Journal of Experimental Botany, Vol. 53, No. 373, pp. 1475–1483, June 2002
High light-induced switch from C3-photosynthesis
to Crassulacean acid metabolism is mediated by
UV-Aublue light
Thorsten E. E. Grams1,2,4 and Stephan Thiel1,3,5
1
GSF-National Research Center for Environment and Health, Institute of Soil Ecology, Department of
Environmental Engineering, Ingolstädter Landstraße, D-85764 Neuherberg, Germany
2
Forest Botany, Department of Ecology, Technische Universität München, Am Hochanger 13, D-85354
Freising, Germany
3
Forschungszentrum Karlsruhe, Institut für Meteorologie und Klimaforschung, Bereich Atmosphärische
Umweltforschung, Kreuzeckbahnstraße 19, D-82467 Garmisch-Partenkirchen, Germany
Received 10 September 2001; Accepted 27 January 2002
Abstract
The high light-induced switch in Clusia minor
from C3-photosynthesis to Crassulacean acid metabolism (CAM) is fast (within a few days) and
reversible. Although this C3uCAM transition has been
studied intensively, the nature of the photoreceptor
at the beginning of the CAM-induction signal chain
is still unknown. Using optical filters that only
transmit selected wavelengths, the CAM light induction of single leaves was tested. As controls the
opposite leaf of the same leaf pair was studied
in which CAM was induced by high unfiltered
radiation (c. 2100 mmol m2 s1). To evaluate the
C3-photosynthesisuCAM transition, nocturnal CO2
uptake, daytime stomatal closure and organic acid
levels were monitored. Light at wavelengths longer
than 530 nm was not effective for the induction of the
C3uCAM switch in C. minor. In this case CAM was
present in the control leaf while the opposite leaf
continued performing C3-photosynthesis, indicating
that CAM induction triggered by high light conditions
is wavelength-dependent and a leaf internal process.
Leaves subjected to wavelengths in the range of 345–
530 nm performed nocturnal CO2 uptake, (partial)
stomatal closure during the day (CAM-phase III), and
decarboxylation of citric acid within the first 2 d after
the switch to high light conditions. Based on these
experiments and evidence from the literature, it is
4
suggested that a UV-Aublue light receptor mediates
the light-induced C3-photosynthesisuCAM switch in
C. minor.
Key words: Citrate decarboxylation, Clusia minor,
C3-photosynthesisuCAM
switch,
spectral
irradiance,
UV-Aublue light receptor.
Introduction
Crassulacean acid metabolism (CAM) is a typical ecophysiological adaptation of plants to arid conditions. Its
characteristics are nocturnal CO2-uptake via the enzyme
phosphoenolpyruvate carboxylase (PEPCase, CAMphase I sensu Osmond, 1978) and subsequent accumulation of the fixed CO2 in the form of organic acids (in
the case of Clusia these are malate and citrate) in the
vacuole. Apart from periods of high stomatal conductance in the morning and evening hours (CAM-phases II
and IV, respectively), CAM plants show a typical daytime
stomatal closure (CAM-phase III) paralleled by decarboxylation of the stored organic acids in the cytoplasm.
Subsequently, the CO2 released is assimilated into
carbohydrates by 1,5-ribulose bisphosphate carboxylaseu
oxygenase (Rubisco) and used in the C3-photosynthesis
carbon reduction cycle. In Clusia the CAM-phases II
and IV are often very pronounced and, therefore,
Present address: Department of Integrative Biology, University of California, 3060 Valley Life Science Building, Berkeley, CA 94720, USA.
To whom correspondence should be sent at: Forschungszentrum Karlsruhe, Institut für Meteorologie und Klimaforschung, Bereich Atmosphärische
Umweltforschung, Kreuzeckbahnstraße 19, D-82467 Garmisch-Partenkirchen, Germany. Fax: q49 8821 183 295. E-mail: [email protected]
5
ß Society for Experimental Biology 2002
1476
Grams and Thiel
CAM-phase III is less obvious (for a review of CAM in
the genus Clusia see Lüttge, 1999).
Several plant species are able to switch from the
more common mode of photosynthesis (C3) to CAM,
either during their ontogenetic development (e.g.
