Oecologia (Bet1.

) 31, 85-94 (1977) Oecologia

9 by Springer-Verlag 1977

Water Relation Parameters

of the CAM Plant Kalancho daigremontiana
in Relation to Diurnal Malate Oscillations

Ulrich Lfittge and Erika Ball

Institut ffir Botanik der TechnischenHochschuleDarmstadt, Fachbereich Biologie,
Schnittspahnstr. 3 5, D-6100 Darmstadt, Federal Republic of Germany

Summary. In the CAM plant Kalancho6 daigremontiana, kept in an environ-

mental rhythm of 12 h L : 12 h D in a growth chamber at 60% relative
humidity and well watered in the root medium, decreasing water potentials
and osmotic potentials of the leaves are correlated with malate accumulation
in the dark. In the light increasing water and osmotic potentials @w and
Os) are associated with decreasing malate levels. Transpiratory H20 loss
is high in dark and low in light.
In continuous light, the CAM rhythm rapidly disappears in the form
of a highly damped endogenous oscillation. Malate levels, and water and
osmotic potentials of the leaves remain correlated as described above. How-
ever, transpiration is very high as malate levels decrease and water and
osmotic potentials increase.
It can concluded, that water relation parameters like total water potential
(~'w) and osmotic potential (ffs) change in close correlation with changes
of malic acid levels. As an important osmotically active solute in CAM
plants, malic acid appears to affect water relations independently of and
in addition to transpiration. The question remains open, whether turgor (Op)
is involved in CAM regulation in intact plants in a similar way as it deter-
mines malate fluxes in leaf slices.

Introduction

Experiments with leaf slices of the CAM plant Kalancho6 daigremontiana have
led to the idea that turgor pressure might be involved in regulation of diurnal
changes between malate accumulation in the vacuoles and re-mobilisation from
the vacuoles. External solutions which increase the turgot increase malate efflux
from slices of leaves harvested at the end of the nocturnal dark phase (Lfittge
et al., 1975, 1977). Solutions reducing turgor increase malate accumulation by
leaf slices obtained at the end of the light phase (von Willert, 1974; L/ittge

Abbreviations. CAM= Crassulacean Acid Metabolism; L = Light; D = Dark

86 U. Liittge and E. Ball

et al., 1975). It remains an intriguing question though, to what extent this

allows conclusions about the role of turgor in CAM regulation in intact plants.
Such a role has been postulated earlier, because the considerable diurnal oscilla-
tions of malic acid levels in the vacuoles are equivalent to considerable oscilla-
tions of osmotically active material (Liittge et al., 1975).
CAM is widely interpreted as a biochemical adaptation of plants allowing
photosynthesis with a high water use efficiency in arid habitats (Kluge, 1976).
A certain small amount of precipitation and not excessively low soil water
potentials are, nevertheless, important for plants to perform photosynthesis
via CAM (Szarek and Ting, 1975 ; Nobel, 1976). CAM plants appear to predomi-
nate in arid regions with regular winter rain, e.g. in North America, where
the desert climate is not extreme (Kluge, 1976). In extreme deserts like the
Negev of Israel, CAM largely is observed only in annuals such as several
Aizoaceae species. These plants germinate only when sufficient winter rain has
fallen. They grow performing C3 photosynthesis and presumably change to
CAM as the soil dries out; CAM possibly allowing them to complete their
life cycle during increasing water stress (Winter and Ltittge, 1976 ; Winter et al.,
1976). So far, only one perennial CAM species, CaralIuma negevensis, has been
found in the Negev. This species is restricted to less exposed sites in rocky
niches (Lange et al., 1975).
In the present study water relation parameters of K. daigremontiana plants kept
under controlled conditions in growth chambers were related to oscillations
of malate levels. Comparisons with C3 plants kept under the same conditions
were also made.

