Journal of Experimental Botany, Vol. 50, No. 331, pp. 253–261, February 1999
Chlorophyll fluorescence during CAM-phases in
Clusia minor L. under drought stress
Eduardo A. de Mattos1,2, Britta Herzog1 and Ulrich Lüttge1,3
1 Institute of Botany, Darmstadt University of Technology, D-64287 Darmstadt, Germany
Received 25 September 1998; Accepted 25 September 1998
Abstract
Some species of Clusia show a high flexibility in regulating carbon uptake during the day–night cycles in
response to environmental conditions. In this study,
individuals of the C -CAM intermediate plant Clusia
3
minor were subjected to drought. The characteristics
of chlorophyll fluorescence, gas exchange and organic
acid content were investigated in individuals performing CAM under controlled laboratory conditions.
The organic acid content increased after 16 d of
drought, however, the malate/citrate ratio showed a
2.6-fold decrease. After 13 d of drought, phase IV of
CAM was completely suppressed. The highest levels
of non-photochemical quenching (measured as q and
N
NPQ) were observed on day 16. However, increased
capacity to dissipate the energy in excess to drive
photosynthesis was not enough to maintain a low
reduction state of the primary electron acceptor of
photosystem II (measured as 1−q ) at late afternoon
p
under drought stress. Sustained decreases in predawn
F /F ratio were observed even though organic acid
V M
accumulation increased after 16 d without irrigation.
Despite non-photochemical quenching remaining high
after rewatering, the decline in F /F ratio was relaV M
tively rapidly reversible. Considering the partitioning
of q into its fast (q ) and slow (q ) components, it was
N
F
S
observed that the proportion of the two components
was dependent on both the number of days without
watering and the different CAM phases.
Key words: Gas exchange, organic acid, phoinhibition,
CAM, Clusia minor.
Introduction
CAM species may make flexible use of the various CAM
phases, where CO is taken up and the organic acids
2
synthesized are accumulated in the vacuole during the
dark period (phase I ), where there is a peak of CO 2
uptake in the early light period (phase II ), where stomata
close during the following major part of the light period
when organic acids are remobilized, decarboxylated and
the CO regained is assimilated (phase III ), and where
2
stomata may reopen for CO uptake during the late light
2
period (phase IV ) (Osmond, 1978). In some species of
Clusia it was found that they may show a remarkable
plasticity in the patterns of carbon uptake and in the
capacity for the accumulation of massive amounts of
malate and citrate ( Franco et al., 1990; Borland et al.,
1992; Haag-Kerwer et al., 1992). In species able to
perform CAM, the ability to change rapidly the amount
of CO taken up during the day and night might help to
2
maintain the integrity of the photochemical apparatus
under adverse environmental conditions (Borland et al.,
1992, 1996; Haag-Kerwer et al., 1992). Moreover, some
evidence was obtained regarding the probable role of
citric acid as a means of increasing the buffer capacity of
the vacuole ( Franco et al., 1992) and for the generation
of a high daytime internal CO concentration by the
2
potential release of three CO , compared to the one
2
released from malate (Lüttge, 1988; Borland and Griffiths,
1996). Thus, it was suggested that the daytime decarboxylation of citrate might confer some protection against
severe photoinhibition (Haag-Kerwer et al., 1992).
Notwithstanding the increase in night-time accumulation of organic acids during the induction of CAM in
Clusia minor, Borland et al. (1992, 1996) observed a
decline in photochemical efficiency as the dry season
progressed in Trinidad. Franco et al. (1992) observed
that for several species of Clusia CO uptake in phase IV
2
of CAM is very sensitive to water deficits, which might
enhance the potential for photoinhibition. Values of F /F
V M
declined significantly during phase IV of CAM in semi-
2 Present address: Departamento de Ecologia Geral, IB, Universidade de São Paulo, C.P. 11461, São Paulo, SP, 05422–970, Brazil.
