Journal of Experimental Botany, Vol. 50, No. 334, pp. 665–675, May 1999
Adaptation of tobacco plants to elevated CO : influence
2
of leaf age on changes in physiology, redox states and
NADP-malate dehydrogenase activity
J.E. Backhausen1 and R. Scheibe2
Pflanzenphysiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany
Received 9 September 1998; Accepted 20 November 1998
Abstract
Transgenic tobacco plants (Nicotiana tabacum L. cv.
Xanthi) with altered chloroplast NADP-malate dehydrogenase (NADP-MDH) content were grown under ambient or under doubled atmospheric CO in order to
2
analyse the effect of elevated CO on the redox state
2
of the chloroplasts. Since large differences exist
between the individual leaves of tobacco plants, gas
exchange characteristics, enzyme capacities and metabolite contents were measured separately for each
leaf of the plants. Large variations between leaves of
different age were found in nearly every parameter
analysed, and the differences between younger and
older leaves were, in most cases, larger than the differences between comparable leaves at ambient or elevated CO . For all parameters (chlorophyll fluorescence,
2
P700 reduction, NADP-MDH activation) that are indicative for the redox situation in the electron transport
chains and in the chloroplast stroma, more oxidized
values were determined under elevated CO . The
2
increased redox state of ferredoxin, observed at ambient conditions in the NADP-MDH-under-expressing
plants, disappeared under elevated CO . It was con2
cluded that the reduced rate of photorespiration under
elevated CO decreases the amount of excess elec2
trons. Interestingly, this lowered not only the activation
state of NADP-MDH, but also the expression of the
enzyme in the wild-type plants. The results are discussed with respect to a possible interaction between
stromal reduction state and gene expression.
Key words: Elevated CO , malate valve, transgenic tobacco
2
plants, regulation of photosynthesis, redox state.
Introduction
In short-term experiments with C plants, a rise in ambient
3
CO leads to elevated substomatal CO concentrations.
2
2
Since more CO is available at the active site of Rubisco
2
(Poorter, 1993), the rate of photorespiration is diminished
(Leegood et al., 1995) and net carbon fixation is stimulated. This affects the redox situation in the chloroplast
stroma, since in the photorespiratory pathway, additional
ATP is required for the re-entry of glycerate. Furthermore,
photorespiration increases the required ATP:NADPH
ratio by increasing the amount of carbon which passes
through the regenerative part of the Calvin cycle, relative
to PGA reduction. Thus, it can be expected that photorespiratory activity will lead to more reduced conditions in
the stroma. On the other hand, fewer excess electrons are
produced under elevated CO , when photorespiration is
2
suppressed.
Since electrons are often in excess of the amount
required for CO fixation or photorespiration (Stitt, 1986;
2
Backhausen et al., 1994), alternative electron acceptors
(poising mechanisms) are present in the chloroplast
stroma to prevent over-reduction and oxidative damages.
Ferredoxin (Fd )-dependent cyclic electron flow around
photosystem 1 (PSI ) (Bendall and Manasse, 1995), the
reduction of O (Polle, 1996a), and the malate valve
2
(Scheibe, 1987; Fridlyand et al., 1998) are known
1 Present address: Robert-Hill Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
2 To whom correspondence should be addressed. Fax: +49 541 969 2265. E-mail: [email protected]
Abbreviations: A−, A, reduced or oxidized acceptor of PSI; a, leaf area; Chl, chlorophyll; DTT, dithiothreitol; Fd, ferredoxin; F , ground fluorescence
O
level; F , maximal variable fluorescence; LHC, light-harvesting complex; mdh, NADP-MDH gene; NADP-MDH, NADP-dependent malate dehydrogenase;
M
OAA; oxaloacetate; PGA, 3-phosphoglycerate; PSI, photosystem 1; PSII, photosystem 2; q , photochemical quenching of the variable chlorophyll
P
fluorescence; q , non-photochemical quenching of the variable chlorophyll fluorescence; W , quantum yield of PSI; W , quantum yield of PSII.
N
I
II
© Oxford University Press 1999
666 Backhausen and Scheibe
specifically to remove excess electrons without affecting
CO assimilation or nitrite reduction (Backhausen et al.,
2
1994). NADP-dependent malate dehydrogenase (NADPMDH ) is a nuclear-encoded enzyme, and from our previous studies with NADP-MDH over- and underexpressing
potato plants ( Faske et al., 1997; Backhausen et al., 1998)
it is evident that NADP-MDH and the malate valve keep
the stromal Fd pool in an oxidized state. These mechanisms clearly stabilize the redox state in chloroplasts in
cases of sudden changes in the amount of excess electrons.
