The role of epithiospecifier protein (ESP) in plant metabolism and its impact on the regulation of anti-cancer isothiocyanates in Brassica crops

David Williams (2010). The role of epithiospecifier protein (ESP) in plant metabolism and its impact on the regulation of anti-cancer isothiocyanates in Brassica crops PhD Thesis, School of Biological Sciences, The University of Queensland.

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Author David Williams
Thesis Title The role of epithiospecifier protein (ESP) in plant metabolism and its impact on the regulation of anti-cancer isothiocyanates in Brassica crops
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2010-09
Thesis type PhD Thesis
Supervisor Christa Critchley
Total pages 216
Total colour pages 30
Subjects 06 Biological Sciences
Abstract/Summary Abstract Glucosinolates are sulphur containing natural plant products found in Brassica vegetables as well as brassicaceous forages and oilseeds. Almost without exception they occur in the plant in conjunction with the hydrolytic enzyme, myrosinase. In intact tissues, the enzyme is stored separately from the glucosinolates. When tissue damage occurs glucosinolates are hydrolysed by the myrosinases to a range of breakdown products such as isothiocyanates, simple nitriles, epithionitriles and thiocyanates. The type of product that is formed depends on pH, structure of glucosinolate side chain and the presence or absence of supplementary specifier proteins such as epithiospecifier proteins (ESPs), thiocyanate-forming proteins (TFPs) and the recently identified nitrile-specifier proteins (AtNSPs). Knowledge of the factors controlling glucosinolate hydrolysis is critical for understanding the ecological roles and human health benefits of these compounds, because it is the hydrolysis products and not the glucosinolates themselves that have been found to be the most active. From a human health perspective, isothiocyanates are the most important because they are major inducers of carcinogen detoxifying enzymes. By contrast, most nitriles are ineffective as inducers of these detoxification enzymes and less effective as anti-proliferative agents. ESP operates in conjunction with myrosinase such that it directs the products of glucosinolate hydrolysis towards epithionitriles. ESP is not present in all brassicaceous plants and several studies have suggested that it is absent from those species that lack glucosinolates with terminally unsaturated side chains. This thesis describes investigations into the role of ESP in the formation of both simple nitriles and epithionitriles at the expense of isothiocyanates, the importance of the terminal alkenyl glucosinolate substrates and factors that influence ESP activity. This study provides strong evidence that simple nitrile formation is under ESP control in most Brassicas although the recent identification of nitrile specifier proteins in Arabidopsis thaliana demands a closer inspection of their possible role in simple nitrile formation. Fe2+ is an essential factor in ESP activity although several recent studies have highlighted discrepancies in our understanding of the ESP and Fe2+ interaction. To investigate further the role iron species play in regulating ESP activity, seedpowders were analysed for ESP and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of iron species, specific chelators and reducing agents. Aged seeds and 3 d seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The ESP-containing plants gave two distinctive responses, thus providing strong evidence that ESPs viii vary markedly in their Fe2+ requirement for activity. The results also suggested that the Fe2+ concentration as indicated by the ratio of ferric to ferrous iron influences ESP activity. Consistent with this, reduction in the Fe2+ concentration was correlated with the loss of ESP activity during seed storage. Apparently the ESP-homologous AtNSPs were not dependent on iron for activity. When the likelihood of AtNSP activity contributing to simple nitrile formation was evaluated in this study the results obtained indicated only a minor, if any, contribution. Contrasting results have also been found regarding nitrile formation at different pH values. Various studies found nitrile production was favoured at low pH, while others reported predominant nitrile formation at neutral or even basic pH. As the measured hydrolysis pHs gave inconsistent results that varied markedly between species, a novel approach was used to describe these fluctuations. It is well known that proteins close to or at their isoelectric point have less biological activity. Using calculated pIs, based on amino acid sequences of several identified ESPs, differences between pH and pI were determined. The pH-pI values closely followed the variations in ESP activity in all the plant species analysed in this study. Epithionitrile formation has been shown to be heat sensitive in several species of Brassicas. Recent studies have shown that mild heating decreased ESP activity, with a subsequent increase in isothiocyanate levels, indicating that the myrosinase was unaffected. However, these studies were restricted to rapeseed and broccoli and the question was whether these temperatures apply to other Brassica vegetables during different stages of development. Experiments confirmed the results of these earlier studies but noted that both ESP and myrosinase activities of the seeds were more temperature resistant than those in the seedlings and mature plants.
Keyword Brassicas, glucosinolates specifier proteins, myrosinase, epithiospecifier protein activity,
Additional Notes Pages printed in colour,1,19,23-24,58,61,64,84,95,99,101,104,126,135,138,142,144,148,151,185,187-189,196-197,199,200-201,212-213.

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Created: Wed, 29 Sep 2010, 15:05:17 EST by Mr David Williams on behalf of Library - Information Access Service