Mesembryanthemum crystallinum; Adams et al., 1998) or
in response to particular environmental conditions (e.g.
drought). Within the tropical tree genus Clusia the
phenomenon of facultative CAM is widespread and manifold (Lüttge, 1999). The highly flexible species C. minor
has proved to be a useful model plant for the study
of CAM induction because the C3uCAM-switch is fast
(within 1 d), reversible, and can be studied reliably by
gas exchange measurements and analysis of accumulated
organic acids. Several environmental stresses such as
water shortage (Franco et al., 1991, 1992; Borland et al.,
1996, 1998; Grams et al., 1997b, 1998), high irradiance,
dayunight temperatures (Haag-Kerwer et al., 1992;
Borland et al., 1993; Grams et al. 1997a; Roberts et al.,
1998; Herzog et al., 1999) and nitrogen supply (Franco
et al., 1991) have been shown to be effective at inducing the C3uCAM transition. Although in C. minor the
exposure to high irradiance is one of the most intensively
studied CAM-inducing factors, to date the nature of the
initial photoreceptor sensing the high radiation has
remained unclear.
Plants employ a series of photoreceptors, absorbing
in different regions of the solar spectrum. The energy
absorbed by a photoreceptor starts a cascade of
physiological processes that enables plants to monitor
magnitude (‘quantity’) and wavelength distribution
(‘quality’) of the radiation environment (photosensing).
For example, in the facultative CAM plant Kalanchoë
blossfeldiana, CAM is induced by short days via red-light
controlled synthesis of PEPCase mediated through the
phytochrome system (Brulfert et al., 1982, 1988). Other
possible photoreceptors or signalling pigments are chlorophyll (light harvesting for photosynthesis) and the blue
light receptor (cryptochrome). The latter is known as a
photoreceptor, for example, for phototropism, stomatal
aperture and entrainment of endogenous rhythms in
plants and animals (Baskin and Iino, 1987; Batschauer,
1998; Christie et al., 1998; Cashmore et al., 1999).
The aim of the present study was to determine which
of these photosensitive pigments elicits the high light
(here photosynthetically active radiation (PAR) of c.
2000 mmol m2 s1) induced C3uCAM transition in C.
minor. To answer this question, the spectral range of the
radiation applied to the leaves was manipulated using
optical filters. The gas exchange patterns and organic acid
accumulation was then studied in response to the light
manipulations as a reliable way to monitor the CAM
induction. Schmitt et al. showed in a rewatering experiment that C. minor is able to perform CAM in one leaf
and C3-photosynthesis in the opposite leaf of the same
leaf pair (Schmitt et al., 1988). This encouraged the use
of one leaf as a control where the leaf was switched from
C3-photosynthesis to CAM using high light conditions,
while the opposite leaf was tested by selecting wavelengths and monitoring leaf responsiveness with respect to
CAM induction.
Materials and methods
Spectroradiometric measurements
Spectral data were recorded with a spectroradiometer system
which consists of a double-monochromator, a photomultiplier,
electronic devices (high voltage supply, amplifier and ADC), and
an entrance optic (quartz fibre with a quartz diffuser plate; for
details see Thiel et al., 1996). The system was calibrated against
a 100 W quartz halogen lamp of a known spectral emission,
determined via comparison with a 1000 W lamp (calibrated by
the Physikalisch Technische Bundesanstalt, Braunschweig,
Germany).
The spectral irradiance of the light source used was monitored
at the distance of the halogen lamp to the upper leaf surface
during the experiments. Two sets of experiments were performed (see below: experimental design) using first UG11,
OG550 (Schott, Mainz, Germany) and LCLS-550 filters (Laser
Components, Olching, Germany, see Fig. 1a for spectral
transmittance) and, second, a series of long pass filters
(WG345, GG395, GG400, GG455, OG530, OG550, all from
Schott, Mainz, Germany, see Fig. 1b for spectral transmittance).
Plants
Clusia minor L. plants were derived from cuttings from four
individuals, all belonging to one clone, which originated from
a plant that was collected in the Sierra de San Luis, Venezuela
(Popp et al., 1987). This clone is well characterized in at
least 15 publications and was therefore chosen for this study.
Cuttings were grown in standard soil (Fruhsdorfer Einheitserde
LD80) in a glasshouse, kept well watered and were fertilized
regularly (using a 1 : 1 mixture of Hakaphos green and
Hakaphos blue, Campo, Münster, Germany). At the age of
3 months, plants were acclimated for 3–6 months in a
programmed climate chamber to low irradiance (12u12 h
dayunight at c. 120 mmol m2 s1), constant temperature of
25 8C and relative humidity of 50%. The experimental plants
were at least 6-months-old and had three to four new leaf pairs
which developed under the low light conditions in the climate
chamber. The first or second fully developed leaf pair was
chosen for the experiments in which plants were kept well
watered.
