitors of PPO-II but not PPO-I. PPO-I is a constitutive isozyme, whereas PPO-II is a novel bifunctional isozyme associated with laccase activity and induced upon infection with Foc or treatment with SA. The role of these isozymes is discussed in the context of chickpea infected with Foc. (C) 2014 Elsevier Ltd. All rights reserved.