Plant Cell Physiol. 38(1): 51-58 (1997)
JSPP © 1997
Phytochrome Control of Phototropism and Chlorophyll Accumulation in
the Apical Cells of Protonemal Filaments of Wildtype and an Aphototropic
Mutant of the Moss Ceratodon purpureus
Tilman Lamparter1, Heike Esch 1 , David Cove 2 and Elmar Hartmann'
1
2
Institute for Plant Physiology, Free University Berlin, Konigin Luise Str. 12-16, D-14195 Berlin, Germany
Department of Genetics, University of Leeds, Leeds LS2 9JT, U.K.
The aphototropic mutant line ptrll6 of the moss Ceratodon purpureus shows characteristics of a deficiency in the
phytochrome chromophore. Photoreversibility measurements indicate an approximately 20 time lower concentration of spectrally active phytochrome compared to wildtype, whereas normal phytochrome apoprotein levels are
found on immunoblots. Feeding with the tetrapyrroles
biliverdin, the proposed precursor of the phytochrome
chromophore, or phycocyanobilin, which may replace
the phytochrome chromophore, resulted in the rescue of
ptrll6 phototropism.
The ptrll6 mutant and the phenotypically-related mutant ptrl contain lower chlorophyll levels than the wildtype. Chlorophyll content of wildtype and mutant tissue
grown under different light conditions was estimated using
conventional spectrophotometry of extracts and fluorimetrically, on single apical cells. Dark-grown tissue contained
about 100 times less chlorophyll than tissue grown under
standard white light conditions. Red light given for 24
h to dark adapted filaments induced an increase in the
chlorophyll content in the wildtype, but not in ptrl16. Blue
light induced an increase in chlorophyll both in wildtype
and in ptrl 16. The red light effect on the wildtype was partially reversible with far-red, \iptrll6 was grown on phycocyanobilin, an increase in chlorophyll was also found when
cells were irradiated with red light.
The results indicate that phytochrome as well as a blue
light photoreceptor regulate chlorophyll accumulation in
C. purpureus protonemata. It can be assumed that in
ptrll6, the synthesis of the phytochrome chromophore is
blocked specifically beyond the synthesis common to chlorophyll and the phytochrome chromophore and affects an
enzymatic step between protoporphyrin and biliverdin.
Key words: Blue light photoreceptor — Ceratodon purpureus — Phototropism — Phycocyanobilin — Phytochrome
(chromophore) — Regulation of chlorophyll synthesis.
Abbreviations: /dJ A, red/far-red reversible change of absorbance difference between two wavelengths; n.d., not determined;
PCB, phycocyanobilin; Pfr, far-red absorbing form of phytochrome; Pr, red absorbing form of phytochrome; s.e., standard error of the mean.
51
Phototropism of the protonemal apical cell of the
moss Ceratodon purpureus is controlled by the plant photoreceptor phytochrome (Hartmann et al. 1983). Following
UV mutagenesis, several aphototropic lines have been isolated that are apparently defective in the biosynthesis of
the phytochrome chromophore (Lamparter et al. 1996). In
such mutants all phytochrome responses are expected to be
defective and a comparison with wildtype lines may point
to other physiological effects in addition to phototropism
that are under the control of phytochrome. Such a comparison has already shown that the gravitropic response is
down-regulated by phytochrome (Lamparter et al. 1996),
and an identification of further phytochrome-controlled responses may help to establish C. purpureus as a model system for phytochrome research.
Since light-grown tissue of ptrl, the mutant characterized initially, shows significantly lower chlorophyll levels
than the corresponding wildtype strain, phytochrome may
have a regulatory role in chloroplast development and/or
chlorophyll biosynthesis. The regulation of these processes
is well analyzed during de-etiolation of angiosperm seedlings. Two different light-dependent steps are involved in
the development of fully intact green chloroplasts. One
step requires the activation of the photoreceptors phytochrome and the blue light receptor. The other step is
the conversion of protochlorophyllide into chlorophyllide,
which is dependent on light absorbance via protochlorophyllide (Virgin and Egneus 1983). It is difficult to dissect
these two light absorbing systems in angiosperms and to analyze the function of phytochrome separately. Often the
effect of phytochrome on chlorophyll accumulation is monitored by estimating the duration of the lag phase of chlorophyll biosynthesis which occurs when etiolated plants are
brought into white light. Protochlorophyllide-chlorophyll
conversion in mosses is not light dependent and occurs in
darkness, allowing a more straightforward assessment of
photoreceptor action. A further advantage is that chlorophyll levels can be analyzed in a single growing cell, the
protonemal apical cell.
