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Fig. 17.2 Room-
temperature fluorescence
emission spectrum of (a) the
hexane-extracted acetone
extract of etiolated control
and etiolated ALA-treated
seedlings and (b) green
control and ALA-treated
seedlings. The acetone
extract containing various
pigments was extracted
with one volume of hexane,
then with a third volume of
hexane to remove the Chl.
The hexane-extracted
acetone residue containing
monocarboxylic
tetrapyrroles such as
Pchlide and dicarboxylic
tetrapyrroles such as
protoporphyrin IX
(if present) was subjected to
high resolution
spectrofluorometric analysis
according to methods
described by Rebeiz ( 2002 )
darkness (Sisler and Klein 1963 ). Protochlorophyllide, a Mg-porphyrin, is the
immediate precursor of chlorophyllide which upon esterification is converted to
Chl. However when green plants were treated in a similar manner, and their acetone
extract was analyzed by absorbance spectroscopy, tetrapyrrole accumulation was
not detected (Fig. 17.1b ). These results reinforced the notion that (a) in green plants
the Chl biosynthetic pathway was not highly functional, and (b) that the
tetrapyrrole-dependent photodynamic herbicide (TDPH) hypothesis was not likely
to succeed. At that stage it was conjectured that the lack of observed tetrapyrrole
accumulation in green tissues treated with ALA might be due to inadequate
analytical techniques than lack of appropriate metabolic activity. To test this
hypothesis, acetone extracts of control and ALA-treated etiolated and green cucum-
ber cotyledons that were previously analyzed by absorbance spectroscopy, were
processed and analyzed by fluorescence spectroscopy as described in (Rebeiz 2002 ).
Surprisingly it was observed that green tissues that had been treated with ALA
had actually accumulated more Pchlide than the etiolated tissues (Fig. 17.2a, b ).
This tetrapyrrole accumulation was not detectable however with classical sample
preparation and absorbance spectroscopy. When a sample preparation was used that
eliminated the Chl from the green extracts prior to analysis by room temperature
fluorescence spectroscopy (Rebeiz 2002 ), a true picture of the tetrapyrrole profile
emerged that indicated that green tissues were actually more active than etiolated
tissues at converting exogenous ALA to tetrapyrroles Fig. 17.2 . This in turn
suggested that the TDPH hypothesis may after all be reduced to practice.
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