Download Stomatal Size, Speed, and Responsiveness

Topical Review on Stomata and Water Use Efficiency
Stomatal Size, Speed, and Responsiveness Impact on
Photosynthesis and Water Use Efficiency1[C]
Tracy Lawson* and Michael R. Blatt
School of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom (T.L.); and
Laboratory of Plant Physiology and Biophysics, University of Glasgow, Glasgow G12 8QQ,
United Kingdom (M.R.B.)
The control of gaseous exchange between the leaf and bulk atmosphere by stomata governs CO2 uptake for photosynthesis and
transpiration, determining plant productivity and water use efficiency. The balance between these two processes depends on stomatal
responses to environmental and internal cues and the synchrony of stomatal behavior relative to mesophyll demands for CO2. Here we
examine the rapidity of stomatal responses with attention to their relationship to photosynthetic CO2 uptake and the consequences for
water use. We discuss the influence of anatomical characteristics on the velocity of changes in stomatal conductance and explore the
potential for manipulating the physical as well as physiological characteristics of stomatal guard cells in order to accelerate stomatal
movements in synchrony with mesophyll CO2 demand and to improve water use efficiency without substantial cost to photosynthetic
carbon fixation. We conclude that manipulating guard cell transport and metabolism is just as, if not more likely to yield useful benefits
as manipulations of their physical and anatomical characteristics. Achieving these benefits should be greatly facilitated by quantitative
systems analysis that connects directly the molecular properties of the guard cells to their function in the field.
In order for plants to function efficiently, they must
balance gaseous exchange between inside and outside the
leaf to maximize CO2 uptake for photosynthetic carbon
assimilation (A) and to minimize water loss through
transpiration. Stomata are the “gatekeepers” responsible
for all gaseous diffusion, and they adjust to both internal
and external environmental stimuli governing CO2 uptake and water loss. The pathway for CO2 uptake from
the bulk atmosphere to the site of fixation is determined
by a series of diffusional resistances, which start with the
layer of air immediately surrounding the leaf (the
boundary layer). Stomatal pores provide a major resistance to flux from the atmosphere to the substomatal
cavity within the leaf. Further resistance is encountered
by CO2 across the aqueous and lipid boundaries into the
mesophyll cell and chloroplasts (mesophyll resistance).
Water leaving the leaf largely follows the same pathway
in reverse, but without the mesophyll resistance component. Guard cells surround the stomatal pore. They increase or decrease in volume in response to external and
internal stimuli, and the resulting changes in guard cell
shape adjust stomatal aperture and thereby affect the flux
1
This work was supported by the Natural Environment Research
Council (studentship to T.L.) and the Biotechnology and Biological
Sciences Research Council (grant no. BB/I001187/1 to T.L. and grant
nos. BB/H024867/1, BB/F001630/1, BB/H009817/1, and BB/
L001276/1 to M.R.B.).
* Address correspondence to [email protected].
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:
Tracy Lawson ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
www.plantphysiol.org/cgi/doi/10.1104/pp.114.237107
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of gases between the leaf internal environment and the
bulk atmosphere. Stomatal behavior, therefore, controls
the volume of CO2 entering the intercellular air spaces of
the leaf for photosynthesis. It also plays a key role in
minimizing the amount of water lost. Transpiration, by
virtue of the concentration differences, is an order of
magnitude greater than CO2 uptake, which is an inevitable consequence of free diffusion across this pathway.
Although the cumulative area of stomatal pores only
represents a small fraction of the leaf surface, typically
less than 3%, some 98% of all CO2 taken up and water
lost passes through these pores. When fully open, they
can mediate a rate of evaporation equivalent to one-half
that of a wet surface of the same area (Willmer and
Fricker, 1996).
Early experiments illustrated that photosynthetic rates
were correlated with stomatal conductance (gs) when
other factors were not limiting (Wong et al., 1979). Low
gs limits assimilation rate by restricting CO2 diffusion
into the leaf, which, when integrated over the growing
season, will influence the carbohydrate status of the leaf
with consequences for crop yield. Stomata of wellwatered plants are thought to reduce photosynthetic
rates by about 20% in most C3 species and by less in C4
plants in the field (Farquhar and Sharkey, 1982; Jones,
1987). However, even this restriction has been shown to
impact substantially on yield. For example, Fischer et al.
(1998) demonstrated a close correlation between gs and
yield in eight different wheat (Triticum aestivum) cultivars. Those studies highlighted the effects gs can have on
crop yield, not only through reduced CO2 diffusion but
also through the impact on water loss and evaporative
cooling of the leaf. Indeed, enhancing photosynthesis
yields by only 2% to 3% is sufficient to substantially increase plant growth and biomass over the course of a
growing season (Lefebvre et al., 2005; Zhu et al., 2007).
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Stomata Impact on Water Use Efficiency
Stomata and their behavior profoundly affect the global
fluxes of CO2 and water, with an estimated 300 3 1015 g
of CO2 and 35 3 1018 g of water vapor passing through
stomata of leaves every year (Hetherington and
Woodward, 2003). Changes in stomatal behavior in
response to changing climatic conditions are thought to
impact on water levels and fluxes. For example, it is estimated that partial stomatal closure driven by increasing
CO2 concentration over the past two decades has led to
increased CO2 uptake and reduced evapotranspiration in
temperate and boreal northern hemisphere forests
(Keenan et al., 2013), with implications for continental
runoff and freshwater availability associated with the
global rise in CO2 (Gedney et al., 2006). Concurrently, the
increase in global water usage over the past 100 years
and the expectation that this is set to double before 2030
(UNESCO, 2009) has put pressure on breeders and scientists to find new crop varieties, breeding traits, or
potential targets for manipulation that would result in
crop plants that are able to sustain yield with less water
input. The fact that stomata are major players in plant
water use and the entire global water cycle makes the
functional and physical attributes of stomata potential
targets for manipulation to improve carbon gain and
plant productivity as well as global water fluxes.
There are several approaches for improving carbon
gain and plant water use efficiency (WUE) that focus on
stomata. It is possible to increase or decrease the gaseous
conductance of the ensemble of stomata per unit of leaf
area (gs) through the manipulation of stomatal densities
(Büssis et al., 2006). In addition, there is potential to alter
the stomatal response or sensitivity to environmental
signals through the manipulation of guard cell characteristics that affect stomatal mechanics (e.g. OPEN
STOMATA [ost] mutants; Merlot et al., 2002). Such
approaches have produced an array of mutant plants
with altered characteristics and varying impacts on CO2
uptake and transpiration, several of which we discuss
in greater detail below. An intuitive measure of the efficacy of such manipulations is the WUE, commonly
defined as the amount of carbon fixed in photosynthesis
per unit of water transpired. In general, higher WUE
values have been observed in plants with lower gs, but
these gains are usually achieved together with a reduction in A and slower plant growth. Plants with
higher gs have greater assimilation rates and grow faster
under optimal conditions, but they generally exhibit
lower WUE. An approach that has not been fully explored or considered in any depth is to select plants for
differences in the kinetics of stomatal response or to
manipulate stomatal kinetics in ways that improve the
synchrony with mesophyll CO2 demand (Lawson et al.,
2010, 2012). To date, the majority of studies assessing
the impact of stomatal behavior on photosynthetic
carbon gain have focused on steady-state measurements of gs in relation to photosynthesis. These studies
do not take account of the dynamic situation in the
field. As we discuss below, a cursory analysis of stomatal synchrony with mesophyll CO2 demand suggests
that gains of 20% to 30% are theoretically possible.
