J Plant Physiol. VOL 155. pp. 86-92 (1999) 1;1 JOUR.

AL OF ~E~
http://www.urbanfischer.de/journals/jpp Plan. Pbysjolo.y
1999 URBAN &. FISCHER

Effects of Stress Conditions and Calcium on the Light-

induced Hydrogen Gas Exchange in Oscillatoria cha/ybea

REFAT ABDEL-BASSET* and KLAus P. BADER

Lehrstuhl ftir Zellphysiologie, Fakultat ftir Biologie, Universitat Bielefeld, Postfach 10 0131, D-3350l Bielefeld, Germany

Received June 20, 1998 . Accepted October 15, 1998

Summary

The flash-induced evolution of molecular hydrogen under various conditions of stress and stress-cal-
cium interactions was followed by means of mass spectrometry in protoplasts from the filamentous, non-
heterocystous cyanobacterium Osciltatoria chalybea. Osmotically equivalent levels of salinity and water
stress inhibited the hydrogen signals approximately to the same level, indicating little difference between
ionic (NaCl) and non-ionic (mannitol) osmotica. Consequently, the inhibition can be attributed rather to
a water deficit stress than to a specific ionic effect. Heat shock at 45C for 5 min dramatically decreased
the hydrogen evolution signals compared with the maximum control recorded at 22 0c. Chemical stress
was imposed to the cyanobacteria by the application of a standard pyrethroid insecticide (deltamethrin) as
an example for the impact of modern plant protective chemicals on plants. This insecticide, which has
recently been shown to exert significant herbicidal activities (Bader and Schuler, 1996), substantially
inhibited the cyanobacterial hydrogen gas exchange, even at very low applied concentrations (1-5 ).Lmol/
L). Although the installation of an anaerobic atmosphere is required by principle in order to observe a sub-
stantial reaction rate of proton reduction, we observed a strong further increase in hydrogen photoevolu-
tion with time. Thus, the effect that can be denominated 'time-dependent increase' (TDI) consisted of an
enhancement up to about 400 % during the incubation time from 40 min to 120 min with continuous
nitrogen flushing. We conclude that this increase might be correlated with the activation of hydrogenase
or even a de novo synthesis of the enzyme. Generally, the time-dependent increase was higher under any of
the investigated stress conditions than in the corresponding controls. Under stress conditions, we observed
a stimulation of the normally relatively small dark evolution of molecular hydrogen from Osciltatoria cha-
lybea and this effect was maximal in the case of salt stress. When we applied calcium ions to the assays suf-
fering from a stress situation we observed an apparent relief, i.e. the absolute hydrogen evolution signals
and also the ratios from the time dependent increase were enhanced depending on the concentration.

Key words: Hydrogen gas exchange, Osciltatoria chalybea, stress, calcium, mass spectrometry.

Introduction 1998). Physiological properties of the resulting gas exchange,

In previous papers we have shown and described by means
of mass spectrometry the capacity of the filamentous non-
heterocystous cyanobacterium Osciltatoria chalybea both to
photooxidize molecular hydrogen and to photoreduce pro-
tons (Abdel-Basset and Bader, 1997; Abdel-Basset and Bader,
* Permanent address: Botany Department, Faculty of Science,
Assiut University, Assiut, Egypt.
which can be detected at mle = 2 in a HID collector of a
mass spectrometric set-up, have been shown to consist of an
initial hydrogen evolution that is rapidly converted to an
uptake signal. The tentative interpretation of such a transi-
tion in terms of the light-induced activation of a dark-inac-
tive Calvin cycle, which would derive electrons from proton
reduction to carbon dioxide assimilation (Greenbaum et aI.,
1995; Lee et al., 1996), has been ruled out at least for Oscilla-
toria chalybea by parallel mass spectrometric measuring of the

