Photodynamic Herbicides - Chlorophyll Biosynthesis and Technological Applications - page 355

energy as fluorescence. These non-fluorescing Chl-polypeptides may become
fluorescent only when their structural relationship to other Chl-polypeptides is
disrupted. For example Chl b does not fluoresce in healthy thylakoid membranes
because it transfers its excitation energy to Chl a . It becomes fluorescent when its
structural organization is disrupted.
A fraction of the light energy absorbed by chloroplast membranes is converted to
chemical energy via the process of photosynthesis. Another fraction of that energy is
dissipated via several mechanisms including fluorescence. As mentioned in Chap. 16 ,
At 77 K, freshly isolated chloroplasts exhibit a deceptively simple three banded
fluorescence emission spectrum with emission maxima at 683-686 nm (F686),
693-696 nm (F696) and 735-740 nm (F740) (Bassi et al. 1990 ; Butler and Kilajima
1975 ). It is believed that the fluorescence emitted at F686 nm arises from the Chl a of
the light-harvesting Chl-protein complexes (LHCII and LHCI-680), that emitted at
F696 nm originates mainly from the Photosystem (PS) II antenna Chl a (CP47 and/or
CP29). That emitted at F740 nm originates primarily from the PS I antenna Chl
a (LHCI-730) (Bassi et al. 1990 ;ButlerandKilajima 1975 ). Under the same experi-
mental conditions, each fluorescence excitation spectrum recorded at emission
wavelengths of 685 (LHCII and LHCI-680), 695 (CP47 and/or CP29) or 740 nm
(LHCI-730) exhibits four excitation bands with maxima at 415-417, 440 nm,
475 nm and 485 nm. The excitation band with a maximum at 415-417 nm is
probably caused by the 1 transition of Chl a , while the 440 nm band corresponds

tothebulkoflightabsorptionbyChl a in the Soret region. The excitation bands
with maxima at 475 and 485 nm are excitation energy transfer bands and corre-
spond to light absorbed by Chl b and carotenoids in the Soret region. In healthy
chloroplasts the photons absorbed at these wavelength by Chl b and by
carotenoids, are transferred to Chl a wheretheyareconvertedtochemicalenergy
or wasted as Chl a fluorescence.
As mentioned above, this simple picture of the fluorescence properties of thyla-
koid membranes is rather deceptive, since thylakoid membranes contain several Chl
a and b -binding polypeptides which may not fluorescence until their structural
organization is disrupted. In this context, the ratio of emission at 739-740 nm relative
to that at 685 nm (F740/F686), as well as F740/F696 have been used to determine
changes in the relative distribution of excitation energy between PSI and PSII which
is mediated mainly by LHCII (Hipkins 1986 ). The magnitude and blue shift of these
fluorescence ratios have also been used to study the onset of chloroplast degradation
that disrupts the normal distribution of excitation energy between the photosystems
and results in a steady decrease in the F740/F696 and F740/F686 fluorescence
emission ratios (Rebeiz and Bazzaz 1978 ). Furthermore disorganization of the
chloroplast structure results in a blue shift of the emission and excitation maxima
to shorter wavelength and eventual disappearance of the emission peaks between
680 and 740 nm, and the excitation bands between 470 and 490 nm. With this
introduction to Chl and Chloroplast fluorescence the effects of exogenous
tetrapyrroles on isolated chloroplasts will now be discussed.

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