Plant Physiol. (1981) 68, 1-4
0032-0889/8 1/68/0001/04/$00.50/0
In Vivo Phytochrome Difference Spectrum from Dark Grown
Gametophytes of Anemia Phyiitidis L. Sw. Treated with
Norflurazon
Received for publication August 7, 1980 and in revised form December 2, 1980
RENATE GRILL AND HELMUT SCHRAUDOLF
Abteilung Aligemeine Botanik, Universitat Ulm, Oberer Eselsberg, D- 7900 Ulm/Donau, B.R.D.
ABSTRACT
An in vivo phytochrome difference spectrum of dark grown Anemia
phyllitidis L. Sw. gametophytes has been measured. The spectral characteristics estimated from the difference spectrum were as follows: red
maximum at 662 nanometers, far red maximum at 737 nanometers, isosbestic point at 695 nanometers, and a shoulder from 620 to 630 nanometers.
To diminish the influence of chlorophyll on phytochrome measurements in
dark grown gametophytes, total chlorophyll content could be reduced by
about 65% by treatment with 1 micromolar Norflurazon. In gametophytes
grown in red light, the inhibition caused by 1 micromolar Norflurazon was
97.5%, based on g spores. Total carotenoid content of dark grown gametophytes was not appreciably different from that already present in
dry spores. Herbicide treatment reduced this level to about 60%. Gametophytes grown in red light showed a nearly 7-fold increase in total carotenoids compared with that of the dark control. In treated light grown
protonemata, no carotenoid accumulation took place, and the level remaining corresponded with that found in treated dark grown protonemata.
Presence of phytochrome has been demonstrated throughout
the plant kingdom. In ferns, only physiological evidence points to
its occurrence, and the pigment has not yet been demonstrated
spectrophotometrically. The reason is that spores of most fern
species require light for germination, and, consequently, only
green material is obtained, which renders phytochrome measurements impossible. Although spores of the fern Anemia phyllitidis
also have an absolute light requirement for germination, they can
be induced to germinate and to develop into long filaments in
total darkness with GA3 (21). Preliminary measurements of phytochrome in such dark grown gametophytes showed that the
gametophytes were capable of forming a considerable quantity of
Chl in total darkness, an amount which proved too high to allow
reliable phytochrome measurements. Inasmuch as Chl has been
shown to be photobleached after treatment with the herbicide San
9789 in the light (14), an attempt was made to inhibit the dark
accumulation of Chl in Anemia gametophytes by treatment with
this herbicide. It has also been shown that the herbicide had no
damaging effect on phytochrome measurements in treated plants
(14).
MATERIALS AND METHODS
Spores of A. phyllitidis L. Sw. (harvest 1979) were sown in Petri
dishes (9.5 cm) with 20 ml nutrient solution after Mohr (17),
including 5 x 10-6 g/ml GA3 and San 9789. For phytochrome
measurements, dishes were kept in total darkness at 22 C for 3 to
4 weeks. For Chl determinations, 50 mg spores were sown per
Petri dish and were kept for about 3 weeks, either in continuous
darkness or in continuous red light (250 ,uw cm-2). Phytochrome
was measured with a dual wavelength spectrophotometer, PerkinElmer model 156. Actinic irradiation was obtained from a Schott
light-source KL 150 supplied with a 150 w halogen lamp, passing
through interference filters (Xmax = 657 nm or 730 nm, respectively)
and was given for a saturating 2-min irradiation. Cooling of the
sample holder was achieved by flowing water from a cryostat set
at 2 C. Sample thickness was 5 mm. The phytochrome difference
spectrum was obtained in the following way: the reference beam
was set at 800 nm throughout, and MA was measured at a number
of wavelengths 5 or 10 nm apart, between 750 and 610 nm.
For Chl extraction, gametophyte material from two Petri dishes
per sample was filtered through a Millipore apparatus, blotted
between filter paper, and weighed. Extraction procedure was
carried out in dim green light using a mixture of acetone, diethyl
ether, methanol, and petroleum ether (1:1:1:1). After centrifugation, the clear supernatant was measured at 648.5, 664.5, and 800
nm in a PMQ 3 spectrophotometer, and Chl concentration was
calculated after Sagromsky (20).