0176-1617/99/155/86 $ 12.0010

CO 2 -gas exchange at mle = 44 (Abdel-Basset and Bader,
1998). A common link between the processes can rather be
seen in context with the carbon dioxide concentrating mech-
anism (CCM), which has been described for cyanobacteria
(Kaplan et al., 1991; Fridlyand et al., 1996; Kaplan et al.,
1997).
The question whether or how the formation of molecular
hydrogen might compete with carbon dioxide fixation for
reducing power can possibly be further approached by expos-
ing the photosynthetic organisms to various stress conditions.
In this sense, stressed plants, algae or cyanobacteria may face
the necessity of additional energy demands with consequen-
ces on the respective electron transport. This might hold for
maintenance processes, growth or production on the one
hand, coupled with a shortage of supplies (e.g. reduced pho-
tosynthetic activity) on the other hand. Numerous kinds (and
levels) of stresses have been repeatedly studied in almost all
aspects of plant physiology (Momonoki et al., 1996; Ruiz-
Lozano and Azcon, 1997; Palta, 1996). Only scattered re-
ports, however, have been devoted to follow the hydrogen gas
exchange as an important physiological activity under stress
conditions. Recently, Schnackenberg et al. (1996) studied the
effect of pH and temperature stresses on hydrogen evolution
in the marine alga Chlorococcum littorale. In this case, the
photohydrogen production that is supposed to be largely me-
diated by PSI appeared transitorily stimulated by an increase
in the pH value (above 7.5) or in temperature (above 25C).
The response to the shifts in pH and temperature showed
distinct differences to photosystem I! reactions. Apparently,
the conditions in Chlorococcum are not comparable to the hy-
drogen gas exchange reactions in Oscillatoria chalybea as we
described the necessity of a rather acidic pH milieu for an op-
timal and stable hydrogen evolution, a condition that can be
mimicked by addition of the protonophore CCCP (Abdel-
Basset and Bader, 1998). In addition, Schnackenberg et al.
(1996) applied high concentrations of NaCI (up to 1 mollL)
to overcome the effects of alkaline stress that may cause CI--
depletion from PSI!. Under nutritional stress, two mutants of
Anabaena variabilis exhibited enhanced levels of hydrogen
evolution when grown under CO 2 or nitrogen starvation
(Sveshnikov et aI., 1997). Due to a general lack of related in-
vestigations, the target of the present work was to assess the
effect of a number of stressors that are of probable or coinci-
dent occurrence in various habitats on the evolution and on
the uptake of molecular hydrogen in Oscillatoria chalybea.
Thus, we investigated the effects of salinity, water stress, heat
shock as well as of the insecticide deltamethrin. Moreover, we
looked into the possible antagonistic or protective effect of
calcium against stress by applying calcium ions to Oscillatoria
protoplasts in addition to the respective treatment.
Materials and Methods
The filamentous non-heterocystous cyanobacterium Osciltatoria
chalybea was originally obtained from the Algal Collection in
Gattingen (Germany) and has been cultivated since then in our lab-
oratory on clay plates as porous mechanical support in large Petri
dishes. The cyanobacteria were grown under a 14 h light/lO h dark
regime in a climatized room at 26C at a light intensity of approx.
H 2-metabolism under Stress 87
12~mol.m-2.s-1. As culture medium we used medium D accord-
ing to Kratz and Myers (1955). Preparation of protoplasts from
Osciltatoria chalybea was carried out following an earlier described
procedure including enzymatic digestion with glucuronidase and
cellulase (Bader et aI., 1983).
Light-induced hydrogen gas exchange in Osciltatoria chalybea was
performed by the direct analysis of molecular hydrogen (H 2) via
