Molecular Characterization of Polyphenol Oxidase Gene Promoter

Abstract

Polyphenol oxidases (PPOs) are the nuclear encoded, copper binding enzymes and are ubiquitous in nature. These enzymes are responsible for oxidation of ortho-and para- diphenols to quinones, which are highly reactive. Oxidized phenolic compounds in plant tissues due to PPO activity results is the formation of undesirable browning leading to unwanted color changes in food items during storage and processing which is one of the main causes of quality loss. However, upon damage or injury to plant, phenolic compounds oxidize to polymeric structure in order to protect against insects or micro- organisms. PPO is the source of quinones that limits the accessibility of alkylating dietary proteins to insects and ultimately starvation may happen. PPOs are of significant interest because of their high capacity to oxidize aromatic compounds. They are also suitable for some biotechnological applications in food, medicine, paper and textile industry. PPOs have a wide range of antioxidant activities and supplements with diet play a significant role in health care. Keeping in view the importance of PPOs, a study was carried out to characterize rice PPO promoter (OsPPOP) region at molecular level. The OsPPO promoter region was amplified, cloned and sequenced. Bioinformatics tools based comparative analysis of OsPPO promoter sequence and six other PPO promoter sequences available in Genbank revealed that PPO promoter regions possess common, novel and distinct cis-regulatory elements. The identified cis-regulatory elements have roles in environmental stresses (biotic and abiotic), activation of pathogen related genes and secondary metabolite path ways. Moreover, light responsive elements, pathogen inducible elements and copper binding elements are also present in the promoter region of PPO genes. Maps of cis- regulatory elements were framed manually in order to know the number, density and location of these elements available both on positive and negative strands of PPO promoters analyzed. Further, blast revealed that small regions of 1020 bp OsPPO promoter region have homology at chromosome 2 with the stretch of regions encoding ethylene gene, 3-isopropylmalate dehydratase small subunit and transposons (superfamily of elements Rim2/Hipa) and conserved sequences of CACTA-like family. xxiThen an expression construct was designed for the functional analysis of OsPPO promoter region in transgenic tobacco plant. The expression construct was transformed into tobacco via leaf disc method. Expression was observed in transgenic tobacco by GUS staining. The microscopic analysis revealed that the PPO promoter region is tissue specific and the GUS expression was observed in leaves; leaf veins and leaf petiole, stem; epidermis, vascular bundles, pith, collenchyma, sclerenchyma, xylem and phloem and roots; xylem and phloem. It is being first time reported that OsPPO promoter region is a strong wound inducible promoter. Further, Real-Time PCR study revealed that higher level of OsPPO promoter expression is at lower temperature and with the increase in temperature the expression level gradually decreases. The observed properties of OsPPO promoter can be used for plant transformation related studies as the OsPPO promoter region is plant origin promoter with wound inducible properties and it can also be expressed at higher level in low temperature conditions. Thus, the findings elucidate that OsPPO promoter is an important promoter which can be exploited for crop improvement and plant transformation studies.