3 To whom correspondence should be addressed. Fax: +49 6151 164630. E-mail: [email protected]
© Oxford University Press 1999
254
de Mattos et al.
exposed and exposed plants of Clusia hilariana in the
coastal sandy plains of Macaé (Brazil ), when lower
internal CO levels probably prevailed (Franco et al.,
2
1996). In a comparison of light response curves of chlorophyll fluorescence in phases III and IV of CAM in C.
hilariana, Mattos et al. (1997) measured much lower
levels of effective quantum yield and a substantial increase
in both 1−q and total non-photochemical quenching
P
with increasing light levels in phase IV. They related these
results to the depletion of the organic acid pool and low
stomatal conductances during this phase. Therefore,
phase IV of CAM in Clusia seems to be most prone to
photoinhibition, as has also been reported for other CAM
plants (Adams et al., 1989; Guralnick et al., 1992; Barker
and Adams, 1997).
The ability to dissipate the energy in excess of that
needed to drive photosynthesis by means of increasing
non-photochemical quenching when external CO supply
2
is restricted may confer some protection on the photochemical apparatus (Demmig-Adams, 1990; DemmigAdams et al., 1996). Depending on environmental conditions and plant material an increase in non-photochemical
quenching may be related to the contribution of different
relaxation-time components ( Krause and Weis, 1991;
Keiller et al., 1994; Scholes et al., 1997). Thiele et al.
(1998) have recently summarized the components of nonphotochemical quenching of chlorophyll a fluorescence
of photosystem II in kinetic terms as follows: reversion
of energy-dependent quenching due to the trans-thylakoid
H+ gradient is <10 min; reversion of quenching due
to xanthophyll-cycle-mediated energy dissipation is <60
min, and quenching due to D1–protein degradation lasts
longer. Thus, in this paper, the following questions are
addressed: (1) Is the increased energy dissipation high
enough to counterbalance the energy in excess of that
driving photosynthesis when phase IV is drastically
depressed under water deficits in Clusia minor performing
CAM? (2) Is there a change in how fast and slow relaxingtime components contribute to total non-photochemical
quenching during water deficit?
Materials and methods
Plant material and environmental conditions
Young plants of Clusia minor L. were collected in Serrania San
Luis, Falcon State, Venezuela in 1987 and maintained in a
greenhouse in Darmstadt, Germany. Cuttings of these plants
were rooted and potted in soil (Fruhsdorfer Erde). After 3–4
months when 3–4 leaf pairs had developed, plants were repotted
to 5 l pots, transferred to an Ecophyt-chamber (Heraus, Vötsch,
Balingen, Germany) and held under controlled conditions for
1 month. Day/night temperatures averaged 30/18 °C while
relative humidity averaged 65/75%. This corresponded to a
constant day/night leaf-to-air vapour pressure deficit ( VPD) of
20/6 kPa MPa−1. A 12/12 h photoperiod was used and the
plants were watered every day. Photon flux density (PFD) at
the level of the leaves used in the measurements of gas exchange
and chlorophyll fluorescence was about 400 mmol photons
m−2 s−1 (l=400–700 nm). Water stress was imposed by
withholding water.
Gas exchange measurements and organic acid analyses
Continuous measurements of net CO exchange and water
2
vapour loss were made in fully expanded leaves by using a CO
2
and H O steady-state porometer (B, Leybold, Heraeus,
2
Hanau, Germany). Leaves of C. minor are hypostomatic. The
gas exchange system and the equations used to calculate CO
2
and H O exchange are described in Lüttge et al. (1986). To
2
maintain the porometer cuvette at the same humidity conditions
as those in the Ecophyt-chamber, the incoming air was
humidified by bubbling it through water and then cooling it to
the desired dew point by a Peltier-cooling system ( Walz,
Effeltrich, Germany). Dawn and dusk leaf samples collected
from four different individuals consisted of four leaves, which
were dried in a microwave oven for subsequent measurements
of dry weight, malate and citrate. Prior to organic acid
determinations, the material was boiled in distilled water.
Malate and citrate levels were determined enzymatically according to Hohorst (1965) and Möllering (1985), respectively. For
each individual leaf sample, all analyses were performed in
triplicate. Results were related to leaf area.