Here, we address the question of the kinetics of the
stomatal response to the naturally fluctuating environment, notably to fluctuations in light that are typical of
the conditions experienced in the field. We focus on
vascular seed plants, to which crop plants belong, and
do not address seedless vascular plants, such as ferns.
The characteristics of the latter, and hence the issues
and challenges they present, are very different. In our
minds, of paramount importance is whether there is
potential for engineering guard cells of crop plants to
manipulate the dynamic behavior of stomata so as to
improve WUE without substantial cost in assimilation.
Of course, in many circumstances, stomata are not the
only factor to limit water flux through the plant (see
other articles in this issue), but they are one of the most
important “gatekeepers” and therefore, serve as a good
starting point for such considerations. Thus, we explore
the physical and functional attributes of stomata, their
signaling, and the solute transport mechanisms that determine pore aperture as targets for potential manipulation
of stomatal responses to changing environmental cues.
INFLUENCE OF ANATOMY ON gs
In addition to stomatal sensitivity and responses to
physiological drivers, gs is also dependent upon anatomical characteristics. Stomatal anatomical features define the maximum theoretical conductance (Dow et al.,
2014a) and also influence the speed of response. Maximum gs is dictated by the size and density of stomata,
which in turn can be influenced by the growth environment (Hetherington and Woodward, 2003; Franks
and Farquhar, 2007). It is generally accepted that stomatal density is altered by atmospheric CO2 concentration (Woodward, 1987; Gray et al., 2000), light (Gay and
Hurd, 1975), and other environmental factors. More recent experimental evidence has demonstrated that stomatal density is negatively correlated with stomatal size
(Hetherington and Woodward, 2003; Franks and Beerling,
2009). The interaction/correlation between stomatal size
and density and the impact on stomatal function have
received much attention, particularly with reference to the
evolution of performance and plasticity in plants (Franks
and Farquhar, 2007). The latest studies have also implied
that physical attributes affect stomatal response times
following environmental perturbations (Drake et al.,
2013). Therefore, although it is possible to manipulate
stomatal function, we must be fully aware of the interactions between stomatal size and numbers and
the impact they can have on rapidity of stomatal
movement.
Stomatal Density
Selecting crop plants with alterations in stomatal
density to improve/reduce plant water use was first
explored in the 1970s and 1980s (Jones, 1977, 1987) in
various breeding programs with limited success. The
original hypothesis was that increasing or decreasing stomatal numbers would, respectively, increase
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Lawson and Blatt
or decrease gs. However, several studies have demonstrated that this approach may be more complex
than the simple argument outlined above. Arabidopsis
(Arabidopsis thaliana) stomatal density and distribution
(sdd1-1) mutants have a point mutation in a single gene
that encodes a subtilisin-like Ser protease that results in
plants with a 2.5-fold higher stomatal density compared
with their wild-type counterparts (Berger and Altmann,
2000). The increase in stomatal density translates to an
increase in gs and 30% greater photosynthetic rate under
high-light conditions (Schlüter et al., 2003). Transgenic
plants overexpressing SDD1 have a 40% reduction in
stomatal densities compared with the wild type (Von
Groll et al., 2002). In a comparative study, Büssis et al.
(2006) showed no difference in gs or assimilation rate
when measured under growth photosynthetically active
photon flux density (PPFD) conditions (180 mmol m22 s21)
between the overexpressing SDD1 plants, the sdd1-1
mutants, and wild-type controls. These findings indicated that increased stomatal aperture compensated
for the lower stomatal density in the SDD1 plants
while reduced aperture in the sdd1-1 plants offset the
greater number of stomata. However, at high light
intensities, low gs in the SDD1 overexpressors and restricted CO2 diffusion limited A to 80% of the wild
type (Büssis et al., 2006). These findings exemplify the
role of both the physical and functional stomatal features in determining gs and that manipulation of
physical attributes may be counterbalanced by modifications in function. However, more meaningfully,
this work illustrates the importance of the surrounding
environmental conditions on stomatal behavior and the
significance of examining gs limitation on A at appropriate light and CO2 concentrations. In the example
above, no significant impacts on carbon gain were observed under growth PPFD; however, when photosynthesis was driven by greater PPFD, A was limited by CO2
diffusion via reduced gs. Such studies also provide an
explanation for why breeding for increased WUE by altering stomatal density has so far been mostly unsuccessful (Jones, 1987). To date, there is only one report
claiming a positive effect of stomatal density on A in
plants maintained in their growth conditions: Tanaka
et al. (2013) used overexpression of STOMAGEN, a positive regulator of stomatal density, to produce plants with
a 2- to 3-fold greater stomatal density than the wild type.
A in these plants was increased by 30% due to greater
CO2 diffusion into the leaf rather than changes in photosynthetic carboxylation capacity (Tanaka et al., 2013).
However, the resultant 30% increase in A was at the expense of transpiration, which was double that observed in
the wild-type plants, resulting in a 50% decrease in WUE.
As stomatal density has been correlated with gs
(Franks and Beerling, 2009), the rationale behind manipulating stomata anatomical characteristics to increase
or decrease stomatal density may be considered relatively straightforward, helped by studies that have illustrated that manipulation of a single gene can alter
stomatal patterning (Doheny-Adams et al., 2012). However, the manipulation of stomatal functional responses
is clearly more complicated. Before such approaches are
possible, we need a full understanding of the metabolic
pathways (and underlying genetics) that form the basis
of stomatal sensing, signaling, and response processes as
well as a comprehension of physiological responses at
the leaf level and at the cellular level. This understanding
will need to include an appreciation for the hierarchical
response of guard cells to internal and external signals
and is likely to benefit from exploring the natural variation that exists in the magnitude of changes between
individual stoma or groups of stomata within and between leaves (Lawson et al., 1998). Additionally, if such
manipulations are directed at improving WUE, it is important to understand the coordination and synchronization of gs responses with mesophyll carbon assimilation
(Lawson et al., 2012).