In contrast to such short-term effects, plants can adapt
towards elevated CO within several weeks (Stitt, 1991;
2
Poorter, 1993), and changes on biochemical, physiological, anatomical and morphological levels occur (Bowes,
1991). An often described consequence of adaptation
towards elevated CO is that, in spite of higher C and
2
i
suppressed photorespiration, the rate of carbon assimilation is decreased (Sage et al., 1989). Long-term growth
under elevated CO was assumed to cause sink limitations
2
and to lead to accumulation of carbohydrates in the
source tissues. This occurs especially when photosynthesis
becomes limited by the ability of the plant to form
additional sink tissues, often caused by a shortfall in the
supply of nitrogen or other nutrients (Stitt, 1991). Using
transient gene expression in maize protoplasts, Sheen
(1990) demonstrated that the accumulation of sugars,
especially glucose, sucrose and fructose, leads to specific
and co-ordinated repression of the transcription activity
of several photosynthetic gene promotors, with hexokinase being an essential mediator (Jang et al., 1997). Van
Oosten and Besford (1994) and Van Oosten et al. (1994)
demonstrated with tomato leaves that the mRNA contents of some nuclear-encoded chloroplast enzymes, such
as rbcS, cab and rca are decreased when the plants were
grown at elevated CO or after feeding with sucrose. The
2
lower Rubisco content is thought to be especially responsible for the decreased assimilation rates in leaves adapted
to elevated CO ( Woodrow, 1994).
2
This raises the question of how the redox state of the
chloroplasts is influenced upon adaptation to elevated
CO . On the one hand, the decreased contribution of
2
photorespiration would, as in short-term experiments,
lead to less reduced conditions in the stroma. However,
when the utilization of the assimilated carbon and its
translocation into the sink tissues becomes problematical
for the cell, the concentration of phosphorylated intermediates will increase in the cytosol and may cause the
opposite situation ( Flügge et al., 1980). Starch synthesis
is stimulated, and the decreased availability of inorganic
phosphate would cause over-reduction, as often studied
in isolated spinach chloroplasts (Furbank et al., 1987).
However, it can be expected that the situation may be
different in sink and in source leaves.
The poising mechanisms that function to stabilize the
stromal redox state clearly act in the short-term range,
but it must be questioned whether they can compensate
for long-term imbalances of the stromal redox state. It is
known that long-lasting alterations of the redox pressure,
as can be expected under elevated CO , can influence
2
gene expression, probably even of enzymes involved in
redox poising. It has been suggested that environmental
changes can be sensed via accumulation of H O ( Van
2 2
Camp et al., 1998), by the redox status of glutathione
( Karpinski et al., 1997), or by the plastoquinone pool
that activates a phosphorylation cascade ( Escoubas
et al., 1995).
A further problem becomes apparent from our earlier
results with wild-type and transgenic tobacco plants
( Faske et al., 1997). In tobacco and in several other plant
species, the capacity of NADP-MDH changes strongly in
leaves of different age. On the one hand, this obstructed
the interpretation of transgene effects, but furthermore it
indicates that physiological differences must exist between
the leaves of different age. The observation that several
parameters, such as metabolite concentrations, enzyme
activities, and chlorophyll and protein contents, vary
greatly in leaves of different age has been made earlier
( Vivekanandan and Edwards, 1987; Merlo et al., 1993).
Thus, it is questionable whether adaptation towards elevated CO occurs to the same extent in each leaf of a plant.
2
In this work, transgenic tobacco plants with altered
expression of NADP-MDH were used to analyse (i)
whether adaptation towards elevated CO differs in the
2
individual leaves of each plant, (ii) whether the adaptation
towards elevated CO affects the amount of excess elec2
trons and the nature of dissipating pathways, and (iii)
whether this influences the malate valve in its role to
stabilize the redox state of the chloroplasts.
Materials and methods
Plant material and growth conditions
Transgenic tobacco plants with increased or decreased NADPMDH capacity were characterized in detail in a previous paper
(Faske et al., 1997). The wild-type and transgenic tobacco
plants (Nicotiana tabacum L. cv. Xanthi) were grown in a
commercial soil mixture (10% sand, 10% pumice, 10% loam,
35% compost, 35% peat) in growth chambers. Up to day 30
after germination the pot size was 5 cm in diameter (70 ml ),
and from then on 14 cm in diameter (1.3 l ). After potting, the
plants were transferred to their final growth regimes at 350
(controls) or 700 ppm CO , kept constant using an ADC 200
2
system (ADC, Hoddesdon, UK ). The light intensity was 300–
450 mmol quanta m−2 s−1 at plant height for a daily period
of 16 h light (22 °C ), 8 h darkness (18 °C ), and 75% relative
humidity.
All experiments were performed during the period of maximal
plant growth (between day 50 and 70 after sowing). The
samples for metabolite measurements were taken at the end of
the light period. Gas exchange and fluorescence measurements
were done under the respective growth conditions, 2–4 h after
the end of the dark period. For each data point, at least five
different plants were analysed. All given values are means±SD.