Chlorophyll fluorescence analyses
The chlorophyll fluorescence measurements were performed
using a pulse-amplitude modulation fluorometer (PAM 101,
Walz, Effeltrich, Germany) in the same position as that used
for gas exchange measurements. The leaf was kept at a constant
distance (c. 2 cm) and angle (60°) to the fibre optics by an
adapted leaf clip of the porometer head. Potential quantum
yield of photosystem (PS ) II, F /F , was determined just before
V M
the onset of the light period and 20 min after the 12 h
photoperiod (see van Kooten and Snel, 1990, for fluorescence
nomenclature). The light-adapted parameters were determined
during the photoperiod by applying at 1 min intervals 800 ms
saturation pulses of white light. After establishment of steadystate conditions, at least three maximum fluorescence yield
values (F∞ ) were recorded. After this time, the leaf was covered
m
with a black cloth and far-red light illumination was given
immediately to determine the minimal level of fluorescence, F∞ .
o
Following 2 min of darkness another saturation pulse was given
to determine the final level of light-saturated fluorescence, F◊ .
m
Photochemical (q ) and total non-photochemical quenching
P
(q ) were calculated according van Kooten and Snel (1990).
N
The fast (q =1−[(F∞ −F∞ )/(F◊ −F )]), and the slow (q =1−
F
m
o
m
o
S
[(F◊ −F )/(F −F )]), components of total non-photochemical
m
o
m
o
quenching (q ) were calculated as described by Keiller et al.
N
(1994). No distinct differences in these two quenching components were found when using 2 or 3 min of darkness to obtain
F◊ . The effective quantum yield of PSII (DF/F∞ ) was determined
m
m
as (F∞ −F )/F∞ (Genty et al., 1989). The term 1−q was used
m
m
P
as an estimate of the reduction state of the primary quinone
electron acceptor of PSII (Q ). The reduction state of Q ,
A
A
reflects the balance between the rate of excitation energy
transfer into PSII centres and the rate of electron transport
beyond PSII (Demmig-Adams, 1990). The Stern–Volmer equation (NPQ)=(F −F∞ )/F∞ was used as an indicator of the
m
m m
activity of energy dissipation in the pigment bed of PSII and it
is proportional to the effective rate constant for energy
dissipation in the antennae as well as the concentration of
quenching centres (Demmig-Adams and Adams, 1994; DemmigAdams et al., 1996).
Chlorophyll fluorescence in Clusia minor 255
Results
CO exchange and organic acid accumulation in response
2
to drought
Under well-watered conditions C. minor showed a typical
CAM pattern of gas exchange ( Fig. 1). Subsequently, as
the soil was drying out, daytime net CO uptake (Fig. 1)
2
and stomatal conductance (data not shown) in phases II
and IV progressively decreased. After 13 d, phase IV of
CAM was completely suppressed, which continued until
the last day prior to rewatering (Fig. 1, day 16). This
decrease in CO uptake during the light period was not
2
compensated by the slight increase in CO uptake during
2
the dark period ( Table 1). Compared with the maximum
values observed on day 3 for daytime CO uptake, a 73%
2
decrease was observed after 16 d of withholding water.
The plants were rewatered at the end of the light period
of day 16. After 12 h of rewatering (day 17) net CO
2
uptake in phase II showed signs of recovery. After 4 d in
well-watered conditions (day 20) CO uptake during
2
phases II and IV increased, but recovery was still not
complete.
Malate and citrate accumulation increased after 16 d
of drought. However, the relative increase in citrate levels
was much larger than that observed for malate. Thus, the
Table 1. Integrated CO uptake for the dark and the light periods
2
in one leaf of C. minor
Day 0 was the last day that the plant received water. Days 3 to 16 are
the number of days that the plant was not watered. At the end of the
light period of day 16, the plant was rewatered.
Days of experiment
J
int. night
CO2
(mmol
m−2)
J
int. day
CO2
(mmol
m−2)
0
3
9
16
17
20
78.1
92.2
98.2
93.9
82.9
84.0
68.3
79.1
31.4
5.0
29.6
40.4
malate/citrate ratio showed a 2.6-fold decrease ( Table 2).
