Localization of Mechanisms Involved in Hydropassive
and Hydroactive Stomatal Responses of Sambucus nigra
to Dry Air1
Hartmut Kaiser* and Nicole Legner
Botanisches Institut der Christian-Albrechts-Universität, D–24098 Kiel, Germany
The response of stomata to a reduction of air humidity is composed of a hydropassive opening followed by active closure.
Whereas the mechanisms behind the hydropassive opening are largely understood, the location and physiological basis of the
sensing mechanisms leading to active closure are not yet known. This study attempts to evaluate the importance of a single
pore’s transpiration on its own response and that of adjacent pores. Selected stomata on attached intact leaves of Sambucus
nigra were sealed with mineral oil and the response to a reduction of humidity was continuously observed in situ. Blocking a
pore’s transpiration had no appreciable effect on hydropassive opening and subsequent stomatal closure. If the adjacent
stomata were additionally sealed, the closing response was reduced, but not the hydropassive opening. On the other hand,
sealing the entire leaf surface, except a small area including the observed stomata, also reduced stomatal closure. These results
indicate that strictly local processes triggered by a pore’s own transpiration are not required to induce stomatal closure. To
describe the effect of one pore’s transpiration on the hydropassive and hydroactive responses of neighboring stomata, a simple
spatial model was constructed. It suggests that 90% of the closing effect covers an area of approximately 0.5 mm2, whereas the
effect on hydropassive opening affects an area of approximately 1 mm2. This divergence may suggest mechanisms other than
or in addition to those involving changes of local leaf water potential.
Plant water loss is tightly balanced with water uptake to maintain beneficial water status. The most important control on water transport is the change of
stomatal aperture, which governs water diffusion from
the leaf interior to the atmosphere, as well as the opposite flow of carbon dioxide (CO2) into the photosynthesizing mesophyll. To balance transpiration and
photosynthesis, guard cells may sense and integrate
many environmental as well as physiological signals
related to photosynthesis, the transpirational demand
of the atmosphere, and the plant’s current hydraulic
status (Buckley, 2005; Roelfsema and Hedrich, 2005).
Atmospheric humidity is one of the key environmental signals that stomata need to sense to adjust water
loss. The sensing mechanisms involved in the stomatal
humidity response are nonetheless still not identified.
This is mainly due to the fact that investigation of
stomatal humidity responses must be done on leaves
with unspoiled water relations. This excludes many of
the recently developed methods of molecular and cell
biology.
1
This work was supported by the Deutsch Forschungsgemeinschaft (grant no. KA1711/1–1).
* Corresponding author; e-mail [email protected]; fax 49–
431–880–5568.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Hartmut Kaiser ([email protected]).
www.plantphysiol.org/cgi/doi/10.1104/pp.106.089334
1068
The typical stomatal response to a sudden decrease
in air humidity is composed of a transient opening
caused by hydraulic mechanisms, which is followed
by a delayed closing movement (Kappen et al., 1987;
Buckley and Mott, 2002). The initial hydropassive
opening occurs because of the mechanical advantage
(Sharpe et al., 1987; Franks et al., 1998) of the epidermal cells over guard cells. This leads to stomatal
opening when guard cell and epidermis turgor equally
decline. The subsequent closing response, however, is
difficult to explain by purely hydraulic mechanisms.
The most parsimonious explanation is an osmotically
driven decrease in guard cell turgor. Although direct
evidence for active closure is still lacking, it is supported by much circumstantial evidence (Buckley,
2005).
The search for possible mechanisms for stomatal
closure in dry air has, for a long time, been motivated
by the hypothesis of a so-called feed-forward response, which stands for a direct response to atmospheric humidity (Schulze et al., 1973; Farquhar, 1978).
Feed-forward responses produce a decrease in transpiration rate despite an increase in the driving gradient. To mechanistically explain the feed-forward
response, a direct sensing of atmospheric humidity
via transpiration through the external guard cell cuticle (peristomatal transpiration) was suggested (e.g.
Cowan, 1977; Farquhar, 1978; Grantz, 1990). According
to some authors, the guard cell cuticle is more permeable for water than the epidermal cuticle (Kerstiens,
1997a). Until now, it has not been shown convincingly
that the effect of the rather small cuticular transpiration
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Spatial Aspects of Stomatal Humidity Response
is sufficiently large to predominate over the much
larger water losses through the stomatal pore. On the
contrary, experimentally increased permeability of the
cuticle did not sensitize stomata to dry air (Kerstiens,
1997b). Maier-Maercker (1983, 1999) used the term
peristomatal transpiration in a broader sense, also including evaporation into the substomatal cavity, and
suggests that total water loss from guard cells may
trigger stomatal responses to dry air. Other mainly
hydraulic explanations assume large hydraulic resistance for the flow to the evaporating site in the vicinity
of the guard cells (Farquhar, 1978; Nonami et al., 1990;
Dewar, 2002), leading to direct sensing of ambient
humidity partly independent of whole-leaf water loss.
