Journal of Experimental Botany, Vol. 52, No. 359, pp. 1303±1313, June 2001
Stomatal oscillations at small apertures: indications for
a fundamental insufficiency of stomatal feedback-control
inherent in the stomatal turgor mechanism
Hartmut Kaiser1 and Ludger Kappen
Botanisches Institut der Christian-Albrechts-UniversitaÈt zu Kiel, Olshausenstraûe 40, D-24098 Kiel, Germany
Received 5 October 2000; Accepted 6 February 2001
Abstract
Introduction
Continuous measurements of stomatal aperture
simultaneously with gas exchange during periods
of stomatal oscillations are reported for the first
time. Measurements were performed in the field on
attached leaves of undisturbed Sambucus nigra L.
plants which were subjected to step-wise increases
of PPFD. Oscillations only occurred when stomatal
apertures were small under high water vapour mole
fraction difference between leaf and atmosphere
(DW). They consisted of periodically repeated opening
movements transiently leading to very small apertures. Measurements of the area of the stomatal
complex in parallel to the determination of aperture
were used to record volume changes of guard cells
even if stomata were closed. Stomatal opening upon
a light stimulus required an antecedent guard cell
swelling before a slit occurred. After opening of the
slit the guard cells again began to shrink which, with
some delay, led to complete closure. Opening and
closing were rhythmically repeated. The time-lag
until initial opening was different for each individual
stoma. This led to counteracting movements of
closely adjacent stomata. The tendency to oscillate
at small apertures is interpreted as being a failure of
smoothly damped feedback regulation at the point of
stomatal opening: Volume changes are ineffective for
transpiration if stomata are still closed; however,
at the point of initial opening transpiration rate
rises steeply. This discontinuity together with the
rather long time constants inherent in the stomatal
turgor mechanism makes oscillatory overshooting
responses likely if at high DW the `nominal value' of
gas exchange demands a small aperture.
The stomatal pore width of an illuminated leaf is
regulated to satisfy the con¯icting needs of maintaining
a suf®cient intercellular CO2 concentration (Ci) for
photosynthesis on the one hand and of preventing
excessive water loss by transpiration (E) on the other
hand. Apart from direct responses, for example, to PPFD,
negative feedback loops serve to maintain stomatal
conductance for water vapour and CO2 in an appropriate
range (Raschke, 1965): A reduction of Ci due to photosynthetic CO2 assimilation stimulates stomatal opening
(Mott, 1988), which increases CO2 diffusion into the
leaf intercellular spaces. Stomata respond to changes in
air humidity in a manner consistent with a feedbackregulation where an increase of transpiration acts as
a negative stimulus (Franks et al., 1997; Monteith, 1995;
Mott and Parkhurst, 1991). Despite the fundamental
importance of these processes the physiological sensing
and transduction involved in both feedback systems is
barely understood (Cousson, 2000; Kearns and Assmann,
1993).
The control by negative feedback is most apparent
when oscillations of stomatal aperture occur (Farquhar
and Cowan, 1974; Raschke, 1979). These oscillations
have fascinated many researchers, giving rise to numerous
reports of oscillations observed by different methods
(Barrs, 1971) with different plant species. Most often gas
exchange techniques were used, but measurements of
chlorophyll a ¯uorescence (Eckstein et al., 1996; Siebke
and Weis, 1995), leaf temperature, leaf water potential
or other methods concerning water relations (Herppich
and von Willert, 1995; Lang et al., 1969; McBurney and
Costigan, 1984; Naidoo and von Willert, 1994) were also
used to record stomatal oscillations.
These methods integrate the responses of large
numbers of stomata and therefore can convey only
limited information on the actual responses at the single
Key words: Stomatal oscillations,
feedback control, turgor mechanism.
1
stomatal
aperture,
To whom correspondence should be addressed. Fax: q49 431 880 1522. E-mail: [email protected]
ß Society for Experimental Biology 2001
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Kaiser and Kappen
guard cell level. The synchronism or variability of the
responses of individual stomata remains obscure. It is
not possible to draw ®rm conclusions on the amplitude of
the underlying stomatal movements unless the relationship between aperture and gS is known. This relationship
has not been determined by parallel measurements of
gas exchange and apertures in the majority of gas
exchange studies. Even the general physical relation
between aperture and stomatal conductance is still in
doubt. Although it is commonly believed and supported
by theoretical models (Lushnikov et al., 1994; Parlange
and Waggoner, 1970), that gS is largely linearly related to
aperture, recent measurements demonstrated a non-linear
relationship, with the steepest increase of gS with aperture
at small degrees of opening (Kaiser, 1999; Kaiser and
Kappen, 2000).