Recycling of respiratory CO was not significant under
2
both conditions.
Chlorophyll fluorescence
Figure 2 gives a detailed set of chlorophyll fluorescence
measurements performed with C. minor. Under wellwatered conditions, variations in DF/F∞ and q remained
m
N
fairly constant during phases II and III (day 3, Fig. 2).
However, at the end of the light period, in phase IV of
CAM, a 34% decrease in DF/F∞ was observed (from 0.63
m
to 0.47). An increase from about 0.44–0.50 at midday to
0.76 at the end of the light period was observed for q .
N
After 9 d without irrigation, marked changes in DF/F∞
m
and q were also observed in phase II, at the beginning
N
of the light period. The highest differences between CAM
phases were observed after 16 d without watering the
plants and when afternoon CO uptake from the atmo2
sphere via open stomata was not expressed ( Figs 1, 3).
The lower values of DF/F∞ (0.14) during phase II (day
m
16) rapidly increased to 0.55 at the onset of phase III,
when the decarboxylation of organic acids probably raised
the internal CO concentration. In contrast, q decreased
2
N
from 0.93 to 0.49. On this day, after 9 h of light period,
DF/F∞ presented the highest decline, i.e. from 0.57 to
m
0.08 at the end of the light period, whereas q increased
N
from 0.54 to 0.96. Although in phase III lower values of
q were always observed, the minimum values were not
N
Table 2. Night-time malate and citrate accumulation (Dmal,
Dcitr) and the malate/citrate ratios (Dmal/Dcitr) under wellwatered conditions and after 16 d without watering the plants
of C. minor
Data expressed as means±SD, n=4 leaves.
Dmal (mmol m−2)
Well watered 55.8±10.3
16th day of
water stress 99.4±1.9
Dcitr (mmol m−2)
Dmal/Dcitr
10.1±4.9
5.55
45.0±0.4
2.14
Fig. 1. Influence of drought and rewatering on daily net CO exchange measured continuously over 20 d for one given leaf of C. minor. Days 3 to
2
16 are the number of days that the plant was not watered. At the end of the light period of day 16, the plant was rewatered. The dark bars indicate
night-time, roman numbers indicate CAM-phases sensu Osmond (1978).
256
de Mattos et al.
Fig. 2. Influence of drought and rewatering on diurnal courses of effective quantum yield (DF/F∞ ), total non-photochemical quenching (q ), Stern–
m
N
Volmer non-photochemical quenching (NPQ), the reduction state of Q (1−q ), and the fast and the slow relaxing-time components of q (q and
A
P
N F
q ) for the same leaf used for gas exchange measurements in Fig. 1. Roman numbers indicate CAM-phases and the arrow at the end of day 16
S
indicates the time of rewatering.
lower than 0.44 during the first 16 d of the experiment.
After rewatering the plants, there was a gradual increase
in the minimum values of DF/F∞ during phases II and
m
IV and a smaller decrease in q was observed during late
N
afternoon.
During the 16 d of drought stress NPQ was lowest
during the middle of the day, but did not show any
systematic variations (from about 0.59 to 0.65) (phase
III of CAM ) (Fig. 2). However, maximum values
obtained in the early morning and late afternoon
increased gradually with drought stress. On day 16 the
maximum value of NPQ in phase II was 4.3, whereas at
the end of the day it reached 5.1. As also observed with
q , NPQ and q declined rapidly with the onset of phase
N
F
III. However, after 9 h of light period a steep increase in
NPQ was observed until maximum values were reached
at the end of the day, whereas an increase in q was
F
observed after midday. This increase was most pro-
nounced after 9 h of light. After rewatering the plants the
high values of NPQ and q observed during phases II
F
and IV showed some decline, but, even after 4 d of
rewatering recovery was not complete. Under wellwatered conditions, low values of 1−q during the whole
P
day were observed (Fig. 2). Similarly, q did not show
S
marked variations during day 3, however, values around
0.44–0.47 were observed during the middle of the day.
