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Regulation of Transpiration to Improve Crop Water Use

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Regulation of Transpiration to Improve Crop Water Use

Chan Yul Yoo a; Heather E. Pence a; Paul M. Hasegawa a; Michael V. Mickelbart a
a
Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, IN, USA

Online publication date: 10 November 2009

To cite this Article Yoo, Chan Yul, Pence, Heather E., Hasegawa, Paul M. and Mickelbart, Michael V.(2009) 'Regulation of
Transpiration to Improve Crop Water Use', Critical Reviews in Plant Sciences, 28: 6, 410 — 431
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 Critical Reviews in Plant Science, 28:410–431, 2009
Copyright © Taylor & Francis Group, LLC
ISSN: 0735-2689 print / 1549-7836 online
DOI: 10.1080/07352680903173175

Regulation of Transpiration to Improve Crop Water Use

Chan Yul Yoo, Heather E. Pence, Paul M. Hasegawa, and Michael V. Mickelbart
Center for Plant Environmental Stress Physiology, Purdue University, 625 Agriculture Mall Drive, West
Lafayette, IN 47907-2010, USA

Table of Contents

I. INTRODUCTION ............................................................................................................................................. 410

II. PHYSIOLOGY OF PLANT WATER USE ......................................................................................................... 412

A. Stomatal Transpiration ................................................................................................................................. 412
1. Stomatal Number (Density) ................................................................................................................... 413
2. Stomatal Aperture ................................................................................................................................ 414
B. Cuticular Transpiration ................................................................................................................................. 415
C. Boundary Layer Conductance ....................................................................................................................... 416
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D. Nighttime Transpiration ................................................................................................................................ 416

III. GENETICS OF WATER USE ............................................................................................................................ 417

A. Genetic Variation for WUE ........................................................................................................................... 417
B. Genes That Regulate Water Use Efficiency ..................................................................................................... 421
C. Determinants That Regulate Transpiration and May Improve Water Use Efficiency ............................................ 422
1. Stomatal Density Determinants ............................................................................................................. 422
2. Stomatal Aperture Determinants ............................................................................................................ 423
3. Nighttime Transpiration Determinants .................................................................................................... 425

IV. CONCLUSION ................................................................................................................................................. 425

ACKNOWLEDGMENTS ........................................................................................................................................... 425

REFERENCES .......................................................................................................................................................... 425

transpirational water loss occur predominantly through stomatal

Decreasing fresh water supplies and increasing agricultural
drought threaten sustainable worldwide crop production. Conse-
quently, there is a global priority to develop crops with higher water
use efficiency (WUE): biomass production or yield per unit of wa-
ter used. Water use efficiency varies substantially among species
and genotypes within a species, and a major effort is now underway
to identify the genetic determinants of WUE. Today, it is known
that genotypes in primary gene pools exhibit allelic variation for
WUE through mechanisms that regulate transpiration, which is the
conductance of water through stomata, the cuticle, and the bound-
ary layer. Because of the differential diffusion properties of water
and carbon dioxide (CO2 ) through these pathways, it is feasible
that WUE could be improved by decreasing transpiration without
a concomitant reduction in CO2 uptake. Since CO2 uptake and
pores, it is not surprising that genes involved in stomatal devel-
opment and stomatal opening/closing impact WUE. Furthermore,
loss- and gain-of-function genetic screens have identified genes that
regulate transpiration and WUE by yet undetermined mechanisms.
This review will discuss the genetic determinants that regulate tran-
spiration and WUE in the context of the modern agricultural goal
of improving WUE while sustaining biomass and yield.
Keywords water use efficiency, stomatal transpiration, cuticular
transpiration, boundary layer, nighttime transpiration,
stomatal movement, stomatal density, water availability,
drought, water conservation

Address correspondence to Michael V. Mickelbart, Center for Plant

Environmental Stress Physiology, Purdue University, 625 Agriculture
I. INTRODUCTION
Mall Drive, West Lafayette, IN 47907-2010. E-mail: mickelbart@ Fresh water availability has shaped human population de-
purdue.edu velopment and distribution throughout history (Jackson et al.,

410
 TRANSPIRATION TO IMPROVE CROP WATER USE 411

2001). Today, population growth and sustainability are threat-
ened by limited availability of water that is satisfactory for hu-
man consumption and/or agriculture (Vörösmarty et al., 2000).
Current worldwide water consumption may be beyond the ca-
pacity of the geological recycling process to provide adequate
supplies of fresh water (Jackson et al., 2001). In 1995, insuf-
ficient water availability adversely affected about 2.3 billion
people, which was 41% of the world population. This number
will likely increase to about 3.5 billion (48% of the population)
by 2025 and to almost 7 billion (75% of the population) by 2050
(Rosen, 2000; Hightower and Pierce, 2008).
Water shortages are limiting crop production, which jeopar-
dizes adequate food production and socioeconomic well-being
(Falkenmark, 1997; Rockström, 2007). Furthermore, industri-
alization and urbanization are reducing availability of arable
land for cultivation (Ramankutty et al., 2008). Consequently,
agricultural production is relocating to more arid areas, which
will likely reduce productivity because crops are not adequately
adapted to these environments. Global warming associated with
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and CO2 uptake is referred to as the transpiration ratio (water
transpired/CO2 assimilated) or water use efficiency (WUE: CO2
assimilated/water transpired) (Lawes, 1850; Briggs and Shantz,
1913; Farquhar and Sharkey, 1982).
Plant WUE can be improved by increasing carbon as-
similation relative to transpiration or reducing transpiration
relative to carbon assimilation (Chaves et al., 2004). Re-
cent reviews detail how crop yield has been improved by
genetic traits that enhance photosynthetic efficiency, nitro-
gen assimilation, seed yield, source-sink balance, and radi-
ation use efficiency (Sinclair et al., 2004; Sharma-Natu and
Ghildiyal, 2005; Long et al., 2006; Zhu et al., 2008). Data
from numerous studies suggest that phenotypic traits that re-
sult in a slight reduction in transpiration can increase WUE
because instantaneous water loss is reduced slightly, long-
term water loss is reduced greatly, and CO2 uptake is not
altered.
It may be feasible to improve WUE in plants by decreas-
ing daytime or nighttime transpiration, as demonstrated by an