The idea of this work was therefore to observe
stomatal oscillations at the aperture level by a combination of microscopic observation and CO2uH2O gas
exchange measurements. Measurements were performed
on intact leaves of an undisturbed Sambucus nigra L.
shrub on a ®eld site. The conditions for the occurrence
of oscillatory behaviour were explored and the responses
described in detail. The results support an hypothesis
which explains oscillations as the consequence of insuf®cient feedback-control inherent in the stomatal turgor
mechanism and stomatal mechanics.
Materials and methods
Experimental site and plant material
Experiments were made on leaves of an approximately 5 m high
shrub of Sambucus nigra growing on the border of a small
stand of shrubs and young trees in the New Botanical Gardens
in Kiel (FRG). The site was irrigated during dry periods
whenever a tensiometer monitored soil water potentials less than
0.5 MPa. The experiments presented here were performed
at soil water potentials between 0.04 and 0.46 MPa. S. nigra
continuously developed new leaves throughout the summer;
thus by using only young but fully expanded leaves, interference
from senescence effects could be avoided. Leaves were enclosed
in the gas-exchange cuvette for about 1 week, during which
several experiments on the same set of stomata were performed.
Except during the experimental periods between 09.00 h and
15.00 h the cuvette received ambient light and tracked the
external humidity and temperature conditions.
Gas exchange and aperture measurements
A combination of microscopic in situ observations and
CO2uH2O gas exchange measurements on the same leaf was
used. The technical details have been described previously
(Kaiser and Kappen, 1997, 2000). In short, it consists of an
inverted video microscope (long distance objective 40 3 , Zeiss,
FRG) inserted in the bottom of a gas-exchange cuvette. A leaf is
attached above the microscopic objective in a leaf holder, which
is driven by a motorized remote controlled microscopic
stage. By computer control it was possible repeatedly to
relocate selected stomata and to capture time series of digitized
video-images for image analysis. Observation of stomata at
low light or in darkness were enabled by the transmitted light
of a GaAlAs emitter infrared diode (model OD880F, Optek,
US). The images of the stomata were taken with the microscope focused to the narrowest part lower down in the pore.
Measurements of the pore area on digitized images were
made manually by delineating the pore edge with the cursor.
Measurement errors as determined by randomly repeated
measurements of a set of images ranged between 0.5 and
3 mm2 ("standard deviation), depending on the image quality.
The relative error is larger at small apertures. Apertures of very
slightly opened stomata cannot be exactly determined. A `zero'
reading therefore cannot completely rule out the presence of
a small stomatal opening, which nevertheless can signi®cantly
contribute to gas exchange (Kerstiens, 1996). Although uncommon in the literature, stomatal opening has been expressed in
terms of pore area instead of pore width (aperture). This does
not affect the interpretation of the results, because area and
width of the elliptic pore are linearly related if the pore length
stays constant during movements. This was con®rmed for
S. nigra. Apertures of stomata of different longitudinal extension
were compared by a relative measure, the `degree of opening',
which expresses pore width as a percentage of pore length.
In some experiments the stomatal complex area (SCA)
between the dorsal cell walls was also measured.
Either 15 or 50 stomata were observed in each experiment,
usually randomly sampled from an area of about 2 cm2 in the
centre of the leaf. The entire leaf surface could not be inspected
as the petiole was not ¯exible enough and was then bent by the
movements of the leaf holder.
The gas exchange equipment (Walz, FRG) is an open ¯ow
system which measures transpiration every minute by means of
the bypass principle and CO2 exchange every 2 min with an
infrared gas analyser. Humidity and temperature in the cuvette
were regulated to a constant value during the experiments.
Leaf temperature was measured by a 0.2 mm thermocouple
attached to the lower leaf surface. The internal fan of the cuvette
was set to a moderate speed, producing a boundary layer
conductance of about 600 mmol m 2 s 1 which was measured
with a water-saturated ®lter paper sealed on one side. Gas
exchange calculations were performed according to Ball
(Ball, 1987). The relatively large cuvette volume (c. 4500 cm3)
necessary to enclose the mechanical components for microscopic
inspection caused some cuvette lag. The calculations described
previously (KuÈppers et al., 1993) were used to estimate the
effective cuvette volume and to calculate a time-corrected signal
of CO2 gas exchange resulting in a temporal resolution of 2 min.