Leaf-age dependent effects of elevated CO on stromal redox state
2
Gas exchange, chlorophyll fluorescence and P700 measurements
in leaves
The measurements of gas exchange, chlorophyll fluorescence
and P700 redox state were done simultaneously. Gas exchange
was measured with an ADC-LCA 4 system (ADC, Hoddesdon,
UK ). Chlorophyll fluorescence quenching and changes in the
leaf absorption at 830 nm (DA ) were measured with PAM
830
fluorometers ( Walz, Effeltrich, Germany). The quantum yield
of photosynthetic electron transport (w ), photochemical (q )
II
P
and non-photochemical quenching coefficients (q ) were deterN
mined by the saturation-pulse method as described by Schreiber
et al. (1986) and Genty et al. (1989). The redox state of
P700, A− and w were calculated from DA
according to
I
830
Klughammer and Schreiber (1994). The maximal amplitude of
the P700 signal (denoted as FR) was determined in the dark by
the changes in leaf absorption at 830 nm (DA ) after
830
illumination with far red light (>700 nm). For better comparison, the amplitude for the largest leaf (relative leaf position
100) of the control plants was set to a FR value of 1. For the
determination of w during illumination, a saturating light pulse,
I
immediately followed by a dark pulse of 5 s duration was
applied. Reduced PSI acceptor (A−) was calculated from the
difference between the FR signal obtained in the dark, and the
amplitude of the light/dark pulse measured during illumination
(Backhausen et al., 1998).
667
Results
Classification of the leaves
Tobacco plants exhibit a typical pattern of leaves with
different sizes which is shown in Fig. 1. The youngest
leaf measuring about 1 cm in length was defined as leaf
number 1. The leaf area increases continuously with leaf
age, and the largest leaves are found in the lower third
of each plant, followed up by several older leaves with
decreasing size. The example in Fig. 1A shows a wildtype tobacco plant at the beginning of the phase of
maximal growth rates. In this state, the plants have 12–14
leaves, and represent the youngest state used for the
experiments. A similar leaf pattern was maintained over
2–3 weeks until a total number of 20–22 leaves per plant
was reached ( Fig. 1B). Afterwards, the number of small
leaves (10–30% of the maximum size) in the apex region
increased strongly, and soon flower buds became visible.
This state was not used for the experiments. A comparison
of plants grown at ambient or elevated CO revealed that
2
Determination of NADP-MDH capacity and in vivo activity
For measurements of the NADP-MDH capacity, 3 leaf discs of
0.9 cm in diameter were cut with a cork borer from both sides
of the leaf midrib (central area) and ground under liquid N in
2
a 1.5 ml microfuge tube. The frozen powder was suspended in
200 ml extraction medium as described by Scheibe and Stitt
(1988). In vitro activation with reduced dithiothreitol (DTT )
and subsequent activity assay was as described by Scheibe and
Stitt (1988).
The samples for the estimation of the in vivo NADP-MDH
activities were obtained by freeze-clamping, using an LCA4 gas
exchange system (ADC, Hoddesdon, UK ) with a modified
PLC2 leaf chamber (Feinmechanische Werkstatt, Universität
Osnabrück). The samples were taken under the respective
growth conditions, and the NADP-MDH activation states were
determined according to Scheibe and Stitt (1988). Aliquots for
measuring chlorophyll and protein content and NADP-MDH
capacity were kept aside from each sample. Chlorophyll was
determined according to MacKinney (1941).
Determination of metabolite contents in the leaves
Samples for the determination of starch, malate and hexose
contents were obtained under the respective growth conditions.
At the end of the light period, 3 samples of 0.9 cm2 leaf area
were cut with a cork borer from both sides of the leaf midrib
(central area) and immediately frozen in liquid nitrogen. For
determination of the malate and hexose contents, the leaf discs
were ground under liquid N in a 1.5 ml microfuge tube,
2
suspended in 300 ml of 20% perchloric acid and incubated for
2 h on ice. Prior to metabolite measurements the samples were
adjusted to pH 7.8 with a mixture of 0.2 M triethanolamine,
2 M KHCO , and 2 M K CO (pH 9.0). Determination of
3
2 3
malate was as described in Backhausen et al. (1994), hexoses
(glucose and fructose) were determined enzymatically according
to Beutler (1985). The starch content of the leaves was
determined enzymatically in ethanolic extracts as described in
Neuhaus and Schulte (1996).
Fig. 1. Leaf area of plants grown under ambient or elevated CO . The
2
youngest (A) and the oldest (B) stage of development used for the
experiments is shown. The youngest leaf measuring about 1 cm in
length was defined as number 1. Plants were grown under ambient (1)
or elevated CO (2). All values are mean (±SD) of at least three
2
different experiments.
668 Backhausen and Scheibe
under elevated CO , the plants possessed 1–2 additional
2
leaves, and the older leaves especially were approximately
10–20% larger.
