Expression Profiling of Reporter Genes Induced by Rice Polyphenol Oxidase Promoter in Transgenic Model Plant

Abstract

Polyphenol oxidase (PPO) enzymes are ubiquitous in plant kingdom that catalyzes the oxidation of phenols to highly reactive quinones. PPO plays important role in plant defense mechanisms against biotic and abiotic stresses but the regulation of PPO promoter in response to stresses remains unclear. Here, Oryza sativa polyphenol oxidase (OsPPO) promoter was fused separately to β-glucuronidase (GUS) and firefly Luciferase (LUC) reporter genes. Agrobacterium strain GV3101 harbouring OsPPOLUC and OsPPOGUS (separate clones) was used to transform Arabidopsis by floral dip method. T1 transgenic seeds were used to obtain T2 lines which were finally checked for the effects of wounding, ABA, MJA applications and drought as well as salt stress on PPO regulation. Expressional profiling of OsPPO promoter by real- time (RT-PCR) in transgenic Arabidopsis has revealed a higher level (11 folds) of expression by wounding. However, OsPPO promoter was also expressed in response to ABA by an induction of 3 fold but it was not induced by MJA. ABA plant hormone which regulates key processes biotic and abiotic stresses ultimately triggering plant defense related genes. ABA and wound inducibility of OsPPO promoter is a strong indicative of its role in plant defense mechanism against biotic and abiotic stresses. Moreover, transgenic T2 plants were also screened against different concentrations of osmotic stress (PEG-6000) and salt (NaCl). Experimental data showed that relative GUS expression of OsPPO gene promoter increased with the increase of osmotic stress. In case of salt stress, OsPPO induction showed similar trend and GUS expression was increased as salt concentration increased. This response of OsPPO to drought and salt stress suggest the x xpossible participation of PPO in plants defense against drought as well as salt stress. Moreover, OsPPO promoter sequence was computationally analyzed by Signal Scan (PLACE), which indicated the presence of different cis- regulatory elements, for example wound responsive elements, ABA signaling complexes responsive to drought and salt responsive regulatory elements. During in vivo experimental work, the induction and expression of reporter genes proved the presence and functioning of these cis-regulatory elements. In summary the role of OsPPO in wounding, ABA application, drought and salt stresses, and the subsequent OsPPO promoter induction indicates that these stresses induce PPO expression in plants as a defense mechanism.