Materials and Methods

1. Plant Material

Plants were grown in the greenhouse; 5-7 m o n t h old plants were transferred to a growth chamber
several weeks before the experiments. Relative humidity in the growth chamber was always kept
close to 60%, Roots of the plants were kept well watered, i.e. pots were watered two times per
week. Controls showed that watering never affected transpiration and water relation parameters
of the plants, i.e. soil water potential never was the limiting factor in the experiments.
The other conditions varied depending on the type of experiment. In one type of experiment,
there was an environmental rhythm of 12 h L, 25 ~ C: 12 h D, 15 ~ C (light 7~176 ~176dark 19 ~176
7~176
In a second type of experiment, temperature was kept constant at 20 ~ C_+ 2 ~ C. The light regime
was 12 h L:12 h D followed by continuous light and, subsequently, again by a L : D rhythm or
by continuous darkness. Light intensity was 10,000 lux from Xenon lamps. For comparison with
the C A M plant K. daigremontiana, the C3 plants Peperomia magnifolia and Tradescantia spec.
were chosen because of their somehow similar fleshy leaf appearance. One of these plants, P.
magnifolia, has high steady malate levels in the leaves; in the other species, Tradeseantia spec.,
malate levels are low (Fig. 2).

2. General Analytic Methods

In extracts of leaves malate was determined enzymatically after Hohorst (1970), osmotic potential
@s) was measured cryoscopically, and pH with a glass electrode.
CO2 and H 2 0 gas exchange were recorded by infrared gas analysers (URAS) in an open
circuit. Two pairs of adult leaves on the intact plant (similar to those leaves used for the other
determinations) were sealed in a perspex vessel of 0.0185 m 3 vol. The whole setup was kept in
 Water Relations of a C A M Plant in Relation to Malate Oscillations 87

110 time ~s real ~s real ~ ]

[ bar] [~mote g4w ] time Cbar~E~umotegqW~
1
9 -4.6
7 ~~ 34 9 7~ -3.6 1.6
7~ A9~176 -4.4 9~176 -3.5
14~ l, O ' ~x\ 14"~
gFW 1600 -4:7 = ~6~176-3.7 2.5
A
q\
ekx~\
1.1 - -
~9~ 1 7 6
/~.o.O-o-O--o--O
9
.... 8 --~---
-
745 am
0 0 ' "=
1.04
---x,%
~-" ~5 p.m. 1.0";
1.05
gFW ~' 20mM mannitol 1.0C I I I I'llj~- ~ k. r
-2 -3 ~,-5 -6
1.00 -2 -3 -4 ','5..'-s
"9 30 60 90 12 min 150 180 [bar] ' e~ Ebar] ~.~.~
0.gE
o~ " ~ 1 7 6 1 7 6 1 7 6 7t'Sa.m.-
1.05 \.

1,1
~o
"o. 200 mM mannito[
0.9E

0.94
/ a
-\
\
-~o-_ 1500p.m._ 0.92 g a l a n c h o ~ Peperomia Tradescantia "o
1.15
2
Fig. 1. Changes of weight of K. daigremontiana leaf slices in solutions of two different mannitol
concentrations (20 m M - ~ - 0 . 5 bar, 200 m M - ~ - 5 bar). Leaves harvested 45 rain after commence-
ment of the light phase (74s) had high malate levels, leaves harvested after 8 h L (15 ~176contained
low a m o u n t s of malate. Initial fresh weight (FW) of sample was 1 g: Dim room light, 25 ~ C.
Points are averages of four measurements, errors are smalIer than symbol size
Fig. 2. Determination of water potentials (~w) with the liquid exchange method in the C A M
plant K. daigremontiana, and in the C3 plants Peperomia magnifolia and Tradescantia spec. at
various times during an environmental r h y t h m of 12 h L, 2 5 ~ 12 h D, 15~ the light phase
began at 7 ~176mal malate, ~'s osmotic potential. Initial fresh weight (FV/) of samples was 1 g.
Points are averages of eight measurements for Kalanchog (with standard errors) and four measure-
ments for Peperornia and Tradescantia

the growth chamber under conditions as given above, and the perspex vessel was flushed with
air from within the growth chamber at a rate o f 0.185 m 3 h-1.