As gas exchange was decreasing with the course of the
experiment, 1−q and q gradually showed increased
P
S
levels during both phases II and IV. After rewatering,
both 1−q and q showed a marked decrease at the
P
S
beginning and at the end of the day approaching the
values observed under well-watered conditions.
Figure 3 shows the effect of drought stress on integrated
CO uptake during phase IV of CAM in C. minor. The
2
response of NPQ and 1−q to decreased levels of CO
P
2
uptake in this phase is also presented. The highest values
Chlorophyll fluorescence in Clusia minor 257
Fig. 4. Relationship between the highest values of Stern–Volmer nonphotochemical quenching (NPQ) and integrated CO uptake obtained
2
during phase IV of CAM from days 0 to 16. The fitted line is NPQ=
4.76527−0.0977CO int phase IV, R2=0.95, P<0.0001.
2
Fig. 3. (A) Integrated CO uptake during phase IV of CAM, (B)
2
highest value of Stern–Volmer non-photochemical quenching (NPQ)
and the reduction state of Q (1−q ) measured during phase IV of
A
P
CAM during the 16 d without watering the plant and from day 16
onwards when the plant was rewatered.
of 1−q and NPQ at the end of the light period were
P
observed concomitantly with the suppression of CO
2
uptake in the late afternoon. However, in contrast to a
constant steep increase in NPQ, 1−q seemed to show
P
two distinct phases of increase. First, a slow increase
during the first part of the experiment and second, a
faster increase after day 9. A strong negative correlation
was obtained between NPQ at the end of the light period
and integrated CO uptake in phase IV of CAM (NPQ=
2
4.76527–0.0977CO int phase IV, R2=0.95, P<0.0001)
2
( Fig. 4).
Predawn values of maximal fluorescence (F ) were
M
higher than values measured 20 min after the end of the
light period, i.e. dusk values ( Fig. 5). Both declined up
to day 16 during drought. Predawn measurements of
minimal fluorescence (F ) showed somewhat irregular
0
changes during the experiment, but dusk values gradually
declined during stress. Thus, the maximum difference in
F /F between predawn and dusk values occurred after
V M
16 d of stress. There was rapid recovery after rewatering.
Discussion
In this study, as already observed in several species of
Clusia including C. minor (Franco et al., 1992), potted
young plants of C. minor subjected to drought stress
showed a gradual decrease of daytime net CO uptake
2
Fig. 5. The time-course of maximal (F ), minimum (F ) and the ratio
M
0
of variable to maximum fluorescence (F /F ) in the same leaf used in
V M
Fig. 1. ‘Predawn’ values were obtained just prior to the light period
and ‘after dusk’ values were taken 20 min after the light was turned
off. Days 0 to 16 indicates the days from the last day that the plant
was watered. The plant was watered after the light period of day
16 onwards.
258
de Mattos et al.
rates and an increase in gross dark CO fixation. However,
2
the latter was not enough to compensate lower daytime
CO uptake in C. minor, in contrast to observations with
2
C. uvitana ( Winter et al., 1992). Thus, integrated CO
2
uptake during 24 h decreased. Phases II and IV of CAM
were curtailed in C. minor under drought stress. The
results obtained in this study, clearly indicate that when
the soil was drying out the main response of leaves of C.
minor to decreased integrated CO uptake in phases II
2
and IV of CAM was to increase non-photochemical
quenching (measured as NPQ or q ), which indicate the
N
engagement of mechanisms leading to thermal dissipation
of excess light. Increased levels of energy dissipation may
help to protect PSII from over-excitation and photodamage, however, it brings about a decline in the effective
quantum yield of PSII photochemistry. It is interesting
to note, however, that the abrupt changes in light intensity
at the beginning and at the end of the light period may
affect the behaviour of the chlorophyll fluorescence parameters. For instance, any gradual increase in q and NPQ
N
at the beginning of the light period could not be observed
because the lights were suddenly switched on in the
growth chamber. However, it should be borne in mind
that PFD at the leaf surface was only about
400 mmol m−2 s−1, with a daily integrated PFD of
17.3 mol m−2, which corresponds to semi-exposed conditions in the field. By using constant irradiance during the
light period it was possible to compare the differences
that might emerge by the characteristic behaviour of the
diurnal patterns of the CAM phases. On the other hand,
variable light intensity under field conditions may mask
the intrinsic differences between the CAM phases.