Experimental set-up and assessment of leaf gas exchange
Gas exchange of individual leaves was assessed using a
minicuvette system (Fa. Walz, Effeltrich, Germany) with two
climate- and light-controlled Plexiglas chambers (volume: 0.7 l)
each enclosing one leaf of the same leaf pair. Measurements
were performed under standardized conditions (cuvette temperature of 25.0"0.1 8C and dew point of 12.0"0.1 8C). Due to
the effective climate control of the minicuvette system the
increase of leaf temperature after the switch to high irradiance
was small (2.4"1.5 8C) and similar for control and treatment
leaves. Hence, the increase of vapour pressure deficit (VPD) with
increasing irradiance was small. However, in C. minor, VPD is
UV-Aublue light induced CAM 1477
Fig. 2. Spectral irradiance of the used light sources (control and
treatment) under high light conditions (c. 2100 mmol m2 s1, open
and closed circles) and low light conditions (c. 120 mmol m2 s1, thin
line and squares). The thick line shows the ratio of spectral irradiance of
both lamps under high light conditions with a deviation between the
lamps in the range of "10%.
Fig. 1. Spectral transmittance of the optical filters used in the
experiments. (a) Filters used in the first experimental set. Circles:
‘UG11’ filter, 2 mm thickness. Dashed line: short pass filter ‘-550 nm’,
3 mm thickness. Solid line: long pass filter ‘)550 nm’, 2 mm thickness.
(b) Cut-off filters used in the second experimental set. From left to right:
WG345, GG395, GG400, GG455, OG530, OG550, all 2 mm thick.
Filter names give, theoretically, wavelength of 50% transmittance.
not effective for the C3uCAM transition (Herzog, 1994). The rates
of leaf gas exchange were calculated according to von Caemmerer
and Farquhar (von Caemmerer and Farquhar, 1981).
As a light source, halogen lamps were used (Master Line,
20W, type no. 17717, Philips, The Netherlands), which were
selected for similar spectral distributions (Fig. 2). Each lamp was
mounted in a light unit at a distance of 15 cm to the leaf. Slots
allowed the installation of optical filters between lamp and leaf.
To avoid external light the Plexiglas chambers were covered with
aluminium foil.
One leaf was switched from C3-photosynthesis to CAM by
high light (control leaf ), while the opposite leaf of the same leaf
pair was used for the induction manipulations using selected
wavelengths. This treatment leaf was measured for its responsiveness to the light manipulation and light-induced C3uCAM
switch. The use of optical filters reduce the spectral irradiance
within the experimental wavelength range (transmittance below
1.0, see Fig. 1). Therefore, the irradiance for the control leaf was
reduced to the same order of magnitude by quartz glass, NG11
or NG4 filters (Schott, Mainz, Germany).
Organic acid analyses
Leaves were sampled at the end of each experiment (end of light
phase) and stored at 75 8C. After drying of the leaf samples in
a microwave oven (Popp et al., 1996), malate and citrate were
determined enzymatically in hot water extracts of the dried leaf
material according to Möllering (Möllering, 1974, 1985). The
titratable Clusia leaf acidity at dusk is known to be negatively
correlated with the CAM capacity (Zotz and Winter, 1993).
Therefore, differences between control and treatment leaves at
dusk were used to evaluate the CAM induction: CAM is not (or
to a lesser extent) expressed in the treatment leaf, if its organic
acid concentration is higher than in the control leaf.
Statistics
Significant levels were tested by means of Student’s t-test.
Experimental design
The aim of a first set of experiments was to contain the spectral
range of the effective radiation. Figure 3 outlines the experimental scheme that was followed. Each experiment is represented by a box that gives the filter used and a number indicating
whether the C3uCAM switch occurs (‘1’) or not (‘0’). In the first
experiment the UG11 filter tested the effectiveness of radiation
from the ultraviolet part of the spectrum and radiation above
670 nm, while most of the photosynthetically active radiation
was blocked. If the radiation that passed through the UG11
filter was effective for the C3uCAM switch (denoted as ‘1’
in Fig. 3), an additional experiment with a filter that transmits
only radiation above 550 nm would be necessary to decide
which of both possible receptors ( phytochrome or UV-B
receptor) was responsible for the CAM induction. If, on the
other hand, the UG11 filter blocked the effective radiation
(i.e. no CAM switch, denoted as ‘0’) further experiments would
be needed as shown in the right branch of Fig. 3: first, a filter
which mainly transmits radiation between 400 nm and 550 nm
(‘-550 nm’) and, secondly, a filter which only transmits
radiation above 550 nm can be used to detect the involved
photoreceptor.
In a second set of experiments several cut-off filters (see
Fig. 1b for spectral transmittance) were employed to study, in
more detail, the spectral response of the CAM induction within
the wavelength range determined in the preceding experiments.