For the following experiments, wild-type plants and
NADP-MDH mutant plants with altered expression of
this enzyme ( Faske et al., 1997) were used. The NADPMDH-cosuppressing mutant plants were preselected
so that they had either 5–30% (underexpression) or
300–800% (overexpression) of the wild-type NADPMDH capacity. In order to simplify the figures, a common
symbol (1 or 2) was used when there was no significant
difference (SD less than 10%) between wild-type and
NADP-MDH mutant plants.
In order to avoid shifts as caused by differences in leaf
numbers or leaf sizes, it was necessary to define a common
basis to which all measured parameters can be related.
Since the leaf sequence did not differ significantly between
developmental stages and growth conditions used over
the time in which the experiments were performed, the
‘relative leaf position’ was defined as standard, using the
leaf area as a basis. From all plants used for the experiments, the areas of the single leaves were determined.
The leaf with the largest leaf area (a) of each plant was
set to a relative leaf position of 100. The younger leaves
were, according to their leaf area, directly related to this
(e.g. leaf no. 3 in Fig. 1A possesses 25% of the area of
the largest leaf and thus is assigned to a relative leaf
position of 25). In order to differentiate between younger
and older leaves with the same area, the relative leaf
position of the older leaves was calculated by
[(100−a)+100], and therefore reaches values above 100
(e.g. leaf no. 12 in Fig. 1A is clearly older than the largest
leaf, but it has only 65% of the area of the largest leaf,
and thus is assigned to the relative leaf position of 135).
Fresh and dry weights, chlorophyll and protein contents of
the leaves
Clear differences between both growing conditions were
observed in the specific fresh and dry weights of the
leaves. With increasing leaf age and leaf size, the specific
fresh weight in the control plants increased from the
younger leaves to the fully developed leaves by about
20%. The plants grown at elevated CO showed a similar
2
course with leaf age, but their leaves were always about
10% heavier (Fig. 2A). These differences between the
growth conditions became more obvious in the specific
leaf dry weight, where especially younger leaves exhibited
a higher specific dry weight (up to 25%) under elevated
CO (Fig. 2B). The reason for this is unclear, but the
2
higher starch content may partially contribute to
the increased dry weight under elevated CO . A compar2
ison of fresh and dry weight indicated that the higher
fresh weight of older leaves is largely caused by an
increase of about 30% in the water content (Fig. 2C ). In
Fig. 2. Specific fresh weight (A), specific dry weight (B) and leaf water
content (C ). The largest leaf of each plant was set as relative leaf
position 100. Plants were grown under ambient (1) or elevated CO
2
(2). All values are mean (±SD) of at least three different experiments.
that respect, control plants and plants grown under
elevated CO had a similar tendency.
2
In Fig. 3, the protein and chlorophyll contents are
shown. The often described effect of elevated CO , a
2
decrease in the leaf protein content caused by a decreased
amount of Rubisco, was also observed. The protein
content of all leaves was generally reduced under elevated
CO . Especially in younger leaves, the protein content
2
was only half of that in comparable control leaves
( Fig. 3A). The protein content further declined with leaf
size under both growth conditions, and in the oldest
leaves, it was reduced to less than half of the content of
the younger ones. The changes in chlorophyll content of
the leaves showed a different pattern. Leaves with about
40% of maximum leaf size had the highest chlorophyll
content under both conditions. The chlorophyll content
declined afterwards (Fig. 3B), but not as much as did the
protein content. However, even the oldest leaves used
(relative leaf position of 130) still appeared green and
healthy, indicating that senescence had not yet started.
Leaf-age dependent effects of elevated CO on stromal redox state
2
669
Fig. 3. Protein (A) and chlorophyll content (B) of the leaves. Plants
were grown under ambient (1) or elevated CO (2). All values are
2
mean (±SD) of at least three different experiments.
At elevated CO , the chlorophyll content was generally
2
lower in all leaves, as compared to ambient CO . Since
2
the above-described changes in both chlorophyll and
protein content with leaf age have the practical consequence that all other parameters related to them will
be influenced by their changes. Enzyme and metabolite
measurements were thus expressed as relative to leaf area.
Leaf metabolite contents and gas-exchange measurements
The contents of hexoses, starch and malate were determined at the end of the light period. The highest amount
of free hexoses was found in the younger leaves, then
declining with increasing leaf age ( Fig. 4A). In contrast,
malate and starch content of the leaves increased with
size. Under ambient conditions, the malate content of
fully developed leaves was nearly 2.5 times higher than
in smaller ones (Fig. 4B). Under elevated CO , the pattern
2
was similar, but the malate content was only half of that of
control leaves in all cases. In contrast to transgenic potato plants with NADP-MDH underexpression
(Backhausen et al., 1998), no significant difference
occurred in tobacco leaves between the wild-type and
NADP-MDH overexpressing mutant plants. The largest
difference between both growing conditions became
apparent in the starch content of the leaves. Under
elevated CO , all leaves contained generally 3–6 times
2
more starch than leaves of control plants, and this may
significantly contribute to the increased dry weight of the
leaves. In addition, the starch content clearly increased
Fig. 4. Contents of free hexoses (glucose+fructose; A), malate (B) and
starch (C ) in the different leaves. Samples were taken at the end of the
light period. The plants were grown at ambient (1) or elevated CO
2
(2). All values are mean (±SD) of at least three different experiments.
with leaf age. Fully developed leaves contained 2–3 times
more starch than younger ones (Fig. 4C ) which was not
significantly degraded in the dark (data not shown).