Stomatal Patterning
Most research has focused on the physical behavioral
aspects of stomata on gaseous diffusion, but investigations using density and patterning mutants have
underpinned the physiological importance of stomatal
patterning on CO2 uptake and water loss. An interesting observation in the Büssis et al. (2006) study was the
high-resolution chlorophyll fluorescence imaging that
revealed two distinct areas of mesophyll in the SDD1
plants: one where mesophyll lay above stomata and the
other where stomata were absent. Measurements of
maximum and actual photosynthetic efficiency were
identical to the wild type in areas with stomata; however, leaf areas without stomata showed lower maximum and actual photosynthetic efficiency, illustrating
that stomatal patterning determined CO2 concentration
and photosynthesis across the leaf lamina and that lateral fluxes of gas could not compensate for reduced
vertical diffusion as a result of reduced stomatal numbers (Büssis et al., 2006). This work agrees with reports
that suggest that lateral fluxes can limit photosynthesis
(Morison et al., 2005) but depend on species (Morison
et al., 2007; Morison and Lawson, 2007) and leaf anatomy (Lawson and Morison, 2006).
There are many known density and patterning mutants (e.g. fama, spch, mute, and tmm) in which specific
gene mutations have resulted in changes to cell division
and differentiation and altered patterns of stomata and
epidermal cells, resulting in stomatal pairing or clustering
in which the “one-cell-spacing” rule is broken (for review,
see Lau and Bergmann, 2012). The one-cell-spacing rule
refers to the fact that stomata are separated from each
other by a minimum of one cell, enabling efficient stomatal operation (Serna and Fenoll, 2000) and maintaining
the efficiency of gas fluxes (Nadeau and Sack, 2002). This
is exemplified in a recent study by Dow et al. (2014b),
who revealed that high stomatal density (due to stomatal
clustering) restricted CO2 diffusion and lowered A. These
findings were explained by the reduced functional capabilities of the guard cells from the reduced availability
of ions required for driving membrane processes as well
as the influence of turgor pressure for adjacent guard cells
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Stomata Impact on Water Use Efficiency
and the disruption of signaling processes due to the close
proximity of the guard cells. This work reiterated the
importance of the one-cell-spacing rule and indicated
that optimal stomatal function depended to a large extent on the patterning of stomata. There is also a good
understanding of environmental regulation (Lake et al.,
2001; Bergmann, 2004; Casson and Gray, 2008; Casson
and Hetherington, 2010) and what determines stomatal
development as well as the underlying genetic pathways that lead to altered epidermal cell patterning
(Bergmann and Sack, 2007; Casson and Hetherington,
2010). However, less is known about how environmental regulation influences stomatal density (e.g. CO2
concentration) and even less about the impact of patterning on leaf transpiration.
Stomatal Anatomy
Doheny-Adams et al. (2012) investigated the impact of
epidermal patterning factor (epf) mutants altered in
density and patterning of stomata on gs, A, and WUE.
Alterations in the expression of the different members of
the EPF family affected stomatal and, in some cases,
epidermal cell densities as well as the spacing of cells,
indicating an impact on division and differentiation. In
general, plants with no expression of EPF1 or EPF2, and
double mutants of epf1 or epf2, showed increases in stomatal density; overexpressors showed reduced stomatal
densities. It is noteworthy that the strong correlation
between stomatal density and size was maintained
within these plants: plants with lower stomatal densities
also showed a greater mean stomatal size, whereas
smaller stomata were found in leaves with greater stomatal densities. Interestingly, those plants with reduced
density and larger stomata also showed reduced transpiration, greater growth rates, and a larger biomass
(Doheny-Adams et al., 2012). The authors ascribed the
improved growth rate to a combination of improved
water status, higher metabolic temperatures, and lower
metabolic costs associated with the development of
guard cells. That growth rate was not enhanced in plants
with greater stomatal densities indicates that resistance
to CO2 diffusion was not limiting in these conditions and
that leaf water status dominated growth. It would be
instructive to determine productivity in these plants
when PPFD is increased and mesophyll demand for CO2
is elevated or the plants are subjected to fluctuating light
conditions. In contrast to the SDD1-expressing and ssd1
mutant plants with altered stomatal densities described
above, the epf1 and epf2 mutants showed no compensatory behavior in stomatal aperture. It is important to
remember that reduced gs often decreases transpiration
at the expense of carbon gain. It is necessary, therefore, to
assess both CO2 uptake and water loss when considering
strategies for improved WUE (McAusland et al., 2013).
An excellent example of enhanced WUE driven by stomatal numbers are the gtl1 mutants of Arabidopsis.
GTL1 is a transcription factor that regulates trichome
and stomatal development through its interaction with
SDD1 expression (Breuer et al., 2009). Physiological
analysis of these plants revealed no difference in photosynthetic rates over a range of light levels but reduced
gs and transpiration (Yoo et al., 2009, 2010), illustrating
that manipulating one gene related to stomatal development can “fine-tune” WUE.
Evidence from several studies has also suggested
that smaller stomata respond faster than larger stomata, an observation that has been explained in the
context of surface-to-volume ratios and the requirement
for solute transport to drive movement (Hetherington and
Woodward, 2003; Franks and Beerling, 2009; Drake et al.,
2013). There are some notable exceptions to this relationship. The stomata of ferns are relatively large compared
with many angiosperm species but nonetheless respond
rapidly to changes in vapor pressure difference (VPD),
which is the difference in water vapor pressure between
inside the leaf and outside; however, their response to
light is much slower than that of angiosperms (Brodribb
and Holbrook, 2004; McAdam and Brodribb, 2012b,
2013). The differentiation in these responses between
modern angiosperms and earlier vascular seedless plants
most likely arises from the passive hydraulic characteristics of ferns (McAdam and Brodribb, 2012a, 2013). It is of
interest that the size-speed relationship holds for the
characteristics predicted of stomatal behavior in Vicia faba
and Arabidopsis guard cells when modeled on the basis
of quantitative information available for these two species
(Chen et al., 2012c; Wang et al., 2012). In this case, the
difference can be ascribed directly to the surface-tovolume ratios, an observation that is broadly consistent
with the suggestions of Drake et al. (2013) and Hetherington and Woodward (2003). However, it should be
borne in mind that stomatal opening is a physicomechanical process and that, in order to open, guard
cells must overcome the pressure exerted by the surrounding subsidiary cells. Franks and Farquhar (2007)
illustrated that stomatal opening in some species was
only possible with a substantial reduction in subsidiary
cell osmotic pressure and that rapid opening in wheat
and other grasses, which have dumbbell-shaped guard
cells and subsidiary cells, was due to complementary
changes in turgor pressure between the guard cells and
these surrounding subsidiary cells. Furthermore, as we
note below, there can be substantial variations in the
transport activities of guard cells, even within one species. So, direct comparisons of surface-to-volume ratios is
likely to be uninformative as often as not.