1478
Grams and Thiel
Results
Representative results of the first set of experiments are
shown in Fig. 4: during the first 24 h all leaves were
exposed to a radiation regime similar to the growth
conditions in the climate chamber (i.e. low photosynthetic
Fig. 3. Processing chart for the first set of experiments. The first
experiment was conducted using the ‘UG11’ filter, which blocks mainly
photosynthetically active radiation (Fig. 1a). If CAM is induced (‘1’), the
following experiment will be performed using the ‘)550’ long pass filter.
If CAM is not induced (‘0’), the next experiment will be conducted with
the ‘-550 nm’ short pass filter (and so on). This sequence of
experiments determines the photoreceptor which is responsible for high
light CAM induction. Please refer to text for details.
photon flux density (PPFD) of about 120 mmol m2 s1,
see Fig. 2 for spectral irradiance). Under these conditions
all leaves showed the typical gas exchange pattern of
C3-photosynthesis: CO2 uptake and stomatal opening
was restricted to daytime and plants displayed respiratory
CO2 loss during the night. At ‘time zero’ the PPFD of the
control leaf (Fig. 4a, c, e) was increased to about
2100 mmol m2 s1 and leaves subsequently switched
from C3-photosynthesis to CAM with nocturnal
CO2-uptake anduor a clearly expressed CAM-phase III.
The corresponding opposite leaf received only radiation
of selected wavelengths. Radiation which passed through
the UG11 filter (mainly -400 nm, see Fig. 1a) was not
effective at inducing the C3uCAM switch (Fig. 4b).
Moreover, since photosynthetically active radiation was
blocked by this filter, plants did not take up CO2 either
during the day, and stomata stayed closed day and night.
By contrast, wavelengths below 550 nm (filter ‘-550’ in
Fig. 1a) induced a switch to CAM with a clearly expressed
CAM-phase III and a small CO2-uptake during the
second night after the irradiance increase (Fig. 4d). The
following experiment (Fig. 4f ) shows that CAM is not
induced by wavelengths above 550 nm (filter ‘)550’
in Fig. 1a): plants remained in the C3-mode of photosynthesis showing only daytime stomatal opening and
Fig. 4. Net CO2 exchange (JCO2, –m–) and stomatal conductance (gH2O, –––) of individual C. minor leaves under increasing irradiance. At ‘time zero’
the light conditions were switched from approximately 120 to 2100 mmol m2 s1 for control leaves (a, c, e), while the corresponding opposite leaves of
the same leaf pairs received only selected wavelengths (b, d, f, respectively). Following the processing chart (Fig. 3) the ‘UG11’, ‘-550’ and ‘)550’
filters were used. Experiments shown are representative for a total of three experiments. Dark bars indicate night-time.
UV-Aublue light induced CAM 1479
CO2 uptake, while stomata closure and some respiratory
CO2 loss was observed during the night.
The second set of experiments is shown in Fig. 5:
similar to the earlier figures, the gas exchange patterns of
control leaves are presented on the left hand side (Fig. 5a,
c, e, g, i, k), while the spectral responsivenesses of high
light CAM induction (corresponding opposite leaves of
the same leaf pairs) are shown to the right (Fig. 5b, d, f, h,
j, l). Under low light conditions (24 to 0 h) all leaves
showed C3-photosynthesis with daytime stomatal opening
and CO2-uptake, while stomata stayed closed during the
night and respiratory CO2 loss was recorded. After the
irradiance was increased all control leaves switched to
CAM with nocturnal CO2-uptake, (partial) stomatal
closure during the day (CAM-phase III; see gH2O) anduor
daytime citrate breakdown (Fig. 6). In some control
Fig. 5. Net CO2 exchange (JCO2, –m–) and stomatal conductance (gH2O, –––) of individual C. minor leaves under increasing irradiance. At ‘time zero’
the light conditions were switched from approximately 120 to 2100 mmol m2 s1 for control leaves (a, c, e, g, i, k), while the corresponding opposite
leaves of the same leaf pairs received only selected wavelengths (b, d, f, h, j, l, respectively). Dark bars indicate night-time.
1480
Grams and Thiel
Fig. 6. Differences (values of control leaves minus values of treatment
leaves) in organic acid concentrations between control and treatment
leaves of the same leaf pair at dusk. Negative values indicate a lower acid
concentration in the control leaf relative to the treatment leaf and, thus,
no or only small CAM capacity of the treatment leaf. Results of the first
set of experiments are shown in (a) and (b) (with n ¼ 3–6"1 SE) while
(c) and (d) give the data for the second set of experiments).
leaves (Fig. 5e, g) the high light-induced C3uCAM switch
was relatively slow and the expression of phase III only
started during the second day under high light conditions.