Microclimatic measurements
To measure the PPFD incident on the observed leaf, a small
GaAsP Photodiode (model G2711±01, Hamamatsu, Japan,
calibrated against a Li-Cor quantum sensor), was mounted
inside the cuvette about 12 mm distant from the observed leaf
region. Soil water potential in the root zone of S. nigra was
measured at a depth of 40 cm by a pressure transducer
tensiometer (Kappen et al., 2000). Microclimate and gas
exchange data were recorded by a datalogger (model 21 XL,
Campbell, Shepshed, UK) which was supplemented by a
multiplexer (model AM 416, Campbell, UK).
Experiments
Stomatal and gas exchange responses to step-wise increases in
PPFD at different levels of DW were observed. Irradiance was
Stomatal oscillations at small apertures
provided by a ®bre optic illuminator (Kaltlicht-Fiberleuchte
FL-400, with Spezial Fiberoptik 400-F, Walz, Effeltrich, FRG)
or ®ve halogen cold-light lamps (50 W) emitting a PPFD of
up to 750 mmol m 2 s 1 through a diffuser. During the experiments ambient light was nearly completely kept off by covering
the cuvette with a black cloth.
Either `full' steps from darkness to about 750 mmol m 2 s 1
were applied, or PPFD was increased step by step (e.g. from
0 to 60 to 130, 270, 510, 750 mmol m 2 s 1). PPFD was always
held constant until a leaf conductance was at steady state or, in
case of oscillations, at least for 1 h. In the `full step' experiments
a set of 15 stomata was observed every 4±10 min. In the step by
step experiments 50 stomata were measured only once at the end
of each PPFD step. During subsequent days the leaves were
subjected to the identical light treatment under different levels
of DW and always the same set of stomata was observed.
Experiments were performed always at the same time of the day,
to avoid interference with diurnal rhythms.
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step (Figs 1, 4, 7) or by step-wise increases (Fig. 2).
Oscillations were generally more likely to occur at higher
DW (Fig. 2). This ®gure also shows that the initial
opening as well as the closing reaction is faster the drier
the air is. Another determining factor for oscillations
appeared in the experiments with step-wise increases of
PPFD. The tendency to oscillate was maintained as long
as the average degree of opening was quite low and was
diminished when stomata opened further (Fig. 2). This
suggested a restriction of oscillations to small apertures,
which was further explored by summing up all PPFD-step
experiments (Fig. 3). This demonstrates that pronounced
oscillatory responses were restricted to both, DW above
Results
Oscillations were visible in both gas-exchange responses
and movements of individual stomata. They could be
easily elicited by step-wise increases of PPFD at DW
higher than about 10 mmol mol 1. The tendency to
oscillate was quite variable. It ranged between damped
oscillations with one single overshooting response and
sustained oscillations over a time period of more than 2 h
(Figs 1, 2, 4, 7).
The conditions for the different oscillation patterns
were explored in a series of experiments at different
levels of DW. Oscillations were elicited either by increase
of PPFD from zero to 850 mmol m 2 s 1 in one single
Fig. 1. Response of stomata and leaf conductance to 1 h light periods
at different levels of DW. DW (mmol mol 1) during illumination is
displayed by numbers in the graph. The experiments were performed on
consecutive days using the same set of 15 stomata. Average stomatal
reactions are shown. The individual responses of stomata at
22 mmol mol 1 (not shown) were similar to the reactions shown in
Fig. 4.
Fig. 2. The response of leaf conductance and 50 stomata (degree of
opening) during a step by step increase of PPFD. Numbers between
dotted lines refer to the PPFD (mmol m 2 s 1) which was held constant
during this time interval. The boxplots show the distribution of apertures
(median, 10th, 25th, 50th, 75th, and 90th percentile, dots present values
outside the 10th or the 90th percentile). The experiment was performed at
cuvette conditions of 19 8C, and a DW of approximately 13 mmol mol 1.
DW outside the cuvette ranged from 7±9 mmol mol 1.