increased greenhouse gas emissions alters microclimates, and
appears to be the basis for recent recurring drought episodes in
the middle and lower latitudes, which are major cereal produc-
tion regions (Kerr, 2007). An imperative objective is to develop
crop germplasm that is more efficient at utilizing water while
sustaining food and biomass production (Howell, 2001; Araus
et al., 2002; Polly, 2002).
Plants require water for biochemical reactions and cell expan-
sion, which is necessary for growth and development (Nobel,
1999). However, up to 97% of water absorbed by plants is
transpired to the atmosphere through stomata (Taiz and Zeiger,
2006; Gosney and Mickelbart, unpublished). Transpirational
flux is critical to the maintenance of plant water status, suitable
leaf temperatures, and essential mineral element uptake from
soils. Stomatal pores are also responsible for the bulk of carbon
dioxide (CO2 ) uptake that is necessary for carbon assimilation
into biomass and yield. The relationship between transpiration
experiment conducted by Smith and Mickelbart (unpublished).
Seedlings of four Cucumis sativus cultivars with similar biomass
had different transpiration rates. The cultivars ‘Muncher’ and
‘Double Feature’ had higher WUE than ‘Pickalot’ and ‘Lemon’
due to significantly lower daytime and nighttime transpiration,
respectively (Figure 1). Other studies have suggested that slight
decreases in daytime transpiration resulting in greater WUE
may also occur at the crop level (Sinclair et al., 2005). The re-
lationship between nighttime water loss and WUE is less clear,
although there does appear to be some relationship between
these traits (Pence and Mickelbart, unpublished). In crop plants,
flowering time is also critical. If a given genotype has lower
instantaneous or even daily WUE, but flowers later than higher
WUE genotypes, there may be no crop-level increase in WUE.
This review will focus on the physiology and associated ge-
netic determinants of WUE, specifically as a result of reduced
transpiration.

FIG. 1. Diurnal transpirations in four cultivars (‘Muncher’, ‘Pickalot’, ‘Double Feature’, and ‘Lemon’) of Cucumis sativus. Transpiration of four cultivars was
measured gravimetrically under a 12 hour photoperiod. All cultivars had similar total dry weight (1.56 ± 0.43 g). However, lower daytime transpiration was
observed in ‘Muncher’ compared to ‘Pickalot’, and lower nighttime transpiration was observed in ‘Double Feature’ compared to ‘Lemon’. Shaded arrows indicate
differences in daytime (A) and nighttime water loss (B) between paired cultivars.
 412 C. Y. YOO ET AL.

TABLE 1
Various expressions of WUE in plants
WUE Instantaneous WUE Integrated WUE

Abbreviation WUEi WUEB WUEY

Carbon assimilation CO2 assimilation (µmol CO2 m−2 s−1 ) Biomass (g) Biomass (g)

Water use Transpiration (mmol H2 O m−2 s−1 ) Water use (kg) Water use (kg)

Unit µmol mmol−1 g kg−1 g kg−1

II. PHYSIOLOGY OF PLANT WATER USE
WUE is expressed by various units based on how CO2 assim-
ilation and transpiration are assessed (Jones, 2004; Tambussi et
al., 2007) (Table 1). Instantaneous WUE (WUEi ) is determined
from the simultaneous measurement of net CO2 assimilation
(µmol CO2 m−2 s−1 ) and transpiration (mmol H2 O m−2 s−1 )
of a leaf or whole plant at a given point in time. WUEi can
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1976; Rosenberg et al., 1983; Timlin et al., 2008). Net CO2
assimilation generally increases linearly at lower conductances,
but saturates at higher conductances due to nonstomatal limita-
tions such as ribulose-1,5-bisphosphate regeneration (Figure 2;
von Caemmerer and Farquhar, 1981; Stuart et al., 1985; Mans-
field et al., 1990; Radin et al., 1994; Liu et al., 2002; Medrano
et al., 2002; Thompson et al., 2007). As stomatal conductance