However, CAM induction of these leaves was confirmed
by low organic acid levels at dusk (see below: Fig. 6).
Wavelengths above 345 and 395 nm (Fig. 5b, d) proved
to be effective for the C3uCAM switch in C. minor:
stomatal conductance (gH20) and CO2 gas exchange
showed all four phases of CAM (Osmond, 1978).
Leaves at ‘)400’ and ‘)455 nm’ (Fig. 5f, h) showed a
slow CAM induction similar to the corresponding control
leaves (Fig. 5e, g). Thus, wavelengths above 400 and
455 nm are still effective, although the C3uCAM switch
was slowed down: CO2-uptake was small during the
second night and CAM-phase III was hardly expressed.
The experiments using wavelengths above 530 and
550 nm (Fig. 5j, l) confirmed the results presented in
Fig. 4: wavelengths above 550 nm are not effective for
the CAM induction.
The results presented above (Figs 4, 5) are summarized
in Table 1 showing integrated nocturnal CO2-exchange
under the different light conditions (low irradiance and
high irradiance, including and excluding blue light). In the
case of low irradiance, both control and treatment leaves
showed CO2-loss during the night. After the irradiance
was increased in the range of 345–530 nm (‘UV-A blue
light’) to high light conditions, treatment leaves had
similar nocturnal CO2-uptake as control leaves under
unfiltered radiation (8.21 and 7.19 mmol m2, respectively; P)0.862). However, if blue light was excluded
after the irradiance increase, integrated nocturnal
CO2-exchange was negative (CO2-loss) for the treatment
leaves, while the opposite control leaves switched to CAM
(3.24 and 4.35 mmol m2 for treatment and control
leaves, respectively; P-0.006).
At the end of the experiments (end of light phase)
leaves of the gas-exchange measurements were harvested
and analysed for organic acid levels (Fig. 6). The
differences in malate or citrate concentrations within
one leaf pair (concentration of control leaf minus
concentration of treatment leaf ) indicates the degree of
CAM induction in the treatment leaves. If the difference
is close to zero, both leaves used about the same amount
of organic acid for daytime decarboxylation as an internal
CO2 source during CAM phase III. A negative value
(higher organic acid level in the treatment leaf at dusk)
indicates that the treatment leaf did not (or to a lesser
extent) switch to CAM since less organic acid was
decarboxylated. Differences in malate concentrations
between control and treatment leaves (Fig. 6a, c) did
not give a clear trend with respect to the high lightinduced C3uCAM switch. However, differences in citrate
concentrations at dusk confirmed that CAM was induced
by wavelengths below 550 nm (Fig. 6b), since the
treatment and control leaves decarboxylated the same
amount of citrate during the day (i.e. ‘-550’-value is not
significantly different from zero). Wavelengths which
passed through the UG11 and ‘)550’ filters were not
effective for the citrate decarboxylation: in both cases
treatment leaves decarboxylated no citrate or decarboxylated less citrate than the control leaves (see negative
values in Fig. 6b; both values are significantly different
from ‘-550’ citrate-value, P-0.05). Similar conclusions
can be drawn from the citrate data of the second
experimental set (Fig. 6d): wavelengths above 455 nm
appear not, or only partially, to be effective for the high
light-induced C3uCAM switch (negative values), whereas
experiments with wavelengths above 345, 395 and 400 led
to no or only minor differences in citrate concentrations
between treatment and control leaves, indicating that
both leaves switched to CAM.
Discussion
Most of the Clusia species studied so far are facultative
CAM plants, in which CAM is induced by various
environmental factors like drought, increased daytime
temperature or increased irradiance (Grams et al., 1998;
Lüttge, 1999). CAM induction caused by high irradiance
is well known and has been studied intensively, especially
in C. minor (Schmitt et al., 1988; Franco et al., 1991;
Haag-Kerwer et al., 1992; Borland et al., 1993, 1996;
Roberts et al., 1998; Herzog et al., 1999). It is shown
here that the photoreceptor responsible for the CAM
UV-Aublue light induced CAM 1481
Table 1. Integrated nocturnal CO2-exchange of control and treatment leaves under different light conditions
Experiments of Figs 4 and 5 are summarized with respect to blue light (345–530 nm) application. ‘Low irradiance’: summary of all experiments under
low light conditions. ‘High irradiance including UV-Aublue light’—High spectral irradiance in the UV-A- and blue range: summary of experiments
with filters ‘-550’, ‘)345’, ‘)395’, ‘)400’, and ‘)455’. ‘High irradiance excluding UV-Aublue light’—No or negligible radiation from the UV-A- and
blue range: summary of experiments with filters ‘UG11’, ‘)530’ and ‘)550’. P-values show if treatment leaves are significant different from control
leaves. Means"1 SE (n) ¼ 10).