For C. purpureus filaments, an effect of light on chlorophyll content and on plastid morphogenesis (Valanne 1971)
implies the participation of phytochrome in these processes. However, the roles of phytochrome and of the blue
light photoreceptors are not yet clear. We have therefore
52
Phytochrome control of chlorophyll biosynthesis
begun to quantify chlorophyll levels from tissue pre-irradiated with different light qualities, using a fluorimetric
approach which allows us to follow chlorophyll levels in
single protonemal apical cells. This has in turn allowed us
to observe the ability of a newly-isolated mutant, which apparently lacks the phytochrome chromophore, to recover
chlorophyll biosynthesis after feeding with PCB, suggesting that PCB can replace for the phytochrome chromophore. The same mutant has also allowed us to distinguish
clearly between blue light and red light induced processes.
Materials and Methods
Moss strains and cultivation—For all analyses, the wildtype
wt4 (Hartmann et al. 1983) and an aphototropic mutant, ptrll6,
derived from the same strain, were used. ptrll6 was isolated during a screen for aphototropic growth following UV-mutagenesis
as described by Lamparter et al. (1996). Filaments were grown on
solid lb medium (1 mM KNO3, 100 /xM CaCl2, 1 mM KH2PO4, 10
nM C6H3FeO7, 27 mM glucose, trace elements, adjusted to pH
5.8 with KOH, and 1.1% Agar (Sigma) (see Lamparter et al.
1996)). Standard growth conditions were: 20°C in a 16 h light (fluorescent tubes Philips MCFE white; fluence rate lOO/rniolm"2
s~' PAR)/8 h dark cycle. For dark adaptation filaments were
grown in black boxes on cellophane overlaying agar medium at
20° C. The agar plates were always placed vertically so that the apical cells aligned parallel on the surface of the cellophane, as a
result of their negative gravitropism.
Tetrapyrrole feeding—Stock solutions of tetrapyrroles were
made as follows. Biliverdin dihydrochloride (Sigma) was dissolved in water at a final concentration of 0.5 mM by continuous stirring, adjusted to pH 6.0 with KOH. Phycocyanobilin (PCB) was
prepared from the blue alga Spirulina geitlerie according to
Kunkel et al. (1993), dissolved with dimethyl sulfoxide to give a final concentration of 2 mM, as monitored spectrophotometrically
using the molar absorption coefficient of 37,900 M" 1 cm" 1 at 680
run, diluted 1/100 with water, and adjusted to pH 6.0 with KOH.
Protoporphyrin IX disodium salt (Sigma) was dissolved in water
to a final concentration of 2 mM, adjusted to pH 7.5 with HC1.
The more alkaline pH was necessary to yield soluble protoporphyrin. Heme (protoheme, Sigma) was prepared in the same way
as described for protoporphyrin. Stock solutions were sterilized
by filtration and then added to equal quantities of melted, doublestrength lb medium at 50°C, to give final concentrations of tetrapyrroles of 0.25 mM for biliverdin, lOjuMforPCB, 1 mM for protoporphyrin and heme. Twenty four hours prior to physiological
assays, the cellophane carrying dark-adapted filaments was transferred to tetrapyrrole-containing medium. Filaments used for controls were transferred to tetrapyrrole-free medium.
Phytochrome measurements and immunoblotting—Protonemal tissue was extracted using a French pressure cell (Mini-Cell
FA003, SLM Instruments, Rochester, NY, U.S.A.) and processed
as described previously (Lamparter et al. 1995, 1996). Photoreversibility was measured in a computer-controlled dual wavelength photometer in which the measuring wavelengths are set to
670 and 780 nm (Lamparter et al. 1994). Actinic irradiation occurred at 660 ± 12 nm and broadband far-red above 725 nm (RG-9
filter, Schott, Mainz, Germany). Spectral assays utilised 10 mm diameter cuvettes containing 400 fil extract mixed with 250 mg
CaCO 3 , a scattering agent enhancing the photoreversibility signal
by extending the light path.
