J. Cell Set. ia, 391-401 (i973)
391
Printed in Great Britain
LIGHT SATURATION OF GROWTH AND
PHOTOSYNTHESIS OF THE SHADE PLANT
MARCHANTIA POLYMORPHA
R. MACHE AND S. LOISEAUX
Laboratoire de Physiologie Vigitale, Universite I de Grenoble (38), France
SUMMARY
The growth rate of the shade plant Marchantia was at its maximum for a low illumination,
2-3 x io8 lx, and was inhibited by an excess of light.
Photosynthesis by intact thalli and by isolated chloroplasts of Marchantia was saturated by a
light intensity of 2-3 x io3 lx. These isolated chloroplasts were able to carry on satisfactory rates
of photosynthesis, up to 35 fiM CO2/h/mg chlorophyll.
The Hill reaction and photosystem II were also saturated by the same light intensities, demonstrating that the factor limiting the light saturation of photosynthesis is located in the chloroplast. The structure of chloroplasts was strongly modified by an excess of light, small grana and
fret membranes being replaced by continuous grana.
INTRODUCTION
Marchantia polymorpha, a liverwort, is found in various climates but always in moist
and shaded areas.
It is well known that shade plants' photosynthesis is saturated by low illumination
as opposed to sun plants which require a high illumination for full photosynthesis.
This phenomenon still remains unexplained. The chlorophyll content is not the limiting factor since it has been shown that the amount of chlorophyll per unit of fresh
weight is higher in shade plants than in sun plants (Kirk & Tilney-Bassett, 1967).
Bjorkman (1968) suggested that the low carboxydismutase activity in the shade plants
is partly responsible for the low light saturated photosynthesis.
We first observed morphological changes on Marchantia due to different light intensities which lead us to study the light saturation of growth and of photosynthesis of this
liverwort. At the cellular level we demonstrate that the factor controlling the saturation
by low light of the photosynthesis is located in the chloroplasts. This point had to be
shown since photoreceptors like the phytochromes are known to be present outside
the chloroplasts.
The effects of excess of light on the structure of the chloroplast are described.
These observations might contribute to our understanding of the low light saturation
effect of shade plants.
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MATERIALS AND METHODS
Culture conditions
Female thalli of Marchantia polymorpha were grown on Petri dishes as previously indicated
(Mache & Loiseaux, 1972) but were illuminated at different light intensities as described under
Results. The material taken for photosyntheric studies was grown on vermiculite in a greenhouse using the same nutrient solution as for cultures on Petri dishes.
Electron microscopy
The method as previously described (Mache & Loiseaux, 1972) was used with the difference
that 0-5 M cacodylate was employed instead of 0-2 M.
Isolation of chloroplasts
The isolated chJoroplasts used for studying the CO, fixation were prepared according to the
method previously used (Mache & Loiseaux, 1972). For the Hill Reaction and study of photosystem II, chloroplasts were extracted as follows: harvested thalli were washed, chilled and ground
for 15 s in a Waring blender with twice their volume of a solution containing 50 mM phosphate
buffer at pH 7-0, 50 mM KC1, 5 mM MgCl2, 0-4 M sucrose. The slurry was filtered through
8 layers of cheese cloth. The filtrate was centrifuged for 1 min at 2000 g. The pellet was resuspended in the grinding solution. All operations were conducted at about 2 °C.
Fixation of CO2
The fixation of CO8 by isolated chloroplasts and adult thalli was studied by the method
previously described (Mache & Loiseaux, 1972), but thalli were immersed in a final volume
of 2 ml.
Chlorophyll determination
The chlorophyll content was determined according to Arnon's procedure (Arnon, 1949).
Hill reaction and photosystem
We followed the methods employed by Hoober, Siekevitz & Palade (1969). Assays were conducted with chloroplast suspensions containing 20-30 fig chlorophyll.
RESULTS
Light saturation curves of growth
Light intensity had an effect on the growth of Marchantia and a saturation curve was
drawn (Fig. 1) using the length of thallus as a measure. The length of thalli at low illumination (below 750 Ix) was not as reliable as other measures since etiolation occurred:
thalli were longer than expected, narrower and thinner. Propagulae of Marchantia
grown under different light intensities, with a photoperiod of 14 h light, 10 h darkness,
showed a maximum of growth for 5-6 x io 3 lx when 15 days old (Fig. 1). After 26 days
of growth, saturation was obtained with 2-3 x io 8 lx as seen in Fig. 2, where length
increments have been reported between the 15th and the 26th days of growth. This
demonstrates 2 phases of growth occurring in our experiments: phase I up to the
15th day and phase II afterwards.