All these hypotheses assume that water loss of guard
cells triggers their responses to changing air humidity.
Until now, there is no clear evidence favoring or disproving these different hypotheses, which claim that
guard cells are the primary sensor for changes in
transpiration rate.
The feed-forward hypothesis has been challenged
by a reevaluation of previous reports of feed-forward
responses (Monteith, 1995) and new data (Franks et al.,
1997), which suggest that the decline of transpiration
rate in spite of increasing humidity gradient is irreversible, possibly caused by water stress to the leaf.
Thus, it is more appropriate to use the term apparent
feed-forward response. Moreover, a number of studies
suggested feedback control of transpiration, which
keeps transpiration below a target value (Mott and
Parkhurst, 1991; Franks et al., 1997). This could either
involve localized sensing mechanisms, which are confined to the guard cells or their direct vicinity, or
spatially distributed mechanisms involving other leaf
tissues. Transpiration-dependent accumulation of substances in the apoplast of guard cells (Lu et al., 1997;
Zhang and Outlaw, 2001) could form a locally confined
sensing mechanism. Apoplastic solutes are assumed to
be transported to the sites of evaporation in the guard
cell apoplast and to become concentrated. This effect
has been confirmed for Suc (Outlaw and De VlieghereHe, 2001).
Much of the evidence derived from gas exchange
and water status measurements points to control of
stomatal responses by local leaf water potential (cl;
Buckley, 2005). The precise location and nature of the
putative sensing mechanisms, however, are not yet
identified, but could involve mechanosensitive channels (Cosgrove and Hedrich, 1991; Garrill et al., 1996)
or osmosensing (Yoshida et al., 2006).
Knowing the location of the involved mechanisms
could enhance the understanding of transpiration
sensing, but only little and mostly indirect evidence
is available.
Microscopic observations of oscillating stomata revealed that stomata of Sambucus nigra with a spacing
of 2 mm oscillated independently and with different frequencies, which resulted in a phase shift of
individual responses (Kaiser and Kappen, 2001). This
pointed to localization of the involved mechanisms of
feedback control on a spatial scale smaller than 2 mm.
The phenomenon of stomatal patchiness also helps to
pin down the spatial scale of transpiration-sensing
mechanisms because it relies on locally coordinated
actions of stomata. Thermal imaging and imaging of
chlorophyll fluorescence, which is taken as a measure
of CO2 supply to the leaf and corresponds to stomatal
opening, suggests that stomata inside an areole may
behave similarly (West et al., 2005). This could point to
a distributed sensing mechanism, which coordinates
neighboring stomata. Because direct microscopic observations are lacking, these methods, like other integrating methods, are not able to assess the actual
degree of the proposed conformity. Consequently, they
remain inconclusive with respect to the question of spatial localization of transpiration-sensing mechanisms.
Therefore, it seemed necessary to assess spatial aspects of humidity sensing on the level of single stomata by direct microscopic observation rather than by
integrating methods. In previous, similar attempts,
small streams of dry air applied by capillaries were
used to modify the local transpiration rate (Lange
et al., 1971; Mott et al., 1997; Mott and Franks, 2001). In
contrast, we used the new method of applying small
amounts of mineral oil, which, by capillary force, are
sucked into the pores and, as a barrier of hydrophobic
liquid, block transpiration. If processes driven by a
pore’s transpiration, which are located in the guard
cells or in their immediate vicinity, were involved, this
should lead to a decrease of the active closing response
of oil-treated pores. Additionally, the inverse experiment was performed: Transpiration of the entire leaf
was blocked by sealing it with adhesive foil, leaving
only small areas to transpire. In the case of strictly local
sensing mechanisms, relying on processes related to
the transpiration stream through individual pores, the
humidity response of stomata in these areas should be
the same as before. These predictions were to be
tested.
RESULTS
Assessment of the Effectiveness of Blocking Pore
Transpiration and Possible Side Effects of Oil
Treatment and Covering the Leaf with Adhesive Foil
To assess the effect of treating pores with oil on leaf
conductance, the entire lower surface of a leaf was
gently brushed with an oil-saturated wiper in the
middle of the day when stomata had their maximal
opening. Possibly not all stomata were sealed by this
treatment. Nevertheless, leaf conductance immediately decreased from 107 to 17.5 mmol m22 s21, which
is comparable to conductance in the middle of the
night (16.5 mmol m22 s21) when stomata were closed.