Integrated nocturnal
CO2 exchange
(mmol m2)
Low irradiance
Treatment leaves
Control leaves
Significance level
8.83"0.96
8.27"0.89
P)0.669
induction by high irradiance is most effective in the
range of 345 to 530 nm (UV-Aublue light, Figs 4, 5, 6;
Table 1). Since light above 530 nm was not effective for
this CAM induction, a phytochrome induction system
can be excluded as the primary chromophore involved in
the high light CAM induction process.
Obviously, CAM induction caused by high irradiance
requires the attainment of a certain threshold value,
which can be estimated from a biologically weighted
radiation quantity as follows:
ð
Ebiol ¼ EL ðlÞ3AF ðlÞ3 Sbiol ðlÞdl
where Ebiol represents the biological effective irradiance
(here for the C3uCAM transition), EL the spectral
irradiance of the radiation source, AF the filter transmittance and Sbiol the action spectrum of the CAM
induction. If the action spectrum of alfalfa hypocotyl
phototropism (mediated by a blue light receptor; Baskin
and Iino, 1987) is taken to estimate Ebiol for the high light
CAM induction, the applied low light level (C3-photosynthesis conditions) equals 167 mW m2, while for
unfiltered radiation under high light conditions (CAM
conditions) a value of 2750 mW m2 is calculated. These
values also show that the amount of UV-Aublue light
passing through the ‘UG11’ filter and resulting in an
biologically effective radiation of 59 mW m2 is below
the threshold value for the CAM induction. On the other
hand the values for ‘)400’ and ‘)455’ (2479 and
1392 mW m2, respectively) are above the threshold,
since CAM was still induced by these wavelengths.
Despite the fact that a threshold value for the CAM
induction has to be reached, the C3uCAM transition
might be a gradual process rather than an all-or-none
response, since the amount of nocturnally fixed CO2 is
known to saturate with the integrated light sum from
the previous day (Schmitt et al., 1988). The fact that
CAM induction does not occur in experiments with
wavelengths higher than 530 nm, shows that the maximum of the action spectrum for CAM induction is in
the range of blue light. However, the investigation of a
High irradiance
Including UV-Aublue light
Excluding UV-Aublue light
8.21"4.22
7.19"4.01
P)0.862
3.24"0.63
4.35"2.30
P-0.006
dose–response relationship above the threshold and the
determination of an action spectrum remains a task for
further research.
High irradiance in the range of 345–530 nm induces
nocturnal CO2 uptake anduor (partial) daytime stomatal
closure (phase III of CAM, sensu Osmond, 1978; see
Table 1; Figs 4, 5) as well as decarboxylation of citric
acids (Fig. 6). Concentrations of malate at the end of the
light phase stayed low (2.1"1.8 mmol m2, data not
shown), since nocturnally accumulated malate will be
broken down completely during the day. However,
these experiments monitored only the beginning of the
C3uCAM transition, which will result, if environmental
conditions remain unchanged, in a higher nocturnal CO2
uptake and organic acid (malate and citrate) turnover.
Similar findings have been reported previously (Herzog
et al., 1999) which showed that in C. minor diurnal
malate oscillations are not present during the first day
under high irradiance (i.e. an increase from 100 to
1000 mmol m2 s1), while citrate breakdown had
already started (cf. Fig. 6). Likewise, a much closer
correlation was found between PPFD and citrate breakdown than malate breakdown during the first days of
CAM induction in C. minor and it was concluded that
light-induced CAM starts with daytime citrate breakdown (Borland et al., 1996). Thus, following the concept
of a limited vacuole storage capacity for Hq, only
daytime citrate breakdown and, thus, a reduced titratable
leaf acidity at dusk allows nocturnal CO2 uptake (Zotz
and Winter, 1993). These findings were confirmed and the
citrate concentrations were used to confirm CAMinduction and to evaluate the effective wavelengths for
the high light-induced C3uCAM switch (Fig. 6).
The direct impact of blue light on stomatal aperture,
as shown for many C3 plants (Briggs and Huala, 1999),
seems to be unlikely if CAM is present. Several studies
(Lee and Assmann, 1992; Mawson and Zaugg, 1994;
Tallman et al., 1997) report that this stomatal blue-light
response is either lost or inhibited in C3uCAM intermediate plants after the transition to CAM. Rather, the
signal transduction pathway of blue light-mediated CAM
1482
Grams and Thiel
induction in C. minor might be similar to the short-day
CAM induction in K. blossfeldiana, where red light
is sensed by the phytochrome system and CAM is
induced via de novo synthesis of PEPCase (or
phosphoenolpyruvate carboxykinase; Brulfert et al.,
1985, 1988).