At the 26th day the saturation curve is an hyperbola as shown by reciprocal plot of
Light saturation of Marchantta
15
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Fig. i. Dependence of the length of thalli on light intensity after 15 days
of growth (phase I).
-5 4
10
Fig. 2. Effect of light intensity on the length increment of thalli between the 15th and
the 26th day of growth (phase II).
lengths and light intensities (Fig. 3, B). This is not the case during phase I (Fig. 3, A).
From these observations it follows that light operates on Marchantia's. growth through
at least 2 light-limited mechanisms at the beginning of growth whereas there is only
one light-limited factor afterwards.
Inhibition effect of excess of light
Growth of Marchantia was inhibited by light intensities of 6x io 3 lx and above,
during phase II (Fig. 2), and its external morphology was modified. This inhibiting
effect has often been observed with Marchantia grown on agar under continuous
illumination, with lower light intensities in this case, 4 x io 3 lx after 6 days of growth.
Thalli became thicker, brittle, yellowish and curved down into the medium.
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Fig. 3. Reciprocal plot of light intensities and lengths, A reported in Fig. i (phase I) and
B obtained after 26 days of growth (phase II).
Electron microscopy was carried out in order to see whether any ultrastructural
change could be detected in plants grown for 3 3 daysat8'5 x io3lx compared with plants
grown at 2 x io 3 lx. Surprisingly, chloroplasts were effectively modified by an excess of
light. Dividing cells at the edge of the thallus were the most interesting as they showed
different aspects and stages of chloroplast transformation. Dividing nuclei are normal
as are mitochondria (Fig. 7) and Golgi bodies but chloroplasts are very different from
those observed in controls. The most striking change is the numerous invaginations of
the inner membrane in the whole surface of the chloroplasts. Invaginations (see arrow
Fig. 10) are tubular and irregular-looking like those of mitochondria when cut tangentially (Fig. n ) . Another, probably more important, change occurs in the organization
of the lamellae which is completely modified. This is particularly evident in young
chloroplasts (Fig. 8) where thylakoids extend from one side of the chloroplast to the
other, grouped in parallel strands. The partitions seem to be maintained as in normal
grana. In older chloroplasts, probably normal at first, starch grains seem to resorb and
normal grana tend to disappear, being replaced by very long groups of thylakoids. Small
globules or tubules of unknown nature are seen (double arrow Figs. 10, 11) scattered
in the plastid stroma. Large lipid globules are found in the cytoplasm.
Only chloroplasts seem to be structurally modified by light in excess.
Light saturation of photosynthesis in vivo
It was interesting to study the saturation of photosynthesis and to see whether saturation occurs at the same light intensity as for growth and whether any inhibition appears
at high illuminations.
If saturation of photosynthesis occurs in vivo, for the same light intensities as it
Light saturation of Marchantia
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o
1
2
3
4
IXX10"
5
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Fig. 4. Dependence of the rate of C0 a fixation by intact thalli of Marchantia
on light intensity.
DO
E
8- 1
Fig. 5. Dependence of the rate of COt fixation by isolated chloroplasts of Marchantia
on light intensity.
occurs in the second phase of growth it would mean that photosynthesis is the
limiting factor.
Experiments were conducted with adult thalli, grown under 2 x io 3 lx and photosynthesis was measured as indicated in Material and Methods. To be sure that limitation of C0 2 diffusion did not occur when thalli of Marchantia were immersed in 2 ml
of solution, we made the same experiments with 6 mM of bicarbonate instead of 3 mM
in the solution and obtained results identical to those reported in Fig. 4. On this figure
it can be seen that photosynthesis was'saturated at 2-3 x i o 3 lx, the same maximum as
for growth in the second phase. It can also be observed that 7 x io 3 lx did not inhibit
photosynthesis. The inhibition effect of high light intensity on the growth of Marchantia is not caused by action on photosynthesis per se, at least in a short while.
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8 30
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Fig. 6. Hill reaction, activity as a function of light intensity. Addition of 0-06 fiM of
3.(3,4-dichlorophenyl)-1,1 -dimethylurea completely inhibited light-dependent dichlorophenol indophenol reduction by chloroplasts.
Table i:. Effect of bicarbonate concentration on carbon dioxide fixation
Concentratiori1 of bicarbonate, mM
fiM CO, fixed/h/mg chlorophyll
% of "CO, in methanol-soluble compounds
1
2
8-4
43
16
2-5
3
15-6
16-5
38
—
10
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42
Light saturation of photosynthesis by isolated chloroplasts
Quantitative illumination experiments were conducted with isolated chloroplasts (see
Materials and Methods). Maximum photosynthesis was obtained with 1-5-2 x io 3 Ix
and no inhibition occurred at higher light intensities (Fig. 5).