To detect short-term effects of the application of
mineral oil, stomatal movements were recorded while
oil was applied with micropipettes under conditions
of high humidity (leaf-to-air mole fraction of water
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Kaiser and Legner
for Dcpass and 21.582% to 1.645% for Dcact, which
shows that even if an effect exists, it is rather slight.
To check whether additionally blocking transpiration in the immediate surrounding of a pore has an
effect on the stomatal humidity response, the observed
pore and six to eight adjacent pores were sealed with
oil, resulting in a nontranspiring area of approximately 0.2 mm2. This treatment had no or very little
effect on Dcpass (Fig. 6B), but Dcact was reduced by 3.7%
circularity, which is about 40% of the total closing
amplitude measured in untreated stomata.
Effect of Allowing Transpiration Only on a Small Area
Figure 1. Short-term responses of stomata to the application of mineral
oil. Ten pores were continuously observed while filling them with
a micropipette under conditions of high humidity (DW 5 2, PPFD 5
500 mmol m22 s21, [CO2] approximately 360 mmol mol21). Individual
responses were aligned to the time of oil application (arrow). The average initial pore area was 75.3 mm2 6 38.9 (SD). For better comparison,
the aperture of each stoma in the moment of oil application was arbitrarily set to 100%.
vapor [DW] 5 2 mmol mol21, photosynthetic photon
flux density [PPFD] 5 500 mmol m22 s21, [CO2] approximately 360 mmol mol21). The stomatal aperture
did not change during oil application and in the
following hour (Fig. 1). One week after the application
of oil to the stomatal pore and their surrounding pores,
the stomatal degree of opening under identical light,
CO2, and humidity conditions (DW 5 2 mmol mol21,
PPFD 5 500 mmol m22 s21, [CO2] approximately
70 mmol mol21) at the same time of day was virtually
unchanged (Fig. 2). To check whether fixing adhesive
foil to the lower leaf surface causes damage, stomatal
responses to reduced air humidity were observed
before attaching the foil, with attached foil, and after
careful removal (Fig. 5). After foil removal, the response was the same as before, which indicates that no
permanent damage had occurred.
The observation that occlusion of an area of approximately 0.2 mm2 only partly inhibited the response to
dry air suggested that a significant portion of the closing stimulus must have originated outside this area. To
confirm this conclusion, the inverse experiment was
performed by preventing transpiration on the whole
leaf except small spots of 0.8-mm diameter, which were
allowed to transpire freely (Figs. 5 and 6C). Whereas
the amplitude of the hydropassive opening response
appeared to be only slightly reduced, the amplitude of
the active closure was halved to 7.8%. After removal of
the adhesive foil, the amplitude of the closing response
was the same as before.
Effect of Sealing Stomata with Oil on the Stomatal
Humidity Response
When leaves were subjected to a sudden decrease of
air humidity, stomata showed the well-known initial
transient hydropassive opening response, followed by
partial or total active stomatal closure (Fig. 3). To
extract the most important characteristics of the humidity response, the amplitudes of the hydropassive
increase of circularity (Dcpass) and actively regulated
decrease of circularity (Dcact) were calculated as shown
in Figure 3. Treating single pores with oil had no
detectable effect on the stomatal response to a switch
from high to low atmospheric humidity. Both Dcpass
and the subsequent course of the active closing movement remained unchanged (Figs. 4 and 6A). The 95%
confidence interval for the difference between the
treated and the untreated state is 20.671% to 0.86%
Figure 2. Long-term effects of oil treatment. Twenty guard cell pairs
were measured under identical high humidity conditions before and
after oil treatment. Box and Whisker plots show the difference of
circularity prior to and 1 week after oil treatment of the observed pores
and all adjacent pores from a circular area with a diameter of 0.5 mm.
The control group was left untreated.
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Spatial Aspects of Stomatal Humidity Response
Model for the Distance-Effect Relationship of Stomatal
Transpiration for Hydropassive Opening and
Active Closing
Experimental data from the different treatments
were pooled to construct a simple spatial model of
the distance-dependent effect of a pore’s transpiration
on neighboring stomata. The model leaf was simplified in that it had evenly spaced stomata with a stomatal density typical for S. nigra of 46 mm22. It was
assumed that the transpiration of each stomatal pore
has distance (d)-dependent effects on Dcpass and Dcact of
its own guard cells and that of the other stomata,
which follow a Gaussian function. Thus, the stimulus
acting on a stoma (S) is calculated as the sum of the
distance (di)-dependent stimuli caused by the transpiration of its own and that of the surrounding pores.