3. Determination of Water Potential tpw

With the smali cells of higher plant leaves, direct measurements of water relation parameters
using the pressure probe developed by Z i m m e r m a n n and Steudle (Steudle et al., 1975) is not possible.
The soft tissue of/(. daigremontiana also does not allow use of the pressure bomb method. Therefore,
it was decided to return to the 'liquid exchange m e t h o d ' as described by Slatyer (1967, pp. 153-157).
Leaves are harvested from the plant, sliced immediately, and 2 m m wide slices are incubated
in a series of solutions with graded water potential, for which mannitol was used as a solute
in 0.1 m M CaSO4 solution. In solutions of higher water potential than that of the leaves, the
tissue gains weight by rapid water uptake. In solutions of water potential below that of the leaves,
the cells lose weight (Figs. 1 and 2). Water potential of the leaves on the intact plant is taken
equivalent to the water potential of a solution in which the leaf slices do not change weight.
Slatyer (1967) discusses several pitfalls of this method. It is suggested that metabolic changes
of solute levels in the cells are minimized by working at low temperatures. Conversely, water
permeability m a y be significantly reduced at Iow temperatures. In several preliminary experiments
with the material it was found that equilibration was rather sIuggish at 0 ~ C and at + 5 ~ C, but
at + l l ~ the same results were obtained as at + 2 5 ~ C. Therefore, I I ~ was used in all further
experiments. As an example, the kinetics of equilibration of leaf slices with H 2 0 in an external
solution obtained at 25 ~ C are shown in Figure 1. This figure reveals a further problem of working
with C A M leaf slices. After an initial rapid water uptake in solutions of high water potential,
slices of leaves harvested in the morning begin to lose some of their weight again (see curve
for 74s a.m. and 2 0 m M mannitoI - ~ - 0 . 5 bar in Fig. 1). This is probably due to the fact that
water uptake from the solution of high water potential is overlapped by the somewhat slower,
but considerable, loss of malate from the tissue which occurs under these conditions. Osmotic
water movements will accompany this flux of solute. Hence, the observed curve of change of
fresh weight is the resultant of two different processes (Li~ttge et al., 1977). However, since malate
88 U. Liittge and E. Bali

efflux is considerai~ly reduced as the water potential of the external solution is lowered by the
addition of mannitol (Liittge et al., 1975, 1977), this should not affect the determination of Ow
by the liquid exchange method. In determinations of Ow as shown in Figure 2, weight changes
obtained after 90 min of equilibration with K. daigremontiana and after 120 min with Tradescantia
and Peperomia leaf slices were plotted. Surface adhesion contributes little to these weight changes.
K. daigremontiana leaf slices of 1.000 g fresh weight soaked in water for 2 rain and then reweighed
after brief blotting gave 1.006 +_0.003 g (n=8, the error is a standard error).
This indirect method of determining tpw has several drawbacks and presumably does not
provide strictly correct absolute values of ~'w. It is supposed, however, that in the case of the
present experiments these are systematic errors. For a comparative investigation the trends in changes
of Ow are probably mirrored adequately. The total water potential is
Ow=Os+~P+OM,
where, in addition to the parameters ~Pwand Os already described above, 0P is the turgor potential
and 0N the matric potential. Neglecting ffM, this theoretically would allow estimation of 0P (Op =
0w-Os). Because of the difficulties of the method mentioned above, it is not claimed that one
can follow changes of turgot pressure in this way. Since both. ~w and tps represent numerically
large values, the difference would not allow to follow smaller changes of Op. Furthermore, it
may not be justified to neglect O~a, inasmuch as K. daigremontiana leaves appear to contain a
slimy substance which may well contribute to a rnatric water potential. Nevertheless, gross changes
of 6w-~'s might be of interest, and this was plotted for some experiments.

Results and Discussion

I. Diurnal Changes of Ow in Leaves

of the CAM Plant K. daigremontiana in Comparison with C 3 Plants
in an Environmental Rhythm of 12 h L, 25 ~ C:12 h D, 15~ C

Figure 2 shows that in an environmental rhythm of 12 h L, 25 ~ C: 12 h D, 15~ C,

Ow in leaves of the CAM plant K. daigremontiana is significantly higher 1 h
before the end of the light phase than at the end of the dark phase (18 oo
and 7o~ This could be due to transpirational water loss during the night,
because of the inverse stomatal rhythm of CAM (i.e. with stomatal opening
during the night and closure for most of the day). If transpirational water
loss were the major factor determining 0w of plants in the growth chamber,
one would expect a decrease of Ow during the light phase in the Ca plants
kept under the same conditions. However, this is not observed. In the two
C3 plants, water potentials do not change significantly. The detailed reasons
for this are not elucidated by the experiment of Figure 2. Leaf resistance for
transpirational water loss must be higher as stomata close during the night,
but root resistance for water supply may also be higher as the temperature
is lowered. Presumably diurnal changes of water flow resistances are balanced
under the conditions of the experiment so that Ow of the leaves is not subject
to significant diurnal changes. This then allows one to argue that the changes
observed in K. daigremontiana leaves may be associated with diurnal malate
oscillations rather than with stomatal control. Malate levels are very different
in Peperomia and Tradescantia. In both cases, there are only slight increases
of malate levels during C3 photosynthesis in the light phase, and osmotic poten-
tials (0s) are very steady (insert data in Fig. 2). In K. daigremontiana, the
typical CAM oscillations of malate levels are observed under the conditions
of this experiment. High malate levels are correlated with low water potential
(~w) and low osmotic potential (Os) (Fig. 3).
 Water Relations of a CAM Plant in Relation to Malate Oscillations 89