Fig. 3. Response type of stomatal reactions (as determined by the course
of leaf conductance) upon step increases of PPFD in dependence on
current DW and average degree of stomatal opening. Degree of opening
was determined by measuring 15, in some cases 50 apertures at steady
state, when the reaction had ®nished, or in case of oscillations at a
medium value of the cycling leaf conductance. Each data point
represents the response to one step increase in PPFD. The symbol type
shows the observed response type: Black squares, responses with at least
two oscillatory peaks; triangles, damped responses with only one peak;
circles, gradual arrival at steady state. Data originate from experiments
on seven leaves.
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Kaiser and Kappen
10 mmol mol 1 and small apertures below 3±4% average
degree of opening. This also entails that a large
proportion (about 50%) of stomata was closed at any
time. One condition alone was not suf®cient to produce
oscillations: If stomata were opened wider than 3±4%
(due to, for example, high PPFD), light changes did not
elicit oscillations even if DW was 15 mmol mol 1.
Damped oscillations with only one single peak were
observed in a larger range of apertures and DW.
The detailed observation of aperture changes allows
an analysis of the processes during stomatal oscillations
(Figs 4, 5, 7). Figure 4 shows the responses of leaf
conductance and net photosynthetic rate together with
the responses of 15 stomata. The period of gas-exchange
Fig. 4. Oscillations of stomatal apertures of 15 stomata, leaf conductance and net photosynthesis following a step increase in PPFD from 0 to
800 mmol m 2 s 1at a DW of 18 mmol mol 1. Arrows (stoma 1) refer to the microscopic images in Fig. 5. Grey bars in the panels of stoma 1 to 4
denote maxima to make clear phase shift of stomatal oscillations as well as different frequencies (4, 5 or 6 oscillations in 4 h). 15 stomata from a leaf
area of 11.1 3 7.2 mm were observed. The average distance between stomata was 5.2 mm. Stomata with phase shifted oscillations had a distance as low
as 1.7 mm (stoma 1u2), 4 mm (stoma 3u4) and 2.7 mm (stoma 4u14).
Stomatal oscillations at small apertures
1307
Fig. 6. The relation between the area of the stomatal complex (SCA)
and the pore area for one individual stoma. The term `Spannungsphase'
marks the state when guard cell volume is below the threshold of
stomatal opening (StaÊlfelt, 1929a). In the `Motorphase' the pore is
opened and pore area is linearly linked to the area of the guard cell
complex which is a surrogate measure of guard cell volume. Area
measurements were performed on two different days under a DW of 7
and 17 mmol mol 1 during opening and closing reactions elicited by
light±dark changes. Lines were drawn by hand.
Fig. 5. Images of stoma 1 from the experiment shown in Fig. 4 during a
peak of the oscillation (a) and in the closed state 6 min later (b). The
time of observation is also marked by arrows in Fig. 4. The arrow in (a)
points to the slightly open pore.
oscillations amounted to about 40 min. After PPFD
was increased the individual stomata needed between
10±40 min to open. The aperture was then oscillating
between a slight opening (2±5%) and complete closure. In
absolute terms the aperture changes were rather slight.
This is illustrated in Fig. 5 which shows the image of the
maximum observed aperture in the experiment of Fig. 4.
It was remarkable that some stomata reacted phase
shifted to the overall course of leaf conductance. Phase
shift even occured between stomata which were only a few
mm apart. Stomata furthermore displayed individually
different frequencies of the oscillations. A more vivid
demonstration of the spatio-temporal action of the
responses displayed in Fig. 4 is given in a videoclip which is available at JXB online as supplementary
material.
To record stomatal responses (volume changes) in the
closed state the area of the stomatal complex between the
dorsal cell walls of the guard cells was measured. As this
measurement, as far as is known, has not previously been
used to record guard cell activity, the close relationship
to stomatal aperture was ®rst con®rmed by simultaneously measuring pore area and guard cell complex area
(SCA) of individual stomata over a period of several
days under different conditions (Fig. 6). The linear
relationship to pore area con®rms that SCA is a suitable
indirect measurement for guard cell swelling. There is,
however, a certain measurement error associated with
both area measurements, which introduces some scatter.
Interestingly SCA in the closed state was observed to fall
to about 10% below the threshold SCA at the point of
stomatal opening, giving an idea of the extent of the
`Spannungsphase'.
Using simultaneous measurements of SCA and pore
area, the events during stomatal oscillations were
observed in detail (Fig. 7).