Immunoblots were prepared as described previously (Lamparter et al. 1995) using SDS-PAGE (1% separating gel). Phytochrome was immunostained with affinity-purified APC1 polyclonal antibody (Lamparter et al. 1995).
Phototropic response—The phototropic curvature was estimated from filaments that have been aligned by negative gravitropic growth on cellophane-overlayed, vertically oriented plates.
After 5 d growth in darkness, phototropism was induced using
red light from a halogen projector applied through a 665 + 12
nm DAL interference filter (Schott, Mainz, F.R.G.) at an intensity of 4/jmolm~ 2 s~' for 24 h. Light was given horizontally,
parallel to the agar surface, so that a maximal phototropic response would give a 90° deviation from the original growth direction. The resulting angle was evaluated using a microscope, a computer-coupled video camera and an imaging software program
(Image P2, H + H MeBsysteme, Berlin, Germany).
Chlorophyll extraction and measurement—Light-grown tissue was taken directly from moss cultures grown for 7 d after
subculturing under standard light conditions. Dark-adapted tissue was obtained by growing filaments at 20° for 14 d on vertically-oriented agar plates in darkness. Red and far-red irradiations were given using the apparatus described for phototropism
analyses with 665 ± 12 nm (red) or 735 ± 12 nm (far red) DAL interference filters. Light intensities were 4/imol m~2 s~' for red and 5
^mol m~2 s" 1 for far-red. The filaments grown during the dark incubation period were carefully separated from the older tissue
below by cutting with a sharp razor blade.
Routinely, 50 mg fresh weight were extracted with 1 ml of
80% acetone by incubating at 4° in darkness for 4 h. The filaments
were separated from the supernatant by centrifugation (48,000 x
g, 15 min) and absorbance was measured using a Kontron
(Neufahrn, Germany) 941 spectrophotometer at 700, 663, 652 and
645 nm with an integration time of 30 s for each wavelength. The
700 nm absorbance was taken as an internal standard and subtracted from the values obtained for 663, 652 and 645 nm. The
concentration of total chlorophyll was obtained from the sum
over chlorophyll a and b as calculated by the formulae given by Arnon (1949) and was routinely compared with the value for total
chlorophyll estimated from the isosbestic point 652 nm. Spectra
were recorded at 100 nm per min using an 80% acetone baseline.
For low absorbing samples, averages were taken from three spectra recorded from the same sample.
Chlorophyll fluorescence imaging and quantification—Moss
filaments were grown either under standard white-light conditions
or on vertically-oriented plates in darkness for 5 d and thereafter
irradiated for 24 h with different light programs as described
above.
For quantification of chlorophyll fluorescence, specimens
were imaged using a confocal laser-scanning microscope consisting of an Axiowert 35 with inverted optics and a 40x PlanNeofluar objective (Zeiss, Oberkochen, Germany) coupled to a
MRC 1024 laserscan system (Biorad, Hemel Hempstead, Great
Britain). The 650 nm red band of the krypton-argon laser was
always selected for excitation; a 680 nm cutoff filter was selected
for the emission light path.
Apical cells were selected arbitrarily through the normal optics of the microscope. Cells that were not'in contact with other
cells were chosen. For all measuring procedures, the high voltage
of the photomultiplier was set to 1,000 V and the aperture was
held maximally open (8.0). To adapt the system to the greatly varying fluorescence signals, the intensity of the excitation beam was
adjusted via neutral density filters in order to generate signals just
below the saturation level. Control experiments have shown that if
Phytochrome control of chlorophyll biosynthesis
several images are taken from one cell at different excitation intensities, the subsequent quantification yielded the same result within
an error of ± 5 % . For each specimen, fluorescence images of 10 or
more cells were stored on hard disk. For each cell a relative value
for the fluorescence intensity within the area of the most apical
100/im was estimated using the imaging software Lasersharp 1.01
(Biorad). This value, divided by 1,000 and divided by the intensity
of the exciting light beam in % is given as "fluorescence units".