Therefore, these leaves seem to function as starch storage
tissues in tobacco, especially under elevated CO .
2
Gas exchange of the leaves was measured in parallel
with chlorophyll fluorescence, and thus it was necessary
to use leaves that had been predarkened for 1 h. After
this dark period, the leaves were illuminated with
400 mmol quanta m−2 s−1 of white light under their
respective growth conditions (380 or 700 ppm CO ) until
2
stable values for assimilation and transpiration were
obtained. The substomatal CO concentration (C )
2
i
was clearly higher at elevated CO . Here, values between
2
380–450 ppm were measured in all leaves, compared to
180–250 ppm under ambient conditions (data not shown).
The transpiration rates declined slightly with leaf age, but
were otherwise very similar in plants grown under both
conditions (data not shown).
670 Backhausen and Scheibe
Although transpiration and substomatal CO concen2
tration were similar in all leaves, large differences were
measured in the assimilation rates of leaves of different
age that cannot be attributed to differences in stomatal
closure. Under both growth conditions, leaves of about
50% of maximal size had the highest assimilation rate,
afterwards it declined strongly. In young leaves, elevated
CO increased the assimilation rate to much higher values
2
compared to control leaves ( Fig. 5A). The often described
reduction of assimilation under elevated CO was
2
observed only in the larger leaves. Their assimilation rates
were only slightly higher or even comparable to ambient
conditions.
Further differences occurred in dark respiration, measured as CO release. The dark respiration was determined
2
at the end of the illumination period, because it was
observed that stomatal closure, as caused by predarkening, strongly influenced the determination of respiration. The highest respiration rates were found in
young leaves of the control plants, and they declined
strongly (to almost zero) with increasing leaf size
(Fig. 5B). Elevated CO clearly suppressed dark respira2
tion, which was particularly evident in younger leaves.
measurements. For photosystem II (PSII ), the ratio
between the ground level of fluorescence (F ) and the
O
maximal variable fluorescence (F ), the F /F ratio of
M
O M
dark-adapted leaves, reflects the number of open PSII
centres, and increasing values for the F /F ratio indicate
O M
a decreasing amount of open PSII centres. In the plants
used in this study, the F /F ratio increased with leaf age
O M
( Fig. 6A), and under elevated CO , the values were about
2
15% higher, indicating less open PSII centres. A similar
situation was found for PSI. The relative number of open
PSI centres was determined by quantifying the amplitude
in DA , induced by far red illumination in the dark
830
( FR), as described under Materials and methods. In
Fig. 7A, the amplitude for the largest leaf (relative leaf
position 100) of the control plants was set to a relative
FR value of 1, and a decreased FR value indicates a
lower number of open PSI centres. The relative amount
of PSI centres changed in the same way as did the PSII
centres. The number of open PSI decreased with leaf age,
and was about 10% lower under elevated CO .
2
Changes in chlorophyll fluorescence (PSII) and P700 (PSI)
The relative amount of open photosystem centres was
determined from chlorophyll fluorescence and DA
830
Fig. 5. Net CO assimilation in the light (A) and dark respiration (B)
2
in the different leaves. Assimilation and dark respiration were determined
under the respective growth conditions. Dark respiration was determined
after a preillumination period of 20 min. The plants were grown at
ambient (1) or elevated CO (2). All values are mean (±SD) of
2
at least three different experiments.
Fig. 6. Chlorophyll fluorescence parameters of PSII. The F /F ratio
O M
(A), q (B) and q (C ) were determined under the respective growth
N
P
conditions at ambient (1) or elevated CO (2). All values are mean
2
(±SD) of at least three different experiments.
Leaf-age dependent effects of elevated CO on stromal redox state 671
2
than under control conditions ( Fig. 7B), and became
more reduced with increasing leaf age.
The measurements of DA
give further information
830
about the redox state at the PSI acceptor site (A−). Here,
a modified method for DA
measurements was used
830
(Backhausen et al., 1998). During illumination, a saturating light pulse that completely oxidizes P700 was applied,
immediately followed by a dark ‘pulse’ of 3 s which allows
P700 to return in its fully reduced state. The difference
between P700 and P700 in the light was compared to
red
ox
the FR signal obtained in the dark (see above). High
values of A− indicate that PSI cannot donate electrons
to the acceptor Fd, probably because it is already partially
reduced. Under ambient conditions, clear differences in
A− were found between wild types and NADP-MDH
mutants. In young leaves of underexpressing plants, the
A− values were around 30%, and increased to over 40%
in the fully developed leaves. In the wild types, A− values
were around 20%, while in the overexpressors values of
only about 15% were determined. In contrast, under
elevated CO the differences between wild-type and
2
mutant plants disappeared, and all A− values ranged
around 15% in young leaves, and slightly increased up to
20% in older leaves (Fig. 7C ).