After the increase of PPFD the guard cells began to
swell. Swelling proceeded for some time without the
appearance of a pore. This introduced a time lag
individually different for each stoma before initial pore
opening. As soon as the pore was slightly open the
swelling stopped and the guard cells de¯ated again, which
in turn led to complete closure. De¯ation thereafter
proceeded for some time until a new swelling of the guard
cells began. These processes were repeated periodically by
some stomata (e.g. stoma C in Fig. 7). This experiment
also revealed a phase shift between closely adjacent
stomata (2±4 mm). This is apparently brought forth by
the individually different lag time (e.g. 1 h in stoma A,
0.5 h in stoma B) before initial opening.
As the conditions at small apertures were obviously
decisive for the development of oscillations, the relationship between pore area and leaf conductance was
explored by simultaneously measuring gas exchange and
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Kaiser and Kappen
Fig. 7. The simultaneous responses of leaf conductance and three
stomata during a phase of stomatal oscillations. Both the response of
stomatal complex area (®lled symbols) and corresponding aperture
changes (open symbols) are shown. Oscillations were induced by
increasing PPFD from 0 to 600 mmol m 2 s 1 (dotted vertical line) at
a DW of 17 mmol mol 1. Arrows point to the individually differing times
of the transition from `Spannungsphase' to `Motorphase'.
apertures of 50 randomly selected stomata during opening responses after transfer to saturating PPFD (Fig. 8).
This brought forth a typical saturation curve with a
steeply rising slope at small apertures which can be ®tted
quite well by a hyperbolic function. As data were
collected over three consecutive days it can also be
concluded that this relationship is repeatable with respect
to time. The dependence of Amax under saturating PPFD
on pore area displayed an even steeper increasing slope at
small apertures, which demonstrates that in S. nigra
even small apertures are suf®cient to allow non-limiting
CO2-supply of the mesophyll.
The controlling effect of aperture changes on
transpiration can be described best by the slope of the
dependence of E on pore area (dEudA) which is
demonstrated in Fig. 9 using the experimentally determined hyperbolic function (Fig. 8). It is obvious that
during continual changes of guard cell volume the effect
on transpiration is most pronounced at high DW at the
state of transition from closed to slightly open.
Fig. 8. The dependence of leaf conductance (a) and net photosynthesis
(b) at light saturation on average stomatal pore area (circles). The ®gure
includes measurements on a sample of the same 15 stomata at different
times of opening and closing movements which were induced by light±
dark changes. To demonstrate the variability of apertures the boxplots
in (a) display typical aperture distributions (median, 10th, 25th, 50th,
75th, and 90th percentiles, crosses represent the maximum aperture of
the sample). Dotted lines mark average pore areas, which yield 90% of
maximum gL respective Amax. Hyperbolic functions were ®tted to the
data (lines). Data originate from experiments on three consecutive days
at different DW (10u12u14 mmol mol 1), a PPFD of 650 mmol m 2 s 1
and a temperature of 19 8C.
Fig. 9. The slope of the relationship between pore area and transpiration area (dEudA) in dependence on pore area and DW (mmol mol 1;
numbers at the curves). Calculations were performed using the
empirically derived hyperbolic function describing the dependence of
gL on average pore area (Fig. 8).
Discussion
These observations of individual stomata add important
information not already contained in the numerous
existing observations of oscillations based on methods
Stomatal oscillations at small apertures
which integrate responses of several stomata. The
observation that in S. nigra stomatal oscillation developed only at very small pore widths and that the aperture
changes in absolute terms were quite small, points to an
important role of the low aperture range near stomatal
closure. Alternative explanations for the lack of oscillations at higher apertures could be ruled out: in the
experiments with the step-wise increases of PPFD the
decreasing tendency to oscillate could also have been
explained by an inhibiting action of high PPFD (Fig. 2).
However, this seems unlikely, as in other experiments
stomata oscillated at high PPFD if other conditions (e.g.
high DW) led to a very small aperture (Figs 4, 7). A
decreasing tendency to oscillate in the afternoon due to
the well-known endogenous rhythmicity of stomata is
also unlikely as stomata showed no damping of oscillations in experiments extending into the afternoon hours
(Fig. 4).