SPEED OF THE STOMATAL RESPONSE
The opening and closing of stomata is driven by a
number of external environmental and internal signaling
cues (Blatt, 2000), and significant variation in sensitivity
and responsiveness is known to exist among different
species (Lawson et al., 2003, 2012; Lawson, 2009). In
general, stomata open in response to light (with the exception of Crassulacean Acid Metabolism stomata), low
CO2 concentration, high temperatures, and low VPD,
while closure is driven by low light or darkness, high
CO2, and high VPD (Outlaw, 2003). In the natural
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Lawson and Blatt
environment, these factors exert compound effects on
stomatal movements (Sharkey and Raschke, 1981; Zeiger
and Zhu 1998; Talbott et al., 2003; Wang et al., 2008);
therefore, stomata must respond to multiple signals in an
integrated and sometimes hierarchical manner (Lawson
et al., 2010). Short-term stomatal responses to changes in
VPD (and to some extent temperature) are often considered to be related to the water status of the plant rather
than the in situ photosynthetic carbon demand, while
responses to CO2 concentration and irradiance are closely
associated and correlated with mesophyll CO2 demand.
Stomatal responses to changes in light, CO2 concentration, and VPD have been studied extensively in
steady-state conditions (Jones, 1994; Lawson et al., 2010).
However, such steady-state situations are rarely observed in nature (Jones, 1994) or in isolation (Lawson
and Morison, 2004). Few studies have examined the
dynamics of stomatal response and photosynthetic output in the face of environmental perturbations (Grantz
and Zeiger, 1986; Knapp and Smith, 1987; Kirschbaum
et al., 1988; Tinoco-Ojanguren and Pearcy, 1993; Barradas
et al., 1994; Lawson et al., 2010; Wong et al., 2012;
McAusland et al., 2013). The majority of these have
concentrated on the impact of sun/shade flecks on carbon gain in understory plants, often without reference to
stomata, and even fewer have looked at the impact on
crop plants. Although grown in a monoculture, crops
will also experience sun and shade flecks across the
canopy from changes in cloud cover and sun angle to
self-shading and shading from neighboring plants (Way
and Pearcy, 2012), overlapping leaves, and wind-driven
movements (Lawson et al., 2010). In the naturally fluctuating environment, stomata and photosynthesis
respond continually to changing environmental cues,
especially light and temperature. However, these responses are not always synchronized, as stomatal
movements can be an order of magnitude slower than
the more rapid photosynthetic responses to the same
environmental stimuli (Pearcy, 1990; Lawson et al.,
2010). For example, over the diel period, plants experience short-term fluctuations in light (sun/shade flecks)
that drive the temporal and spatial dynamics of carbon
gain and water loss (Fig. 1A). The temporal disconnect
between gs and A means that under natural fluctuating
environmental conditions, the coordination between
carbon gain and water loss (and, therefore, WUE) is far
from optimal (Fig. 1B) and can result in spatial heterogeneity over individual leaves (Lawson and Weyers,
1999) as well as throughout the crop canopy (Weyers
et al., 1997). In C3 plants, photosynthetic rate can adjust
in seconds to changes in irradiance (e.g. a sun fleck), but
the lag in stomatal responses will constrain photosynthesis by limiting CO2 uptake (Tinoco-Ojanguren and
Pearcy, 1993; Barradas et al., 1998; Lawson et al., 2010,
2012). The example in Figure 1 is of natural changes in
irradiance, A, and gs in a bean (Phaseolus vulgaris) leaf
over a 70-min period and clearly shows periods in which
gs and A were uncoordinated, others in which the slower
stomatal reopening limited A, and periods in which gs
was greater than required for the A achievable under the
available light. Intrinsic water use efficiency (WUEi;
which used gs as an estimation of water loss rather than
transpiration [A/gs]) over this period was between 0.03 and
0.11 mmol CO2 m22 s21/mmol water m22 s21 (Fig. 1B)
and yielded a time-averaged value of 0.071. When
recalculated assuming instantaneous responses and
coordination of gs with A for the theoretical maximum
WUE (determined from steady-state measurements of
A and gs at different irradiances; Fig. 1B), this timeaveraged value was improved by 22%, effectively accounting for the WUEi deficits illustrated by the shaded
regions in Figure 1B. This simple experiment demonstrates the impact that the rapidity of stomatal responses
to changing light environment has on carbon gain and
water loss and illustrates the possible improvement in
WUE if stomata respond rapidly (almost instantaneously)
and in synchrony with mesophyll demands for CO2.
Pearcy and coworkers pioneered research on the
impact of sun flecks on carbon gain and stomatal dynamics and dissected the photosynthetic response into
several different phases, attributing the initial induction
phase, periods of up to 10 min, to biochemical limitations (Barradas and Jones, 1996); induction was followed by a period dominated by stomatal limitation to
the photosynthetic maximum; a third phase was identified in which gs remained high, effectively exceeding
that needed for maximum assimilation rates under the
given light conditions (Kirschbaum et al., 1988; TinocoOjanguren and Pearcy, 1993) and, hence, out of synchrony with A (Lawson et al., 2010). Figure 2 shows an
example of gs limiting photosynthesis, post induction,
when irradiance was increased during a 2-min sun
fleck. As the PPFD declined at the end of this period, gs
continued to rise over several minutes, leading to a
surplus in transpiration without the benefit of enhanced
assimilation. If we consider the impact of this short increase in illumination on instantaneous WUE (Fig. 2,
gray squares), it is clear that gs has less impact on the
measurements of WUEi after transition to the higher
light intensity. However, it is also evident that assimilation was limited by gs during the sun fleck, with A
rising by 0.6 mmol m22 s21, roughly 10% over the second half of the 2-min period, together with gs. Finally,
Figure 2 illustrates the continued rise in gs following the
sun fleck, leading to a significant decline in WUEi relative to the mean starting value.
The effects of shade flecks are less well studied (Lawson
et al., 2010), but they yield dynamics similar to those
observed during sun flecks. A decrease in light results in
an immediate drop in A, to a new value imposed by the
light level. gs rarely responds immediately, demonstrating a lag phase, before the start of closing. Although
closing is often faster than the opening (Hetherington and
Woodward, 2003), it can still take several tens of minutes
to establish a new steady state in gs. Figure 3 illustrates
dynamic responses in bean and V. faba subjected to 1-, 5-,
and 15-min shade flecks. In agreement with previous
observations, a decrease in light intensity of 1 to 2 min
resulted in very little or no change in gs and a near-instant
recovery in photosynthetic rate when light was restored.
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Stomata Impact on Water Use Efficiency
Figure 1. A, Variations in PPFD, A, and
gs over a 1-h period in bean experiencing natural light fluctuations in a
glasshouse in Essex. PPFD is represented by the solid gray line, A by the
dashed black line, and gs by the solid
black line. Average temperature within
the glasshouse was 25˚C and humidity
was 46%. Readings were recorded every 60 s. Shaded areas illustrate periods
when (a) gs and A responses are uncoordinated and responding in opposite directions, (b) A is limited by the slow
stomatal opening in response to increasing PPFD, and (c) gs is greater than necessary for the prevailing irradiance, due to
a lag before reduced PPFD induced
closing. B, WUEi calculated for the data
collected in A (solid gray line) and theoretical maximum WUE (dashed black
line), assuming total synchrony and coordination in A and gs responses. Maximum
WUE i was determined from lightresponse curves that were of a sufficient duration to ensure that both steady
stomatal conductance and A were achieved. Values for coordinated responses
were determined from steady-state
measurement of A and gs in response
to changing irradiance (T. Lawson,
unpublished data). [See online article
for color version of this figure.]