Capacity and activation state of NADP-MDH
Fig. 7. Absorbance changes at 830 nm (DA ). Parameters of PSI are
830
calculated as FR (A), w (B) and %A− (C ). Open symbols represent
I
growth under ambient CO ; closed symbols represent growth at elevated
2
CO . ((, ,), NADP-MDH underexpression; (#, $): wild-type;
2
(6,+): NADP-MDH overexpression. No significant difference between
mutant plants and wild-types: (1, 2). Open symbols were used for
plants grown under ambient conditions, and closed symbols indicate
growth under elevated CO . All values are mean (±SD) of at least
2
three different experiments.
The q is often taken as an indicator for the transN
thylakoid proton gradient (DpH ). In young leaves, q
N
was slightly lower in control plants, as compared to the
plants grown under elevated CO , indicating a lower DpH
2
in these leaves ( Fig. 6B). This difference disappeared in
the fully developed leaves, and under both growing conditions, q strongly increased when the relative leaf position
N
was above 100. Further differences were measured in the
redox states of both photosystems. For PSII, q showed
P
generally between 5% and 10% higher values in the plants
from elevated CO , indicating more available PSII
2
acceptor (Fig. 6C ). Under both growing conditions, the
q values decreased with leaf age, especially in older
P
leaves. The redox state of PSI can be directly deduced
from w , and it showed a similar pattern as did q . At
I
P
elevated CO , P700 in PSI was 10–15% more oxidized
2
Elevated CO had two different effects upon NADP2
MDH, the key enzyme of the malate valve. First, the
activation state of the enzyme was influenced. The samples
for the determination of the activation state were obtained
using the freeze-clamp method and the activity was calculated as a percentage of V . Under control conditions,
max
the highest activation states were measured in the NADPMDH underexpressing plants. Activation states around
80% were measured in young leaves, and the enzyme was
almost fully activated in older leaves. In wild-type plants,
NADP-MDH was activated to about 30% (Fig. 8A),
while in NADP-MDH overexpressing plants the activation state was slightly lower (around 20%). In plants
grown under elevated CO , generally lower activation
2
states were found, especially in younger leaves of underexpressing plants. This is consistent with the measurements
of q and especially A−, which also indicate lower redox
P
states in the stroma and in the electron transport chains
( Figs 6C, 7C ).
The second effect of elevated CO on NADP-MDH
2
was a decrease in the enzyme capacity. The NADP-MDH
capacity was determined in leaf samples after in vitro
reduction of the enzyme with reduced DTT. In Fig. 8B,
only the capacities of untransformed wild-type plants are
shown. Under ambient conditions, a typical decrease of
the capacity with leaf age was found. In plants grown
under elevated CO , a similar course can be seen, but in
2
all leaves the enzyme capacity was roughly 50% lower
than under ambient conditions. The same results were
672 Backhausen and Scheibe
suppression and promotion of CO assimilation within
2
the same plant. Thus, the effects of elevated CO strongly
2
depend on the developmental state of the leaves, and they
have to be regarded separately. However, both, ageing of
leaves and the adaptation towards elevated CO , caused
2
clear changes in the contents of chlorophyll and of several
metabolites, and altered the protein composition of the
cells ( Webber et al., 1994). It was further intended to
find out to what extent, together with the altered contribution of photorespiration, these effects influenced electron
transport properties and electron distribution in the
chloroplast stroma.
Fig. 8. Activation state (A) and capacity (B) of the NADP-MDH. The
same symbols as in Fig. 7 are used. All values are mean (±SD) of at
least three different experiments.
obtained when protein content or leaf area were used as
a basis for calculation (data not shown). Therefore, it
cannot be regarded as an artefact caused by an altered
chlorophyll content. No significant influence of elevated
CO on transgene expression in the over- or underexpress2
ing plants was found, probably as a consequence of the
use of the constitutive 35S promotor in the transgenic
plants ( Faske et al., 1997). Furthermore, the preselection
procedure used in this study and the comparably wide
range of NADP-MDH capacities used here, between 5%
and 30% of the wild-type capacity for the underexpressors,
and between 300% and 800% for the overexpressors may
further have led to this result.
Discussion
In this work, it was intended to study the effects of longterm exposure of plants to elevated CO . Several, some2
times even contrasting, effects caused by elevated CO
2
have been described earlier (Bowes, 1991; Poorter, 1993).