In the habitats of C. minor, increases of irradiance
(and simultaneous water shortage) are relatively frequent (e.g. due to leaf senescence of the surrounding
vegetation during drought periods). Switching from
C3-photosynthesis to CAM is an important and useful
mechanism to cope with excess radiation energy (Herzog
et al., 1999; Lüttge, 1999, 2000). Therefore, the sensing of
high irradiance via UV-A blue light and the subsequent
switch to CAM gives C. minor a larger ecological
amplitude and, in general, a better performance under
highly variable radiation conditions.
Acknowledgements
The authors are grateful to Dr H-D Payer, Dr HK Seidlitz
and the staff of the EPOKA team at the GSF-National
Research Center for Environment and Health for technical
assistance and the opportunity to perform this study in the
EPOKA phytotrons. Dr R Matyssek and Dr TE Dawson are
thanked for valuable comments on an earlier version of
the manuscript.
References
Adams P, Nelson DE, Yamada S, Chmara W, Jensen RG,
Bohnert HJ, Griffiths H. 1998. Growth and development
of Mesembryanthemum crystallinum (Aizoaceae). New
Phytologist 138, 171–190.
Baskin TI, Iino M. 1987. An action spectrum in the blue and
ultraviolet for phototropism in alfalfa. Photochemistry and
Photobiology 46, 127–136.
Batschauer A. 1998. Photoreceptors of higher plants. Planta
206, 479–492.
Borland AM, Griffiths H, Broadmeadow MSJ, Fordham MC,
Maxwell C. 1993. Short-term changes in carbon-isotope
discrimination in the C3uCAM intermediate Clusia minor L.
growing in Trinidad. Oecologia 95, 444–453.
Borland AM, Griffiths H, Maxwell C, Fordham MC,
Broadmeadow MSJ. 1996. CAM induction in Clusia minor
L. during the transition from wet to dry season in Trinidad:
the role of organic acid speciation and decarboxylation.
Plant, Cell and Environment 19, 655–664.
Borland AM, Tecsi LI, Leegood, RC, Walker RP. 1998.
Inducibility of crassulacean acid metabolism (CAM) in
Clusia species: physiologicalubiochemical characterization
and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different
degrees of CAM. Planta 205, 342–351.
Briggs WR, Huala E. 1999. Blue-light photoreceptors in higher
plants. Annual Review of Cell and Developmental Biology
15, 33–62.
Brulfert J, Kluge M, Güclü S, Queiroz O. 1988. Interaction of
photoperiod and drought as CAM inducing factors in
Kalanchoë blossfeldiana Poelln. cv. Tom Thumb. Journal of
Plant Physiology 133, 222–227.
Brulfert J, Müller D, Kluge M, Queiroz O. 1982.
Photoperiodism and Crassulacean acid metabolism. I.
Immunological and kinetic evidence for different patterns
of phosphoenolpyruvate carboxylase isoforms in photoperiodically inducible and non-inducible Crassulacean acid
metabolism plants. Planta 164, 326–331.
Brulfert J, Vidal J, Keryer E, Thomas M, Gadal P, Queiroz O.
1985. Phytochrome control of phosphoenolpyruvate carboxylase synthesis and specific RNA levels during photoperiodic induction in a CAM plant and during greening in
a C4 plant. Physiologie Ve´ge´tale 23, 921–928.
Cashmore AR, Jarillo JA, Wu Y-J, Liu D. 1999. Cryptochromes:
blue light receptors for plants and animals. Science
248, 760–765.
Christie JM, Reymond P, Powell GK, Bernasconi P,
Raibekas AA, Liscum E, Briggs WR. 1998. Arabidopsis
NPH1: a flavoprotein with the properties of a photoreceptor
for phototropism. Science 282, 1698–1701.
Franco AC, Ball E, Lüttge U. 1991. The influence of nitrogen,
light and water stress on CO2 exchange and organic acid
accumulation in the tropical C3-CAM tree, Clusia minor.
Journal of Experimental Botany 42, 597–603.
Franco AC, Ball E, Lüttge U. 1992. Differential effects of
drought and light levels on accumulation of citric and malic
acid during CAM in Clusia. Plant, Cell and Environment
15, 821–829.
Grams TEE, Borland AM, Roberts A, Griffiths H, Beck F,
Lüttge U. 1997a. On the mechanism of reinitiation of
endogenous crassulacean acid metabolism rhythm by temperature changes. Plant Physiology 113, 1309–1317.