For the result of a single experiment the average value of the
readings from these single cell images was calculated. From 4 or
more single experiments the average and the standard error were
calculated. Calibration of fluorescence was done with protoplasts,
prepared according to Cove et al. (1996), washed and concentrated by 100 x g centrifugation. Chlorophyll concentration of the
preparations was measured spectrophotometrically as above and
fluorescence signals of defined volumes were quantified under
same conditions as for C. purpureus cells. Dilution series showed
linearity up to 0.5 ng chlorophyll in a volume of 5 nl, the highest
concentration tested (r 2 =0.977). One fluorescence unit was equivalent to 0.04 pg chlorophyll.
Results
Phototropism and phytochrome content of the aphototropic mutant, ptrl!6—The newly isolated aphototropic
mutant ptr!16 is derived from the wildtype strain wt4,
while the mutant ptrl, that was analyzed previously is derived from the wildtype strain wt3. As wt3 shows a reduced
phototropic response compared to wt4 (Lamparter et al.
1996), the difference between mutant and the corresponding wildtype with respect to phototropism is higher for
ptrl 16 than for ptrl.
Table 1 shows that during a 24 h unilateral red light irradiation, no phototropic bending was induced in ptrl 16.
Under those conditions the wildtype showed an 85° response. Because p/r/7 6 was phenotypically similar to ptrl,
which had been identified as a mutant lacking the phytochrome chromophore (Lamparter et al. 1996), the recovery
of ptrl 16 phototropism was tested with the four tetrapyrroles protoporphyrin, heme, biliverdin and phycocyanobilin. In higher plants, it is proposed that the biosynthesis of the phytochrome chromophore, phytochromobilin,
follows the synthesis of chlorophyll and branches at the
position of protoporphyrin (see Weller et al. 1996). It is sug-
53
Table 2 Phytochrome photoreversibility of dark adapted
wildtype and ptrl 16
FW)
wt4
8.8±0.4
ptrll6
0.3±0.2
gested that heme is formed via Fe 2+ chelation, which is
transformed into biliverdin and finally phytochromobilin.
A very weak positive phototropic response of around 5°
(Table 1) was observed if ptrl 16 was grown on protoporphyrin (1 mM) or heme (1 mM). Both biliverdin (0.25 mM)
and PCB (10 /uM), which may replace the phytochrome
chromophore, rescued the phototropism of ptrl 16 almost
totally; in both cases the bending curvature was around
60°. The phototropic response of wt4 was not affected by
PCB (Table 1).
Extracts of ptrl 16 contained very low levels of spectrally-active phytochrome (Table 2). The value for ptrl 16 was
around the detection limit of the measuring instrument,
and at least 20 times lower than the wildtype value. On immunoblots, the anti-phytochrome-antibody APC1 stained
an apophytochrome band in wt4 and in ptrl 16 (Fig. 1).
Chlorophyll content of light and dark-grown tissue—
The chlorophyll content of ptrl 16, grown under standard
white light conditions, was about 5 times lower than the
content found in wildtype (Table 3). In both strains, darkgrown filaments contained lower chlorophyll levels than
filaments grown in white light. After a 2 week dark adaptation, both wildtype and mutant contained a similar low
amount of chlorophyll, around 5//g per g fresh weight
(Table 3). An example of absorbance spectra is shown in
Fig. 2. The extract of dark-grown tissue still exhibited an
absorbance maximum at 663 nm characteristic of chlorophyll a. Similar spectra were obtained from filaments that
were grown for 4 weeks in darkness.
A constant 24 h red light irradiation given to dark-
Table 1 Phototropic response of wildtype and ptrl 16
Standard medium
wt4
ptrll6
Curvature 0
Protoporphyrin
Heme
Biliverdin
PCB
85±1
n.d.
n.d.
n.d.
84±1
0±l
5±2
6±2
62±3
57±2
Filaments were dark adapted for 5 days and then irradiated with unilateral red light for 24 h. Prior to phototropic stimulation, filaments
were transferred to protoporphyrin (1 mM), heme (1 mM), biliverdin (0.25 mM) or PCB (10//M). Mean values±standard error of 100
or more cells.