Therefore, the plants were first analysed in order to find
typical adaptational effects towards elevated CO . One
2
point that will be discussed below in more detail is the
finding that most reported adaptational effects are
dependent upon leaf age. In general, all known phenomena of adaptation towards elevated CO could be
2
confirmed in our plants, but with large differences between
the individual leaves. Leaf-specific differences in chlorophyll and protein content, and in starch and metabolite
accumulation were found, and elevated CO caused both
2
Influence of leaf age on adaptation towards elevated CO
2
During the transition from sink to source into storage
leaves, roughly four different developmental stages can
be separated which differred in their physiological characteristics and their degree of adaptation towards elevated
CO . The youngest leaves with less than 30% of the
2
maximum leaf area (relative leaf position <30) behave
as green sink leaves. Their starch and malate content was
low, but they contained the highest amounts of free
hexoses. They contained much protein, but comparably
little water. In wild-type plants, the NADP-MDH capacity was very high in these leaves. Compared to older
leaves, their chlorophyll content and their CO assimila2
tion rate were lower, but they showed the highest rates
of dark respiration. Elevated CO had strong effects on
2
these leaves. It nearly doubled the assimilation rate, and
strongly suppressed dark respiration, as described earlier
by Bunce and Ziska (1996). Both chlorophyll and protein
content were much lower under elevated CO than in the
2
controls.
The leaves in the middle of the plants (relative leaf
position 30–80) showed the highest assimilation rates,
and assimilation was clearly stimulated under elevated
CO . Some other striking differences between leaves of
2
plants grown under ambient and elevated CO are a
2
decreased protein and chlorophyll content, and a strongly
increased leaf starch content under elevated CO .
2
In the fully developed leaves (relative leaf position
80–100), the assimilation rate declined and was no longer
stimulated by elevated CO . In chlorophyll fluorescence,
2
P700 and NADP-MDH, the differences between both
growing conditions were still visible, but clear changes
compared to younger leaves became apparent. The F /F O M
ratio and especially q increased, while the light-use
N
efficiency of both photosystems (w and w ) decreased.
I
II
This probably reflects the fact that these leaves function
more and more as storage organs for starch. This tendency
continued in the oldest leaves with a decreasing leaf area
(relative leaf position >100). In some parameters, especially with chlorophyll fluorescence and P700, very strong
changes occurred.
Leaf-age dependent effects of elevated CO on stromal redox state 673
2
In conclusion, most of the effects caused by elevated
the decreased quantum yield of the photosystems prevents
this situation in vivo.
CO are not really unique. Most of the differences between
2
It is concluded that the reason for the more oxidized
control plants and plants grown under elevated CO
2
redox state under elevated CO is the decrease in the rate
resemble more or less the changes that occurred during
2
of photorespiration. This is evident from the high C
the normal transition from younger to older leaves, such
i
values (400–450 ppm) that were measured in all leaves of
as loss of protein and chlorophyll, reduced rates of
plants grown under elevated CO . As stated earlier, the
assimilation and respiration, or changes in metabolite
2
ratio in which ATP and NADPH are consumed is lower
contents. The leaves of CO -adapted plants behaved in
2
for CO assimilation than for the photorespiratory pathmost cases simply as if they were larger or older, but
2
way. This means that, on the one hand, photorespiration
without any indication of earlier senescence. Only the
can in fact consume excess energy under ambient condistrong stimulation of CO assimilation in growing leaves
2
tions and thus prevent photoinhibition, as was suggested
and the accumulation of large amounts of starch in older
earlier (Osmond et al., 1997). On the other hand, the
leaves can be attributed as typical CO effects. It cannot
2
differences in A− and in the NADP-MDH activation
be excluded, however, that this may be an indication for
state indicate that photorespiration alone would lead to
N-limitation or for a limiting pot size.
over-reduction, and it can only fulfil its function when
additional sinks for excess electrons such as the malate
Consequences for the stromal redox situation
valve are available. Using over- or underexpression of
NADP-MDH, it was demonstrated that NADP-MDH
Moreover, clear differences between leaves of different
is in fact required to keep Fd in an oxidized state
age, and between both growth conditions were measured
(Backhausen et al., 1998). These results indicate that
in all chloroplast-related parameters that yield informaunder ambient conditions the leaf malate content is higher
tion about the redox state of the various components of
( Fig. 4B), possibly because more electrons are used for
electron transport. First, the number of open PSI and
malate formation, but that dark respiration is even
PSII centres, as deduced from FR values and F /F
O M
increased ( Fig. 5B) and may act as a sink for malate.
ratios, was different. It decreased with leaf age, and was
However, the more oxidized stromal redox state under
generally lower in plants grown under elevated CO . This
2
elevated
CO had consequences for the role of NADPdoes not necessarily mean photoinhibition, but may as
2
MDH
and
of the malate valve. Under elevated CO ,
well be the consequence of the altered (reduced) chloro2
consistently a lower value for A−, i.e. less reduced Fd,
phyll and protein content, indicating an altered composiwas found even in the NADP-MDH underexpressing
tion of the electron transport chains. In all parameters
plants, and the small difference between NADP-MDH
that reflect the redox situation in the electron transport
mutants and wild types determined under ambient condichains (q , w , A−), lower values were measured, and the
P I
tions disappeared.