(,1_
%120-
e
100-

~80

60 ~
r
A / ~A
-7 1/.0 40- 5
go

!
E
j
-6 120"~ -6-
o
-5 o 100 | -5-
"-2 b~t -O,
~-4
,/;. ~ 60 _m -3~

"
~at~c 40
>+
_.o

-1 20 --~ ~-1 i--n ,m-

D, 15~ L, 25~ E P~ 1
0 0-
19+~ 7 ~176 h 19~ 4 7~ 7o0 7~ 7~176
[h]
Fig. 3. Correlation between diurnal variations of malate levels and water and osmotic potentials
(~w and Os)in K. daigremontianaleaves. Closedsquares (average of numerous individual experiments
with standard errors) and closedcircles (average of two measurements): ~s; closed triangles (average
of numerous individual experiments with standard errors): 4'w; open squares and open circles: malate
levels from the same experiments as closed squares and circles
Fig. 4. Malate levels (real), pH, and water and osmotic potentials (~'w and Os) in leaves of K.
daigremontianaat a constant temperature of 20~ C during light: dark changes and during continuous
light in a growth chamber. Light periods: open bars of the abscissa; dark periods: bold black
bars. Points are averages of four measurements, errors are smaller than symbol size

An attempt was made to measure possible volume changes of K. daigremon-

tiana leaf cells during the rhythm, by recording the thickness of the leaves.
But, leaf thickness remained unchanged:
730 1.916+_0.024mm (n=60),
18oo 1.919_+0.031mm (n=60).

2. Changes o f ~w, ~#s, Malate Levels, pH, C02, and H 2 0 Gas Exchange
in K. daigremontiana Leaves at Constant Temperature (20 ~ C)
in a Light:Dark Rhythm and in Continuous Light
F o r the e x p e r i m e n t s o f F i g u r e s 4 a n d 5, K. daigremontiana p l a n t s were p r e t r e a t e d
for at least one week in 12 h L : 1 2 h D at a c o n s t a n t t e m p e r a t u r e o f 20 ~ C.
Although higher temperatures (25 ~ C) during the light phase and lower tempera-
tures d u r i n g the d a r k p h a s e (15 ~ C) are f a v o u r a b l e for C A M ( B r a n d o n , 1967;
O s m o n d e t a l , , 1973; Neales, 1973), the typical r h y t h m o f C A M persists at
aconstant temperature of 20 ~ C. As can be seen at the beginning of the experi-
ments o f F i g u r e s 4 'and 5, m a l a t e levels increase d u r i n g the d a r k p h a s e a n d
decrease d u r i n g the light phase, while ~#s, ~#w a n d p H decrease a n d increase,
respectively. As characteristic for CAM, there are predominant COz-fixation in
the d a r k a n d higher t r a n s p i r a t o r y w a t e r loss (open s t o m a t a ) in the d a r k t h a n in
the light.
W h e n the light is left on after the last r e g u l a r light phase (after 19~176
there is o n e further, t h o u g h highly d a m p e d , C A M oscillation in c o n t i n u o u s
light. Leaves a c c u m u l a t e m a l a t e a n d acidify between 19 oo a n d 7 ~176they re-
 90 U. L/,ittgeand E. Ball

f-!l . . . . . .