For bean, longer shade flecks led to progressive declines
in gs and a slowing in the return of A to the initial value
when light was restored (Fig. 3B). With a shade fleck of
15 min, the resulting drop in gs subsequently restricted
the photosynthetic rate by some 35% in the first minutes
after light was restored (T. Lawson and L. McAusland,
unpublished data). The same light regimes had little effect on short-term gs in V. faba (Fig. 3, D–F). Consequently,
these plants showed little evidence of CO2 limitation
when irradiance was restored, but the much more
Figure 2. Effects of sun flecks on A
(white circles), gs (black circles), and
WUEi (gray squares). PPFD was increased from 100 to 900 mmol m22 s21 for
a period of 120 s at time zero (yellow
highlighted section). Measurements of
A and gs were made every 10 s with
cuvette CO2 concentration maintained
at 400 mmol mol21, temperature at 25˚C,
and VPD at 1.34 kPa (T. Lawson and
L. McAusland, unpublished data). [See
online article for color version of this
figure.]
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Lawson and Blatt
sluggish response of the stomata and gs meant a proportionally higher rate of transpiration during the periods
of reduced CO2 demand. In short, the longer stomata take
to close and reach a new gs value appropriate for the light
level and A, the greater the surplus in transpiration and
reduction in WUE (Fig. 1B).
Of course, the speed and magnitude of changes in gs
in response to sun and shade flecks are species specific
(Lawson et al., 2010) and may be dependent on differences in stomatal sensitivity or signaling mechanisms between species (Weyers et al., 1997; Mott and
Peak, 2007; Mott, 2009). They will also depend on plant
water status (Fig. 4), the history of stress (Pearcy and
Way, 2012; Porcar-Castell and Palmroth, 2012; Wong
et al., 2012; Zhang et al., 2012), leaf age (Urban et al.,
2008), and the magnitude and duration of the change
Figure 3. Effects of shade flecks on A and gs in P. vulgaris (A–C) and V. faba (D–F). PPFD was decreased from 900 to 100 mmol m22 s21 at
time zero for 1 min (A and D), 5 min (B and E), or 15 min (C and F). Measurements of A (white circles) and gs (black circles) were
made every 10 s with cuvette CO2 concentration maintained at 400 mmol mol21, temperature at 25˚C, and VPD at 1.34 kPa
(T. Lawson and L. McAusland, unpublished data). Insets show schematic diagrams that illustrate the difference in number and
size of stomata between the two species (not to scale). [See online article for color version of this figure.]
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Stomata Impact on Water Use Efficiency
in irradiance (Lawson, 1997). As illustrated in Figure 3,
V. faba showed little response to changes in irradiance.
These plants were well watered and grown under
moderate light intensities and temperatures of 18°C to
20°C to minimize stress. However, when the plants were
challenged with the same 15-min shade fleck following
4 d without watering (Fig. 4), gs was significantly lower
compared with the well-watered plants (Fig. 3F) and
showed an immediate and more rapid decrease during
the shade fleck. Furthermore, A recovered in parallel
with the relatively slow increase in gs when the light was
restored to its initial level, both A and gs taking more
than 30 min to recover (Fig. 4). Experiments of this kind
illustrate the range of variations in speed and amplitude
in gs dynamics, and they suggest that stomatal size and
density are often of secondary importance. Indeed, bean,
which has smaller and more numerous stomata, here
responded faster when well watered than well-watered
V. faba, which has larger and fewer stomata, but when
water stressed, gs of V. faba responded in a manner similar
to that of bean.
One feature of note is an apparent correlation between opening and closing rates. Vico et al. (2011) used
60 published gas-exchange data sets of the stomatal
response to light variations to determine the impact of
stomatal delays on photosynthesis and transpiration
and its relationship to the energetic costs of stomatal
movement. Unfortunately, the latter are compromised
by the use of incorrect stoichiometries for the coupling
of ATP to transport and by the assumption that stomatal closure is entirely passive, which it is not. However, among other outcomes, their studies show a
general parallel in rates of stomatal movement across
data sets: faster opening rates were generally associated
with correspondingly faster closing rates. These findings agree with others (Hetherington and Woodward,
Figure 4. Effects of 15-min shade flecks on A and gs in V. faba plant
subjected to water stress by withholding water for 3 d. PPFD was
decreased from 900 to 100 mmol m22 s21 at time zero for a period of
15 min. Measurements of A (white circles) and gs (black circles) were
made every 10 s with cuvette CO2 concentration maintained at 400
mmol mol21, temperature at 25˚C, and VPD at 1 kPa (T. Lawson and
L. McAusland, unpublished data).
2003). Vialet-Chabrand et al. (2013) recently developed
a new dynamic model to describe the diurnal time
course of gs using empirical parameters to accurately
predict daily variation in WUEi. This model uses nonlinear methods to describe temporal changes in gs
driven by the fluctuating environment, and it underlines a coupling between rates of opening and closing.
Overall, from these various studies, we have learned a
great deal about the manipulation of stomatal numbers
and guard cell responses to environmental perturbations,
but almost nothing is known about the mechanisms that
control stomatal speed. However, research to date suggests that manipulating stomatal number and size is not
necessarily the simplest or best approach, and the idea
that manipulating the stomatal response to changing
environmental conditions could provide a means to both
improve the WUE of plants and, at the same time, increase the integrated photosynthetic carbon gain. Most
important, they indicate that the rapidity of the stomatal
response influences both daily carbon gain and WUE and
that it is essential to consider the dynamics both of closing
and opening together (Raschke, 1970; Kirschbaum et al.,
1988; Knapp, 1993; Tinoco-Ojanguren and Pearcy, 1993;
Cardon et al., 1994; Allen and Pearcy, 2000; Noe and
Giersch, 2004; Pearcy and Way, 2012; Smith and Berry,
2013). With these points in mind, guard cell ion transport
is an obvious target for exploration with the aim of altering stomatal dynamics and the rates of opening and
closing.