also be expressed as the ratio of net CO2 assimilation rate to increases from near zero, CO2 assimilation increases at a very
stomatal conductance of water vapor (mol H2 O m−2 s−1 ), that rapid rate, so WUEi increases, but only if nonstomatal water
is, the diffusion rate of water vapor through the stomatal pore loss is negligible. However, as nonstomatal limitations to CO2
(Jones, 2004). Integrated WUE is the ratio of carbon dry matter assimilation occur (C’), WUE decreases.
gained per unit water lost (g kg−1 ) and is measured over a pro-
longed period. Integrated WUE may be expressed as biomass
accumulation (WUEB ) or yield (WUEY ) per unit of water used.
An understanding of these relationships and how they can be
improved should begin with an understanding of the primary
processes that affect long-term carbon assimilation and water
loss in plants: leaf level CO2 and H2 O gas exchange.
Transpiration is determined by the vapor pressures of water
inside and outside the leaf, and the resistances (the reciprocal
of conductance) to the movement of water from the inside to
the outside (Farquhar and Sharkey, 1982). Resistances to water
movement include stomatal, cuticular, and boundary layer resis-
tances (Farquhar and Sharkey, 1982; Nobel, 1999). The primary
route of water and CO2 movement along this pathway is via
the stomata. Beyond this primary route, the plant cuticle and
the boundary layer provide resistances that may influence WUE
(Nobel, 1999). For most plants, stomatal opening and closing
follows a diurnal cycle associated with light and dark periods,
respectively (Figure 1; Shimazaki et al., 2007). The bulk of tran-
spirational water loss (70–95% depending on the species) occurs
during the day in C3 and C4 plants. Nighttime transpiration also
contributes (5–30%) to the total diurnal water loss. FIG. 2. Relationship among transpiration, net CO2 assimilation, and WUE.
Illustration indicates how instantaneous WUE (WUEi , bar) is influenced by
stomatal conductance through differential responses of net CO2 assimilation
(closed square), and transpiration (open square) (Jones, 1976; Rosenberg et al.,
A. Stomatal Transpiration
1983; Mansfield et al., 1990; Radin et al., 1994; Liu et al., 2002; Medrano
Daytime transpiration begins with stomatal opening pro- et al., 2002; Thompson et al., 2007; Avola et al., 2008; Timlin et al., 2008).
cesses in response to blue and red light specifically (Shimazaki Transpiration rate increases linearly with stomatal conductance. Net CO2 as-
et al., 2007), increasing stomatal conductance, resulting in in- similation also linearly increases with stomatal conductance, but saturates at
creased water loss (Nobel, 1999; Jones, 2004). Transpiration higher conductance. WUEi increases with stomatal conductance at low stom-
atal conductance because the slope of net CO2 assimilation is steeper than the
increases linearly with stomatal conductance assuming the fol- slope of transpiration. A reduction in stomatal conductance from A to B will
lowing conditions: 1) vapor pressure deficit (VPD) is constant improve WUE because transpiration is reduced (A′′ → B′′ ), but the reduction
and 2) boundary layer resistance is nearly zero (Figure 2; Jones, of net CO2 assimilation is minimized (A′ → B′ ).
 TRANSPIRATION TO IMPROVE CROP WATER USE
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FIG. 3. Cellular division and morphogenesis leading to stomatal complex development. Stomatal development processes include entry, amplifying, and spacing
divisions, and guard cell differentiation (reprinted from Bergmann and Sack, 2007 with permission from Annual Reviews).
High WUE is observed when stomatal conductance is lower
than the potential maximum for a genotype (Figure 2; A →
B)—for example, under low water availability (Thompson et al.,
2007). However, at very low stomatal conductances (A → C),
net CO2 assimilation is also lower (A’ → C’), which results in
reduced growth (Blum, 2005). Therefore, higher WUE is often
associated with low crop production (Condon et al., 2002; Blum,
2005).
It should be possible to improve WUE without substantial
limitation of CO2 assimilation (Figure 2). At high stomatal con-
ductances (A → B), net CO2 assimilation is nearly saturated
(A′ → B′ ), while the increase in transpiration rate is still linear
(A′′ → B′′ ) (Jones, 1976; Geber and Dawson, 1990; Martin and
Ruiz-Torres, 1992; Radin et al., 1994; Franks, 2006; Thompson
et al., 2007; Avola et al., 2008). Consequently, WUE can be im-
proved by reducing water use through the mechanisms that reg-
ulate or determine stomatal conductance to water vapor, which
involves stomatal number (density), stomatal aperture, the cuti-
cle, and the boundary layer.
1. Stomatal Number (Density)
Stomata are pores on the surface of leaves formed as a re-
sult of the anatomy of two identical specialized epidermal cells:
the guard cells. Guard cell development is initiated by differ-
entiation of a postprotodermal cell to a meristemoid mother
cell (MMC) followed by subsequent asymmetric cell division
413
(entry division) that produces a guard cell meristemoid and a
sister cell. Then, the meristemoid may directly produce a guard
cell pair by a symmetric division (see reviews by Bergmann,
2006; Bergmann and Sack, 2007; Figure 3). Alternatively, the
sister cell may undergo asymmetric division producing another
meristemoid and a sister cell, i.e., two meristemoids and one
sister cell (Figure 3). In Arabidopsis, at least one epidermal
cell separates guard cell pairs, a phenomenon known as the
one cell spacing rule (Bergmann, 2006). The sister cell eventu-
ally differentiates into an epidermal cell. A third possibility is
that the meristemoid may undergo further asymmetric cell divi-
sions (amplifying division) to produce additional sister cells that
differentiate into epidermal cells (Figure 3). Finally, the stom-
atal complex is formed by cellular morphogenesis and pore
formation.
Stomatal number in leaves is determined by regulation of
guard cell differentiation, the asymmetric division of guard cell
meristemoids, or epidermal cell expansion (Bergmann, 2006;
Bergmann and Sack, 2007). If the differentiation of a meriste-
moid to a guard cell is arrested, the frequency of asymmetric
division of meristemoids or MMC is decreased, or if epider-
mal cells expand and do not divide, stomatal density will de-
crease. In Arabidopsis, nuclear DNA content in all guard cells
is 2C (1C indicates the DNA content of the haploid genome),
whereas other epidermal cells (including trichomes) have 2C to
32C (Melaragno et al., 1993; Traas et al., 1998). DNA content
 414 C. Y. YOO ET AL.
increases with DNA replication without cell division through
a mechanism called endoreduplication, which is generally as-
sociated with cell size (Sugimoto-Shirasu and Roberts, 2003).
This suggests that endoreduplication may increase epidermal
cell size, thereby increasing the distance between guard cells and
decreasing stomatal density (De Veylder et al., 2002; Bergmann,
2004).
Stomatal density has the potential to affect plant water loss
because the total pore area represents the principal diffusion
pathway for the movement of water vapor, which is driven
by VPD between the leaf and the atmosphere. Reductions in
stomatal density are sometimes correlated with reduced tran-
spiration (Miskin et al., 1972; Monson and Grant, 1989; Lake
and Woodward, 2008). A positive correlation between stomatal
density and transpiration was observed in seven barley popu-
lations; there was no correlation between stomatal density and
photosynthesis in these populations (Miskin et al., 1972), thus
implying that WUE may increase with reduced stomatal den-
sity only to the minimum number that is necessary for sufficient
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CO2 uptake. This notion is supported by evidence showing that
stomatal density was negatively correlated with WUEi in 29
genotypes of poplar (Monclus et al., 2006). Genetic (discussed
later) and environmental interactions also regulate stomatal den-
sity (Woodward et al., 2002; Bergmann, 2006; Bergmann and
Sack, 2007; Casson and Gray, 2008). Higher light intensity
and lower CO2 concentration both increase stomatal density
in leaves that develop under these conditions, an effect that is
presumed to be an adaptive response that reduces the CO2 limi-
tation of photosynthesis (Lake et al., 2001; Thomas et al., 2004;
Casson and Gray, 2008). Exposure to UV-B radiation (Dai et al.,
1995; Visser et al., 1997; Gitz et al., 2005), elevated tempera-
tures (Beerling and Chaloner, 1993; Luomala et al., 2005), and
elevated salinity (Romero-Aranda et al., 2001) all reduce stom-
atal density. These reductions might be necessary to maintain
leaf water status. A reduction in stomatal density due to UV-B
treatments decreased transpiration and increased WUEi overall
(Dai et al., 1995; Gitz et al., 2005). Muchow and Sinclair (1989)
suggest that stomatal density may affect cuticular water loss be-
cause water loss occurs from guard cell teichodes, even when
stomata have minimum apertures. However, the relationship be-
tween stomatal density and transpiration was not examined in
this experiment.
Water deficit conditions also alter stomatal density in sev-
eral species, although the relationship between water deficit and
stomatal density is not clear. Higher stomatal density was ob-
served in leaves of nonirrigated vs. irrigated apple trees (Elias,
1995) and Pseudoroegneria spicata also exhibited an increase in
stomatal density in response to decreased water supply (Fraser
et al., 2009). Severe water stress reduced stomatal density, but
moderate stress increased stomatal density in leaves of the xe-
rophilous grass Leymus chinensis (Xu and Zhou, 2008). In-
creased stomatal density in response to drought stress may act to
increase transpiration to promote an increase in nutrient uptake.
This notion is partly supported by the fact that reduced stomatal
density of Vigna sinesis in response to water deficit occurred
only in phosphorous sufficient conditions, but not in phospho-
rous deficient conditions (Sekiya and Yano, 2008). However, it
is likely that factors that slightly reduce stomatal density with
respect to “normal” stomatal density under optimal conditions
for a given genotype, without nutrient limitations, will decrease
transpirational water loss while maintaining sufficient CO2 up-
take to sustain biomass and yield.
2. Stomatal Aperture
Several recent reviews detail the signal transduction cas-
cades and effectors that mediate stomatal opening and closing
(see reviews by Outlaw, 2003; Roelfsema and Hedrich, 2005;
Nilson and Assmann, 2007; Pandey et al., 2007; Shimazaki
et al., 2007; Sirichandra et al., 2009). These reviews describe
the critical mechanisms in guard cell turgor pressure regulation
that ultimately control stomatal aperture. In stomatal opening,
stimuli such as light, low CO2 concentration, and high humid-
ity promote stomatal opening processes (Outlaw, 2003). Blue
light induces a cytosolic Ca2+ increase and activates the plasma
membrane H+ -ATPase, which couples energy from ATP hy-
drolysis to vectoral transport (cytosol to apoplast) of H+ , re-
sulting in membrane hyperpolarization. Hyperpolarization ac-
tivates inward-rectifying K+ (K+ in ) channels for electrophoretic
K+ uptake into guard cells that results in a lowering of os-
motic potential, water uptake into the guard cell, and a subse-
quent increase in turgor pressure. The resulting change in guard
cell volume mediates stomatal opening. Blue light also induces
starch degradation, which is necessary to synthesize malate2− ,
a critical counter-ion for K+ accumulation in guard cells. Red
light promotes CO2 fixation, which decreases intercellular CO2
concentration (ci ). Guard cells perceive a decrease in ci and
deactivate anion channels, a process that also contributes to
membrane hyperpolarization. The accumulation of sucrose in
the guard cells during the afternoon period is also implicated in
stomatal opening.
Stomatal closure is promoted by darkness, high CO2 concen-
tration, low humidity, high temperature, and abscisic acid (ABA)
(Outlaw, 2003). Generally, the processes that induce stomatal
closure are the reverse of those processes that promote open-
ing. To induce closure, H+ pumps are inactivated and anion
efflux channels are activated, resulting in membrane depolar-
ization. Membrane depolarization activates outward rectifying
K+ (K+ +
out ) channels resulting in K efflux and subsequent loss
of turgor pressure via water efflux; this reduction in guard cell
volume results in stomatal closure (Fan et al., 2004). ABA acti-
vates signal transduction pathways that mediate stomatal clos-
ing in diurnal and water deficit–induced stomatal movements
(Fan et al., 2004; Tallman, 2004). ABA binding to ABA recep-
tors initiates signal cascades through several signaling cascades
which involve changes in cytosolic pH, Ca2+ , reactive oxygen
species (ROS), and nitric oxide (NO), followed by posttransla-
tional modifications such as phosphorylation of proteins in these
signaling pathways (Fan et al., 2004).
 TRANSPIRATION TO IMPROVE CROP WATER USE
Plants have evolved the capacity to control WUE via the regu-
lation of stomatal opening and closing in response to various en-
vironmental changes (Abdulrahman and Williams, 1981; Ball,
1988; Flanagan and Jefferies, 1988; Mobley and Marini, 1990;
Marcelis and Van Hoojidonk, 1999; del Amor and Marcelis,
2006). For example, in environments with a sufficient amount
of water, high water loss is not detrimental to plant growth
and/or survival, and may facilitate other processes such as evap-
orative cooling and nutrient uptake; thus, WUE is typically low.
Water-limited conditions cause a reduction in stomatal con-
ductance, due to ABA accumulation in guard cells. This is a
water-saving strategythat enhances plant survival and increases
WUE, but often causes decreased plant growth and yield overall
because total plant CO2 assimilation may decrease substantially
(Martı̀nez et al., 2003).
B. Cuticular Transpiration
The surface of most plant tissues is coated with a thin cuticle
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layer consisting of a polymer matrix (cutin) and lipids (cutic-
ular waxes) (Riederer and Schreiber, 2001). Cuticle structure
and thickness, and wax amount and composition are variable
among plant species from different habitats, which may result
in varying cuticular transpiration (Schreiber and Riederer, 1996;
Riederer and Schreiber, 2001; Burghardt and Riederer, 2006).
The existence of a “tortuous” hydrophilic pathway through the
larger lipophilic pathway of the cuticle is thought to be the route
for cuticular water loss (Popp et al., 2005). However, the rela-
tionship between cuticle composition or structure and cuticular
transpiration has not been established (Riederer and Schreiber,
2001; Jenks et al., 2002; Burghardt and Riederer, 2006).
The most common method for estimating cuticular transpira-
tion is to measure water loss from plants under dark conditions
or dry-down curves of detached leaves or shoots. Steady-state
water loss rates from plants in darkness or from detached leaves
after the initial rapid decrease in water loss rate (due to transpi-
ration through stomata) are assumed to be representative of cu-
ticular transpiration. However, studies of water loss under dark
conditions and after desiccation have not excluded the possibil-
ity of water loss from incomplete stomatal closure (Kerstiens
et al., 2006).
Cuticle permeability is estimated by the rate of chlorophyll
extraction in an organic solvent, e.g., 80% ethanol. However,
wax itself can dissolve in an organic solvent and the solvent can
infiltrate into nearly closed stomata, which also affects perme-
ability (Kersteins et al., 2006). Furthermore, water molecules
are much smaller than chlorophyll, so a direct relationship be-
tween permeability of the two molecules with respect to move-
ment through the cuticular layer cannot be assumed (Kosma and
Jenks, 2007).
The amount of water loss due to cuticular transpiration is