Dc stimulus S 5 +i e
20:5ðdi =sÞ2
The only parameter determining the shape of the
function, s, was estimated by the least-squares optimization procedure implemented in Excel (solver)
using 89 individual stomatal responses to a step
change from high to low humidity showing the effects
of different treatments (26 responses from two leaves
with only one pore sealed; 27 responses from three
leaves showing the effect of treating a pore and its
adjacent pores; 13 responses on three different leaves
with only the surrounding stomata sealed [data not
shown]; and 21 responses from three leaves from a
circular transpiring area of 0.8-mm diameter, whereas
the rest of the leaf was prevented from transpiring by
adhesive foil). Confidence intervals for s were estimated by a bootstrap approach (Press et al., 2002): The
parameter estimation procedure was repeated 2,000
times for both the hydropassive and hydroactive responses with synthetic datasets generated by randomly drawing (with replacement) sets of stomatal
responses from the original dataset. From the resulting
set of simulated s, the 0.025 and 0.975 percentiles were
taken as limits of 95% confidence intervals. The simulation yielded a shydr 5 0.347 for the hydropassive
opening movement with a 95% confidence interval
from 0.276 to 0.501 and sact 5 0.244 for the active
closing movement with a 95% confidence interval
from 0.215 to 0.283 (Fig. 7A). The confidence interval
for shydr is larger because of the smaller amplitude of
the hydropassive response, resulting in larger relative
measurement errors. Bootstrap analysis showed that,
with P 5 0.002, shydr was larger than sact. According to
this parameter estimation, 90% of the effect of a
stoma’s transpiration covers an area of approximately
1 mm2 (hydropassive response) and 0.5 mm2 (active
closure).
DISCUSSION
Experiments were designed to explore the localization of processes transmitting transpiration-related
signals to guard cells. For this purpose, selected pores
or defined areas of the leaf were prevented from
transpiring and the humidity response was observed.
Oil sealing of pores proved to be a reliable and
rather simple method to block transpiration of selected
pores without mechanical disturbance. In comparison
to applying streams of air (Lange et al., 1971; Mott
et al., 1997; Mott and Franks, 2001), oil treatment
provides a spatially better-defined manipulation of
transpiration. The oil, due to capillary force, stays
in place even if stomata open and close for at least
1 week. Coating the entire leaf with oil reduced leaf
conductance to the same degree as stomatal closure.
Therefore, it is justified to assume that sealing a pore
with oil effectively suppresses transpiration. Oil treatment did not seem to interfere with stomatal responses
because, during application with a micropipette, stomata did not respond in either direction and in the
long term did not change their response characteristics
under nontranspiring conditions (Figs. 1 and 2). The
same is true for covering the leaf with adhesive foil:
After removal, the response was essentially the same
as before shielding (Fig. 6). The drawback of these
methods is the possible spatial inhomogeneity of leaf
internal CO2 concentration caused by hindered CO2
diffusion into the photosynthesizing leaf. This problem was addressed by conducting all experiments at
approximately the CO2 compensation point when,
regardless of stomatal opening, all leaf areas should
have equal internal CO2 concentration. The general
degree of opening in these experiments is possibly
somewhat higher than under ambient [CO2], but this
effect was equal in all treatments. Moreover, a control
experiment comparing humidity responses under ambient and reduced [CO2] revealed no difference in the
course of the response to reduction in humidity (data
not shown).
The most important result of these experiments is
that blocking a single pore’s transpiration does not
reduce the amplitude of passive opening and active
closure in response to a decrease in air humidity (Figs.
5 and 6A). The response of a guard cell pair to dry air
does not require transpiration through its pore as long
as the other pores on a leaf are transpiring. This
suggests that transpiration from all other surrounding
pores may still have reduced water potential locally to
the same degree as for no oil treatment. This argues
against a number of hypothetical mechanisms that
suggest a response of guard cells to water loss from
their own substomatal surface or substomatal cavity.
First of all, this result is incompatible with purely
hydraulic mechanisms driven by differential transpiration between guard and adjacent epidermal cells.
Different authors have suggested that direct evaporation from the substomatal face of the guard cells could
draw guard cell turgor down relative to the epidermal
turgor if there is a sufficiently large hydraulic resistance between guard cells and epidermis (Nonami
et al., 1990; Dewar, 1995, 2002). This mechanism, however, requires complicated additional assumptions to
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Kaiser and Legner
Figure 3. Typical stomatal response to a sudden increase in DW from
2 to 18 mmol mol21 (at time 5 0). Arrows indicate the amplitude of
hydropassive opening (Dcpass) and hydroactive closure (Dcact). Dcpass is
defined as the difference between circularity prior to the humidity
change and the maximal circularity (cmax). Dcact is the difference
between cmax and the final circularity 1.5 h after the humidity change.
be able to explain the initial wrong-way response to
an increase of transpiration (Buckley and Mott, 2002;
Buckley, 2005). Our results add evidence contrary to
this hypothesis because stomata perform the unchanged biphasic response to reduced humidity even
when their pores do not transpire and no direct
evaporation from the guard cells apart from cuticular
transpiration occurs.