Grams TEE, Haag-Kerwer A, Olivares E, Ball E, Arndt S,
Popp M, Medina E, Lüttge U. 1997b. Comparative measurements of chlorophyll a fluorescence, acid accumulation and
gas exchange in exposed and shaded plants of Clusia minor L.
and Clusia multiflora H. B. K. in the field. Trees—Structure
and Function 11, 240–247.
Grams TEE, Herzog B, Lüttge U. 1998. Are there species in the
genus Clusia with obligate C3-photosynthesis? Journal of
Plant Physiology 152, 1–9.
Haag-Kerwer A, Franco AC, Lüttge U. 1992. The effect of
temperature and light on gas exchange and acid accumulation
in the C3-CAM plant Clusia minor L. Journal of Experimental
Botany 43, 345–352.
Herzog B. 1994. Der Einfluss der Wasserdampfdruckdifferenz
(VPD) zwischen dem Blattinneren und der Atmosphäre auf
die C3uCAM-Umstellung bei Clusia minor L. Dipl.-Biol.thesis, Technische Universität Darmstadt.
Herzog B, Hoffmann S, Hartung W, Lüttge U. 1999.
Comparison of photosynthetic responses of the sympatric
tropical C3 species Clusia multiflora H. B. K. and the C3-CAM
intermediate species Clusia minor L. to irradiance and drought
stress in a phytotron. Plant Biology 1, 460–470.
Lee DM, Assmann SM. 1992. Stomatal responses to light
in the facultative Crassulacean acid metabolism species,
Portulacaria afra. Physiologia Plantarum 85, 35–42.
Lüttge U. 1999. One morphotype, three physiotypes: sympatric
species of Clusia with obligate C3 photosynthesis, obligate
CAM and C3-CAM intermediate behaviour. Plant Biology
1, 138–148.
Lüttge U. 2000. Light-stress and Crassulacean acid metabolism.
Phyton 40, 65–82.
Mawson BT, Zaugg MW. 1994. Modulation of lightdependent stomatal opening in isolated epidermis
following induction of crassulacean acid metabolism in
Mesembryanthemum crystallinum L. Journal of Plant
Physiology 144, 740–746.
UV-Aublue light induced CAM 1483
Möllering H. 1974. Malat. Bestimmung mit MalatDehydrogenase und Glutamat-Oxalacetat-Transaminase. In:
Bergmeyer HU, ed. Methoden der Enzymology. New York:
Academic Press, 1636–1639.
Möllering H. 1985. Citrate. determination with citrate lyase,
MDH and LDH. In: Bergmeyer HU, ed. Methods of
enzymatic analysis. New York: Academic Press, 2–12.
Osmond CB. 1978. Crassulacean acid metabolism: a curiosity in
context. Annual Review of Plant Physiology and Plant
Molecular Biology 29, 379–414.
Popp M, Kramer D, Lee H, Diaz M, Ziegler H, Lüttge U. 1987.
Crassulacean acid metabolism in tropical dicotyledonous
trees of the genus Clusia. Trees—Structure and Function
1, 238–247.
Popp M, Lied W, Meyer AJ, Richter A, Schiller P, Schwitte H.
1996. Sample preservation for determination of organic
compounds: microwave versus freeze-drying. Journal of
Experimental Botany 47, 1469–1473.
Roberts A, Borland AM, Maxwell K, Griffiths H. 1998.
Ecophysiology of the C3-CAM intermediate Clusia minor L.
in Trinidad: seasonal and short-term photosynthetic
characteristics of sun and shade leaves. Journal of
Experimental Botany 49, 1563–1573.
Schmitt AK, Lee HSJ, Lüttge U. 1988. The response of the C3CAM tree, Clusia rosea, to light and water stress. I. Gas
exchange characteristics. Journal of Experimental Botany
39, 1581–1590.
Tallman G, Zhu J, Mawson BT, Amodeo G, Nouhi Z, Levy K,
Zeiger E. 1997. Induction of CAM in Mesembryanthemum
crystallinum abolishes the stomatal response to blue light and
light-dependent zeaxanthin formation in guard cell chloroplasts. Plant and Cell Physiology 38, 236–242.
Thiel S, Döhring T, Köfferlein M, Kosak A, Martin P,
Seidlitz HK. 1996. A phytotron for plant stress research:
how far can artificial lighting compare to natural sunlight?
Journal of Plant Physiology 148, 456–463.
von Caemmerer S, Farquhar GD. 1981. Some relationships
between the biochemistry of photosynthesis and the gas
exchange of leaves. Planta 153, 376–387.
Zotz G, Winter K. 1993. Short-term regulation of crassulacean
acid metabolism activity in a tropical hemi epiphyte, Clusia
uvitana. Plant Physiology 102, 835–841.