Partitioning total non-photochemical quenching (q )
N
into its fast (q ) and slow relaxation-time components
F
(q ) indicated that, in spite of a relatively high constant
S
level of q around 0.50 during phase III of CAM, there
N
were changes in q and q . A similar behaviour was also
F
S
observed in leaves of Mesembryanthemum crystallinum
( Keiller et al., 1994). After 16 d of drought stress, q
F
increased towards the end of phase III, whereas q showed
S
a marked decline. In contrast to this behaviour during
the middle of the day, both components of q showed
N
increased values after 9 h of photoperiod under drought
stress conditions. Demmig and Winter (1988) described
q as a sensitive indicator of the point at which light
F
started to become excessive. This fast-relaxing component
of q is thought to be largely related to the trans-thylakoid
N
pH gradient ( Krause and Weis, 1991). Thus, as light
intensity was constant during the photoperiod, the
increased levels of q towards the end of phase III may
F
indicate a decrease in the capacity to attain high internal
CO concentration (c ) by decarboxylation of the organic
2
i
acid pool at this time. This behaviour of q seemed to
F
become most important when drought stress progressed.
However, at the same time marked changes in DF/F∞ , q ,
m N
NPQ or 1−q were not observed during phase III of
P
CAM. By contrast, the pronounced changes in the
quenching of chlorophyll fluorescence after 9 h of photoperiod during drought stress suggest that the restricted
supply of exogenous CO , due to stomatal closure at the
2
time when phase IV would have occurred under wellwatered conditions and the depletion of organic acids,
predispose the leaves to a higher excitation energy
pressure upon PSII.
It is worth mentioning, however, that high values of q
N
and NPQ were also observed in phase II during drought
stress. Despite chlorophyll fluorescence parameters behaving in a relatively similar manner in both phases II and
IV, it appears that high values of energy dissipation
occurred during a shorter period of time in phase II than
in phase IV. Under well-watered conditions in the field,
an extended phase II of CAM is generally observed in C.
minor (Borland et al., 1993; Borland and Griffiths, 1996).
However, the extension of phase II can be shortened by
the presence of a higher malate content in the vacuoles
( Kluge et al., 1981; Borland and Griffiths, 1997). Thus,
potentially photoinhibitory conditions, as evidenced by the
occurrence of high values of 1−q in phase II under
P
drought stress, may last just for the lag-phase of passive
malate efflux out of the vacuoles and the commencement
of phase III of CAM. Indeed, decreased F /F ratios were
V M
only observed during late afternoon at the onset of phase
IV of CAM in Clusia hilariana, an obligate CAM plant,
under field conditions (Franco et al., 1996).
Confirming the results obtained by Franco et al. (1992)
with various species of Clusia, the contribution of citrate
in relation to malate to the overall accumulation of
organic acids during the night, increased in C. minor after
16 d of drought stress. However, bearing in mind that
light intensity was constant during the photoperiod, maximum apparent electron transport rates (DF/F∞ ×PFD=
m
ETR) during phase III of CAM were slightly lower (7%)
after 16 d of drought stress than under well-watered
conditions. However, if the higher amount of organic
acid accumulation after 16 d of drought stress is taken
into account a higher photon utilization on day 16 of the
experiment would have been expected. Several studies
have shown that organic acid accumulation can be
extended to the early hours of daylight during phase II
of CAM (Ball et al., 1991; Franco et al., 1994, 1996;
Haag-Kerwer et al., 1996) and the evidence for phosphoenolpyruvate carboxylase (PEPc) activity at this time was
obtained, for instance, by measuring on-line carbon isotope discrimination (Borland et al., 1993; Borland and
Griffiths, 1997). However, although the higher amount
of CO uptake (44 mmol m−2) in phase II of CAM under
2
well-watered conditions is going directly to organic acid
accumulation, due to PEPc activity rather than CO
2
fixation via ribulose 1,5-bisphosphate carboxylase/
oxygenase (Rubisco), the differences in potential
Chlorophyll fluorescence in Clusia minor 259
endogenous CO generation by the decarboxylation of
2
organic acids are still large between both days.