Another possible mechanism acting in the immediate surrounding of the guard cells is the accumulation
of substances in the guard cell apoplast caused by
evaporation from the guard cell walls, which was proposed by Grantz (1990). For the apoplastic solute Suc,
Outlaw and De Vlieghere-He (2001) found transpirationdependent accumulation in the guard cell apoplast,
which should result in a sufficient decrease of apoplastic osmotic potential to decrease stomatal aperture.
Such an effect, however, appears unlikely because the
neighboring epidermal cells should also lose turgor,
thus at least nullifying the opening effect. In any case,
this observation bolstered the idea of transpirationdriven accumulation of abscisic acid (ABA), which
was also supported by microanalytical determination
of ABA in the guard cell apoplast (Zhang and Outlaw,
2001; Zhang et al., 2001). Our results add evidence
against such a distillation-dependent effect in S. nigra
because the humidity response was not changed when
direct evaporation from the guard cell apoplast was
largely prevented.
However, the possibility remains that peristomatal
transpiration from the outer surface of guard cells
mediates the humidity response. This would explain
the lacking effect of oil treatment of single pores on
their humidity response. There are three reasons why
this explanation does not appear valid.
First of all, stomata of S. nigra are known for their
tendency to oscillate in dry air (Kaiser and Kappen,
2001), which in itself points to a prominent role of feed
back versus feed forward. It has been shown in detail
that, during oscillations, the onset of guard cell deflation strictly follows the moment of initial opening of
the pore, whereas in the closed state, guard cells inflate
even if the air is very dry. Closure, therefore, is initiated by pore transpiration rather than cuticular transpiration of guard cells.
Second, if peristomatal transpiration had been a
major signal, sealing the adjacent pores or blocking
transpiration outside a small area should have left the
response unaffected, which it did not (Fig. 6B).
Third, in the presented experiments, stomata generally had a high degree of opening. The majority of
stomata did not fully close in dry air, possibly due to
lowered [CO2]. Therefore, in the untreated leaf, the
portion of cuticular (peristomatal) transpiration from
guard cells must have been negligible compared to stomatal transpiration. The treatment had an unknown,
but presumably small, effect on peristomatal transpiration, whereas it had a large and substantial effect on
transpiration through the pore. Thus, it appears justified to assign treatment effects to the change in pore
transpiration.
Figure 4. Stomatal response to dry air before and after sealing pores
with oil. The graph shows responses of six stomata (mean 6 SD) to an
increase of DW from 2 to 18 mmol mol21 (vertical line) before (A) and
after (B) sealing the pores with mineral oil (black symbols) compared to
an untreated control group (white symbols).
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Spatial Aspects of Stomatal Humidity Response
shaped function seemed appropriate because both the
lateral spreading of water potential disturbances and
potentially involved diffusion of molecules in the
gaseous and aqueous phase of the tissue follow thermodynamic and therefore stochastic processes. As a
first guess, the Gaussian function was used. The shape
parameter s describes the decreasing effect with distance to the transpiring pore, with a decline to 50% in
the distance s. According to the parameter estimation
by this model, the lateral effect of a pore’s transpiration
Figure 5. The effect of restricting leaf transpiration to small areas of
0.5 mm2 on the responses of stomata to dry air. The graph shows the
responses (mean 6 SD) of nine stomata to an increase of DW from 2 to
18 mmol mol21 before and after attachment of perforated foil to the
lower leaf surface and, finally, after removal of the foil. Measurements
were performed on three consecutive days.
The responses of oil-treated pores indicate that
transpiration-related events leading to stomatal closure in dry air are not located at single pores, but, to a
greater or lesser degree, distributed in the leaf lamina.
In other words, the transpiration of a pore must have a
closing effect on neighboring pores. The other two
experiments were designed to determine the lateral
extension of this effect. Sealing the surrounding pores
in addition to the observed pore led to a decrease of
the amplitude of active closure by about 40% (Fig. 6B).
Apparently, a significant portion of the total closing
stimulus must have originated from the now sealed
area of approximately 0.2 mm2, but a larger part was
still provided by the rest of the leaf.
This conclusion is confirmed by the inverse experiment: When transpiration was restricted to small
areas of 0.5 mm2 by attaching perforated foil, the
amplitude of active closure was reduced by approximately 50% (Fig. 6C). This means that, in these experiments, roughly one-half of the closing stimulus
originated inside the still transpiring leaf area and the
other half was removed by preventing transpiration
from the rest of the leaf.