Extraction and assay of carotenoids was performed according
to Davies (6). Homogenization of dry spores was achieved in a
dismembrator (Braun, Melsungen) with glass beads 1.0 mm in
diameter in ethanol. Gametophyte material was homogenized by
grinding with quartz sand in ethanol. After centrifugation, the
supernatant was saponified for 20 min at 57 C. The carotenoids
were extracted from the saponified material by phase separation
with diethyl ether. For calculation of total carotenoid content, the
average extinction coefficient at 450 nm used was as reported by
Davies (6).
San 9789 (4-chloro-5 - [methylamine] -2- [a,a,a-trifluoro-mofficial common name Norflurazon,
tolylJ-3-[2HI-pyridazinone),
was dissolved in boiling water and was used at concentrations of
1 [LM and 10$M.
For structure analysis, the KMnO4 fixation of dark grown
protonemata was carried out according to Mollenhauer (18). After
dehydration in a graded ethanol series, the samples were infiltrated
with propylene oxide (99.5%) and then embedded in Epon 812.
Ultrathin sections, prepared with a diamond knife on an Ultratome III microtome (LKB, Stockholm), were examined in an EM
10 (Zeiss, Oberkochen).
RESULTS
First, two concentrations of Norflurazon, 1 ,iM and 10 gm, were
used to establish the effect of the herbicide on Chl accumulation
in gametophytes grown in total darkness or in continuous red
light. Since herbicide treatment reduces both Chl a and b, only
total Chl concentration is given. Table I shows that, in dark grown
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GRILL AND SCHRAUDOLF
2
Plant Physiol. Vol. 68, 1981
Table I. Effect of Norflurazon on Chl Accumulation in 21-day-old Dark Grown or Red Light Treated
Gametophytes of A. phyllitidis
Each value is derived from gametophyte material grown from 100 mg spores. Values given are mean ± SE.
,ug Chla + b/g
Gameto- Norflura-
phytes
Da
D
D
zon
Freshwt
Fresh wt
concn.
mg
489 ± 11
487 ± 10
318 ± 3
77.68 ± 1.7
27.12 ± 5.8
28.37 ± 1.8
0
lUM
lOIMM
Control
Chla
Spores
Control
%
34.9
36.5
132.33 ± 13.7
90.48 ± 7.3
34.8
23.8
ratio
3.5
3.4
2.2
4.6
4.8
3472.38 ± 19.1
88.98 ± 18.4
64.09 ± 34.8
2.5
1.8
3.2
2.5
2.1
%
902 ± 3
385.09± 7.3
0
R
502 ± 25
17.93 ± 4.5
R
IMLM
344 ± 0.8
18.62 ± 10.1
R
lOtUM
a D, dark grown; R, red light treated (250 ,uw cm-2).
380.31 ± 22.6
Table II. Influence of Short Periods of White Light on Chl Content in 20-day-old Dark Grown Gametophytes of
A. phyllitidis Treated with or without Norflurazon
Each value is derived from gametophyte material grown from 100 mg spores. Values given are mean ± SE.
,ug Chl a + big
Chl ba
FChl
White lighta Norflurazon Fresh wt
Chl b
Fresh wt
CtoOnCtoOnSpores
hour
0
0
1P
+
-
trol
%
mg
360 ± 60
410 ± 97
81.07 ± 8.5
27.49 ± 3.7
trol
%
ratio
33.9
280.88 ± 37.7
101.96 ± 16.7
36.6
3.5
3.7
0.5
0.5
+
359 ± 40
424 ± 80
79.54 ± 3.1
27.87 ± 3.4
34.9
281.74 ± 22.9
110.36 ± 9.0
39.2
3.7
3.3
1.0
1.0
+
388 ± 37
440 ± 66
73.47 ± 3.5
27.95 ± 3.5
38.0
282.73 ± 18.1
117.78 ± 9.2
41.6
3.5
3.6
al2Wcm-2
Table III. Total Carotenoid Content in Gametophytes of A. phyllitidis
Grown for 20 Days in Darkness or Red Light with or without Norjlurazon
Each value represents total carotenoid content in gametophytes grown
from 100 mg spores. The carotenoid content of 100 mg dry spores was 28
± 2 jig. Values given are mean ± SE.
