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Cloning and expression profiling of polyphenol oxidase from eggplant (Solanum melongena L.).

Santoshkumar, M. Shetty (2012) Cloning and expression profiling of polyphenol oxidase from eggplant (Solanum melongena L.). Doctoral thesis, University of Mysore.

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Abstract

Plant polyphenol oxidases (PPOs) have been extensively studied from various plant sources for their ability to oxidize phenolic compounds to produce brown pigmentation upon tissue damage. Eggplant or brinjal (Solanum melongena), an important member of solanaceae exhibits severe browning reaction upon tissue injury and has been reportedly manifested by PPOs. Though PPO genes from tomato and potato have been extensively studied, information about PPO genes and their promoters in eggplant is lacking. Using a combination of biochemical, molecular and immunological approaches the present study aims at understanding the structural and functional aspects of eggplant PPOs. Chapter 1 provides a retrospective account on plant polyphenol oxidases: its distribution, physico-chemical properties, structural features in relation to enzymic action, functional roles and its applications. It also envisages the importance of molecular characterization of eggplant PPOs, the information conspicuously missing so far. Research on PPO in plants has been multi dimensional as it strives to understand the in vivo roles, post harvest losses of agricultural produce and its potential biotechnological applications. The isolation of six eggplant PPO genes (SmePPO1–6) by RACE and genome walking as well as its comprehensive characterization is described in Chapter 2. These PPO genes (~1.8 kb) were found to be intronless similar to most of the dicotyledonary PPOs and correspond to eight eggplant PPO unigenes. Though SmePPO genes exhibit considerable variation in the transit peptide regions, copper-binding regions and UTRs, they can be clearly distinguished into two distinct structural classes (SmePPO1-3 and SmePPO4-6) based on predicted structural features. A deletion of 7 amino acids in copper binding region B of SmePPO4-6 is most likely responsible for their functional diversity. None of the SmePPOs exhibited proclivity for membrane association; however propensity for N-glycosylation was predicted with number of potential glycosylation sites ranging from 0 to 3. Comparative sequence analyses and collinearity of SmePPO1 and 2 has indicated organization of multigenic SmePPOs in tandem arrays on eggplant chromosome 8 akin to tomato and potato PPO genes. Chapter 3 deals with the heterologous expression of eggplant PPO1 and immunological investigations performed in order to deduce the localized upregulation in eggplant tissues. SmePPO1 was expressed in Escherichia coli as a GST fusion protein primarily found in insoluble fraction; immunoblot using rabbit polyclonal antiserum to GST–SmePPO1 detected a major protein band (~70 kDa) and a minor band (~67 kDa) in eggplant fruit extract. Solubilizing and refolding of the recombinant SmePPO1 did not Abstract of the Ph. D. Thesis yield enzymatically active protein thereby precluding further biochemical and biophysical characterization. Tissue printing indicated the predominant presence of PPOs in the exocarp and the areas surrounding the seeds in the mesocarp of eggplant fruits. Immunolocalization of PPOs in eggplant infested with shoot-and-fruit borer revealed localization of the PPO at the site of infestation in tender shoots and fruits, and further inside the mature tissues. Chapter 4 discusses the transcriptional regulation of multigenic eggplant PPO genes. During normal growth and development, SmePPO1 and 2 are expressed in roots, whereas the transcript levels of all the eggplant PPO genes vary considerably in leaves, flowers and fruits. The upregulation of eggplant PPO gene transcripts following mechanical injury shows that all the genes excepting SmePPO2 are induced in the fruit over 6 h. On the contrary, the transcripts of SmePPO2 and PPO3 are not detectable in the stem, and expression seems to be prominent over a 2 h period for SmePPO1 and SmePPO4–6. Transcriptional profiling of SmePPOs in eggplant seedlings has indicated differential response to methyl jasmonate (MeJA) or salicylic acid (SA) treatment. In planta, while MeJA elicited expression of all the six SmePPOs, SA was only able to induce the expression of SmePPO4–6. Interestingly, in dual treatment, SA considerably repressed the MeJA-induced expression of SmePPO 1-3 genes. Chapter 5 describes the isolation and characterization of SmePPOPROMOTERs. Genome walking was successfully adapted to clone SmePPOPROMOTERs and their transcription start sites (TSS) were determined by RLM-RACE. Extensive sequence analyses of SmePPOPROMOTER1 and 2 revealed the presence of evolutionarily conserved and over-represented putative cis-acting elements. The TSS for SmePPO genes are located 9–15 bp upstream of ATG with variable lengths of 5′ untranslated regions. Functional dissection of SmePPOPROMOTER1 by deletion analyses using Agrobacterium-mediated transient expression in tobacco leaves has shown that MeJA enhances the SmePPOPROMOTER1-β-glucuronidase (GUS) expression in vivo, while SA does not. It is concluded that similar to the coding regions of multigenic SmePPOs, the regulatory elements are also evolutionarily conserved and fall into two distinct classes based on their responses to MeJA and SA Chapter 6 summarises the significant outcome of this study on eggplant PPOs and also discusses the scope for further investigations and possible applications of resources and information generated from this research investigation.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: Eggplant, brinjal, Solanum melongena, Plant polyphenol oxidases
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
600 Technology > 08 Food technology > 23 Vegetables
Divisions: Dept. of Biochemistry
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 14 Nov 2013 08:54
Last Modified: 11 Oct 2018 06:39
URI: http://ir.cftri.com/id/eprint/11299

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