Together, the experiments point to an extension of
the lateral effect of a pore’s transpiration smaller than
1 mm. To describe this spatial relationship more precisely, all datasets under the three different treatments
were used to estimate the shape parameter of a spatial
model (Fig. 7A). This model is based on the assumption that the effect of a pore’s transpiration on its own
guard cells and on other stomata decreases with distance. The function that best describes this relationship
is unknown and open to discussion. The use of a bell-
Figure 6. Effect of different spatial restriction schemes on the stomatal
response to an increase of DW. This plot compares the amplitudes of the
hydropassive opening and the active closing response between stomata
in their untreated (white bars) and treated (gray bars) states. A, Effect of
oil treatment of individual pores on their humidity response (26
individual responses from two leaves). B, Effect of oil sealing of pores
and their adjacent pores (27 responses from three leaves). C, Effect of
restricting the transpiration to circular areas of 0.8-mm diameter (21
responses from three leaves). A and B display the amplitude of the
circularity of stomatal pores; C displays the amplitude of the circularity
of the guard cell pairs. Insets show significance level (n.s., P . 0.05;
***, P , 0.001) and 95% confidence intervals for the difference of
means between treatments.
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Kaiser and Legner
Figure 7. Modeled effect of a pore’s transpiration on the responses of
other stomata in dependence on the distance. The estimated effects
together with their 95% confidence intervals on hydropassive opening
(dashed line/gray fill) and active closure (black line/hatched fill) are
displayed. Drop lines mark the radius of the areas that receive 90% of
the total stimulus. For further explanation, see text.
on active closure of other pores on the leaf decreases to
50% in a distance of 0.24 mm and 90% of the total effect
cover an area of 0.5 mm2 and affects only approximately the 23 nearest stomata.
This result is in general agreement with Xanthium
strumarium measurements by Mott et al. (1997), who
subjected small groups of stomata to different humidity from the rest of the leaf by directing a small stream
of air to the leaf surface by a needle. Similar to the
results presented here, they found that, even if stomata
were under constant humidity, they responded to reduced humidity in their vicinity. The extension of the
lateral effects is roughly in the same range as reported
here, although the small number of observed stomata,
together with the less well-defined border between
regions of high and low humidity, allows no exact
estimation.
Comparable experiments were also performed by
Lange et al. (1971), who directed streams of different
humidity to closely neighboring regions of epidermal
strips of Polypodium vulgare. Somewhat contradictory
to our results, stomata strictly followed the local humidity conditions and even closely neighboring stomata showed opposite responses. The reason for the
discrepancy could be the use of epidermal strips or differences between species. The epidermis of P. vulgare
is connected to the mesophyll only at the main veins,
which may result in different hydraulic conditions in
the epidermis influencing properties of the stomatal
humidity response.
The conclusion drawn, that the primary processes of
humidity sensing must be small-scale distributed processes in the leaf tissue, seems to agree with the
hypothesis that cl triggers stomatal responses to humidity. Much indirect or circumstantial evidence
shows a close correlation between cl and stomatal responses (for review, see Buckley, 2005).
This hypothesis is supported by the frequent observation that any perturbation of the balance between
water supply and loss that influences cl causes similar
stomatal responses. Nonetheless, it is still uncertain
whether this is a causal relationship. With this question in mind, we will now discuss the hydraulic aspects revealed by the hydropassive opening response.
Following the concept of the mechanical advantage
of epidermal cells over guard cells (Sharpe et al., 1987;
Franks et al., 1998), the amplitude of the transient
wrong hydropassive opening can be taken as a surrogate measure for the decline of local cl if the hydraulic
conductivity between guard and epidermal cells is
sufficient for an equilibration during the dynamic
changes in local cl caused by increased transpiration.
Typical relaxation half times of plant cells are mostly
around 1 to 60 s (Kramer and Boyer, 1995), which would
be sufficient to allow hydraulic equilibration during
the hydropassive opening, which lasts for 5 to 10 min.
Apparently, hydraulic resistance of guard cell membranes has not yet been measured systematically, but
K.A. Mott and J.C. Shope (unpublished data), reported
by Buckley (2005), point to similar relaxation times in
guard cells of Vicia faba. The theoretical possibility that
hydraulic resistance of the guard cell membrane is temporarily increased (e.g. by regulation of aquaporins;
Sarda et al., 1997; Huang et al., 2002), which would
prevent quick equilibration, appears counterproductive because this would impede turgor loss of guard
cells in a situation that demands stomatal closure.