6 I H~O I -o.o
i ~.~
! _
r'-
_ c;:: , .0.025._
,:,oV!/ 9
.4

/',::i-i/;i 0:[6
"5. -8 I

~~-E~ , ~ //x4~54,,I
n

o~

8,

g~
.~-
"~_"

,..~
~ ,9~ 19~
19"*] 19o~I 19"* I 19"0
7 ~176 7 ~176 7"* 7"* 7'* 7 ~176 7 *~ 7 *~ 7*~
2.1.77 3.1.77 ~.I.77 5.5.77 6.1.77 4.577 5.1.77 6.1.77

Fig. 5. As Figure 4, but in addition transpiration (dotted lines in the left part of the diagram)
and CO2 exchange (solid lines in the left part of the diagram) were recorded. CO2 and H20
exchange curves were integrated to give the amount of CO2 and H20 net exchange per one
light or dark period of 12 h (upper curves in the left part of the diagram). CO2 and H20 curves
are individual recordings from four mature leaves. Averages and errors of points in right part
of the diagram as for Figure 4

mobilize malate and deacidify between 7 oo and 19 ~176This was observed in

three independent experiments (Figs. 4 and 5, one experiment not shown). A
similar observation has also been made by Kluge (1969) with isolated phyllodia
of Kalancho~ tub!florum. Warren and Wilkins (1961) showed a comparable highly
damped endogenous oscillation of 14CO2 fixation by Bryophyllum fedtschenkoi
in continuous darkness, while CO2 gas exchange showed more persistent endoge-
nous oscillations in continuous darkness (see also Bfinning, 1973).
The present study stresses the close correlation of water relation parameters
with malate levels during continuous light. Changes of 0w and 0s remain corre-
lated with changes of malate levels. As the CAM rhythm disappears in contin-
uous light, malate levels tend to be low, and concomitantly 0re and 0s are
high. However, transpirational water loss is increased during continuous light
and there is much CO2 uptake. Hence, presumably stomata are open and there
is considerable C3 photosynthesis. Direct fixation of atmospheric CO2 by ribu-
lose-l,5-diphosphate carboxylase is known to occur in CAM plants in the light
when no CO2 is available internally from malate decarboxylation (Osmond
and Allaway, 1974). At the particular conditions applied, i.e. with the plants
kept well watered via the soil and with an ambient relative humidity of 60%,
0w and 0s appear to be more closely related to malate levels than to transpira-
tion. While in the light:dark rhythm increasing malate levels are correlated
with decreasing 0w and 0s and with high transpiration (and vice versa), in
continuous light high transpiration does not lead to lower 0w and 0s. As
malate levels remain low, 0w and 0s remain high. This confirms the conclusion
already drawn from the comparative experiment of Figure 2, that at least under
Water Relations of a CAM Plant in Relation to Malate Oscillations 91

the growth chamber conditions used here, Ow and Os to a considerable degree

are determined by the level of malate as an osmotically active solute in the CAM
leaf cells. Environmental conditions during experiments appear to be very impor-
tant for these observations. By contrast to our findings, Osmond et al. (1976,
Table 2) observed decreasing leaf water potentials in K. daigremontiana during
the light and increasing leaf water potentials during the dark phase. However,
these authors had relative humidities in the atmosphere varying around 50% and
a very different watering scheme, imposing water stress on the plants. Thus,
these experiments are not strictly comparable.
Rhythmic changes of ~ w - O s were not observed in any one of the experi-
ments performed in this study. It may be interesting though, to note that ~'w-Os
generally decreases as the CAM rhythm disappears in continuous light in the
experiments of Figures 4 and 5. When dark is given again and the CAM rhythm
is re-established (see next notion) O w - ~ s rapidly increases again.

3. Re-Establishment of the C A M Rhythm Following Continuous Light

The experiments of Figures 4 and 5 differ by the conditions given at the end.
In Figure 4, the first dark phase after continuous light was given at 19~176i.e.
at the correct time of the rhythm preceding the treatment with continuous
light. Malate accumulation in the dark and a normal CAM rhythm are imme-
diately restored. This rules out the possibility that the enzymatic machinery
of CAM is lost and has to be resynthezised after the period during which
the rhythm is absent. In Figure 5 the first dark phase after continuous light
was given at 7 ~176i.e. at the wrong time, 12 h out of phase of the original
rhythm. However, this shift of phase had no effect. The response is identical
to that of Figure 4; the CAM rhythm re-appears immediately. This appears
to rule out the contention of Queiroz (1974; Brulfert et al., 1975), and Wilkinson
and Smith (1976) that the enzymatic machinery of CAM (phosphoenol pyruvate
carboxylase and malate dehydrogenase) is subject to endogenous oscillations
and that this endogenous enzymatic rhythm is responsible for the metabolic
oscillations of CAM. More investigations of re-establishment of CAM after
continuous light may provide further understanding of this problem.