not clear, largely because of measurement limitations, although
Boyer et al. (1997) reported that cuticular conductance ac-
counted for ∼2–30% of total conductance in Vitis vinifera,
415
which has an astomatous adaxial surface. In this study, conduc-
tance of CO2 and H2 O was measured after sealing the abaxial
(bottom) surface with silicone vacuum grease. Although some
exchange of CO2 does occur through the cuticle, CO2 conduc-
tance through the cuticle constitutes only 6% of H2 O vapor
conductance in leaves of Vitis vinifera and was undetectable in
Impatiens coralfutura, suggesting that cuticular conductance
is more critical for water loss than CO2 uptake. However,
the amount of cuticular water loss in stomatous leaves is still
unknown.
Quantitative trait loci (QTLs) for variation in epicuticular
wax production in rice (Oryza sativa L.) co-locate with shoot
and root drought resistance traits (Srinivasan et al., 2008). Sev-
eral mutants with altered wax and/or cutin accumulation and/or
composition were also reported to have altered transpiration.
However, the correlation between altered cuticular traits and
transpiration is weak in these mutants (Jenks et al., 2002; Kosma
and Jenks, 2007). The total wax amount decreased with the wax2
mutation (Chen et al., 2003b) and was increased in the shn mu-
tation (Aharoni et al., 2004). However, both mutations resulted
in reduced transpirational water loss from detached leaves; this
may be due to reduced stomatal density rather than altered wax
accumulation in both mutants (Chen et al., 2003b; Aharoni et al.,
2004). Overexpression of Medicago truncatula WXP1 (wax pro-
duction) in Medicago sativa and Arabidopsis thaliana increased
total wax accumulation, altered wax composition, and resulted
in reduced water loss (Zhang et al., 2005, 2007a). Similar results
were also obtained by overexpression of Medicato truncatula
WXP2 in Arabidopsis thaliana (Zhang et al., 2007a). A mutation
in the very-long-chain fatty acid elongase β-ketoacyl-CoA syn-
thase decreased n-alkanes and aldehydes in cuticle very-long-
chain aliphatics, and resulted in increased cuticular transpiration
in tomato fruit (Vogg et al., 2004). Ristic et al. (2002) showed
that the maize line ZPL 389 had higher concentrations of C32
aldehydes and C32 primary alcohols and had increased tran-
spirational water loss from detached leaves in the dark. These
studies showing the possible influence of altered cuticular traits
on transpiration have not demonstrated the impact of cuticular
transpiration on WUE to date.
In some species, the surface of the substomatal chamber is
covered by a cuticle layer. Some leaves have a stomatal or cutic-
ular ridges that consist of cuticle and cell wall at the outer rim of
the stomatal pore (Field et al., 1998; Kosma and Jenks, 2007).
These cuticular characteristics may affect stomatal transpira-
tion. The wax2 mutation results in a reduction of the stomatal
ridge as well as the reduction of cutin monomers and disruption
of cutin ultrastructure. This genotype also has increased daytime
transpiration (Chen et al., 2003b; Kosma and Jenks, 2007).
Cuticular transpiration may play a more significant role dur-
ing the nighttime portion of the diurnal transpiration cycle,
when stomatal conductance is lower or negligible and cutic-
ular water loss is a larger portion of water lost (Dawson et al.,
2007); however, this hypothesis has yet to be experimentally
tested.
 416 C. Y. YOO ET AL.
C. Boundary Layer Conductance
The final resistance associated with water vapor movement
from the leaf to the bulk air is the boundary layer, which is de-
fined as an air layer adjacent to the leaf surface where wind speed
is reduced by surface friction, creating a resistance to diffusion
of gases (Schuepp, 1993). The boundary layer thickness is in-
creased in larger leaves and under low wind speed conditions
(Nobel, 1974, 1999). Consequently, a reduction of the boundary
layer conductance may decrease transpiration. This resistance is
not likely to be important in many field crop species (e.g., corn,
soybean, etc.), but may be important in some species that are
grown in more protected environments (e.g., coffee, tea, etc.),
or under cover (e.g., greenhouse crops).
Any increases in boundary layer resistance will limit tran-
spiration, but may also result in an increase in leaf temperature
because of the effect of boundary layer on heat conduction from
the leaf to the surrounding air (Gates, 1968; Nobel, 1999). How-
ever, this is likely to be a problem only in extreme environments,
since most metabolic enzymes are stable at relatively high tem-
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peratures (Sage and Kubien, 2007). CO2 movement from the
air to the leaf is also influenced by the boundary layer conduc-
tance. However, the stomatal and mesophyll resistance to CO2
are usually greater than the boundary layer resistance to CO2 ,
suggesting that the boundary layer is not a limiting factor on
total conductance of CO2 (Farquhar and Sharkey, 1982; Nobel,
1999). Because marginal reductions of instantaneous water loss
may lead to significant water savings over a longer period of
time, decreasing boundary layer conductance has the potential
to improve WUE by reducing transpiration without affecting
CO2 uptake, but most likely only in a few specific crops and
situations.
The impact of the boundary layer conductance on transpira-
tion is dependent on stomatal conductance. If the boundary layer
conductance is greater than the stomatal conductance, overall
transpiration is determined mainly by stomatal transpiration as-
suming that cuticular transpiration is negligible. However, if the
boundary layer conductance is smaller than the stomatal con-
ductance, VPD near the leaf surface will be decreased, which de-
creases overall transpiration (Meinzer and Grantz, 1991). Jarvis
and McNaughton (1986) described this effect by the decoupling
coefficient ("); decreased boundary layer conductance will de-
couple the vapor pressure between the leaf surface and the bulk
air.
Boundary layer conductance in plants is primarily deter-
mined by leaf size and shape and leaf surface roughness
(Schuepp, 1993). The boundary layer increases with leaf size
(Nobel, 1999). For example, boundary layer conductance was
lower in the larger leaves of Acer pseudoplatanus than the
smaller leaves in Quercus robur (Stokes et al., 2006), and was
very low in large leaves of timber species (Grace et al., 1980).
Geller and Smith (1982) modeled the effect of leaf size on
boundary layer conductance using leaf and microclimatic pa-
rameters from field measurements. Their model predicts that
increases in leaf size reduce transpiration, possibly through in-
creased boundary layer resistance. Lobes and serration in leaves
reduce the boundary layer resistance as compared to leaves
with smooth margins (Baker and Myhre, 1969; Gottschlich and
Smith, 1982; Schuepp, 1993).
Leaf surface roughness also influences boundary layer con-
ductance. In Populus, air over the rough abaxial leaf surface
was more turbulent than the air over the smooth adaxial leaf
surface, resulting in an increased boundary layer resistance
(Grace and Wilson, 1976). Therefore, leaf surface ultrastruc-
ture such as epicuticular wax structures or trichomes can influ-
ence boundary layer, and subsequently, water loss from the leaf.
Epicuticular wax layers may decrease the boundary layer con-
ductance by increasing surface friction (Pritchard and Amthor,
2005), or may provide a smooth surface that allows for the
development of a substantial boundary layer. Reicosky and
Hanover (1976) analyzed wax height and calculated the bound-
ary layer, concluding that the height of leaf surface waxes of
Picea pungens was too small to influence the boundary layer.
However, since very few direct measurements have been made,
the effect of surface waxes on boundary layer conductance is
unresolved.
Trichomes have been understood theoretically and experi-
mentally to decrease water loss by either increasing light re-
flection or decreasing boundary layer conductance in some
species (Ehleringer, 1984; Schuepp, 1993). Kenzo et al. (2008)
demonstrated a slightly higher midday maximum transpiration
rate in depilated leaves compared to hairy leaves of Mallotus
macrostachyus, while stomatal conductance and photosynthesis
were not altered, resulting in lower WUEi . Benz and Martin
(2006) examined the transpiration and CO2 uptake in 12 species
of epiphytic Tillandsia ranging in leaf trichome size and dis-
tribution. Although trichomes increased the thickness of the
boundary layer and decreased the boundary layer conductance
based on theoretical calculations, there were no differences in
transpirational water loss (Benz and Martin, 2006). The authors
concluded that this might be due to other factors contributing
to transpiration such as stomatal and cuticular components that
mask the small differences of boundary layer conductance (Benz
and Martin, 2006). Furthermore, the structure of trichomes in
the Bromeliacea are highly specialized (Benzing et al., 1976;
Pierce et al., 2001); the specialization of these structures likely
affects water uptake and loss in ways other than changes to
the boundary layer (Schmitt et al., 1989; Reinert and Meirelles,
1993).
D. Nighttime Transpiration
Nighttime transpiration occurs in most C3 and C4 plants
(Mickelbart, unpublished; Musselman and Minnick, 2000; Sny-
der et al., 2003), and accounts for approximately 5–30% of total
diurnal water loss (Richards et al., 1986; Green et al., 1989;
Benyon, 1999; Bucci et al., 2004; Gosney and Mickelbart, 2006;
Caird et al., 2007a, b). Several studies have suggested that night-
time water loss is primarily due to incomplete stomatal closure
 TRANSPIRATION TO IMPROVE CROP WATER USE
(Howard and Donovan, 2007; Caird et al., 2007b), although ac-
tual stomatal aperture was not assessed in these studies. The
physiological necessity, if any, of nighttime transpiration in
plants is not known. Since nighttime water loss can be a sig-
nificant percentage of total water loss, a reduction in nighttime
transpiration has the potential to increase WUE without dimin-
ishing biomass and/or yield.
The majority of studies on the functional role of nighttime
water loss have focused on the relationship between nighttime
transpiration and nutrient uptake and assimilation (Caird et al.,
2007a, 2007b). Holbie and Colpaert (2004) demonstrated that
WUE was correlated with nitrogen availability. Therefore, sup-
pressing nighttime transpiration to reduce water loss may not
result in increased WUE if nighttime transpiration is neces-
sary for sufficient N uptake. Snyder et al. (2008) found that
plants with suppressed nighttime stomatal conductance exhib-
ited consistent patterns of decreased N uptake. Ludwig et al.
(2006) showed that in Helianthus anomalus, fertilization de-
creased both daytime and nighttime stomatal conductance and
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transpiration. However, Howard and Donovan (2007) found that
in Helianthus, nutrient limitations resulted in reduced shoot and
reproductive biomass and leaf N concentration, but no signif-
icant change in nighttime conductance. Likewise, Christman
et al. (2009a) showed that altering VPD to reduce nighttime
conductance had no effect on N, Ca, or K uptake at both high
and low N levels. However, Ealson and Richards (2009) showed
that increased nighttime conductance was associated with in-
creased accumulation of nitrate and leaf starch in Vicia faba
under short day conditions. The long-term effects of selecting
for reduced nighttime transpiration as a means for improving
overall WUE, especially with respect to nutrient flux, are not
known.
When water is limited, substantial water loss at night could
lead to a decrease in WUE (Caird et al., 2007b). Similar to day-
time conditions, water loss at night increases with VPD (Oren
et al., 2001; Gosney and Mickelbart, 2006), and when VPD
is high, substantial crop-level nighttime water loss can occur
(Snyder et al., 2003; Caird et al., 2007a, 2007b). This would be
true if nighttime water loss was the result of incomplete stomatal
closure and/or if cuticular water loss is significant. Incomplete
stomatal closure also results in nighttime ozone exposure and
could result in lower WUE because of ozone injury and dam-
age that is significant during the night due to the lack of ROS
detoxification through photosynthetic electron transport (Re-
ich and Lassoie, 1984; Matyssek et al., 1995; Musselman and
Minnick, 2000). Daley and Philips (2006) found that of three tree
species (Betula papyrifera, Quercus rubra, and Acer rubrum)
grown in the same environment (e.g., same VPD), B. papyrifera
had markedly different nighttime conductance, as estimated by
nocturnal sap flux. Discrepancies in the literature, specifically
independence (or dependence) of regulatory mechanisms for
daytime and nighttime stomatal conductance, illustrate the need
for further research to identify the major physiological and ge-
netic determinants responsible for the regulation of nighttime
417
transpiration and to determine the extent to which nighttime
transpiration affects overall plant WUE.
III. GENETICS OF WATER USE
Genetic variation for the transpiration ratio (reciprocal of
WUE) was determined almost 100 years ago in a survey of
62 different plant species (Briggs and Shantz, 1913). Subse-
quently, research has established that genetic variation for WUE
exists among genotypes within species, indicating that WUE
can be improved by genetic introgression (Alonso-Blanco and
Koornneef, 2000). The genomics era has led to the compilation
of numerous genetic determinants involved in photosynthesis
and transpirational water loss. It is now feasible to link genetic
determinants to physiological mechanisms that are involved in
transpirational water loss. In this section, we summarize evi-
dence of genetic variation for WUE and discuss genetic determi-
nants that are or may be regulators or effectors of transpirational
water loss.
A. Genetic Variation for WUE
Reported values of WUE in different groups of plants are
consistent with their carbon assimilation mechanisms: mean
values of WUEB reported in the literature of C3 and C4 plants
are 3.9 and 5.7 g kg−1 , respectively (Table 2). C4 plants have
consistently higher WUE because they are generally more
efficient with respect to CO2 fixation (Ogren, 1984; Nobel,
1999; Leegood, 2002). The mean WUEB of crassulacean acid
metabolism (CAM) species is 12.4 g kg−1 , two to three times
higher than other species, due to very low daytime transpiration,
as stomata are closed during the day and open at night for CO2
fixation (Chaves et al., 2004).
However, our survey of published results (Table 2) revealed
that reported WUEB values of some C3 plants (e.g., Glycine max
and Phaseolus vulgaris) are higher than those of some C4 plants
(e.g., Amaranthus tricolor, Miscanthus sacchariflorus, Miscant-
hus sinensis, and Sorghum bicolor) as well as some CAM plants
(e.g., Euphorbia tirucalli, Kalanchoë daigremontiana, Kalan-
choë pinnata, and Sedum nuttallianum). This deviation from
expected values may be the result of comparisons that were
made under different environmental conditions or could be the
result of adaptations that improve WUEB (Monson et al., 1986;
Liang et al., 1989; Gravatt and Martin, 1992). The higher pho-
tosynthetic capacity of C4 plants appears to exist only at higher
temperatures and adequate water availability (Monson et al.,
1986; Liang et al., 1989; Ripley et al., 2007). For example,
net CO2 assimilation in C4 plants is similar to C3 plants under
drought conditions; this similarity is mainly due to metabolic
limitations rather than stomatal limitations (Ripley et al., 2007).
A comparison of Sedum species concluded that WUE of S. ter-
natum (C3 ) is similar to S. wrightii (CAM), and higher than
S. telephioides and S. nuttallianum (both CAM cycling) under
both well-watered and drought-stressed conditions (Gravatt and
Martin, 1992). Orthonna opima (C3 ) also had similar WUE
 418 C. Y. YOO ET AL.

