PNL Volume 14
Schwarz, H. P.
Institute of Genetics, University of Bonn, Federal Republic of Germany
The applicability and usefulness of in vivo absorption measurement
of intact leaves were tested using two chlorophyll mutants from Dr.
Gottschalk's collection in comparison with the initial line (IL) 'Dippes
Gelbe Viktoria'. Mutant 1206 A lacks chlorophyll b and has only 50% as
much chlorophyll a as the IL. Mutant 29 is deficient in both
chlorophyll a and chlorophyll b, having only 40% and 23% of the mother
variety, respectively.
The opal glass method of Shibata (1) minimizes the scattering er-
rors that occur when Intact leaves are irradiated by light. In this
study, strips of opal plastic material (perspex) were used instead of
opal glass. The plant material whole etiolated leaves or pieces of
green leaves were affixed to the plastic strips by transparent ad-
hesive tape. In vivo absorption spectra were measured in a Beckman
model 25 spectrophotometer. Depending on the absorption of the leaves,
one or two strips of opal perspex were placed into the spectrophotometer
to serve as reference and also to reduce light intensity. The plastic
strips themselves do not exhibit absorption, as indicated by the base
line (dotted line, Fig. 1b-d).
In Fig. 1, in vivo absorption curves of mutants and the initial
line are compared with the absorption of chlorophyll a extracted by
acetone after separation by thin layer chromatography. The absorption
maxima of in vivo spectra are situated at 678 nm wavelength, in contrast
to 663 nm of chlorophyll a dissolved and measured in acetone. This
spectral shift of the absorption maxima is caused by the interaction of
the chlorophyll molecules with their protein parts in the chlorophyll-
protein complexes integrated in the thylakoids of intact chloroplasts.
Apart from the different absorption maxima, the in vivo spectrum of the
chlorophyll b-less mutant 1206 A (Fig. 1b) exhibited the same
spectrophotometric properties as chlorophyll a (Fig. 1a), i.e. charac-
terized by the clearly visible minor band of chlorophyll a at 62 5 nm in
the in vivo spectrum corresponding to 620 nm in the acetone spectrum.
The distinct minor peak at 625 nm disappears in the spectra of
mutant 29 and the initial line. This phenomenon can be explained by the
presence of chlorophyll b in the leaves of these genotypes. Because of
the higher chloropohyll b content of the mother variety (chlorophyll a/b
ratio: IL = 3.5:1, mutant 29 = 6.1:1), a shoulder at 654 nm is produced
in the in vivo spectrum (Fig. 1d). Thus, this simple and rapid method
.seems to be very useful in distinguishing mutants lacking chlorophyll b
from other chlorophyll deficient genotypes according to their charac-
teristic absorption curves.
PNL Volume 14
Fig. 1. In vivo absorption spectra of intact green leaves recorded at
room temperature compared with the absorption of chlorophyll a
dissolved in acetone.
The in vivo absorption method also allows one to obtain information
about intact chlorophyll biosynthesis. Etiolated leaves of higher
plants accumulate protochlorophyllide in the dark, which is reduced to
chlorophyllide by light treatment only. As shown in Fig. 2a,
protochlorophyllide is detectable in etiolated leaves of mutant 1 206 A
by the absorption maximum at 650 nm. The photoreduction of
protochlorophyllide to chlorophyllide induced by brief irradiation with
white light (1 min, incandescent light, 100 W, 2500 lux) was evidenced
by the decrease of the absorption at 650 nm and by a spectral shift of
the absorption maximum from 650 nm to 680 nm. In the dark following the
light pulse, a second shift (the Shibata shift) occurred, moving the ab-
sorption maximum back to 672 nm. After the Shibata shift, the
absorption at 672 nm increased, indicating an accumulation of
chlorophyll for a limited time. About 60 minutes after the light treat-
ment, protochlorophyllide is detectable again in the dark leaves
(Fig. 2a, arrow).
PNL Volume 14 1982
Mutant 29 and the initial line show the same spectrophotometric
properties of etiolated leaves as mutant 1206 A. The increase of the
absorption at 672 nm after the Shibata shift is similar for the IL and
mutant 1206A. However, in mutant 29 the increase is slower (Fig. 2b).
These in vivo absorption measurements suggest that the first steps of
chlorophyll biosynthesis are not totally blocked in the mutants.
1. Shibata, K. 1957. J. Biochem. 44:147-173-