mass spectrometty. Our set-up consisted of a modified 'delta' Stable
Isotope Ratio Mass Spectrometer from Finnigan MAT (Bremen,
Germany). This device has been principally modified with respect to
both the inlet system and the analyzer, leading to a substantial
increase in the sensitivity and to an improvement of the response
time for dynamic measurements in particular. Details of the modifica-
tions have been described (Bader et aI., 1987). The calibration of the
set-up for the precise quantification of the respective gas signals
(oxygen, hydrogen, nitrogen and carbon dioxide) has also been out-
lined (Bader et al., 1992; Bader and Raben, 1995). We detected and
registered the dynamic changes in the cyanobacterial hydrogen gas
partial pressures at m/e = 2 in a HID collector and recorded the sig-
nals on a SE 130-03 three-channel recorder from Goertz Metrawatt.
The signals were obtained by illumination of the cyanobacterial
assays containing 30 ~g ChI in 0.15 mollL Tricine/0.3 mol/L KCI
(pH 7.5) with sequences of 10 consecutively fired 5 ~s Xenon flashes
spaced 300 ms apart. Hydrogen evolution was followed under the
following conditions of stress, which were imposed directly at the
time of measuring: Salinity: 0, 50, 100, and 150 mmollL NaCl,
respectively. Water stress was simulated by the addition of 0, 100,
200, 300 mmollL mannitol (these concentrations are osmotically
equivalent to those of NaCl). Heat shock was done by exposing the
protoplasts to 22, 30, 35, 45C for 5 min (room temperature was
22 C and served as control). Moreover, we treated the cyanobacteria
with various concentrations of the insecticide deltamethrin (0, 1, 3
and 5 ~mollL). These concentrations of the insecticide have been
reported to exert a strong herbicidal activity by inhibiting photosyn-
thetic electron transport reactions (Bader and Schuler, 1996). In
order to investigate an antagonistic positive effect of calcium on
stressed thylakoids we used a wide range of calcium (CaCI 2) concen-
trations (1-1,000 ~moI/L); only effective ones are shown in the fig-
ures, depending on each stress. The Osciltatoria protoplasts were pre-
loaded in the light with the respective concentrations of Ca2 + 5 min
before being subjected to a stress condition. This preincubation
turned out necessary as preliminary experiments showed that addi-
tion of calcium just prior to or after stress imposition was not effec-
tive.
Results
Light-induced changes in the partial pressure of molecular
hydrogen in protoplasts of the filamentous, non-heterocyst-
ous cyanobacterium Oscillatoria chalybea were induced by a
train of 10 short (5I1s) saturating light flashes and detected at
mle = 2 via the HID collector of our mass spectrometric de-
vice. We described in a previous paper that following an ex-
tensive dark adaptation Oscillatoria chalybea showed an initial
fast hydrogen evolution that was followed by a substantial hy-
drogen uptake signal under continuous illumination for
1 min. The ratio evolutionluptake could be influenced and
controlled by various parameters, e.g. a shift in the pH value
or addition of protonophores (Abdel-Basset and Bader,
1998). With these observations at issue we scrutinized the hy-
drogen gas exchange signals and discovered that time appar-
ently plays an additional role beside the installation of a com-
plete anaerobiosis in order to optimize the hydrogen evolu-
 88 REFAT ABDEL-BASSET and KLAus P. BADER
OmUatoria cita/ybea t = 60'
m/e =2
t=40'
Imin
Fig. 1: Mass spectrometric recording of the hydrogen gas exchange
signal of Oscillatoria chalybea. The tracings represent the cumulated
signals as the consequence of 10 short (5 liS) Xenon flashes spaced
300 ms apart and detected at mle = 2 in a HID-collector. Following
the installation of an artificial anerobic gas atmosphere the signals of