Intrinsic differences in the capacity for the generation
of a high internal CO concentration (c ) may exist when
2
i
comparing obligate and C -CAM intermediate plants, as
3
observed by Borland and Griffiths (1997). It was suggested that c was maintained at a lower level in the C i
3
CAM intermediate Clusia minor than in the obligate
CAM plant Kalanchoë daigremontiana despite the potential generation of CO from organic acid decarboxylation
2
being some 3–4 times higher in C. minor, largely as a
result of citrate breakdown. As stomatal closure during
phase III in CAM plants results from the generation of
a high c by the onset of the decarboxylation of organic
i
acids ( Kluge et al., 1981; Borland and Griffiths, 1996,
1997), the results obtained with C. minor in this study
may indicate that after 16 d without irrigation, the onset
of phase III was about 3 h earlier than under well-watered
conditions. This may indicate that instead of increasing
maximum photon utilization during phase III under
drought conditions, leaves of C. minor may increase the
extent of phase III by an earlier decarboxylation of a
higher organic acid pool. By contrast, chlorophyll fluorescence changes indicate that decarboxylation of organic
acids was always completed at around 9 h of the photoperiod. Thus, the increased levels of organic acid accumulation after 16 d without irrigation may not alleviate the
potential high levels of excess excitation energy in the late
afternoon when phase IV of stomatal opening was not
expressed and a potentially dangerous high reduction
state of Q may have taken place.
A
Demmig and Winter (1988) suggested that, in spinach
leaves, increased levels of q predominantly occur when
S
leaves are subjected to potentially photoinhibitory conditions for prolonged periods of time. This also seems to
be the case in C. minor. After 16 d of drought stress, q
S
markedly increased during late afternoon and this was
accompanied by the highest decrease in both after dusk
and predawn F /F ratios. After rewatering, leaves of C.
V M
minor, despite lower values of 1−q , still showed high
P
levels of non-photochemical quenching, mainly attributable to high values of the fast relaxing component (q ).
F
Due to the effect of low temperatures, sustained decreases
in photochemical efficiency have been observed in some
species. It has been suggested that this was the result of
the retention of antheraxanthin and zeaxanthin, the
de-epoxidized forms of the xanthophyll cycle, and their
sustained engagement in energy dissipation (Adams
et al., 1995).
Keiller et al. (1994) suggested that high levels of
q during the middle of the day in leaves of
S
Mesembryanthemum crystallinum may reflect an increase
in ATP demand during phase III of CAM and that q
S
may be related to state-transitions. State-transitions may
increase the level of excitation of PSI and enhance the
generation of ATP by cyclic photophosphorylation
( Krause and Weis, 1991). The observed changes in q
S
and q towards the end of phase III in C. minor when
F
drought stress had progressed were in accordance with
the results of Keiller et al. (1994) who observed an inverse
relationship between q and q . However, this was not
S
F
the case during late afternoon. The increase in both q
S
and q after 9 h of the photoperiod under drought stress
F
may suggest that q at this time was composed of more
S
than one quenching component and that photoinhibitory
quenching, q , ( Krause and Weis, 1991) may also contriI
bute to q and q . It is interesting to note that Scholes
S
N
et al. (1997) observed that even though the leaves of the
C plant Dryobalanops lanceolata do not have the ability
3
to increase photosynthetic capacity when transferred to
an exposed condition, it may survive under high light
levels by increasing the slowly relaxing phases of nonphotochemical quenching. In its natural habitat, C. minor
seems to show a preference for semi-exposed conditions
(Grams et al., 1997), and any marked increases in ETR
or maximum integrated 24 h CO uptake were not
2
observed when plants of C. minor were growing under
light intensities of up 1200 mmol m−2 s−1 (Herzog, 1997).
