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<h1>Biotechnology and Tomato Fruit Ripening</h1>
<p><img src="images/lab23.jpg" alt="Genetically modifying tomato" align="left" width="250"
height="183">In 1990 I started a B.Sc. In Plant Biotechnology at <a
href="http://www.wye.ic.ac.uk/" title="Wye College" target="_blank">Wye College</a>, part of
Imperial College, London (but actually in Kent). I graduated in 1993, whereupon I started
a PhD studentship at the <a href="http://www.nottingham.ac.uk/"
title="University of Nottingham" target="_blank">University of Nottingham</a> Department of Applied
Biochemistry & Food Science, Sutton Bonington Campus. I graduated from Nottingham in
the Summer of 1997.</p>
<p>My Ph.D. work concerned the characterisation of Pectinesterase
isoforms in tomato. The project was funded jointly by the <a
href="http://www.bbsrc.ac.uk/"
title="Biotechnology & Biological Sciences Research Council" target="_blank">Biotechnology &
Biological Sciences Research Council</a> and <a href="http://www.zeneca.com/"
title="Zeneca Plant Science" target="_blank">Zeneca Plant Science</a>.</p>
<p>The enzyme Pectinesterase (EC 3.1.1.11) is found in all plants,
and many plant pathogens. The enzyme is especially abundant in tomato, where it has been
most extensively studied. In tomato, the enzyme has been detected in fruit, leaves and
roots. In particular, the fruit contain very high levels of the enzyme, leading to the
theory that it has an important role in fruit ripening and softening [<a
href="biotechnology.asp#1">1</a>].</p>
<p>Column chromatography has been able to separate PE into distinct
isoforms. The dominant isoform in tomato is called PE2. This isoform is fruit specific,
and appears to increase during ripening. Most work on PE has concentrated on this isoform,
due to the large amount of it in tomato fruit, and its ease of extraction. The enzyme has
been purified, and genes coding for the enzyme have also been isolated. PE2 has been
previously 100% downregulated in tomato using antisense inhibition [<a
href="biotechnology.asp#2">2</a>]. Fruit from these downregulated plants still displayed
some PE enzyme activity however, so it was thought that other isoforms unaffected by the
antisense inhibition were active in the fruit.</p>
<p>The technique of antisense inhibition has now been used to
"switch off" many genes in plants. The technique has been used to downregulate
the gene corresponding to the main form of PE in tomato, PE2. These plants do not contain
the PE2 enzyme, and have much reduced levels of PE activity compared to normal tomatoes. <img
src="images/bunch-o-toms.jpg" alt="Genetically modified tomatoes" align="right"
width="201" height="250">Fruit from the antisense tomatoes ripen
normally, and the effect on softening of downregulating the PE2 enzyme is marginal. The
small amount of PE activity present in the PE2 antisense fruit is due to the activity of
other isoforms unaffected by the antisense gene. So far, there is good evidence for at
least two minor isoforms of PE, PE1 and PE3. Unlike the main PE2 isoform, the other
isoforms are not fruit specific. They may be important in cell wall biochemistry
throughout the plant.</p>
<p>Previous to my project, the PE isoforms were re-purified and the
proteins sequenced at the N-terminal end. By screening a cDNA library with a degenerate
probe designed from one of the N-terminal sequences, a cDNA clone encoding one of the two
minor isoforms of PE was identified. Using a combination of both manual and automatic
sequencing I have fully sequenced this cDNA [<a href="biotechnology.asp#3">3</a>], which
has no sequence similarity to the major PE isoform previously isolated. It also has no
sequence homology to PEs isolated from a range of other plant, fungal and bacterial
species.</p>
<p>I have produced a construct in which a fragment of the novel PE
cDNA was amplified using PCR, then cloned into a plant transformation vector. This vector
was used to transform tomato plants in order to downregulate the isoform using antisense
inhibition. I have screened the transformed plants using both PCR and Southern blotting to
show the existence of the introduced cDNA fragment within the plants. To examine fruit
from these plants for altered PE isoform profiles, a rapid method of separating<img
src="images/mutant-toms.jpg" alt="An evil looking mutant tomato" align="left" width="287"
height="204"> the fruit PE isoforms using column chromatography, and a more efficient PE
assay were developed. This procedure has given very good, reproducible separation of the
isoforms. Unfortunately the isoform work has shown there to be no clear evidence that the
enzyme has been downregulated. Northern blots have been able to detect the endogenous mRNA
for the isoform, but not the artificially introduced antisense mRNA, so it is most likely
that the chimeric cDNA is not being properly expressed.</p>
<p>Additionally, some of the enzymology developed as a part of this
project is currently being used by other members of the cell wall research team at
Nottingham. The work developed using tomato is being applied to enzyme extracted from
other fruit (such as strawberry and mango) as it is eventually hoped to isolate and
characterise PE genes from a range of fruit.</p>
<p>The ethics of using genetic engineering techniques for the
improvement of food products are complex. What cannot be disputed is the immense value the
techniques have in determining the role of individual enzymes in the complex processes of
fruit ripening and softening.</p>
<p><small>[<a name="1"></a>1] Tucker, G.A., Robertson, N.G. and
Grierson, D. (1982) Purification and changes in activities of tomato pectinesterase
isoenzymes. J. Sci. Food. Agric. 33: pp. 396-400.</small></p>
<p><small>[<a name="2"></a>2] Hall, L.N., Tucker, G.A., Smith, C.J.,
Watson, C.F., Seymour, G.B., Bundick, Y., Boniwell, J.M., Fletcher, J.D., Ray, J.A.,
Schuch, W., Bird, C.R. and Grierson, D. (1993) Antisense inhibition of pectin esterase
gene expression in transgenic tomatoes. Plant Journal 3: pp. 121-129.</small></p>
<p><small>[<a name="3"></a>3] The sequence for this cDNA clone
appears in the EMBL database as accession no. <a
href="http://www.ebi.ac.uk/cgi-bin/emblfetch?Z94058" title="EMBL entry Z94058" target="_blank">Z94058</a>.</small></p>
<h2 align="left">Related Pages</h2>
<ul>
<li><a href="PE_default.asp"
title="Multiple Forms of Pectinesterase are Present in Tomato and Other Fruit">Multiple
Forms of Pectinesterase are Present in Tomato and Other Fruit</a> poster presentation.
</li>
<li><a href="MicrobiologyReagents.asp"
title="Standard Microbiology Reagents">Standard Microbiology Reagents</a>.
</li>
</ul>
<p align="left"><img src="images/demandfreshtomatoes.gif" alt="Demand Fresh Tomatoes!"
width="88" height="31"> </p>
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