54
Phytochrome control of chlorophyll biosynthesis
B
0,70
0,030
0,60
i
0,025
light grown
•
0.50
dark grown
0,020
~
0,40
0,015
0,30
0,010
0,20
<***
0,005
0,10
Fig. 1 Protein pattern (B, C) and phytochrome immunoblot (D,
E) with wildtype (B, D) and ptrll6 (C, E) extracts. Lane A shows
marker proteins 180, 116, 84, 58 and 49 kDa. The position of the
phytochrome band is marked with an arrow.
0,00
300
400
500
600
700
0,000
800
wavelength nm
Fig. 2 Typical absorbance spectrum of an acetone extract of 50
mg C. purpureus wildtype tissue. Light-grown: filaments grown
under standard white light conditions. Dark-grown: filaments
grown during 14 d in darkness.
adapted wildtype filaments increased the chlorophyll level
6-fold over the dark level (Table 3). An almost equivalent
increase was obtained when red light irradiation was given
in 5 min pulses at hourly intervals. Far-red pulses resulted cell, which divides serially to generate the protonemal filain only a slight increase, and far-red pulses immediately ment. This cell is expected to show the most pronounced refollowing red pulses reversed the effect of the red light par- sponses to different environmental stimuli.
Examples of fluorescence images are shown in Fig. 3.
tially (Table 3). Red light given to dark-adaptedptrl 16 filaments did not result in an increase in chlorophyll (Table 3). From such images, qualitative differencies between darkThese results point to phytochrome as one photoreceptor adapted and red light-pretreated wildtype cells are already
obvious. Plastids from dark-adapted cells (Fig. 3A) still
of light-induced increase in chlorophyll synthesis.
Chlorophyll quantification in single apical cells—Meas-display chlorophyll fluorescence; they appear smaller than
uring the chlorophyll content of extracts does not allow the the chloroplasts of light-grown cells and less pigmented. In
differential content of the different cells that are formed dark-grown C. purpureus apical cells, plastids show a typiduring a given incubation period to be determined. The age cal intracellular distribution (Meske and Hartmann 1995,
of the cells within one filament varies and initial observa- Walker and Sack 1990): plastids in the apical dome are sepations indicated that the distribution of plastids and the rated from distal plastids by a plastid-free zone. This charcontent of chlorophyll may vary between cells of different acteristic distribution is observed in the fluorescence images. Thus quantification of chlorophyll at the level of the ages. Apical cells grown for 24 h in red light (Fig. 3B) loose
single cell was undertaken. Studies focussed on the apical this zonation, they are densely filled with chloroplasts,
Table 3 Chlorophyll content of wildtype and ptrll6 extracted after different light treatments
:
LI]ght
T
treatment
Cont. white
14 d dark
13 d dark, 1 d
13 d dark, 1 d
13 d dark, 1 d
13 d dark, 1 d
Chlorophyll /,<g (g FW)-'
wt4
ptrll6
620 ±20
140±30
4.,7± 0.2
3± 2
cont. red
28 ± 1
3± 1
26 ± 2
red pulses
n.d.
7
far-red pulses
± 0.4
n.d.
red/far-red pulses 11 ± 1
n.d.
Light pulses were given for 5 min at hourly intervals. Mean
values±s.e. of 3 extractions.
which appear disk or leaf-shaped with a diameter of approximately 4/im. Images of dark-grown ptrl 16 apical
cells were comparable to dark-grown wildtype apical
cells, showing similar intracellular distribution and similar
plastid size (Fig. 3C). Images of red light-treated ptrl 16
filaments were comparable to dark-adapted filaments
(Fig. 3D) as were those grown in darkness in the presence
of PCB (Fig. 3E). Following red irradiation of tissue grown
in the presence of PCB, the plastids appear slightly enlarged and look intermediate between plastids of dark-grown
and light-treated wildtype cells (Fig. 3F). It should be noted
that during the imaging process, the features of the confocal laserscanning microscope were adjusted for every image in order to gain maximum contrast. For this reason,
the images can only be taken for qualitative and not for
Phytochrome control of chlorophyll biosynthesis
55'
Fig. 3 Fluorescence images of tip cells of wildtype (A and B) and ptrll6 (C, D, E, F). A, C, E: dark adapted for 5 d. B, D, F: grown
for the last 24 h in red light. E and F: grown for the last 48 h on PCB. Images were recorded through the 63 x objective (Plan Achromat,
Zeiss) with oil immersion. The aperture was set to 3.0, fluorescence intensity was set to 100% and the laserscanhead was adjusted to 2 x
zoom. For each image the high voltage of the photomultiplier was adjusted to obtain maximal contrast; thirty optical sections were imaged in 0.5 (im intervals. Final images were reconstructed from those section images using the Lasersharp 1.01 software. The border of
each cell is shown by superimposing a low contrast transmission image.