TABLE 2
Natural genetic variation for water use efficiency.
Species WUEB WUEY WUEi Agea Locb Ref
C3 Herbaceous
Abutilon theophrasti 4.3 4.7 4 GH (1)
Anoda cristata 4.0 4.2 4 GH (1)
Arabidopsis thaliana 2.1 (31) 10 GC (2)
Cassia obtusifolia 4.0 4.4 4 GH (1)
Cucumis sativus 10.0 12 GH (3)
Datura stramonium 4.4 4.2 4 GH (1)
Glycine max 16.7 4.9 12 GH (4)
5.3 (4) 5 GH (5)
1.3 12 F (6)
4.0 3.6 4 GH (1)
Gossypium hirsutum 1.9 27 F (7)
5.0 8 GC (8)
3.0 9 GH (9)
Helianthus annuus 1.8 23 F (7)
Phaseolus vulgaris 8.7 (6) 3 GC (8)
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v 15 F (10)
Sida spinosa 4.8 3.8 4 GH (1)
Solanum lycopersicum 4.5 4 GH (11)
15.4 24 F (12)
Sonchus arvensis 11.2 6 GH (13)
Thellungiella halophila 3.4 (4) 12.7 (6) 8 GC (14)
Vicia faba 4.8 (3) — F (15)
Xanthium pensylvanicum 1.8 12 F (6)
4.4 3.9 4 GH (1)
C3 Non-herbaceous
Annona squamosa 2.4 2y GH (16)
Atriplex canescens 2.3 (3) 16 GH (17)
Atriplex halimus 1.4 (2) 13.8 (2) 12 GH (18)
Carica papaya 5.7 8 F (19)
Coffea ! rabica × C.canephora 2.5 13.7 — GH
C. ! rabica × C. racemosa 1.7 14.5 (20)
Malus domestica 14.0 (2) — GH (20)
Picea glauca 17.0 2y F (22)
Populus simonii 5.9 12.2 1.5y GH (23)
Populus davidiana 4.5 28 GH (24)
Populus × euroamericana 2.5 10.2 — GH (25)
Prunus persica 8.4 (2) 2y F (26)
Pseudotsuga menziesii 2.5 11.8 2.5y GH (25)
Salix burjatica × S. dasyclados 4.9
S. schwerinii × S. viminalis 5.3 24 F (27)
S. viminalis 6.3
S. schwerinii × S. viminalis × S. aegyptiaca 6.7 24 F (27)
Solanum muricatum 8.1 24 F (27)
Swietenia macrophylla 3.9 24 GH (29)
Tectona grandis 2.7 20 GH (29)
Vitis vinifera 6.6 (2) 10y F (30)
C3 Grasses
Avena fatua 1.2 — F (31)
Hordeum vulgare 2.8 12 GH (32)
2.3 (6) 20 F (33)
Oryza sativa 3.8 (34) 12 GH (34)
 TRANSPIRATION TO IMPROVE CROP WATER USE 419

