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

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.