quantitative analysis.
The chlorophyll-fluorescence of light grown ptrll6
cells was about 7 times lower than of light grown wildtype
cells (Fig. 4). This difference correlates qualitatively with
the difference found in corresponding extracts (Table 3).
After a prolonged dark adaptation of 30 d, the longest peri1000
*
o
5
WU
--•••p(Mie
3
theoretical slope
100 j
o
• * • • -
10
15
20
25
days in darkness
Fig. 4 Chlorophyll fluorescence of wildtype and ptrll6 tip cells
after different dark incubation periods. The dashed gray line gives
a theoretical value of chlorophyll, assuming that during each cell
division, 95% of the plastids are retained in the apical cell and
assuming that no net chlorophyll synthesis occurs (see text).
od tested, the chlorophyll fluorescence of both lines dropped to values of around 1/100 of light grown cells. The
decrease was most rapid during the first days of dark adaptation but rather slow between the 12th and the 30th day of
growth in darkness. The ptrll6 fluorescence was always
about 2 to 8 times lower than the wildtype fluorescence
(Fig. 4). The biggest difference between both lines was
found after 6 days of dark growth, a prolonged dark incubation reduced the difference. These results are in contrast to the results found for extracted chlorophyll of dark
grown filaments, where only a slight difference was found
between wildtype and mutant (Table 3). However, this discrepancy may be explained by the fact that fluorescence
measurements were made with the apical cells and chlorophyll extracts were made from the entire filament. In the latter case the apical cells only contribute to a small extend to
the amount of tissue analyzed, the majority consisting of
older intercalary cells. During division of the apical cell,
chloroplasts are distributed unequally between the new
cells (data not shown); the majority being retained in the apical cell. In addition, chlorophyll synthesis, which may also
occur in darkness and which may differ between wildtype
and mutant (see below) is likely to be higher in the metabolically active tip cell. Therefore, after transfer to darkness,
the older intercalary cells may achieve a low steady state
56
Phytochrome control of chlorophyll biosynthesis
level of chlorophyll content much earlier than the tip cells.
The slow decrease of chlorophyll fluorescence during
late stages of dark adaptation, where growth and cell division continues, implies that at least during this period, net
chlorophyll synthesis occurs also in darkness. We tried to
calculate a slope of the decrease of chlorophyll, assuming
that chlorophyll were only lost by the "dilution" of plastids
following cell-growth and -division and assuming that neither synthesis nor degradation of chlorophyll occurs. The
result of this calculation is presented in Fig. 4 as dashed
gray line. For this estimation, it was assumed that three
new cells are formed every day and that during every cell
division, plastids are distributed at a 95 : 5 ratio between
the apical and the basal cell. The cell division rate was estimated by microscopical observations following 24 h of
gravitropical re-orientation (data not shown); this was in accordance with estimations from the final length of the filaments after 30 d growth in darkness. This was between
20 and 24 mm; the mean cell length is about 250/im
(Schwuchow et al. 1990 and Lamparter, unpublished data)
and so this is equivalent to about 3 cells per day. The 95 : 5
ratio of plastid distribution was not measured directly, but
should be taken as an upper estimate, as at least one out of
20 plastids is retained in the sub-apical cell. The actual
slope of chlorophyll decay after 12 d growth in darkness is
significantly lower than this theoretical decay (Fig. 4) which
implies that indeed net synthesis of chlorophyll occurs in
the apical cell. An alternative explanation for the rather
slow decrease of chlorophyll fluorescence is that a concomitant increase in fluorescence yield may cover a faster loss of
chlorophyll. Such a change in the fluorescence yield, which
might be the consequence of adaptive mechanisms on the
level of the photosynthetic apparatus, should however occur during early stages of dark adaptation and is unlikely
during the late stages of dark growth.