the light-induced hydrogen evolution still increased with time.
iii 14
....
~ 79/-lgChl
~ 10
o
~ 8
o 1994; Bader and Schmid, 1988). Under identical but aerobic
>
III 6 conditions the light induced oxygen evolution remained com-
c
e
Q/
'0
4
26/-lgChl
~ 2
0~---2~0~~4~O~L-6~0~--8~O----~lrO~--12~O~~140
Sedimentation and Dark Adaptation Time(min)
Fig.2: Effect of time on the mass spectrometrically recorded hydro-
gen gas exchange in Oscillatoria chalybea. Independent of the respec-
tive chlorophyll concentration, the maximum signals were observed
only after 2 h. Other conditions as in Fig. 1.
tion signals from Oscillatoria chalybea. The increase in the
amplitudes of hydrogen signals by extended adaptation pe-
riods, which is shown in Fig. 1, can by principle not be the
result of an improved anaerobiosis (or an optimized sedimen-
tation on the teflon membrane) as the oxygen partial pressure
in our mass spectrometric reaction chamber appears to be
near zero already after 30-40 min. The phenomenon of the
additional enhancement of the hydrogen signals is referred to
as time-dependent increase (,TDI') and it was calculated as
the ratio between the evolution signal amplitude at 60 min
divided by the corresponding one at 45 min. The principal
possibility of an improved sedimentation as a possible reason
for the TDI can easily be ruled out as an interfering factor for
two reasons. The installation of strictly anaerobic conditions
is achieved not only by extensive and continuous nitrogen or
argon flushing of the respective assay but also by the perma-
nent gas suction through the teflon membrane, which is in
direct connection with the main vacuum system, i.e. the ion
source of the mass spectrometer. Thus, the protoplasts are di-
rect~ exposed to . the high negative pressure of ~bout
10- mbar for 45 mm before the measurements and thIS can
be regarded to be more than sufficient for the protoplasts to
fully settle on the cuvette membrane. Figure 2 shows the de-
pendency of the hydrogen evolution on time up to 120 min
using three different chlorophyll concentrations. After the es-
tablishment of totally anaerobic conditions (recognized from
the very low and constant oxygen background after about
30-40 min.) the signals, nevertheless, continued to increase,
reaching a steady state value only after about 2 h. We suggest
that during this second phase an optimal activation of the
inactive hydrogenase or even a de novo synthesis of the en-
zyme takes place. The question about the time constants of
the hydrogenase synthesis and/or activation in Oscillatoria
chalybea will be analyzed in the forthcoming biochemical in-
vestigations isolating the enzyme(s} from this cyanobacte-
rium. For reasons of comparison, Fig. 3 shows the photosyn-
thetic oxygen evolution in Oscillatoria chalybea under similar
conditions. Firstly, it is observed that under anaerobic condi-
tions the photosynthetic oxygen evolution is almost com-
pletely inhibited in Oscillatoria chalybea (Fig. 3; left trace), a
phenomenon that is clearly distinct from overreduced redox
components inside the electron transport chain but has rather
to be interpreted in terms of a basic requirement of photosys-
tem II functions for catalytic amounts of oxygen (Bader,
c
o
~
:J
o
>
III
N
o
anaerobic
Q/
~
2
a.
::::>
N Imin
o
Fig. 3: Mass spectrometric recording of the oxygen gas exchange
signal of Oscillatoria chalybea. Preilluminations did not contribute to
the ratio aerobiosislanaerobiosis by interfering with photosynthetic
oxygen, as under anerobic conditions Oscillatoria chalybea does not
photooxidize water (left tracing). It should be noted that an addi-
tional sedimentation effect with the resting assay can be excluded
because the oxygen evolution levels did not change from 40 to
60 min (middle and right tracings) in contrast to the hydrogen gas
signals depicted in Fig. 1.