4. Conclusions on the Regulation of the C A M Rhythm in the Intact Plant

CAM is a metabolic rhythm with substantial C O 2 dark fixation and diurnal

oscillations of malate levels. This phenomenon is closely associated with an
inverse stomatal rhythm, i.e. opening during the night and closure during the
day (Wolf, 1932, 1960; Ranson and Thomas, 1960). Some features of this
rhythm can be observed to continue as circadian oscillations endogenously
in continuous darkness or in continuous light (Bfinning, 1973; Warren and
Wilkins, 1961; Wilkinson and Smith, 1976; Kluge, 1969; present paper). This
has led to the search for a 'key point' in the system where the rhythm is
regulated.
It has been envisaged that such a basic oscillator could be an enzyme such
as phosphoenol pyruvate carboxylase (PEPC) with its central role in CAM
and its regulatory properties (e.g. the malate inhibition studied by Kluge and
92 U. Liittgeand E. Ball

stomata[ enzymatic
machinery machinery

water relations metabolite

machinery "I transport and Fig. 6. Participation of various mechanisms
compar tmenta-
tion machinery
in the regulationof CAM in the intact
plant

Osmond, 1972). Recent investigations in our laboratory of the change of Mesem-

bryanthemurn crystallinum plants from C3-photosynthesis to CAM when subject
to water stress add to the importance of the enzymatic machinery (Greenway
and Winter, 1977). However, with Sephadex purified extracts of M. crystallinum
leaves, it could not be confirmed that PEPC activity varies diurnally as suggested
by Queiroz (1974; Morel and Queiroz, 1974; Brulfert et al., 1975), and Wilkinson
and Smith (1976) for KalanchoO species. Our results instead imply a regulatory
role of diurnal variations of PEPC properties, e.g. in response to pH and malate.
Furthermore, the investigations of Greenway and Winter (1977) on the nature
of the slow change of M. crystallinum from C3 photosynthesis to CAM during
which malate levels first begin to rise and only later show diurnal oscillations,
rule out a singular regulatory role of an enzymatic machinery. This is also
supported by the present experiments on reappearance of the CAM rhythm
after extended periods of continuous light. An additional metabolic machinery
such as malate transport and compartmentalization must be important, as argued
already for other reasons by Kluge and Osmond (1972) and Ltittge et al. (1975).
Furthermore, parameters determining water relations can also participate in
regulation of CAM: The work of Winter (1973, 1974a, b; Winter and yon
Willert, 1972; Winter and Ltittge, 1976; Greenway and Winter, 1977) shows
that CAM occurs as a response to limited water supply. Water relations are
highly important for the behavior of CAM in the field (Allaway et al., 1974;
Osmond, 1975; Szarek and Ting, 1975; Hartsock and Nobel, 1976; Nobel,
1976). Malate mobilization from the vacuoles of leaf slices of K. daigrernontiana
and efflux into an external splution depend on turgor potential (Op) of the
cells (Ltittge et al., 1975, 1977). The present study suggests that in the intact
leaves of K. daigremontiana, independent of the stomatal rhythm, water relation
parameters can participate in the control of CAM. CAM itself continues inde-
pendently of stomata in K. daigremontiana leaves when the epidermis is stripped
off (Kluge and Fischer, 1967). This allows speculation on a possible relation
of stomatal movements to mesophyll PEPC. Stomata are very sensitive to COa
concentration (Raschke, 1975). Governing COz fixation, mesophyll PEPC affects
partial pressure of CO2 and, thus, may act on stomatal movements.
In conclusion, there does not appear to be one singular regulator of CAM.
There seem to be a number of interconnected mechanisms (Fig. 6) which can
Water Relations of a CAM Plant in Relation to Malate Oscillations 93

be identified with the stomatal machinery, enzymic machinery, water relations

machinery and the machinery of metabolite transport and compartmentation.
Evidently this multiplicity of mechanisms makes CAM more responsive to
environmental factors than would be possible if it were regulated by a singular
mechanism.

Acknowledgements. We are very indepted to Dr. Karl Fischer for his help with the infrared gas
analyser experiments and to Helmut Immig for technical constructions. Our work was supported
by the Deutsche Forschungsgemeinschaft.

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Received May 14, 1977