Experiments investigating the effect of different light
pretreatments on the chlorophyll fluorescence of tip cells
are summarized in Table 4. Most data were obtained from
tissue which had been dark adapted for 5-6 d, which is also
the standard adaptation period for phototropism analyses.
As mentioned above, the chlorophyll level of the dark control differs between ptrll6 and wt4. Any light-induced increase in chlorophyll was found to be relative to this dark
level, so the data for wildtype and ptrll6 cannot be compared directly.
In wildtype tissue, a 24 h constant red irradiation increased the chlorophyll fluorescence about 10 fold above
the dark control; giving values similar to apical cells grown
under white light. A similar increase was found after irradiation with 24 h blue-light. Red light of 5 min given at hourly intervals also increased the chlorophyll fluorescence of
wildtype cells, but to a lesser extend than continuous red irradiation. The effect of red pulses was partially reversed if
they were immediately followed by pulses of far-red.
In dark-adapted ptrll6 tissue no induction of chlorophyll fluorescence was found after a constant 24 h red irradiation (Table 4). However, chlorophyll-fluorescence was
increased 10 fold over the dark control by a constant 24 h
blue irradiation. If red-light irradiation was given toptrl 16
filaments grown in the presence of PCB, fluorescence increased 9 fold over the dark control. Control experiments
with filaments grown in the presence of PCB in darkness
yielded results similar to those for PCB-free medium, indicating that PCB alone neither contributes to a fluorescence signal nor alters the chlorophyll content. PCB control experiments with wildtype filaments showed that there
was no influence of PCB on the fluorescence signal, irrespective of whether filaments were kept in darkness or
brought into red light (Table 4). Protoporphyrin-feeding
had no effect on the chlorophyll fluorescence of ptrl 16, irre-
Table 4 Chlorophyll fluorescence of wildtype and ptrl 16 tip cells after different light pretreatments, PCB (IOJUM) and
protoporphyrin (1 mM) feeding
nyfpHm FY^
1V1CU1UII1
Standard medium
Ligm preueairoem
cont white
6 d dark
5 d dark, 1
5 d dark, 1
5 d dark, 1
5 d dark, 1
d
d
d
d
blue
cont. red
red pulses
red + far-red pulses
Chlorophyll fluorescence, units
wt4
ptrl 16
360 ±80
40± 8
300 ±20
300 ±30
18O±3O
80 ±10
PCB
6 d dark
5 d dark, 1 d cont. red
26± 8
290 ±40
Protoporphyrin
6 d dark
5 d dark, 1 d cont. red
n.d.
n.d.
Light pulses were given for 5 min at hourly intervals.
45 ±9
2 ±1
24 ±3
2.3±0.3
n.d.
n.d.
1.9±0.3
21 ±2
2.5±0.6
1.3±0.1
Phytochrome control of chlorophyll biosynthesis
spective of whether tissue was kept in total darkness or
brought into red light.
Discussion
The aphototropic mutant ptrll6 shows similarities to
those already reported for ptrl (Lamparter et al. 1996) with
respect to phytochrome photoreversibility, phototropism
rescue with biliverdin and low chlorophyll content. It
seems reasonable to assume that both mutants show defects in the synthesis of the phytochrome chromophore.
In this study it was shown that besides biliverdin, phycocyanobilin (PCB) was also able to rescue phytochromecontrolled phototropism of ptrll6. From in vitro studies
with higher plant phytochromes, it is known that PCB
assembles with apophytochrome to yield a photoreversible
product which is similar to the product formed with phytochromobilin (Li et al. 1995, Kunkel et al. 1993, Weller et al.
1996). In physiological studies with the phytochromedeficient Arabidopsis thaliana mutant hyl (Parks and
Quail 1991) PCB, biliverdin and phytochromobilin could
all partially rescue phytochrome-controlled suppression of
hypocotyl growth. The results reported here that PCB and
biliverdin lead to the rescue of phytochrome-controlled
phototropism, support those results and point to parallels between moss phytochrome and higher plant phytochromes.