Following this consideration, it seems justified to
use the amplitude of the hydropassive response as a
surrogate measure for the local decline of cl. Sealing
the observed pore and its adjacent pores with oil had
no effect on the hydropassive opening. This shows that
direct evaporation from a pore’s substomatal cavity or
the adjacent substomatal cavities is not necessary to
cause local disturbance in local cl, resulting in transient hydropassive opening. Obviously, hydraulic coupling of the epidermis is sufficient to allow lateral
water transport, which equalizes these local differences in cl. According to the model estimation (Fig. 7),
the hydraulic effect of stomatal transpiration declines
to 50% in a distance of 0.35 mm. This agrees with
epidermal turgor reductions measured by Mott and
Franks (2001) in different distances to a small region
influenced by a stream of dry air applied through a
needle. Turgor pressure effects dropped to roughly
50% in a distance of 300 to 400 mm from the needle tip.
Conspicuously, the effect of a pore’s transpiration on
hydropassive opening extends further than the effect
on active closure. This was most obvious when both
the observed and the surrounding pores were treated
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Spatial Aspects of Stomatal Humidity Response
with oil (Fig. 6B): In this case, the active closure was
clearly reduced, although the constant hydropassive
effect indicates virtually the same local reduction of cl
as before. From the hypothesis that guard cells respond to local cl, we would expect, however, that both
effects extend to the same distance. The observation
that the lateral effect of a pore’s transpiration on stomatal closure is more locally confined than the effect
on cl suggests that guard cells may not exclusively
respond to the disturbance of local cl, but may also
utilize other sensing mechanisms driven by transpiration. These could act instead of or in addition to direct
sensing of local cl. Another physical property related
to transpiration that could be sensed is, for example,
the rate of hydraulic flow through a tissue, which
should better reflect local transpiration conditions
than steady-state cl.
Although the mechanisms remain unclear, these
results suggest an important role of the leaf tissue in
humidity/transpiration sensing as other results also
indicate (Cochard et al., 2002). One possibility for such
an indirect sensing mechanism is the production of an
intermediate signal that is transported to guard cells.
This two-step mechanism is also consistent with the
often extended lag phase, which precedes stomatal
closure in response to dry air (H. Kaiser, unpublished data; Buckley and Mott, 2002). This lag phase
can be attributed to the time necessary for producing
and releasing the closing agent into the apoplast and
for diffusion to the guard cells. Other stimuli that are
sensed directly by the guard cells (e.g. blue light
and CO2), in contrast, generally elicit much faster responses.
The putative sensitive tissue and the intercellular
signals are not yet identified. As the results presented
here allow only indirect conclusions on the involved
sensing mechanisms, they will be discussed only
briefly. Much evidence suggests an important role of
mesophyll cells (Lee and Bowling, 1995). Grantz and
Schwartz (1988) found that guard cells need the presence of mesophyll cells to be able to respond to osmotic stress. Leaf tissues may produce signaling
molecules that diffuse to the guard cells or modify
the composition of the transpiration stream. This could
involve changes in the pH (Roelfsema and Prins, 1998)
and ionic composition (Atkinson et al., 1989; Ruiz et al.,
1993) of the apoplastic solution or release and recompartmentation of ABA (e.g. Trejo et al., 1993; Hartung
et al., 1998; Wilkinson and Davies, 2002). Although
ABA is the favorite candidate for an intermediate signal, there is also evidence for an ABA-independent
signaling pathway (Assmann et al., 2000; Luan, 2002;
Yoshida et al., 2006).
In conclusion, by combining different experimental
procedures, we have established that guard cells do
not respond to their own transpiration, but to a locally
averaged water loss in the surrounding of the pore.
The observation that disturbances of cl as measured by
the hydropassive opening response do not correlate
with the hydroactive closure could point to the exis-
tence of controlling entities other than local cl. The
results support the hypothesis that the primary and
initial transpiration-sensing processes are not located
in the guard cells, but involve transpiration-related
processes in the leaf tissue.