ION TRANSPORT, STOMATAL RESPONSE,
AND WUE
There is no question that stomatal movements of seed
plants, including crop plants, arise from the transport,
accumulation, and release of osmotically active solutes. A
very large body of experimental evidence supports the
collective role of ion transport across the plasma membrane and tonoplast in both stomatal opening and closing
(Willmer and Fricker, 1996; Blatt, 2000; Chen et al., 2012c;
Hills et al., 2012). The primary inorganic species transported are K+ and Cl2, which, with the organic anion
malate22 (Mal) and Suc, comprise the bulk of solute that
drives water flux and guard cell turgor (Willmer and
Fricker, 1996; Roelfsema and Hedrich, 2005; McAinsh
and Pittman, 2009). Because mature guard cells lack
functional plasmodesmata (Wille and Lucas, 1984), these
solutes must be transported across the plasma membrane. Much of this solute uptake must also be transported across the tonoplast. The guard cell vacuole, like
that of most mature plant cells, makes up the bulk of the
cell volume and, hence, plays a very important role as a
“repository” for osmotically active solutes (Gao et al.,
2005; MacRobbie, 2006; Chen et al., 2012c). Mal metabolism (notably its synthesis within the guard cell cytosol)
makes a substantial contribution to the osmotic content of
the guard cell, while Mal loss during stomatal closure
occurs largely via efflux across the plasma membrane
(Willmer and Fricker, 1996; Wang and Blatt, 2011). For
this reason, questions of the extent and speed of stomatal
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Lawson and Blatt
responses to environmental cues are intimately connected
with characteristics of the guard cells, the most important
being (1) the capacity for solute transport and exchange
with the surroundings, and its relationship to the volume
of the guard cells, and (2) the speed with which transport
responds to environmental cues that determine stomatal
movements.
Transport capacity is determined primarily by the
density and activity of the various transport proteins at
the membrane and is related secondarily to guard cell
turgor and stomatal opening and closing by the surfaceto-volume ratio of the cellular compartments. As noted
before, guard cell size (and geometry) has been indicated
to affect the speed of stomatal movements, with larger
stomata often exhibiting slower responses. The importance of the size of the stomatal complex, as concluded
from these studies, is predicated on the assumption that
solute fluxes are largely uniform when normalized to the
unit surface area; as a consequence, the time needed to
adjust solute content within the cell volume is expected
to decrease roughly in proportion with guard cell
volume-to-surface ratio and, hence, the linear dimensions
of the guard cell. However, quantitative data from which
to compare solute fluxes are very limited (Willmer and
Fricker, 1996) and depend on factors such as the history
of water stress (Hiron and Wright, 1973; Figs. 3 and 4).
The few studies that have examined specific transport
activities directly, for example, of outward-rectifying K+
currents in intact guard cells of V. faba, tobacco (Nicotiana
tabacum), and Arabidopsis (Blatt, 1988b; Armstrong
et al., 1995; Blatt and Gradmann, 1997; Blatt et al., 1999;
Ache et al., 2000; Bauly et al., 2000; Johansson et al.,
2006; Chen et al., 2012b; Eisenach et al., 2012, 2014), indicate substantial variations between species independent of surface area. These comparisons call in question
the validity of assuming a priori that transport activity
remains constant on a unit-surface-area basis.
There are a large number of studies reporting on the
rates of net ion transport across the plasma membrane as
the principle barrier to solute flux. Attention has often
turned to changes in pH, as, arguably, the rates of H+ flux
are a good measure of membrane energization by primary H+-ATPases (Marre, 1979; Shimazaki and Kondo,
1987; Goh et al., 1996; Kinoshita and Shimazaki, 1999;
Merlot et al., 2007). By their nature, pH measurements
report the net H+ flux rather than the true H+-ATPase
activity and, therefore, are indirect, subject to the availability of other ions to balance charge during transport
(Blatt and Clint, 1989; Clint and Blatt, 1989; Gradmann
et al., 1993). This caveat aside, the rates of H+-ATPase
activity recorded directly under voltage clamp (Blatt,
1987, 1988a; Lohse and Hedrich, 1992), and the corresponding fluxes of K+ and Cl2 (MacRobbie, 1981, 1983,
1984; Clint and Blatt, 1989), have generally proven consistent with the changes in solute content (MacRobbie
and Lettau, 1979, 1980; Blatt, 2000; for a quantitative
summary and comparison, see Hills et al., 2012).
Relating these data to the transport capacity of guard
cells in mechanistic terms has proven much more
complex, however. Difficulties arise in part because the
predominant transport pathways for H+, K+, Cl2, and
Mal are strongly dependent on membrane voltage over
the physiological voltage range, especially at the plasma
membrane. A second complication arises from the fundamental physical requirement for charge balance across
all biological membranes. As a result, the flux of any one
ionic species is necessarily connected to that of all others
across the same membrane, unless this coupling is broken
by introducing a “shunt” through the circuit of a voltage
clamp (Blatt, 1991, 2004). Experiments in which membrane voltage has been brought under experimental
control address the question of transport capacity directly
and have proven quite revealing on several accounts.
First and foremost, the dominant membrane transporters,
including the K+ and Cl2 channels, remain functional
under most physiological conditions. This observation
contrasts with a common misconception that transporters
either activate or shut down fully in response to various
stimuli. Their operation may be kinetically limited by
substrate (ion) availability and especially by membrane
voltage, but the activity of each transporter is generally
evident under voltage clamp. For example, currents
through the inward- and outward-rectifying K+ channels
are present when voltage is clamped, both in the absence
and presence of the water-stress hormone abscisic acid
(ABA) that triggers stomatal closure (Blatt and
Armstrong, 1993; Romano et al., 2000; Garcia-Mata et al.,
2003). ABA affects the K+-carrying capacity of both
channel populations; however, membrane depolarization
is the overriding factor in driving K+ efflux and stomatal
closure (Thiel et al., 1992; Romano et al., 2000). In other
words, guard cells probably rarely, if ever, exist in a state
in which the net flux of an osmotically active solute is
zero. Instead, solute uptake and loss, and stomatal
movements, arise through a dynamic balance in solute
flux (Blatt and Armstrong, 1993; Gradmann et al., 1993;
Chen et al., 2012c). A second point arises from the general
finding that the ion transport underpinning stomatal
movements reflects a small fraction only of the maximal
capacity of the several transporters mediating these fluxes
(Blatt et al., 1990; Thiel et al., 1992; Hamilton et al., 2000;
Pottosin and Schönknecht, 2007; DeAngeli et al., 2009).
The activities of these transporters are kinetically limited
by their inherent gating or other kinetic properties within
the range of voltages typical for the plasma membrane
and tonoplast. Two general conclusions may be drawn
from these observations. First, the capacity for transport,
especially through the several ion channels, is not inherently limiting for solute flux, although the balance of
transport often is. Second, any attempt to manipulate
solute accumulation and loss for accelerated stomatal
movements must therefore address the balance between
ionic fluxes at the plasma membrane and tonoplast, both
individually and coordinately between membranes.