The general validity for other species of the assertion
that small apertures are an essential precondition for
oscillatory responses is uncertain. The experimental
treatments leading to oscillations as, for example, the
increase of DW, blocking of water supply, increase of
PPFD at high DW, changing CO2 concentration of the
air, treatment with abscisic acid (Cardon et al., 1994;
Eckstein et al., 1996; Naidoo and von Willert, 1994;
Raschke, 1965; Siebke and Weis, 1995) all tend to induce
stomatal closure. It thus appears that the formerly
observed stomatal oscillations mostly occurred in the
lower aperture range. There are, however, some reports
demonstrating oscillations at higher apertures with larger
amplitudes (Barrs and Klepper, 1968; Bunce, 1987).
However, even if oscillations may occur at higher degrees
of opening in other species, the observed tendency to
oscillate at the threshold of stomatal opening in S. nigra
suggests a phenomenon of general importance.
The reasons for oscillations are clari®ed by the
observation that many of the observed stomata cycled
between the slightly open and the closed state. The
instability of the reaction is obviously caused by the fact
that here small movements have a large effect on gas
exchange, making it likely that feedback responses
surpass the narrow appropriate aperture range leading
either to complete closure or to a too large aperture.
This interpretation is supported by measurements of
guard cell activity in the periods when the pores were
closed. This was implemented by measurements of the
area of the stomatal complex (SCA). Although similar
measures such as `peristomatal groove distance'
(Eckstein, 1997; Lawson et al., 1998) and `width of
stomatal complex' (StaÊlfelt, 1929b, 1963) have already
been used as a surrogate measure for stomatal aperture,
SCA has hitherto not been used to measure guard cell
movements continuously. Therefore, it had to be con®rmed ®rst that a close and reproducible relationship
1309
between SCA and pore area exists (Fig. 6), and that
continuous changes of SCA could be measured in the
closed state, as can be seen in Fig. 7. These measurements
illustrate the two phases of the stomatal responses ®rst
described by StaÊlfelt as the `Spannungsphase' with a
guard cell turgor not suf®cient to open the pore and as the
`Motorphase', the status when the opened pore changes
its aperture according to the turgor changes (StaÊlfelt,
1929a).
The continuous observation of SCA together with
pore area shows that the transition between swelling and
de¯ation phases is closely linked to the moments of
opening and closing of the pore: light-induced swelling
continues until the pore opens, which after some delay is
followed by de¯ation. This de¯ation continues until the
pore is closed, after which it again swells. This behaviour
can be explained by the action of negative feedback loops
at the level of single guard cell complexes: the opening of
the pore(s) increases local intercellular CO2 concentration
(Ci) and transpiration which act as closing stimuli. Due to
the lag time of stomatal responses, which is inherent in
the relatively slow turgor mechanism, these opening and
closing responses periodically shoot beyond the appropriate `nominal value' of stomatal aperture. It cannot
de®nitely be concluded from these experiments to
what extent the two feedback loops are involved in the
oscillations. It appears, however, that the feedback
related to transpiration plays a major role, because in
some experiments stomata oscillated at low PPFD
although Ci was relatively high and nearly constant
(H Kaiser, unpublished data).
In addition, there exists an hydraulic effect, which
increases the tendency of stomata to oscillate: any
increase of transpiration, for example by an increase of
DW, lowers epidermis turgor more than guard cell turgor
(Shackel and Brinckmann, 1984) and thus supports
stomatal opening (Raschke, 1970). The following increase
of transpiration in turn facilitates further opening by
a positive feedback loop. The reverse effect acts when
stomata close. This physical effect acts quite fast and
precedes the slower, reversely directed physiological
feedback response (Kappen et al., 1987). The strength
of this hydraulic effect is correlated to DW and causes the
different opening and closing rates at different air
humidities, which have previously been observed in
different species (Assmann and Grantz, 1990; Barradas
et al., 1994; Kaiser and Kappen, 2000) and also appear in
the current results with S. nigra (Fig. 1). This component
of positive feedback should be strongest in the lowest
aperture range, where aperture changes have the largest
effect on transpiration. For stomata at the transition from
`Spannungsphase' to `Motorphase' this means that the
opening movement is accelerated and an overshooting
response becomes likely. Conversely, shrinking of the
guard cells is accelerated and may proceed over the
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Kaiser and Kappen
point of complete closure, because the decline of
transpiration boosts epidermis turgor more than guard
cell turgor.