MATERIALS AND METHODS
Plant Culture and Experimental Setup
Experiments were performed on attached leaves of potted Sambucus nigra
plants of approximately 50 to 80 cm in size. Plants were drawn from cuttings
and cultivated in 40-cm pots in a climatic chamber at a PPFD of 220 mmol m22
s21 (16-h light/8-h dark) and a temperature of 20°C. Plants were amply
supplied with water and nutrients. Stomatal movements were observed on
mature leaves in a gas-exchange chamber designed for simultaneous measurement of CO2-water gas exchange and microscopic observation of stomatal
movements under controlled light, humidity, temperature, and CO2 conditions (Kaiser and Kappen, 2001). Temperature, leaf-to-air mole fraction of
water vapor (DW) and [CO2] in the cuvette were set to 20°C to 22°C, 2 mmol
mol21, and approximately 360 mmol mol21, respectively. Irradiance (500 mmol
m22 s21, 16-h light/8-h dark) was provided by a fiber-optic illuminator
(Kaltlicht-Fiberleuchte FL-400 with Spezial Fiberoptik 400-F; Walz). After
mounting the leaf in the gas-exchange cuvette, the plant was allowed to adjust
to measuring conditions for at least 24 h. Leaves were fixed with the adaxial
side to a Perspex plate with double-sided transparent adhesive tape (Tesa
56661–2; Tesa) to allow micromanipulation. Subsequently, the plate was
mounted inside the cuvette, which allows observation of the lower leaf surface with a long-distance microscope lens (503) led through the bottom of the
gas-exchange cuvette (Kaiser and Grams, 2006). The microscope (Axiovert
25CFL; Zeiss) is mounted on a motorized translation stage, which allows
repositioning of samples of selected stomata. Digital images of stomata were
recorded with a video camera, digitized, and stored for subsequent measurement of aperture with custom image analysis software. The aperture of oiltreated pores can no longer be measured due to refraction of the oil. In these
experiments, the area of the guard cell pair between the anticlinal walls was
measured. This measure is linearly related to aperture when pores are open
(Kaiser and Kappen, 2001) and can be taken as a surrogate measure of stomatal opening. Pore area or guard cell pair area were converted to circularity
(c 5 width 3 100/length) to allow comparison between differently sized
stomata. The humidity control by a bypass compensation system was used to
perform quick changes in air humidity from DW 5 2 to approximately
18 mmol mol21 by switching the humidity of the incoming air to lower
humidity. Within approximately 90 s, DW arrived at its new steady state
(Fig. 3). One hour before increasing DW, [CO2] was reduced to approximately
60 to 70 mmol mol21, which is approximately the CO2 compensation point for
C3 plants (von Caemmerer and Farquhar, 1982). This avoids intercellular
[CO2] gradients due to locally suppressed CO2 diffusion into the mesophyll.
Stomatal responses to a reduction in air humidity were observed before and
after the treatment with oil or adhesive foil on subsequent days, always
beginning at the same time 4 h after illumination was switched on. Stomatal
apertures were observed from at least 30 min before to 1.5 h after reduction of
air humidity. Images were taken every 3 to 4 min if apertures changed fast,
otherwise at longer intervals of up to 10 min.
Oil Treatment of Pores
The cuvette allows micromanipulation on the lower leaf surface by micropipettes inserted through small holes in the cuvette wall. Micropipettes were
drawn from 1.5-mm borosilicate glass capillaries with a pipette puller
(L/M-3P-A; List). Tips were ground to a diameter of approximately 5 mm.
Mineral oil (M8662; Sigma-Aldrich) was applied to pores by approaching the
oil-filled pipette to a pore and gently pressurizing it manually by pressing a
rubber ball. In each experiment, six to 20 pores with a spacing of at least 2 mm
were selected. Either the observed pore or the observed pore plus the adjacent
pores was sealed with oil. In an additional experiment, which was only used
for the model parameter estimation, only the adjacent pores were sealed
(leaving the observed central pore free). In all experiments, a control sample of
the same size as the treatment sample was observed to detect and correct for
interday variability, which, however, was found to be small.
Plant Physiol. Vol. 143, 2007
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Kaiser and Legner
Shielding of the Leaf with Perforated Adhesive Foil
To shield the entire leaf except circular areas of 0.8 mm, thin polyethylene
plastic foil (20 mm) was punched with a 0.8-mm syringe needle, which was
ground squarely and sharpened. The correct size of the holes was confirmed
microscopically after attachment to the leaf. Very thin double-sided adhesive
tape (Pritt permanent) was used to attach the foil to the leaf, allowing only the
epidermis inside the circular holes to transpire. In some cases, the foil was not
attached firmly to the epidermis at the edge of the punched hole, leaving a gap
between leaf and foil. These stomata were excluded from the experiments. Up
to six holes with a distance of at least 10 mm were punched into a foil in one
experiment. The foil was at first attached provisionally in its final position to
select stomata located inside the holes. The foil was then removed to observe a
control response to a decrease in air humidity of selected stomata. Thereafter,
the foil was attached firmly in the same position as before to measure the
humidity response of the sample of stomata on the next day. In some
experiments, the foil was carefully removed afterward and another control
response was measured on the next day to test for permanent damage due to
experimental treatment.
ACKNOWLEDGMENTS
We want to thank Annika Hagemann for help with image analysis,
Ludger Kapppen and three anonymous reviewers for helpful comments on a
former version of the manuscript, and Patricia Schoone for language editing.
Received September 1, 2006; accepted November 27, 2006; published December 8, 2006.
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