Plant Physiol. (1976) 57, 400-402
Interactions between Mitochondria and Chloroplasts in Cells
I.
ACTION OF CYANIDE AND OF 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA ON THE SPORE OF
FUNARIA HYGROMETRICA
Received for publication September 19, 1975 and in revised form November 11, 1975
DANIEL CHEVALLIER AND ROLAND DOUCE
Laboratoire de Physiologie Vegetale, Universite Scientifique et Medicale de Grenoble, B.P. 53-Centre de
Tri. 38041 Grenoble-Cedex France
ABSTRACT
The effects of cyanide and 3-(3,4-dichlorophenyl)-1,1-dimethylurea
(DCMU) on photosynthesis and respiration of intact chlorophylic moss
(Funaria hygrometrica) spore was investigated. Thirty micromolar cyanide strongly inhibited dark respiration, was without effect on photosynthesis at high light intensities (above the saturation plateau values), and
stimulated photosynthesis at low light intensities (below the saturation
plateau values). Three hundred nanomolar DCMU inhibited the photosynthesis and was without effect, even under light conditions, on the dark
respiration. It seems likely, therefore, that in the chlorophyllic moss
spore the cytochrome oxidase pathway is not functioning under high light
intensities unless the pbotosynthesis is inhibited by DCMU.
It is now well established that during photosynthesis dihydroxyacetone-P and glycerate-3-P are rapidly transferred across the
chloroplast envelope (2, 9, 10, 17, 22, 23). This shuttle mechanism induces a rapid increase in the cytoplasmic ratio ATP/ADP
which is transmitted to the mitochondria by the mitochondrial
adenylate translocator (5, 9, 21). Under these circumstances, the
Cyt oxidase pathway is probably inhibited in the light. Effects of
light on respiration of autotrophic green cells have repeatedly
been described but have been clouded with conflicting reports
(6, 7, 9, 12, 19).
In the present study, we describe the effects of KCN and
DCMU on the chlorophyllic spores of the moss Funaria hygrometrica in order to elucidate under light or dark conditions the
possible interactions between mitochondria and chloroplasts.
were measured at 25 C in the same cell, using a glass electrode
(Beckman, Century SS pH meter). The velocity of the change of
pH between 6.9 and 7.2 was recorded. The scale was calibrated
by known quantitites of HCI. Light was provided by a 300-w
projector lamp through a 10-cm layer circulating water. Except
when otherwise stated, the light intensity was 7.5 x 105 ergs
sec-1 cm-2. All reaction mixtures for spores were prepared and
used at 25 C and contained: 0.3 M mannitol and 0.5 mm
NaHCO3, pH 7.2.
Spores were prepared for ultrastructural studies by fixation for
1 hr with 4.5% glutaraldehyde in cacodylate buffer at pH 7.4.
Following several washes in cold buffer, the samples were postfixed with 1 % OSO4. Dehydration in an ethyl alcohol series was
followed by embedding, in Araldite. The Araldite blocks were
trimmed and sectioned with a Sorval Porter-Blum MT-I ultramicrotome. Specimens were stained with uranyl acetate and Reynold's lead citrate. Electron microscopy was performed with a
Siemens Elmiskop 1 A microscope.
Chlorophyll was extracted from spores in 80% acetone and
measured according to Arnon (1). Protein was determined by
the Folin-Ciocalteau phenol reagent (18).
RESULTS
Figure la shows a thin section of an ungerminated dry spore.
The mitochondria are numerous, contain a number of well developed cristae, and range in length from 0.6 to 1 gm. The
spheroidal typical proplastids range from 1.5 to 3.5 /Lm in
diameter and contain several starch granules. The cytoplasm is
rich in lipid bodies. Figure lb shows a thin section of a 20-hr-old
spore. The spore remains a single cell. There is, however, evidence of cracking of the outermost spore wall layer. The shape
and the size of the mitochondria are unchanged. The chloroplasts are fully developed and contain a well developed grana
fret-work system. Starch is present in the chloroplasts in relative
abundance. At this stage, the chloroplasts are as numerous as the
proplastids of the dry spore.
Photosynthesis appears very rapidly in moss spores (Fig. 2).
Photosynthesis is perceptible after 10 hr of growth and reaches
its maximum value at 20 hr. The spore presumably have low
rates of photorespiration because net photosynthesis is not increased in 5 ,M 02 as compared with 240 ,UM (24).
Examples of 02 electrode tracing of 02 concentration are
shown in Figure 3a. Upon illumination, 02 production starts
rapidly and proceeds at a remarkable constant rate for a long
period of time (at least for 2 hr). The same is true for the dark
respiration. Figure 3b shows that upon addition of 30 gM KCN
to dark spores, a clear inhibition of the 02 uptake rate occurs.
The shape of the curve obtained seems to indicate that KCN
passes through the cell wall very slowly. In fact this hypothesis is
unlikely. We have systematically observed that the final inhibi-
MATERIALS AND METHODS
Shoots of Funaria hygrometrica Hedw. bearing mature sporophytes were collected in the spring of 1974 near Grenoble. The
sporophytes were stored dry until needed.
The basic nutrient medium used for the culture of the spores
was made up according to Kofler (15). The medium which
contained 12 g/l of Noble agar Difco was poured into sterilized
Petri dishes. The agar surface was covered with a sterilized
cellophane membrane (cellulose membrane, Socidtd "La Cellophane", IIO Bd Haussman, Paris) (16). The Petri dishes were
placed in a growth chamber at 24 C under continuous white light
(10 w.m-2). The spores were rapidly collected by filtration on a
Millipore filter (25 mm diameter, pore size: 5 Mm).