Isolated chloroplasts fixed an average of 10 JIM COj/h/mg chlorophyll under an
illumination of 3-5 x io 3 lx. Higher figures, up to 35 /JM C02/h/mg chlorophyll were
obtained for some experiments. Replacing pyrophosphate by orthophosphate in
solution C did not improve the photosynthetic rate as it did for spinach (Jensen &
Bassham, 1966). Under this illumination, 2 mM of bicarbonate in the assay solution
allowed maximum fixation of COE, as shown in Table 1. The fraction of photosynthetic
products soluble in hot 80 % methanol after 10 min of photosynthesis is about 40 % of
the total.
Observations with the light microscope showed that a few entire cells were still
present after filtration of the chloroplast suspension. These cells, ovoid, with a diameter
of 15—30/jm, containing about 10 plastids each, are typical of the ' assimilatory filaments' of the aerial chambers of Marchantia and are easily detached one from each
other and from the aerial chamber's floor. They represented at the very most 2 % of
the total number of chloroplasts in the chloroplast suspension and could not account
for the 8-15 % of the activity of intact thalli shown by the chloroplast suspension in
these experiments.
These experiments strongly suggest that the factor controlling the low light saturation of photosynthesis is situated in the chloroplast. This point was confirmed by the
light saturation curve (Fig. 6) of the Hill reaction. Photoreduction of the artificial
electron acceptor was saturated with low light intensities, around 2x io 3 lx as for
CO2 fixation by intact thalli and isolated plastids. A few experiments gave identical
Light saturation of Marchantia
397
s
values of photoreduction for the photosystem II at 4, 16 and 24 x 10 lx and a lower
value at 2 x io 3 lx. It seems that saturation is obtained at about 3 x io 3 lx.
DISCUSSION
We have examined the light saturation of Marchantia at two levels, growth and
photosynthesis.
The light saturation curve of growth, if we except the beginning of the development
was found to be an hyperbola, thus indicating one light-limited factor influencing
growth. Saturation was obtained for the same light intensities for growth as for photosynthesis (2-3 x io 3 lx) suggesting that growth is limited by photosynthesis. The rate of
photosynthesis obtained with isolated chloroplasts of Marchantia is of the same order
of magnitude as the rate obtained for isolated chloroplasts of higher plants (Gibbs,
Latzko, O'Neal & Hew, 1970). This rate is lower than in vivo by a factor of about 10.
The same maximum of light intensity obtained for photosynthesis in vivo and fixation
of CO2 by isolated chloroplasts strongly suggests but does not demonstrate that the
limiting factor of the photoreaction is located in the chloroplasts. Saturation curves of
the HOI reaction and of system II show that both photosystems are responsible for low
light saturation and therefore demonstrate that the surrounding cell is not responsible
for the light saturation of Marchantia.
Growth of Marchantia under different light intensities varies according to the stage
of development of thalli. Two different phases occur. The first, at the beginning of
development, is saturated at a high level of illumination, 6 x io 3 lx with a daily light
period of 14 h. The duration of this first phase seems to be dependent either on the
total amount of light received and/or of the photoperiod as indicated by assays with
continuous illumination in which case this phase is shorter and is saturated at lower
light intensity. The second phase, later in the development of thalli, is saturated at
lower light intensities.
More experiments are needed to find out the role of photoperiod, amount of light
received, and nature of light required.
Another observation was the damaging effect on growth and morphology of Marchantia of an excess of light. Contrary to this finding, Gauhl (1969) did not observe
any signs of photo-inhibition when a shade-adapted plant, Solanum dulcamara, was
exposed to high light intensities. This emphasizes differences between shade and
shade-adapted plants.
An excess of light inhibits growth, changes external morphology and modifies the
chloroplast structure. The result of this modification consists of the elaboration of
another type of chloroplast. Light, which is known to be necessary for assembling
thylakoids into grana, seems to continue, in this case, its action by assembling all
thylakoids into long grana, thus suppressing fret membranes. Moreover, a great number of vesicles are formed from the inner membrane of the envelope. These invaginations can be compared to those of maize (Rosado-Alberto, Weier & Stocking, 1968).
It is interesting to point out that both the light-limited factor of photosynthesis and the
structural result of an excess of light are situated in the chloroplast of this shadow plant.
We suggest that the photosynthetic function supported by grana is involved in the light
398
R- Mache and S. Loiseaux
saturation of the photosynthesis of Marchantia. Our experiments on light-saturation of
photosystem II support this hypothesis, because photosystem II is localized in the
grana (Arnon, Chain, McSwain, Tsujimoto & Knaff, 1970) and does not exist in fret
membranes.