The reasons for the promotion of oscillations by high
DW can now be expressed more precisely: On the one
hand high DW leads to a higher controlling impact of
aperture on transpiration (Farquhar and Cowan, 1974),
on the other hand the aperture which is required to arrive
at the `nominal value of gas-exchange' is shifted to very
low pore areas, where volume changes have the largest
impact on gas exchange (Fig. 9) and closing movements
may even completely close the pore. This causes feedback
responses to surpass the narrow appropriate aperture
range. The situation becomes worse by the component of
positive feedback, brought forth by the previously
mentioned hydraulic effect, which is also strongest at
small apertures.
These unfavourable characteristics of regulation are
inseparably linked to the physiological and physical
mechanisms of stomatal functioning and, therefore,
represent a basically inavoidable insuf®ciency of the
stomatal feedback regulation.
The counteracting movements even of closely adjacent
stomata point to the action of a spatially limited feedback
system within a distance of less than 2 mm. It could even
be possible that individual stomata act as autonomous
feedback systems, independent of the gas exchange
through the adjacent stomata. But to con®rm this,
further experiments are required. The action of smallscale responses to humidity changes has been described
previously (Lange et al., 1971). This does not exclude
possible feedback mechanisms acting on larger scales.
Co-ordinated oscillations of stomata have been observed
by chlorophyll a ¯uorescence imaging on the level of
`stomatal patches' (Cardon et al., 1994; Siebke and Weis,
1995). Even oscillations at the whole plant level were
observed (McBurney and Costigan, 1984; Naidoo and
von Willert, 1994). These were apparently co-ordinated
by hydraulic signals from the xylem which are believed
to act mainly in woody plants and as a signal in the
feedback regulation of whole plant water status (Fuchs
and Livingston, 1996; Saliendra et al., 1995; Whitehead
et al., 1996). A very sensitive stomatal response to high
DW reduces the risk of xylem embolism in stenohydric
woody species (Vogt, 1998; Vogt and LoÈsch, 1999) in
limiting whole plant transpiratory water loss. Obviously
S. nigra is of the type of plant that makes use of this
feedback mechanism.
These observations, however, were made on single
attached leaves in the cuvette on a well-watered plant and
oscillations occurred even in cool and cloudy weather at
probably high xylem potential. They thus show that, in
addition to a possible large-scale feedback regulation of
water status, a feedback control of gas exchange is located
on a very small spatial scale. Integration of stomatal
responses into the regulation of whole plant water status
obviously occurs within a wide range of spatial scales.
An interesting phenomenon is that an apparently
coherent gas exchange response was produced by nonsynchronous stomatal movements (Fig. 4), which indicates a more or less synchronous action of the bulk
of stomata. This could be caused by some co-ordination
between the stomata. A more realistic, stochastic explanation is, however, that the statistical distribution of
stomatal properties led to a simultaneous action without
functional synchronization.
As a result, the actual stomatal reponses as presented
in Fig. 4 can by no means be deduced solely from the gS
response. This raises some doubt as to the data base on
which previous attempts to analyse the stomatal control
system (Cowan, 1972; Jarvis et al., 1999) were founded.
These analytical approaches were based on time series of
gS which were assumed to be a reasonable measure for the
average stomatal response. This requires that the
responses must be largely uniform and that the relation
of gS to stomatal aperture is known. Results in this
study show that both prerequisites cannot be taken
for granted. Uniformity can only be proven by visual
observations of apertures, otherwise it is impossible to
exclude counteracting movements concealed by the gas
exchange. Uniformity cannot simply be deduced from the
absence of conditions favouring stomatal patchiness
(Jarvis et al., 1999), because variability on the microscale has to be taken into account, as has been
demonstrated here. The assumption of a linearity between
gS and apertures which was demonstrated analytically is
contradicted by the experimental data presented in Fig. 8
which show a strongly non-linear relationship (see also
Kaiser and Kappen, 2000; Nonami et al., 1990). This nonlinearity makes it dif®cult to conclude directly from gS on
aperture because the shape of the aperture distribution
(Laisk et al., 1980) has to be considered, which is not the
case when linearity is assumed. In particular, changes of
gS do not directly re¯ect stomatal activity when a large
fraction of stomata is closed, because then only the
responses of the still open stomata contribute to changes
of gS, while the action of closed stomata, being in the
`Spannungsphase', is completely ignored. These results
demonstrate, however, that these responses are of high
importance for the understanding of the stomatal control
system.