TABLE 2
Natural genetic variation for water use efficiency. (Continued)
Species WUEB WUEY WUEi Agea Locb Ref
Pascopyrum smithii 1.7 9 GH (35)
Solanum tuberosum 6.2 13 F (36)
Triticum aestivum 2.3 (12) 1.9 (12) 21.4 (7) 32 GH (37)
2.6 1.0 36 F (38)
2.7 1.1 29 F (39)
1.1 (3) — F (40)
Triticum turgidum 3.3 (2) 5 F (41)
3.1 (2) 8 F (41)
3.0 (2) 10 F (41)
2.5 (2) 14 F (41)
C4 Grasses
Amaranthus tricolor 3.3 8.3 4 GH (42)
8.7 14.6 4 GH (1)
A. hypochondriacus 4.9 7.6 4 GH (42)
A. cruetus 5.0 7.6 4 GH (42)
Bouteloua gracilis 3.1 9 GH (35)
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Digitaria ciliaris 9.3 4 GC (43)

Echinochloa crus-galli 8.7 4 GC (43)
Eleusine indica 9.8 4 GC (43)
Miscanthus × giganteus 2.1 12 GH (36)
M. sacchariflorus 4.1 12 GH (44)
M. sinensis 2.9 12 GH (44)
Panicum virgatum 5.3 (11) 11 GH (45)
Sorghum bicolor 3.8 24 F (7)
Zea mays 2.6 12 F (46)
2.1 (4) — F (47)
C3 Succulent
Sedum integrifolium 9.7 — GH (48)
Sedum ternatum 15.0 — GH (48)
CAM Succulent
Aloe vera 18.5 40 GH (29)
Ananas comosus 15.9 12 GH (29)
Euphorbia tirucalli 9.5 36 GH (29)
Kalanchoë daigremontiana 9.2 40 GH (29)
Kalanchoë pinnata 9.4 32 GH (29)
Sedum telephioides 11.0 — GH (48)
Sedum nuttallianum 8.9 — GH (48)
Sedum wrightii 16.9 — GH (48)
Values for WUE (WUEB , WUEY , and WUEi ) of plant species grown in well-watered conditions are summarized as C3 (herbaceous, non-
herbaceous, grass, succulent), C4 (grass), and CAM (succulent) photosynthesis. All units are converted to g kg−1 . Numbers in parentheses after
respective WUE values indicate the number of ecotypes or cultivars used to calculate the average WUE.
Abbreviations: Ref, reference; GC, growth chamber; GH, greenhouse; F, field.
a
Age is given in weeks, except where y indicates years.
b
Location (Loc) where plants were grown and measured.
(1) Patterson and Flint, 1983; (2) Nienhuis et al., 1994; (3) Janoudi et al., 1993; (4) Rosadi et al., 2007; (5) Gitz et al., 2005; (6) Geddes et al.,
1979; (7) Perniola, 1994; (8) Hoffman and Phene, 1971; (9) Plaut and Federman, 1991; (10) Muñoz-Perea et al., 2007; (11) Thompson et al.,
2007; (12) Oliveira et al., 1996; (13) Zollinger and Kells, 1991; (14) Gosney and Mickelbart, unpublished; (15) Avola et al., 2008; (16) Marler
and Zozor, 1996; (17) Glenn and Brown, 1998; (18) Martı̀nez et al., 2003; (19) Clemente and Marler, 1996; (20) Dias et al., 2007; (21) Massonnet
et al., 2007; (22) Wang and Zwiazek, 1999; (23) Liang et al., 2006; (24) Zhang et al., 2004; (25) Ripullone et al., 2004; (26) Glenn et al., 2006;
(27) Wikberg and Ögren, 2007; (28) Chen et al., 1999; (29) Winter et al., 2005; (30) Medrano et al., 2003; (31) Van Wychen et al., 2004; (32)
Grumet et al., 1987; (33) Ouda et al., 2007; (34) Impa et al., 2005; (35) Morgan et al., 1998; (36) Liu et al., 2006; (37) Górny and Garczy~ñski,
2002; (38) Xue et al., 2006; (39) Zhang et al., 1998; (40) Fan et al., 2008; (41) Richards et al., 1986; (42) Omami et al., 2006; (43) Patterson,
1986; (44) Clifton-Brown and Lewandowski, 2000; (45) Byrd and May, 2000; (46) Kang et al., 2000; (47) Massinga et al., 2003; (48) Gravatt
and Martin, 1992.
 420 C. Y. YOO ET AL.
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FIG. 4. Relationships between CO2 assimilation, transpiration, and WUEB . Plots demonstrate the relationship between CO2 assimilation or transpiration rate
and WUEB among 10 cultivars of Triticum aestivum (A and B; Van den Boogaard et al., 1997) and 34 diverse germplasm accessions of Oryza sativa (C and D;
Impa et al., 2005). (Adapted and modified from Van den Boogaard et al., 1997 with permission from Blackwell Publishing Ltd and reprinted from Impa et al.,
2005 with permission from Crop Science Society of America).