Gametophytes Norflurazon
hILM
Da
D
_
+
Carotenoid Content
%
0.3
0.
02
Mg
22.3 ± 4.0
13.8 ± 3.7
111.1 ± 21.2
R
+
16.5 ± 2.0
R
a
D, dark grown; R, red light grown.
61.9
400
450
500 550 nm
Wavelenth
14.8
gametophytes, the higher herbicide concentration slightly reduced
the fresh weight; in light grown gametophytes, both concentrations
considerably reduced the fresh weight, presumably because of the
absence of photosynthesis. Thus, calculations for total Chl on a
per g fresh weight basis showed similar reductions in Chl for both
herbicide concentrations whereas calculations based on g spores
gave a slightly larger effect of the higher herbicide level. Based on
g spores, total Chl content of dark grown gametophytes was
reduced to about 24 and 35% by the herbicide treatment and, in
light grown gametophytes, to 1.8 and 2.5%. Thus, although the
herbicide works in the dark, the effect is more pronounced in red
light. Inasmuch as nothing is known about the mode of action of
the compound on Chl formed in darkness, it can only be assumed
that the greater efficiency in the light is due to photobleaching of
FIG. 1. Absorption spectrum of an acetone-diethyl ether-methanol-petroleum ether extract from Anemia gametophytes (243 mg fresh weight)
grown in red light for 19 days with I ,UM Norflurazon. Total Chl content
in this sample was 15.91 ,g/g fresh weight. Chromatographically purified
a-carotene measured in the same solvent mixture also showed prominent
peaks at 473 and 445 nm and a marked shoulder at 422 nm, thus
corresponding closely with the absorption characteristics of the Anemia
extract.
Chl, as has been suggested by a number of investigators (1, 3, 9,
14).
Next, it was established indirectly whether Pchl was also present
in dark grown gametophJrtes. Dark grown material was exposed
to white light (12 w cm- ) for 0.5 and 1.0 h before extraction in
order to convert any existing Pchl to Chl. As can be seen from
Table II, no Pchl appears to be present in dark grown material
with or without herbicide treatment.
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PHYTOCHROME IN NORFLURAZON TREATED ANEMIA
Plant Physiol. Vol. 68,1981
I
;
+X>
11-1
.1
lw
r!
"
,
3
whereas, in treated plants, no carotenoid accumulation took place.
The remaining level, for which a representative absorption spectrum is shown in Figure 1, was comparable with that of treated
dark grown gametophytes.
In contrast to the dry spore (results not shown), the plastids in
dark grown Anemia gametophytes contain no prolamellar body.
The membrane array is different from that of chloroplasts from
light grown protonemata. Dark chloroplasts show a structure
reminiscent of plastids from dark grown embryos or leaflets from
quiescent buds of conifers (15, 16). They show an arrangement
which can best be described after Mustardy and Brangeon (19) as
an aligned lamellar structure (Fig. 2).
concentration of the herbicide did not reduce
Since the
the fresh weight of gametophytes in darkness and was similarly
effective in inhibiting Chl formation, this concentration was used
for growing gametophytes for phytochrome measurements. However, phytochrome in gametophyte material is extremely difficult
to measure due to the extremely delicate material, to its very low
phytochrome level, and to a large decrease in AA following actinic
irradiation, which is not due to photoconversion of phytochrome
inasmuch as, during the subsequent period of measurement, the
AA gradually increases again until a more or less stable level is
reached, and a reading can be made. This effect might be caused
by the small amount of Chl still present (11). The difference
spectrum (Fig. 3) has, thus, to be regarded with caution. Surprisingly, the characteristics of the difference spectrum (red maximum
at 662 nm, far red maximum at 737 nm, isosbestic point at 695
anm, shoulder from 620 to 630 nm) were found to be more
comparable to those of angiosperms than to those of gymnosperms
I-liM
K
,
*
Zi
-.f
...
v-,4u**O*w*
..
-
-,
..
1-
.f.
"
.r -L.-..L
FIG. 2. Electron micrograph of a thin section of plasstids from dark
grown Anemia gametophytes, showing an aligned laimellar system.
x 10,000.
or lower plants.