It is concluded that any analysis based on integrating
methods such as measurements of gS should be more
cautiously interpreted with respect to responses of the
guard cells. While important for the understanding of
responses on leaf level and their ecophysiological effect,
they may probably convey only limited information on
the feedback processes acting in single guard cells.
The responses observed in these experiments may
also suggest a comparison with the dynamics of patchy
Stomatal oscillations at small apertures
stomatal oscillations as observed by chlorophyll a
¯uorescence imaging (Cardon et al., 1994; Eckstein et al.,
1996; Haefner et al., 1997; Siebke and Weis, 1995). The
patterns of patchwise counteracting ¯uorescence changes
were interpreted as a spatially co-ordinated movement of
stomata. A direct comparison of the direct observations
used here with this indirect as well as integrating method
is dif®cult. The ®rst question is whether the amplitude
of the underlying stomatal movements is comparable
with these results. A simultaneous recording of chlorophyll a ¯uorescence images and stomatal apertures
indicated that patchy ¯uorescence only develops at very
small apertures (Kaiser et al., 1999). This is supported
by a calculation of gS from images of chlorophyll
¯uorescence, with gS being in the range from
0±40 mmol m 2 s 1 (Meyer and Genty, 1998), which
certainly implies quite a low aperture level. This is not
surprising if the type of relationship between aperture
and photosynthetic capacity (Fig. 9) is considered, which
points to the fact that Ci should be suf®ciently reduced
to increase non-photochemical quenching only at very
small apertures. Therefore the oscillating ¯uorescence
patterns are likely to be based on small aperture
differences at a generally low degree of opening. This
suggests that patchy stomatal closure for similar reasons
as stomatal oscillations may be functionally linked to the
particular properties of the lowest aperture range.
The results of this study indicate a functional explanation for oscillations which has already been proposed
(Hopmans, 1971). The question arises, however, are the
supposedly unavoidable oscillations advantageous or
harmful? As most of the time transpiration and photosynthetic rate are far from the supposedly `optimal'
nominal value, oscillations should generally impair plant
performance. However, calculations were presented
which showed that oscillations may improve water use
ef®ciency under certain conditions (Upadhyaya et al.,
1988). This contradicts the assertion that `optimality' is
necessarily linked to steady-state responses and raises
the question whether stomatal oscillations may even
serve a `purpose' (Cowan, 1972). However, even if
stomatal oscillations in some cases are regarded as
bene®cial, the advantage is obviously restricted to
moderate amplitudes. It seems necessary to avoid large
amplitudes with wide ¯uctuations of gas exchange. While
there appears to be no simple escape from the described
insuf®ciency of feedback control at single stoma level, the
responses of populations of stomata could be dampened
by a high variability of stomatal responses. In these
results it is obvious that individual stomata react
differently as can be seen by phase shift and different
frequences. The phase shift was caused by a variation in
the lag-time necessary to overcome the `Spannungsphase'
(Laisk et al., 1980), leading to different starting times
for the oscillations (Fig. 7). It is the variation in the
1311
properties `degree of initial guard cell swelling' and `rate
of volume increase' which is transduced into a temporal
variation and causes non-synchronous initial opening
and subsequent phase-shifted oscillations. It is therefore
proposed that stomatal variability reduces negative
effects on gas exchange by avoiding synchronous closure
and opening of all stomata. Variability should reduce the
tendency to oscillate by lowering possible resonance
effects caused, for example, by hydraulic coupling.
Obviously, stomatal variability does not completely prevent oscillations observable at the leaf level. The observed
oscillation in gL (Fig. 4) is presumably already levelled
off and would have been much more pronounced if
opening and closing movements of stomata had been
synchronous.
The variability of stomatal responses has hitherto been
mainly treated with respect to the effect on steady-state
gas exchange (Laisk, 1983; Mott, 1995), as a phenomenon
of biological variation (Weyers and Lawson, 1997) or
a methodological problem annoying researchers with
the need to use large samples (Kubinova, 1994; Weyers
and Meidner, 1990). It now appears that this variation
may also be advantageous to the plant by improving the
regulation characteristics of the stomatal feedback
system.
Acknowledgements
The authors wish to thank the staff of the Botanical Garden of
the University of Kiel for generous practical support of the
experiments, Mr Thomas Walter for excellent technical assistance and Mrs Shamim Lenz for correcting the manuscript.
The work was supported by a grant from the Deutsche
Forschungsgemeinschaft (Ka 390u12-1).
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