Feeding ptrll6 with protoporphyrin and heme leads
only to a weak positive-phototropic response. For higher
plants, where the biosynthesis of the phytochrome chromophore phytochromobilin has been better analyzed, the proposed pathway proceeds from protoporphyrin, which is
also a precursor of chlorophyll biosynthesis, via heme and
biliverdin, to phytochromobilin (Weller et al. 1996).
If the pathway is the same in mosses, the most likely explanation for the results of feeding experiments is that in
ptrll6, the biosynthesis of the phytochrome chromophore
is blocked between heme and biliverdin. InptrJ 16 the same
step in biosynthesis may be affected as in the pcd mutant of
pea (Weller et al. 1996). The weak positive phototropic response found after heme or protoporphyrin feeding may be
explained by leakyness of the mutant block or by some
non-enzymatic conversion of heme into biliverdin.
After blue light irradiation and after red light irradiation of PCB-grown filaments, chlorophyll levels are increased considerably compared to the control level. Although chlorophyll of ptrll6 never reach wildtype levels,
these results clearly show that the capacity to form protoporphyrin, which is also a precursor of chlorophyll biosynthesis (Castelfranco and Beale 1983) is not the limiting step
of ptrll6 phytochrome chromophore biosynthesis. These
observations give further support to the conclusion that biosynthesis of the phytochrome chromophore in ptrll6 is
blocked between protoporphyrin and biliverdin.
57
Like the phytochrome chromophore-deficient mutants
of higher plants (Parks and Quail 1991, Chory et al. 1989,
Weller et al. 1996), the low chlorophyll levels found in
ptrll6 may partially be explained on the basis of the regulation of chlorophyll accumulation via phytochrome. Two
independent results point to a central regulatory role of
phytochrome in chlorophyll accumulation in C. purpureus.
The first evidence comes from chlorophyll estimations in
wildtype filaments (Table 3, 4), where it was found that in
dark-adapted wildtype cells, red light induces a drastic increase in chlorophyll content and that the effect of red light
pulses can be reversed with subsequent far-red pulses.
Results of the PCB-rescue experiments with ptrll6 (Table
4) confirm the role of phytochrome in chlorophyll biosynthesis since red light only induced an increase in chlorophyll content in this mutant when grown in the presence of
PCB. Because during prolonged dark incubation of up to
4 weeks chlorophyll fluorescence of ptrll6 tip cells is
always lower than of wildtype (Fig. 4), and under these conditions phytochrome is inactive, dark chlorophyll synthesis
in the mutant seems to be negatively affected in a phytochrome-independent fashion: a block between heme and
biliverdin may cause a reduced rate of chlorophyll-synthesis by feedback inhibition.
The action of blue light can either be mediated by
phytochrome, which also absorbs in the blue region of the
spectrum, or by a separate blue light photoreceptor. Since
blue light induces chlorophyll accumulation in ptrll6,
while red light is inactive, it is evident that in C. purpureus
the regulation of chlorophyll accumulation is also mediated by a separate blue light photoreceptor.
The findings reported here on the light regulation of
chlorophyll accumulation in moss protonemata point to a
tight homology between mosses and seed plants. Chloroplast maturation during light irradiation, which can be
monitored by imaging chlorophyll fluorescence in the moss
cells, also appears to parallel chloroplast development during de-etiolation in seed plants. Since mosses are thought to
stand at the base of the evolution of land plants, it seems
that the regulation of chlorophyll synthesis and of chloroplast formation have evolved during the evolution of land
plants or even before that evolution had taken place. Although much more is known about the regulation of chlorophyll synthesis in higher plants compared to mosses, the advantages of the moss system may still help to elucidate the
molecular basis of the signal transduction cascade. These
advantages lie in the ease of monitoring single cells without
the need for microscopic preparation and in the possibility
to analyze photomorphogenesis in a continuously-growing
system which does not die.
This work was supported by the Deutsche Forschungsgemeinschaft (DFG, La 299/2-1). We thank Viola Eckl and Sabine
Artelt for skillful technical assistance.
58
Phytochrome control of chlorophyll biosynthesis
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(Received May 20, 1996; Accepted October 31, 1996)