125
A) Salt Stress
~ 100
~
= 75
~p
tE 50
=
Q)
.a....
OJ)
25
:I:
0 0
0 50 100 150
NaCl(mM)
Fig. 4: Effect of various stresses on the light-induced hydrogen evolution in Oscillatoria chalybea: Salinity; the cyanobacterial protoplasts
were exposed to increasing concentrations of sodium chloride (A), water stress as mimicked by increasing concentrations of mannitol (B).
The data are presented as percent of the controls.
pletely unchanged in the time range from 40 to 60 min or
more (Fig. 3; middle and right tracings), which can be re-
garded as additional evidence that these conditions, the sedi-
mentation effect in particular, are not involved in the TDI ef-
fect. It should be noted, in addition, that prolonged darkness
alone without gas flushing was not sufficient to induce the
signals at their full amplitudes: only 10 % of the average hy-
drogen signals came up in this case, albeit the fact that dark
respiration contributes to the installation of the necessary an-
aerobic conditions (results not shown). Only in the case of
flushing with an inert gas (nitrogen or argon) were maximal
hydrogen signals obtained with Oscillatoria chalybea. In a pre-
ceding paper, we have quantified the oxygen sensitivity of the
hydrogenase in this cyanobacterium and found an I50-value
of 6 ~mollL (Abdel-Basset and Bader, 1997). A contribution
to the increase in the evolution signals might also be seen in
the observation that, anyhow, the hydrogen uptake signal de-
creased with time (result not shown).
Hydrogen evolution signals, the evolution/uptake ratio
and the time dependent increase (TDI) were followed under
various stress conditions. Moreover, we examined the ques-
tion whether calcium can, at least to a certain degree, restore
the physiological activity that has been impaired by the stress
situation. Since salinity, water stress, heat shock and plant pro-
tection chemicals are stresses of widely different natures, the
relevant concentrations of all parameters as well as their effects
vary to a great extent. NaCl-salinity at a concentration of
50 mmol/L inhibited the hydrogen signals by about 50 % (Fig.
4A); higher concentrations of salt had little additional effect.
Under these conditions, however, we observed a net stimula-
tion of the small dark production rate and this enhancement
was highest in the case of salt stress (result not shown). NaCl
is known to impose, simultaneously, two types of stress: an
ion toxicity with a consequently concomitant ion imbalance
stress (due to the accumulation of Na + and Cl-) in addition
to a water stress (water deficit stress). Mannitol, which impo-
ses only the water deficit stress, inhibited the signals nearly to
the same levels by osmotically equivalent concentrations (Fig.
4 B). Such comparable levels of inhibition caused by either
NaCl or mannitol imply that the preponderant accumulation
Hz-metabolism under Stress 89
125
B) Water Deficit
.g
100
~
'"
75
=
0
-:>
.s
50 ~
=
Q)
.a....
OJ)
25
:I:
o 100 200 300
Mannitol (mM)
of Na + and Cl- exerted little, if any, toxic effects on the for-
mation of hydrogen molecules. The recorded inhibition is
then due, essentially, to a common effect exerted by both os-
motica, which is the water-deficit stress (i.e. limited availabil-
ity of free water molecules). The time-dependent increase ex-
hibited slight enhancements (relative to that of the control)
by salt application and by mannitol, respectively (results not
shown). In the marine alga Chlorococcum littorale, however,
NaCl had not been found to exert any inhibitory effect on
hydrogen evolution. Moreover, at very high concentrations
(500 mmollL) NaCl could even overcome the alkali (pH
ll)-induced depletion of PSII chlorides (Schnackenberg et
al., 1996). In contrast to the photoevolution of hydrogen it
was reported that the photosystem II activity in Oscillatoria
chalybea as determined by flash-induced oxygen evolution
was stimulated by 0.1 mol/L KCl (Bader, 1994). Heat shock
(30, 35, and 45C for 5 min) inhibited the hydrogen evolu-
tion signals in Oscillatoria chalybea in comparison with the
maximum signal recorded at 22C. However, none of the
tested temperatures removed the signal competely; the re-
maining value at 45C amounted to about 30 % of the one
measured at 22 C (Fig. 5A). Heat shock is known to inhibit
PSII activity; 3-10 min at 45-50 C can cause a complete
removal of bound manganese with the total reduction of Hill
reaction activity (Kimimura and Katoh, 1972). Based on this
knowledge, the remaining value at 45C indicates that vari-
ous and different electron donors have to be assumed to sup-
ply electrons for proton reduction in Oscillatoria chalybea. In
this respect, the participation of respiration and the quinone
pool in hydrogen evolution has been documented (Abdel-
Basset and Bader, 1997). Schnackenberg et al. (1996) re-
ported similar results in the marine alga Chlorococcum littorale
and ascribed the fraction of hydrogen evolved at temperatures
above 25C to the mere PSI activity. Accordingly, they pos-
tulated the significance of the water-splitting system of pho-
tosynthesis in the absence of other electron sources. Strong
debate concerning the direct participation of the oxygen
evolving system of photosynthesis, i.e. photosystem II in the
process of hydrogen liberation, however, is under way
(Greenbaum et al., 1995).
90 REFAT ABDEL-BASSET and KlAus P. BADER