Information on the speed with which transport responds to environmental cues, especially to light and
CO2, is similarly complicated by the connections between
transport at each membrane. Again, deconstructing the
sequence of events in mechanistic terms must draw on
the voltage clamp to separate the individual transporter
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Stomata Impact on Water Use Efficiency
currents (Blatt, 2004) and is the focus of a number of
recent reviews (Blatt, 2000; Hetherington and Brownlee,
2004; Blatt et al., 2007; Pandey et al., 2007; Melotto et al.,
2008; Wang and Song, 2008; McAinsh and Pittman, 2009;
Hills et al., 2012). In general, however, stomatal movement arises as the cumulative sum of the net solute
fluxes (i.e. of the rates of flux), especially for K+, Cl2, and
Mal integrated over time. Therefore, the stomata aperture responds over substantially longer time scales, typically orders of magnitude greater than that needed for
changes in transport activity and membrane voltage of
the guard cell. For example, membrane depolarization in
ABA stimulates K+ efflux within seconds through
outward-rectifying K+ channels, in Arabidopsis the
GORK K+ channel (Hosy et al., 2003; Suhita et al., 2004),
and these K+ currents are enhanced during the subsequent 3 to 5 min as a consequence of a rise in cytosolic
pH (Blatt and Armstrong, 1993; Grabov and Blatt, 1997).
Stomatal aperture responds more slowly, typically with
half-times of 10 to 20 min, reaching a new stable, (near)
closed state after 45 to 60 min (Raschke et al., 1975;
Roelfsema and Prins, 1995; Zhang et al., 2001). Thus,
making a connection to the speed and efficacy of stomatal movements is necessarily indirect.
One promising way forward draws on quantitative
systems modeling to relate transport and metabolic
activities to stomatal movements. Wang et al. (2014a),
in this issue, have undertaken such an approach using
OnGuard models for V. faba and Arabidopsis (Chen
et al., 2012a; Hills et al., 2012; Wang et al., 2012).
OnGuard models incorporate all of our knowledge of
molecular, biophysical, and kinetic characteristics of
guard cell transport, Mal metabolism, and H+ and Ca2+
buffering, and they link this knowledge to stomatal
aperture. These models have demonstrated true predictive power in uncovering previously unexpected
and emergent behaviors of guard cells, several of
which have been verified experimentally. OnGuard
analysis of the Arabidopsis slac1 mutant exposed an
unexpected connection between the Cl2 channel and
the plasma membrane K+ channels that was subsequently confirmed by experiments (Wang et al., 2012).
This connection accounted fully for the effect of the
slac1 mutant in slowing ion uptake and stomatal
opening, even though the SLAC1 Cl2 channel contributes directly only to solute loss and stomatal closure. Similarly, analysis of the Arabidopsis ost2
mutant, which affects H+-ATPase activity at the plasma
membrane, has demonstrated an uncanny “communication” of transport between the plasma membrane
and tonoplast (Blatt et al., 2013), predictions that now
warrant investigation. Most importantly, the modeling underlines a commonality of kinetic limitations
that arises from the sharing of substrates and membrane voltage across each membrane. These connections underpin the emergent characteristics identified
so far.
So how might we approach the problem of improving water use by the plant without a cost in carbon gain? Different ion flux pathways are responsible
for solute uptake during the opening and solute loss
during the closing of stomata (Blatt, 2000; Roelfsema
and Hedrich, 2005; Shimazaki et al., 2007), and at first
glance, this separation should be a simplifying factor.
However, a primary difficulty is that of transport interactions, such as those described above. Anticipating
fully the consequences of such manipulations is generally beyond intuitive understanding and can only be
addressed through quantitative modeling. Wang et al.
(2014a) have explored systematically the most promising targets for genetic manipulation that will enhance
the speed of stomatal opening and closing using the
OnGuard platform. Their results indicate that, with
few exceptions, modest changes to the populations of
individual ion channels are largely ineffective. Only
primary H+ transport, and those transporters affecting
Ca2+ homeostasis directly, are predicted to have any
substantial effects on stomatal movements. Surprisingly, the effects of manipulating almost all transporter
populations were compound and, in part, at odds with
the desired effects of accelerating opening and closing
while maintaining the dynamic range of apertures. To
the extent of our current knowledge, the results confirm past observations from a number of mutants, including the K+ channel gork (Hosy et al., 2003), the
anion channel slac1 (Negi et al., 2008; Wang et al.,
2012), the ost2 H+-ATPase mutant (Merlot et al., 2007),
the clca H+-Cl2 antiporter mutant (De Angeli et al.,
2006), and the tpk1 K+ channel mutant (Gobert et al.,
2007). They are supported, additionally, by the work of
Wang et al. (2014b), who concurrent with this publication reported the effects of overexpressing the
several K+ channels and the AHA2 H+-ATPase in
Arabidopsis guard cells. Their results confirm the lack
of effect on channel overexpression; they also demonstrate enhanced apertures, gs, and A with H+-ATPase
overexpression. However, these gains come with a
corresponding reduction in WUE, suggesting that the
acceleration in stomatal opening was not reflected in
an increase in the rates of stomatal closure. Again,
these findings confirm modeling predictions (Wang
et al., 2014a) that interactions between transporters
preempt most, if not all, of the intuitive solutions to
accelerating stomatal movements. The most promising
targets for manipulation to arise from these studies are
the voltage-dependent characteristics of the two classes
of K+ channels at the plasma membrane, rather than
simple changes to the populations of these transporters.
Wang et al. (2014a) note that a 218-mV shift in the
gating of the outward-rectifying K+ channel (GORK in
Arabidopsis) could be sufficient to accelerate closing
by more than 30%, whereas doubling the number of
these K+ channels counterintuitively slows the rate of
closing in simulations. Other effective solutions may
include manipulations of more than one transport
pathway. In short, even at this simplified level, the
problem of manipulating stomatal behavior throws up
a surprising degree of complexity that is likely to
challenge practical solutions to future efforts in “reverse engineering” of stomata.
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Lawson and Blatt
ENGINEERING STOMATAL SIGNALING
AND METABOLISM
Many of the challenges associated with manipulating
ion transport in guard cells have analogs in efforts focused on their signaling, cell biology, and metabolism.
Indeed, there are obvious functional overlaps, for example in the contributions of organic acid synthesis to the
osmotic content of the cells. Thus, it is not surprising that
altered Mal metabolism and transport will affect stomatal
movements (Gruber et al., 2011). More still, a survey of
the literature shows that the lessons learned from the
systems biological approach of OnGuard about the (often
counterintuitive) connections between transport and
metabolism (Wang and Blatt, 2011; Wang et al., 2012)
have direct bearing here, too. A thorough discussion of
this literature is not possible here. Thus, we have selected
a few of the more recent articles addressing leaf-level gs
and A that illustrate both the difficulties and the potential
for manipulation of stomatal responses, especially in relation to stomatal kinetics. We stress that, to date, no
single mutations of any structural gene products have
surfaced that enhance stomatal function to improved
WUE without a cost to carbon gain.