to Cotyledon orbiculate (CAM) at certain periods (Eller and
Ferrari, 1997). These data suggest that predicted WUE based
on photosynthetic pathway (C3 , C4 , and CAM) is not always
consistent with actual WUE under certain environmental con-
ditions, especially water-deficit stress.
Genetic variation for WUE exists in both natural and agri-
cultural populations as demonstrated by studies on various
species, accessions, ecotypes, cultivars, and hybrids (Takeda
and Matsuoka, 2008). WUEB of Triticum aestivum cultivars
ranged between 9.7 to 11.7 g kg−1 (Van den Boogaard et al.,
1997) and WUEi of Sorghum bicolor inbred lines ranged from
2.91 to 3.49 mmol mol−1 (7.11 to 8.53 g kg−1 ) (Balota et al.,
2008). Natural genetic variation for WUE has also been docu-
mented in Arabidopsis thaliana ecotypes and recombinant in-
bred lines (Nienhuis et al., 1994), Glycine max cultivars and
inbred lines (Hufstetler et al., 2007), Oryza sativa accessions
(Impa et al., 2005), Panicum virgatum cultivars (Byrd and May,
2000), Salix inbred lines (Wikberg and Ögren, 2007), Thel-
lungiella halophila ecotypes (Pence and Mickelbart, unpub-
lished), and Triticum aestivum cultivars (Górny and Garczyński,
2002; Chen et al., 2003a).
Evidence supports the notion that adaptive variation for WUE
is correlated with plant water loss in some plant species. A neg-
ative correlation between WUE and transpiration, and a lack of
correlation between WUE and CO2 assimilation have been re-
ported in Cicer arietinum and Vigna unguiculata (Udayakumar
et al., 1998), Oryza sativa (Figure 4D; Impa et al., 2005), Thel-
lungiella halophila (Pence and Mickelbart, unpublished), and
 TRANSPIRATION TO IMPROVE CROP WATER USE 421