In spite of the increased levels of organic acid accumulation after 16 d without irrigation, sustained decreases in
predawn F /F ratio were observed. Moreover, after 9 d
V M
without irrigation the decrease of after dusk F /F ratio
V M
and the lowering capacity for F /F recovery during the
V M
12 h night period may indicate that protective mechanisms
have been exhausted. Despite some increase in predawn
initial fluorescence (F ), which is thought to be indicative
0
of damage of PSII centres (Osmond and Grace, 1995),
the observed decline in both predawn and after dusk
F /F ratio was related to a greater extent to the decrease
V M
in maximal fluorescence (F ). These chlorophyll fluoresM
cence characteristics suggested that the increase in energy
dissipation was the main cause for the depression in
photon efficiency in C. minor under drought stress. It is
clear that these mechanisms may provide some protection
and may permit rapid recovery of photosynthetic competence following rewatering, as actually observed.
Haag-Kerwer et al. (1992) suggested that the massive
night-time accumulation of malate and especially citrate,
which seems to be a common characteristic of Clusia
species (Popp et al., 1987; Franco et al., 1990, 1994;
Borland et al., 1992), may help to alleviate photoinhibition. In contrast to malate, where the decarboxylation of
1 mol during the day provides only 1 mol of CO , the
2
decarboxylation of each mol of citrate may provide
theoretically between 1–6 mols of CO (Lüttge, 1988;
2
Franco et al., 1992; Haag-Kerwer et al., 1996). In a field
study in Trinidad, Borland et al. (1992, 1996) observed
that the expression of CAM in C. minor was substantially
enhanced at the onset of the dry season. However, despite
pronounced accumulation of citric acid in exposed leaves
260
de Mattos et al.
of C. minor, a marked decline in photochemical efficiency
was observed, suggesting some degree of photoinhibition.
In this way, in spite of the pronounced reduction in
effective quantum yield (DF/F∞ ) along with the capacity
m
to increase markedly the radiationless energy dissipation
(by means of NPQ or q ) when drought stress progressed,
N
leaves of C. minor were not able to keep a low reduction
state of the primary quinone electron acceptor, Q , of
A
PSII (as estimated by 1−q ) in the late afternoon. A high
P
reduction state of PSII centres is indicative of overexcitation of these centres and is thought to result in
photoinhibitory damage over longer periods of time
(Demmig-Adams, 1990).
In conclusion, the results obtained with C. minor in
this study, corroborate the hypothesis that when phase
IV of stomatal opening of CAM is not expressed due to
drought stress, the leaves are most prone to sustained
decreases in photochemical efficiency. Moreover, the relatively strong photoinhibition at dusk after 16 d of drought
( Fig. 5) when phase IV is not expressed ( Fig. 1), but
nocturnal accumulation of organic acids is much increased
( Table 2), does not support a putative role of organic
acid decarboxylation to alleviate photoinhibition in the
absence of phase IV CO -uptake. On the other hand,
2
leaves of Clusia minor showed an increased capacity to
dissipate the energy in excess of that driving photosynthesis when phase IV was drastically suppressed during
drought stress. This also seems to show a limited capacity
of effectively maintaining a lower reduction state of the
primary electron acceptor of PSII, and moderate chronic
photoinhibition was observed as drought stress progressed. However, despite non-photochemical quenching
remaining high after rewatering, the reduction of F /F
V M
ratios was relatively rapidly reversible. This indicates that
further studies must clarify the mechanisms and causes
of the differential contribution of the fast and slow
relaxing-time components to q and NPQ, during the
N
development of drought stress in Clusia minor.
Acknowledgements
This work was supported by a 4 month fellowship from
CAPES-DAAD to EAM and by the programme PROBRAL
from Fundação Coordenação de Aperfeiçoamento de Pessoal
de Nı́vel Superior, Brazil and Deutscher Akademischer
Austauschdienst, Germany. We wish to thank TEE Grams
(GSF ) for many useful discussions during the experiments, AC
Franco and two anonymous referees for their valuable comments
and suggestions on the manuscript.
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