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

•Two PPOs were purified and characterized from chickpea infected by Fusarium oxysporum.•Induced PPO was shown to be associated with laccase activity.•Laccase activity was modulated by salicylic acid.•Native PPO was insensitive to salicylic acid.

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 °C, respectively. Using catechol as a substrate, Km for PPO-I and PPO-II were 5 mM and 2.5 mM, respectively, while Vmax 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 μM) 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.

Graphical abstractPolyphenol oxidase isoforms (I and II) were purified and characterized from the chickpea plants infected by Fusarium oxysporum f. sp. ciceri. PPO-I was a native whereas, PPO-II was induced and associated with laccase activity. Salicylic acid has no effect on PPO-I whereas, PPO/laccase activity was increased by 20%.Figure optionsDownload full-size imageDownload as PowerPoint slide