Direct Observations of Stomatal Movements
233
potential (Schulze 1986) or by a direct ABA signal from the root (Zhang
and Davies 1989; Schurr 1992). Water stress response can be induced in
many plant species by a decrease in the water content in the ambient air
(Lange et al. 1971; Schulze et al. 1987; Grantz 1990). Stomata respond
promptly to air humidity changes and therefore experiments based on
humidity alterations are very useful in testing stomatal movements.
In most cases, stomatal movement or conductance has been estimated
by calculations using water relations, transpiration, and temperature as
parameters (Ball 1987; Von Caemmerer and Farquhar 1981). Rarely, stomatal arerture has been observed directly (Elkins and Williams 1962; Losch
1977; Omasa et al. 1983), but it was not possible to observe stomatal
responses in darkness. In the present chapter, we demonstrate to what
extent visual inspection of stomatal responses in situ, coupled with simultaneous measurements of CO2 and water fluxes, are capable of giving direct
evidence of the hitherto reported stomatal responses to plant internal and
external factors. Moreover, it is demonstrated that our method allows more
insight into the mechanism of opening and closing of the stomata. So,
for instance, a transient phase in the response to changed air humidity
clearly indicates the role of a hydraulic component in the stomatal response
mechanism.
11.2 The Methodical Approach
The methods used here were described in detail by Kappen et al. (1987) and
Kappen and Haeger (1991). Principally, the system consists of a conditioned
gas exchange cuvet (7 dm 3 ) with CO2 and H20 analysis units (H. Walz,
Effeltrich, Germany). The system operates with an open gas stream and
records transpiration, net photosynthesis, and dark respiration. The leaf,
still attached to the intact plant, is sealed within the gas exchange cuvette
and fixed by a clamp that can be moved by a microscopic gliding stage
(Kappen et al. 1987). This is hand-operated by three external adjustment
knobs. Motor-driven remote control was mounted recently and will be used
in future experiments (see Omasa et al. 1983).
The conditioning unit of the gas exchange cuvette is laterally mounted so
that the microscope device could be inserted through the bottom part of
the chamber (Kappen et al. 1987; Fig. 11.2). Images of the stomata are
taken either in light from incandescent lamps illuminating the gas exchange
cuvette or in physiological darkness by means of IR light (780-1200 nm)
that is directed through the microscope objective to the leaf surface. Images
are taken by a TV camera and are visible on a computer monitor. Instead
of the previously described image-analysis method (area determination
from photographic pictures), we are now using digital picture analysis as
described by Omasa and Onoe (1984) and Van Gardingen et al. (1989). Our
233
potential (Schulze 1986) or by a direct ABA signal from the root (Zhang
and Davies 1989; Schurr 1992). Water stress response can be induced in
many plant species by a decrease in the water content in the ambient air
(Lange et al. 1971; Schulze et al. 1987; Grantz 1990). Stomata respond
promptly to air humidity changes and therefore experiments based on
humidity alterations are very useful in testing stomatal movements.
In most cases, stomatal movement or conductance has been estimated
by calculations using water relations, transpiration, and temperature as
parameters (Ball 1987; Von Caemmerer and Farquhar 1981). Rarely, stomatal arerture has been observed directly (Elkins and Williams 1962; Losch
1977; Omasa et al. 1983), but it was not possible to observe stomatal
responses in darkness. In the present chapter, we demonstrate to what
extent visual inspection of stomatal responses in situ, coupled with simultaneous measurements of CO2 and water fluxes, are capable of giving direct
evidence of the hitherto reported stomatal responses to plant internal and
external factors. Moreover, it is demonstrated that our method allows more
insight into the mechanism of opening and closing of the stomata. So,
for instance, a transient phase in the response to changed air humidity
clearly indicates the role of a hydraulic component in the stomatal response
mechanism.
11.2 The Methodical Approach
The methods used here were described in detail by Kappen et al. (1987) and
Kappen and Haeger (1991). Principally, the system consists of a conditioned
gas exchange cuvet (7 dm 3 ) with CO2 and H20 analysis units (H. Walz,
Effeltrich, Germany). The system operates with an open gas stream and
records transpiration, net photosynthesis, and dark respiration. The leaf,
still attached to the intact plant, is sealed within the gas exchange cuvette
and fixed by a clamp that can be moved by a microscopic gliding stage
(Kappen et al. 1987). This is hand-operated by three external adjustment
knobs. Motor-driven remote control was mounted recently and will be used
in future experiments (see Omasa et al. 1983).
The conditioning unit of the gas exchange cuvette is laterally mounted so
that the microscope device could be inserted through the bottom part of
the chamber (Kappen et al. 1987; Fig. 11.2). Images of the stomata are
taken either in light from incandescent lamps illuminating the gas exchange
cuvette or in physiological darkness by means of IR light (780-1200 nm)
that is directed through the microscope objective to the leaf surface. Images
are taken by a TV camera and are visible on a computer monitor. Instead
of the previously described image-analysis method (area determination
from photographic pictures), we are now using digital picture analysis as
described by Omasa and Onoe (1984) and Van Gardingen et al. (1989). Our