In some of the plants exhibiting-the photosynthetic pathway of Slack & Hatch the
chloroplasts of the bundle sheath contain only single lamellae, without any grana.
These plants have no photorespiration. The opposite situation is occurring in Marchantia grown with an excess of light. Their chloroplasts have no single lamellae and
only very developed 'grana'. They probably possess a photorespiration metabolism
(Bjorkman & Gauhl, 1969) which could be stimulated under high illumination.
The above observations suggest that one component of photosystem II is related to
the photorespiration metabolism. This hypothetical mechanism has also been proposed
by Plaut & Gibbs (1970) for the formation of glycolate in spinach chloroplasts.
REFERENCES
D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta
vulgaris. Plant Physiol., Lancaster 24, 1-15.
ARNON, D. I., CHAIN, R. K., MCSWAIN, B. D., TSUJIMOTO, H. Y. & KNAFF, B. (1970). Evidence
from chloroplast fragments for three photosynthetic light reactions. Proc. natn. Acad. Sci.
U.S.A. 67, 1404-1409.
BJ6RKMAN, O. (1968). Carboxydismutase activity in shade adapted and sun adapted species of
higher plants. Physiologia PI. ai, 1-10.
BJORKMAN, O. & GAUHL, E. (1969). Effect of temperature and oxygen concentration on photosynthesis in Marchantia polymorpha. Carnegie Instn Wash. Yearbook 67, pp. 479-482.
GAUHL, E. (1969). Differential photosynthetic performance of Solanum dulcamara. Ecotypes
from shaded and exposed habitats. Carnegie Instn Wash. Yearbook 67, pp. 482-487.
GIBBS, M., LATZKO, E., O'NEAL, D. & HEW, C. S. (1970). Photosynthetic carbon fixation by
isolated maize chloroplasts. Biochem. biophys. Res. Commttn. 40, 1356-1361.
HOOBER, J. K., SIEKEVITZ, P. & PALADE, G. E. (1969). Formation of chloroplast membranes in
Chlamydomonas reinhardi y-i. Effect of inhibitors of protein synthesis. J. biol. Chem. 244,
2621-2631.
JENSEN, R. G. & BASSHAM, J. A. (1966). Photosynthesis by isolated chloroplasts. Proc. natn.
Acad. Sci. U.S.A. 56, 1095-1101.
KIRK, J. T. O. & TILNEY-BASSETT, A. E. I. (1967). The Plastids, p. 479. London and SanFrancisco: Freeman.
MACHE, R. &LOISEAUX, S. (1972). Action of rifampicin on the liverwort Marchantia polymorpha.
J. Cell Sci. 10, 821-831.
PLAUT, Z. & GIBBS, M. (1970). Glycolate formation in intact spinach chloroplasts. Plant
ARNON,
Physiol., Lancaster 45, 470—474.
J., WEIER, T. E. & STOCKING, C. R. (1968). Continuity of the chloroplast
membrane system in Zea mays L. Plant Physiol., Lancaster 43, 1325-1331.
ROSADO-ALBERTO,
(Received 5 June 1972)
Fig. 7. Cell of Marchantia grown at 8 x io3lxfor33days.Thiscellissituated in the growing tip of the thallus and the nucleus is dividing. All organelles are normal except for
the chloroplasts which show long strands of thylakoids instead of the normal grana
and fret membranes, x 15 200.
Fig. 8. Young chloroplast in Marchantia grown for 33 days at 8-5 x io 3 lx. Thylakoids
grouped in parallel strands extend from one side of the plastid to the other. Partitions
are maintained. /, lipid globule; n, nucleus, x 62000.
Fig. 9. Grana in a normal chloroplast. Marchantia grown in a greenhouse with little
illumination, x 138000.
Light saturation of Marchantia
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R- Mache and S. Loiseaux
Figs, io, I I . Two sections of the same group of chloroplasts of Marchantia grown for
33 days at 8'5 x io 8 bt. Note the numerous invaginations of the inner membrane (single
arrows), the long strands of thylakoids and globules of unknown nature (double arrows).
x 28000.
Fig. 12. Another chloroplast oi Marchantia grown with an excess of light (same conditions as in Figs. 10, 11). Grana tend to be replaced by long strands of thylakoids. p,
plasmodesm with probably some reticulum inside, x 39000.
Fig. 13. Normal chloroplast in a greenhouse-grown Marchantia. Note the grana, fret
membranes and absence of invaginations of the inner membrane, x 24000.
Light saturation of Marchantia
401