Effects of Potato Polyphenol Oxidase Gene Silencing in Transgenic Tobacco

Abstract

Polyphenol oxidases (PPOs) are nuclear encoded copper metalloenzymes that catalyze the oxidation of ortho-diphenols to ortho-quinones. PPO genes play an important role in plant defense mechanisms against biotic and abiotic stresses as well as responsible for undesirable browning in some fruits and vegetables. In the current study, ninety five PPO gene coding sequences from thirty six different plant species were analyzed in silico for multiple sequence alignment and phylogenetic relationships to gain insight into the structure-function relationships within the plant PPO family with special emphasis on potato. The obtained results indicated highly conserved copper binding domain in all the studied sequences. Phylogenetic clustering of monocot and dicot PPO proteins into separate groups reflects the lineage specific gene family expansion and duplication events through evolution. Interestingly, grand average of hydropathicity values proposed that all potato PPO proteins are hydrophilic in nature. To evaluate the stress induced expression of PPO gene, in vitro grown tobacco plants were subjected to wounding, abscisic acid (ABA) and methyl jasmonate (MeJ) stresses. Transcript levels measured by qRT-PCR for endogenous tobacco PPO gene showed an up-regulation of 14 folds by wounding and 6 folds by MeJ application, while no significant induction was observed with ABA treatment. An up-regulation of endogenous PPO gene in response to wounding and MeJ suggested a possible role of PPO in biotic stresses. Keeping in view the role of tobacco endogenous PPO induction by wounding and MeJ as well as already studied reports of PPO in other plants, Agrobacterium tumefaciens mediated genetic transformation of in vitro grown tobacco plants was carried out. The expression construct containing a copper binding domain segment of PPO gene ligated in anti-sense orientation downstream to a wound inducible OsRGLP2 promoter in p1391Z backbone vector was used for this transformation event. Expression analysis of transgenic tobacco plants with OsRGLP2 driven anti-sense segment of PPO gene was carried out with qRT-PCR in response to various stresses e.g. wounding, MeJ, ABA and whitefly (Trialeurodes vaporariorum) infestation. Expression analysis of PPO gene in transgenic tobacco showed a significant higher suppression (3.5 folds) of PPO in response to wounding. Similarly, MeJ application down-regulated the PPO expression up to 4.7 folds, while no significant expression of PPO was observed in vii case of ABA treatment. Moreover, considerable level of PPO reduction (6 folds) was observed with whitefly feeding assay in transgenic tobacco. These results indicated that expression of potato PPO in an anti-sense orientation has down-regulated the PPO activity. This down-regulation of PPO by wounding, MeJ and whitefly infestation clearly links the role of PPO in biotic as well as abiotic stresses. PPO gene suppression in transgenic plants by using anti-sense potato PPO gene construct suggested that this tool can be used to inhibit the enzymatic browning in fruits and vegetables e.g. potato