Photosynthetic 02 production and respiratory 02 uptake were
measured at 25 C in a 3 ml stirred cell, using an 02 electrode.
The 02 concentration in air-saturated medium was taken as 240
and respiratory CO2 production
Mm. Photosynthetic CO2 uptakeDownloaded
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401
Plant Physiol. Vol. 57, 1976 MITOCHONDRIA AND CHLOROPLAST INTERACTIONS
b
FIG. 1. a: Section of an ungerminated dry spore; b: Section of a 20-hr-old spore. Spores were grown on a mineral medium under continuous white
light. c: chloroplast; ex: exine; in: intine; 1: lipids; m: mitochondria; n: nucleus; p: proplastids; s: starch.
Ott
Off
Ott
of
off
Off
Ott
25pM
off
off
OttaIfI
00
0c
Ot
0exi00
t
0
0
n~~~~~~~~~~)
2min
thOtaesree
darkrespratonogrwnmossoe.Teubrson
25r#A
16ah
12h
20h
-8h
FIG. 2. Evolution Of 02
dark respiration of growing
concentration during
moss
to nmoles
Of
02 consumed
spores
and the time of
exine;
off:
time of
24h
Te
spores.
photosynthesis
numbers
protein-hr.
or produced/mg
are given under
growth
each trace. in: intine;
light
light was switched off;
growth indicated the Chl/protein ratios
was
on:
and
the traces refer
The shape of the
on
switched
are
on.
For
ex:
each
successively: 0, 0.7,
1.2, 1.9, and 2.1 %.
dependent on the 02 concentration in the reaction
The half-maximal inhibition Of 02 uptake rate is attained at 10 ,UM KCN. Upon illumination, although the dark
respiration is inhibited, the 02 production remains unaffected
tion
is
strongly
medium.
and
be
proceeds at
pointed
constant rate for a long period of time. It has to
out that, above 60 Am KCN, the 02 production is also
affected (Fig.
Figures
dependent
a
3,
c
4 and
on
the
and
5
d).
show that the effects of 30
light intensity.
the saturation plateau values
addition of 30
on
the
AmM
At
(7.5
high
x
mM
KCN
light intensities
105 ergs sec-'
are
above
CM-2), the
KCN to the reaction medium is without effect
02 production and CO2 uptake rates (Figs. 4a and 5a). On
light intensities (1.5 x 105 ergs sec-' CM-2),
the contrary, at low
the addition of 30
lmM
KCN induces
a
marked increase of
theO02
production and CO2 uptake rates (Figs. 4b and 5b). This effect is
less pronounced at intermediate light intensities (results not
FIG. 3. Effect of various concentrations of KCN on the 02 evolution
of 20-hr-old spores. The concentrations given are the final concentrations in the reaction medium. The numbers on the traces refer to nmoles
Of 02 consumed or produced/mg protein-hr. off: light was switched off;
on: light was switched on.
conditions. The rate of 02 uptake is stimulated by an uncoupler
(FCCP)' and is inhibited by 30 AM KCN.
DISCUSSION
These results indicate that, at least in moss spore, the lightsaturated photosynthetic rate is not affected when the 02 uptake
catalyzed by the Cyt oxidase is specifically inhibited by 30 AM
KCN (4, 13). Conversely, the Cyt oxidase pathway is not altered
under light conditions when the electron flow between the two
photosystems is specifically inhibited by DCMU. These results
seem to be in good agreement with the work of, Heber (9)
showing that in full light the Cyt oxidase pathway, which is under
the control of the cytoplasmic ATP/ADP ratio, is not operative.
shown).
Photosynthetic 02 production by moss spores is inhibited by
300 nm DCMU (Fig. 6). On the contrary, 02 uptake remains
' Abbreviation: FCCP: carbonyl cyanide p-trifluoromethoxyphenunaffected even after a long period
of timefrom
andonunder
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402
Plant Physiol. Vol. 57, 1976
CHEVALLIER AND DOUCE
a
FIG. 4. Effects of light intensity and KCN on the 02 production of 20hr-old spores. The numbers at the arrows represent light intensities (ergs
sec- cm-2). The numbers on the traces refer to nmolesof 02 consumed
or produced/mg of protein-hr. off: light was switched off; on: light was
switched on.
the chloroplasts(11, 20) is operative under light conditions. This
could explain the fact that the Krebs cycle is operating during
light conditions(19). Furthermore at the onset of photosynthesis
the triose-P/glycerate-3-P shuttle transfer energy as ATP and
NADPH (3, 8, 9, 14, 23). In the cytoplasm, the NADH formed
is oxidized either by the oxaloacetate/malate shuttle (9) or by
cyanide-insensitive respiration.
The fast growing spores represent a good material for the
studies of the interactions between the chloroplasts and the
mitochondria inside the cell. The arguments in favor of the use of
this material are numerous. First, the 02 production and uptake
proceed at a remarkable constant rate for a long period of time.
Second, the cyanide insensitive 02 uptake is not considerable as
it is often the case for the green leaves (Chevallier and Douce,
unpublished data). Third, photorespiration does not seem to be
operative.
Acknowledgment-We wish
to thank
copy.
Fran,oise
LITERATURE
off
Nurit for her advice
on
electron micros-
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FIG. 5. Effects of light intensity and KCN on the CO2 uptake of 20hr-old spores. The numbers at the arrows represent light intensities (ergs
sec-I cm-2). The numbers on the traces refer to nmolesof CO2 consumed
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FIG. 6. Effect of DCMU on the 02 evolution of 20-hr-old spores. The
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