decreased NADP-MDH activation state indicates that
These results so far indicate that a decreased rate of
even the stromal NADPH/NADP ratio is lower under
photorespiration leads to a decreased production of excess
elevated CO . This means that the complete electron
2
electrons and thus, the demand for mechanisms for their
transport chain, from PSII through PSI into the stroma,
removal, such as the malate valve, is lower. In the simplest
is kept in a more oxidized state. Under both growing
case, this would cause a lower activation state of the
conditions, all parameters were more reduced in the older,
NADP-MDH, but interestingly, the long-term adaptation
starch-storing leaves.
to elevated CO even decreased the expression of the
2
It must be noted that in neither case was evidence
enzyme in the untransformed wild-type plants ( Fig. 7A).
found for another effect of elevated CO that is likely to
It is known that several factors such as chlorophyll
2
occur, namely over-reduction caused by metabolite accucontent, Chl a/b ratio and the expression of various
mulation and subsequent P limitation. In several cases,
enzymes are regulated by endogenous factors, such as the
i
mostly observed with isolated chloroplasts, the rate of
developmental state, and by external factors. Both translaCO fixation is inverse to the amount of excess electrons.
tion and transcription of several chloroplast-encoded pro2
This situation could be expected in the older leaves where
teins are activated by light (Danon and Mayfield, 1994).
the assimilation rate did not differ much between ambient
The extent of the light-induced increase in translation is
and elevated CO , and where the starch content had
often 50–100-fold (Fromm et al., 1985), but the respective
2
increased significantly. Starch accumulation is often
mRNA contents are mostly not increased (Levings and
regarded as an indicator for P limitation (Stitt and Quick,
Siedow, 1995). Possibly, light regulates the binding of an
i
1989). Although the redox state of the fully developed
activator protein, by thioredoxin-mediated reduction of
leaves with the highest amount of starch was rather
regulatory disulphide bridges within this activator protein
reduced, no evidence was found for over-reduction. It is
(Danon and Mayfield, 1994).
assumed that the altered electron transport composition,
Furthermore, redox effects on gene expression of
the decreased Rubisco content, the high q , and especially
several nuclear-encoded genes influencing transcription
N
674 Backhausen and Scheibe
or translation have been reported. There is some evidence
that H O and other reactive oxygen species act as signals
2 2
to increase the expression of superoxide dismutase and
of ascorbate peroxidase during stress responses (Schreck
et al., 1991). Karpinski et al. (1997) suggested a signal
transduction pathway mediated by reduced glutathione.
In the nucleus, DNA-binding transcriptional factors of
the PEBP-type are known in mammalian cells which
possess a specific, highly conserved domain (Runtdomain) (Bae et al., 1995). The Runt-domain contains
two cysteines which can be reduced by thioredoxin. This
redox change alters the DNA-binding properties of the
DNA-binding proteins (Akamatsu et al., 1997).
Alternatively, phosphorylation cascades have been
suggested as signal transducers. The synthesis of the lightharvesting complex (LHC ) II protein is under transcriptional control; a largely reduced plastoquinone pool in
the chloroplasts inhibits LHCII transcription ( Escoubas
et al., 1995). The authors suggested that reduced plastoquinone activates a phosphorylation cascade that is
passed from the stroma into the cytosol and controls
LHC expression.
The capacity of the NADP-MDH showed clear changes
correlating with both leaf and plant age. The highest
capacities were determined in young sink leaves, and
during the sink/source transition ( Vivekanandan and
Edwards, 1987; Merlo et al., 1993; Faske et al., 1997),
when the ability of the leaves to fix CO is not yet fully
2
developed. Several antioxidative enzymes such as ascorbate peroxidase or superoxide dismutase, show changes
dependent upon leaf age that appear to follow the same
pattern (Polle, 1996b). Superimposed on this regulation
is a further modulation that clearly depends on the actual
redox situation, as is the case under elevated CO . It has
2
been observed earlier that the decreased oxidative stress
under elevated CO leads to a decreased expression of
2
antioxidative enzymes (Polle, 1996b; Polle et al., 1997).
Although both enzyme sets have not yet been measured
simultaneously, this can be taken as evidence that the
expression of enzymes related to poising or removal of
excess electrons is regulated by a common signalling
system.
Acknowledgements
We thank Susanne Vetter, Marielle Singer-Bayrle and Ilka
Haferkamp (Osnabrück) for valuable help with the experiments,
and Dr M Faske (Osnabrück) for critical discussion. This work
was financially supported by the Deutsche Forschungsgemeinschaft (DFG; Sche 217/5).
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