The overlaps with ion transport and the importance of
stomatal dynamics are well illustrated by the vesicletrafficking protein SYP121 in Arabidopsis. Eisenach
et al. (2012) reported that the syp121 mutant impairs
stomatal reopening following closing signals associated
with elevated cytosolic free Ca2+ concentration. The
mutant mimicked the phenomenon of so-called “programmed closure,” previously ascribed to a “memory”
of stress that leads stomata to open only slowly thereafter. The authors demonstrated the underlying mechanism that arises from cycling of the KAT1 K+ channel
between the plasma membrane and endosomal membranes, thereby suppressing channel-mediated K+ uptake by the guard cells and slowing stomatal reopening.
Stomatal reopening was slowed from a half-time of
approximately 20 min to half-times of more than 60 min,
sufficient to reduce long-term carbon assimilation and
slow growth at low relative humidity. Slowed stomatal
opening in the light has also been observed in the high
leaf temperature1 mutant (Hashimoto et al., 2006), which
exhibits alterations in responsiveness to CO2 and thus
may have important implications for synchronizing
stomata with mesophyll-based photosynthesis. Finally,
as noted above, Wang et al. (2012) analyzed the slac1
mutant that eliminates the predominant anion channel
in Arabidopsis guard cells and greatly slows stomatal
closure. They found that the mutation also slowed stomatal opening by altering the two K+ channel currents.
These observations were traced directly to changes in
the baseline of cytosolic free Ca2+ concentration and elevated cytosolic pH, the latter arising from the accumulation of Mal and suppression of its synthesis. In
other words, mutation of a Cl2 channel has unexpected
and profound effects that extend well beyond Cl2
transport, both to other ion channels and to organic acid
metabolism.
Manipulation of photosynthetic carbon metabolism
has likewise revealed some informative interactions between mesophyll and stomatal guard cells, although the
conclusions are often seemingly contradictory. Transgenic
plants with reduced photosynthesis showed that gs was
largely unaffected (Quick et al., 1991; Stitt et al., 1991;
Hudson et al., 1992; Evans et al., 1994; Price et al., 1998),
thereby breaking the long-standing correlation between
A and gs that is usually conserved. More recent studies
have similarly demonstrated that gs and A can be uncoupled
when carbon fixation via Rubisco (von Caemmerer et al.,
2004) and electron transport (Baroli et al., 2008) are
down-regulated. However, accelerated stomatal opening
was observed when the regeneration capacity of ribulose
1,5-bisphosphate was suppressed in transgenic tobacco
with reduced levels of Sedoheptulose-1,7-bisphosphatase (Lawson et al., 2008). Overexpression of maize
(Zea mays) NAD-malic enzyme in tobacco resulted in
plants with a decrease in gs but gains in biomass, suggesting that manipulation of both stomata and mesophyll
processes could improve plant WUE (La Porte et al.,
2002). Finally, Kelly et al. (2012) increased the expression
of hexokinase in guard cells with the counterintuitive effect of decreasing gs. Hexokinase drives starch breakdown
through the phosphorylation of Glc, which then feeds into
glycolysis, the tricarboxylic acid cycle, and malic acid
synthesis. Analysis of these transgenics suggested a Suc
feedback mechanism that coordinated stomatal behavior
with mesophyll photosynthesis. Other, seemingly more
indirect, manipulations can have unexpected consequences for stomata and WUE. Some examples include
manipulations to SUC2 that increased guard cell invertase
activity (Antunes et al., 2012). Araújo et al. (2011) observed
an elevated gs and a 25% increase in A in tomato (Solanum
lycopersicum) plants with altered succinate dehydrogenase
levels. This response was only observed on ectopic overexpression with a 35S promoter, not when expression was
restricted to the guard cells only. In short, these studies
highlight the potential for manipulating metabolic processes, both of mesophyll and guard cells. However, they
also underscore the need, in addressing complex behavior
such as WUE, for quantitative systems approaches on a
tissue-wide scale if we are to effectively target and exploit
stomatal guard cell physiology.
CONCLUSION
Improving plant WUE and a plant’s ability to cope
with reduced water availability is high on the scientific
agenda. Stomata ultimately control 95% of all gaseous
fluxes between the leaf and the environment. It follows
that stomata represent an attractive target for manipulations aimed at reducing water loss. Given that stomata
also regulate CO2 access to the photosynthetic tissues of
the leaf, the challenge will be to achieve this goal without
compromising carbon gain (Lawson et al., 2012). Equally
important, any manipulations to stomatal function, sensitivity, or responsiveness should not render the plants
more vulnerable to environmental extremes or biotic
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Stomata Impact on Water Use Efficiency
challenge. Progress to these ends is most likely to come
now from combinations of physiological and molecular
genetic methods together with those of quantitative
systems analysis and, therefore, will benefit from additional information about the quantitative kinetics, especially of signal transduction.
Significant advances have been made in our understanding of the biochemical response pathways of guard
cells and the underlying molecular biology and genetic
basis that underpin these functional responses and anatomical characteristics. Recent advances in molecular
biology tools and approaches to manipulate key biochemical processes or select for desirable traits mean that
we can reconsider these earlier ideas and the manipulations of guard cell metabolism or specific stomatal traits
as tangible targets, with real potential to deliver plants
with improved WUE and yield. For example, transcriptional analysis of guard cells from epidermal fragments
(Wang et al., 2012) or microdisection of individual guard
cells (Gandotra et al., 2013), along with single-cell metabolic profiling (Burrell et al., 2007), have greatly assisted
in our understanding of the metabolic signaling and response pathways within guard cells. The identification of
highly specific guard cell promoters (Müller-Röber et al.,
1994; Cominelli et al., 2005) and transcription factors
(Cominelli et al., 2010; Yoo et al., 2010), the production of
guard cell enhancer trap lines (Gardner et al., 2009), and
the ability to induce transient expression in guard cells
(Rusconi et al., 2013) open up new exciting potential
approaches to not only fully understand signaling and
transduction pathways in guard cells but also to provide
us with the tools to manipulate guard cell metabolism in
order to improve plant WUE. Such tools have greatly
improved our understanding of the molecular networks
that control guard cell perception of and response to
internal and external environmental cues and have provided possible candidates for manipulation (Galbiati
et al., 2008; Rusconi et al., 2013).
It is equally important that plant phenotyping
approaches keep pace with the molecular and genetic
technical developments described above to deliver
rapid and practical screening approaches and tools to
identify plants with faster stomata. McAusland et al.
(2013) recently used combined chlorophyll fluorescence
and thermal imaging to develop the first imaging approach to screen plants for alterations in intrinsic WUE.
This approach provides both spatial and temporal images of gs and A as well as dynamic protocol capabilities. Using this technique, stomatal responsiveness and
speed can be assessed simultaneously with rates of
photosynthesis, which would be ideal for identifying
plants with faster stomata with no impairment in carbon assimilation.
Received February 1, 2014; accepted February 25, 2014; published February
27, 2014.
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