125~----------------------------~----------------------------~ 125

A) Heat Shock B) Chemical Stress

.~
g
100 100
I
g
0: 75 75 0:

j j
? 0
~ 50 50 &:i
0: 0:
"
OJ)
"8
OJ)

.a>. 25 25 "">.
::c ::c

0 0
22 30 40 o 2 3 4 5 6

Temperature (ec) Deltamethrin (~

Fig. 5: Effect of various stresses on the light-induced hydrogen evolution in Oscillatoria chalybea: Cyanobacteria were subjected to a heat
shock, i.e. to various temperatures (A) or to increasing concentrations of the insecticide deltamethrin (B). The data are presented as percent
of the controls.

Heat shock, which caused a severe inhibitition of the hy-

drogen evolution signals (approx. 70 %) (Figs. 5A and 6), in- 5
duced a very high time dependence increase in comparison
with the other stresses examined (Fig. 7). The enhanced in- 4)

crease of the (inhibited) signals by time indicates that struc- '"0: 4

tural damage (proposed e.g. for PSII) is not likely to be the e
.s
0

reason for the strongly inhibited hydrogen evolution. A pos-

1: 3
sible explanation, however, would be a time-dependent delay 4)
""0
of the development of the hydrogen signals induced by the c::
4)
a.
heat shock in the sense that the cyanobacteria suffered from a
Q2
4)
E
f=:;
,./
60 m/e=2
,-.,
,.. '?>, ~
......
oj
..
~
'-'
..
c::
0 40 ....
..
~
'.p
= ~ ..
..
"0 .. .. "':':'
~ -= ...... .. ....
>
~
c:: ~/ .. ...... ..
..
..
.. .... ..
~
4)
00
e .... F ~
.. ..
..
Fig.7: Effect of salt-(NaCl), water deficit-(mannitol), heat-(temper-
20 .... .... ..
""0
.... ..
I""""
....
ature) and chemical (deltamethrin) stresses on the mass spectromet-
~ ~ ..
.. ......
F ..
..
......
..
....
rically determined time dependent increase (TDI) in the hydrogen
evolution signals and the influence of additional calcium supply in
r/.: ...... ...... ..
.. .. .. Oscillatoria chalybea. Conditions were as in Fig. 6 .
:;1E1 .... F;;:;:
..
.. ~
.. ~ .. .. ry
0
~
~~ ~p c.# /:6- c.# ~'U~:r;,.v'. ~
. W~ #
~ ~v... (reversible) impairment of the hydrogenase, which can be
cJ v~ v~ Ci # xG
x ~ x x ~ overcome with time, reaching an about normal level. It is
~~ ~. # <:)'U -<:).
well-known that, in comparison to other organisms, cyano-
~
bacteria are specifically resistant to various kinds of stress.
Plants are increasingly exposed to artificial and anthropo-
Fig. 6: Inhibitory effect of salt-(50 mmol/L NaCl) , water deficit-
(300 mmol/L mannitol), heat-(45 C) and chemical (5 JlmollL delta-
genic stresses by the use of different kinds of plant protective
methrin) stresses on the mass spectrometrically detected hydrogen chemicals like herbicides, insecticides, fungicides and others.
gas exchange signals and the calcium-dependent recovery of the hy- For the present study we have selected the effect of the often
drogen evolution in Oscillatoria chalybea. Optimal recovery was ob- used pyrethroid insecticide deltamethrin as a representative
tained with calcium ion concentrations of 10-100 JlmollL. for the enormous amount of plant protective chemicals