I~~~~~~~
I
+4
+3
+2
*+1
o
<
_1
-2~
-3
-4
610 630 650 670 690 710 730 750
Wavelength (nm)
FIG. 3. Phytochrome difference spectrum of dark-grown gametophytes
of A. phyllitidis treated with 1 ,UM Norflurazon. Points represent
values from seven experiments.
mean
Because herbicide treatment has been shown to inhibit carotenoid biosynthesis in light (3, 13) as well as in darkness (1, 2, 9,
25), it was of interest to determine its effect on carotenoid accumulation in the Anemia system. First, dry spores were assayed for
their total carotenoid content to see what changes occurred in
gametophytes grown in light or darkness and treated with or
without 1 ,UM Norflurazon. The amount of carotenoids already
present in the dry spore is considerable (Table III) and remains
practically unaltered during gametophyte growth in darkness.
Herbicide treatment reduced this level to about 60%. In red light
grown gametophytes, a nearly 7-fold increase was observed,
W%fC_N-4 TCNCNV4%IT
DISCUSSION
To our knowledge, this is the first report of in vivo spectrophotometric demonstration of phytochrome in fern gametophytes.
The assay has been possible only since the discovery (21) that
spores of A. phyllitidis can be induced to develop long filaments in
total darkness under the influence of GA3. The difference spectrum of phytochrome appears to have characteristics resembling
those of angiosperm phytochrome (4, 8, 11, 22). This is surprising,
because phytochrome maxima and isosbestic point for some lower
plants (10, 24) and gymnosperms (12) were found at considerably
shorter wavelengths. However, the phytochrome difference spectrum of Anemia has to be considered as preliminary. It serves
mainly to demonstrate spectrophotometric evidence of phytochrome in fern gametophytes in vivo.
In this system, Chl accumulation, not only in the light but also
in darkness, can be strongly inhibited by Norflurazon, although
it has been stated that this herbicide "does not inhibit Chl synthesis
per se" but "only affects stability of Chl in the light provided that
the light is strongly absorbed by Chl" (8). This conclusion was
drawn from the findings that, in the mustard seedling, Chl accumulation was not inhibited in continuous far red light, and, in
etiolated oat seedlings, no inhibition was found on Pchl or its
conversion to Chl (5, 13, 25). Also, in Anemia gametophytes, Pchl
does not need to be taken into consideration, because it seems to
be absent in darkness. Concerning Chl formation in darkness, this
observation merits special consideration, because, except for some
algae and conifers, the existing information is very scanty. In
certain algae grown in darkness and on media in which Chl
synthesis is greatly reduced, Pchl was found also not to accumulate
in measurable quantities (7). In conifers, dark conversion of Pchl
to Chl varies from more or less complete in Pinus, intermediate in
Picea, to rather incomplete in Larix (23). Further research on the
effect of the herbicide in these plants forming Chl in darkness is
very desirable.
Carotenoids are already present abundantly, mainly in nonplastid compartments of the dry spore, with a-carotene as the
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4
GRILL AND SCHRAUDOLF
major component. During growth of gametophytes in darkness,
total carotenoid content did not increase, although some turnover
is indicated by the fact that herbicide treatment does result in a
decrease of the already existing carotenoid level. In light grown
protonemata, a large increase in total carotenoids occurred, which
is completely prevented by treatment with Norflurazon; only a
level remained which is comparable to that of treated dark grown
gametophytes. Thus, inhibition of carotenoid accumulation in
Anemia is observed more clearly in the light.
These results imply that in the Anemia system inhibition of Chl
accumulation by Norflurazon is not dependent on light and that
the existing carotenoids during gametophyte growth in darkness
only undergo some turnover whereby they become susceptible to
herbicide inhibition.
Acknowledgments-We wish to thank Sandoz AG, Switzerland, for generously
supplying us with a sample of Norflurazon. We are indebted to Mrs. U. Paukner for
assisting with the embedding and sectioning of the material. We are also grateful to
the "Sektion fur Elektronenmikrokopie" of the University of Ulm for providing the
electron microscope. Thanks are due also to Prof. H. Kayser, Abteilung Ailgemeine
Zoologie, University of Ulm, for chromatographically confirming a-carotene as the
major component in Anemia spores.
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