(other than herbicides) that plants are exposed to in modern
agriculture. It is generally believed that those compounds can
be applied under the assumption that they are only effective
(harmful) for the respective target organisms but harmless for
the treated plants. From Fig. 5 B it can be derived that the in-
secticide deltamethrin (at very low concentrations of
1-5IlmoIlL) even strongly inhibited the hydrogen evolution
signal of Oscillatoria chalybea by about 70 %. The same con-
centrations were found to strongly inhibit the photosynthetic
electron transport in Oscillatoria chalybea (Bader and Schuler,
1996), which casts doubt on the specificity of this compound
as a strict insecticide. The time dependent increase was
strongly enhanced by an increase in the temperature and even
by the lowest concentration of deltamethrin (lllmoIlL) (Fig.
5 B). Deltamethrin-induced inhibition was recovered follow-
ing the addition of calcium; 5001lmol/L Ca2+ restored about
30 % of the lost evolution capacity (Fig. 6). ci+, also, rela-
tively counteracted the deleterious effects of deltamethrin by
a slight decrease in the stress-induced increase of the time de-
pendent increase. In this case, the positive effect of calcium
was most pronounced following heat stress (Fig. 7).
Generally, the time-dependent increase 'TDI' was in all
cases of stress higher than those of their corresponding con-
trols. Such enhancement of the time dependent increase, un-
der stress conditions, opposes the hypothesis that molecular
hydrogen is produced as a mechanism to remove electrons in
cases of surplus energy availability. The cells in situations of
stress are usually in need of energy to cope with the imposed
structural and metabolic disturbances, i.e. they suffer short-
age of energy. A model example of energy expenditure to
cope with stress conditions is the maintenance respiration
that is diverted to repair stress-induced damages or turnover
(see for example Banuls et a!., 1997; Huang and Redmann,
1996). Combinations of NaCl and CaCl 2 decreased respira-
tion and enhanced photosynthesis of Chlorella vulgaris (Ab-
del-Basset, 1993). Calcium usually interferes by counterac-
ting the stress-induced injuries (e.g. to membranes), and
thereby energy consumption for maintenance processes is
saved.
Calcium ions, when combined with NaCl, slightly but
consistently restored the NaCl-inhibited signal of hydrogen
evolution (Fig. 6). The recovery amounted to approximately
35 % relative to the rate under NaCl-stress without supple-
mental Ca2 +; additional supply in the concentration range
between 0.5 - 500 Ilmol/L yielded no or only marginal addi-
tional differences. In the case of mannitol stress, however, the
inhibited signal (about 50 % as in the case of NaCl) could be
completely restored by addition of 100 IlmollL Ca2+ (Fig. 6).
Hence, the response to calcium addition was completely dif-
ferent in the presence of the two compounds, which suggests
that, albeit the identical inhibition rate, other and different
recovery mechanisms by calcium have to be regarded for ion-
ic (NaCl) or non-ionic (mannitol) osmotica.
In the case of heat shock, additional Ca2 + (l00 Ilmol/L) re-
covered only 15 % of the lost capacity; a further increase of
the concentration did not improve the signals (Fig. 6). The
deltamethrin-induced inhibition could be compensated to a
somewhat higher extent (Fig. 6). The principal capability to
restore the stress-induced inhibited signals can be regarded as
an additional indication that drastic and irreversible struc-
Hz-metabolism under Stress 91
tural damage can not have taken place as a consequence of
the respective stress situation. We assume that rather rever-
sible conformational changes are responsible for the decrease
in the proton reduction capacity with the overall activity
being unaffected.
From the foregoing results it can be deduced that the evo-
lution of hydrogen was decreased by stress conditions and
Ca2 + could relatively ameliorate the four examined stresses to
various extents, i.e. increased the hydrogen evolution. How-
ever, the meaning of amelioration, in the case of hydrogen
metabolism, in particular, is a challenging question. From the
physiological aspect, hydrogen evolution is a loss of electrons
and energy but when considered as a promising fuel it turns
into an interesting metabolic activity to be enhanced. There-
fore, a decrease in evolution or evolution/uptake ratio may
mean that the ability of the cells to re-use (oxidize) molecular
hydrogen to generate energy is enhanced.
Acknowledgements
The generous financial support provided by the 'Alexander von
Humboldt-Stiftung' (R.A.-B.) and by the 'Deutsche Forschungsge-
meinschaft' (KP.B.) is gratefully acknowledged.
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