TABLE 3
Genes regulating WUE under well-watered conditions. Genes that are linked to WUE and related physiological traits such as
transpiration (E) and net CO2 assimilation (A).
Gene Function Modification WUE E A Phenotypes Ref
Stomatal opening/closing, ABA biosynthesis/signaling
MRP5 ABC transporter null mutation ↑i ↓ - DT (1)
ABP9 bZIP TF overexpression ↑i ↓↓ ↓ ↑ABA (2)
NCED1 9-cis-epoxycarotenoid deoxygenase overexpression ↑B ↓↓ - ↑ABA (3)
Stomatal density, drought-tolerant determinants
ERECTA LRR-like kinase null mutation ↓B ↑gs ↓ ↑SD (4)
HDG11 Homeodomain-ZIP TF overexpression ↑B ↓ ↑ DT, ↓ SD (5)
GTL1 trihelix TF null mutation ↑B ↓ - DT, ↓ SD (6)
DREB1A AP2/EREB TF inducible expression ↑B ↓ - DT (7)
HARDY AP2/ERF-like TF overexpression ↑i ↓ ↑ DT (8)
ESB1 unknown null mutation ↑B ↓ ↓↓ DT (9)
Abbreviations: gs , stomatal conductance; E, transpiration; A, net CO2 assimilation; DT, drought tolerance; SD, stomatal density; Ref,
reference; TF, transcription factor; i, instantaneous WUE; B, biomass WUE.
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(1) Klein et al., 2003; (2) Zhang et al., 2008b; (3) Thompson et al., 2007; (4) Masle et al., 2005; (5) Yu et al., 2008; (6) Yoo et al., 2008; (7)
Bhatnagar-Mathur et al., 2007; (8) Karaba et al., 2007; (9) Baxter et al., 2009.

Triticum aestivum (Figure 4B; Van den Boogaard et al., 1997).
These results indicate that crops, at least in some plant species,
exhibit genetic variation for WUE that involve adaptations that
regulate plant water use (Blum, 2005) and that mechanisms exist
in plants to improve WUE by modifying transpiration.
B. Genes That Regulate Water Use Efficiency
The existence of natural genetic variation within species has
provided the basis for identification of QTLs responsible for im-
portant agricultural traits, including WUE (Mitchell-Olds and
Schmitt, 2006; Shindo et al., 2007; Takeda and Matsuoka, 2008).
QTLs linked to WUE have been identified in different species
such as Arabidopsis thaliana (Hausmann et al., 2005; Juenger
et al., 2005; Masle et al., 2005), Brassica oleracea (Hall et al.,
2005), Glycine max (Mian et al., 1996, 1998), Hordeum vul-
gare (Teulat et al., 2002), Solanum lycopersicum (Martin et al.,
1989), and Stylosanthes scabra (Thumma et al., 2001). Ara-
bidopsis ERECTA, which encodes a putative leucine-rich repeat
receptor-like kinase (LRR-RLK), was the first published gene
that regulates WUE to be identified by QTL mapping (Masle
et al., 2005). A loss-of-function erecta allele increases stomatal
conductance due to increased stomatal density and decreased
photosynthetic capacity due to a reduced rubisco carboxyla-
tion rate, which results in decreased WUE (Masle et al., 2005).
Eight other genes have been reported to directly regulate WUE
in different species (Table 3).
Other loci that regulate stomatal function have been linked to
alterations in WUE. The Arabidopsis ABC-transporter MRP5
mediates stomatal regulation through ABA-, Ca2+ -, and auxin-
dependent pathways, although the mechanisms involved are not
understood (Klein et al., 2003). An mrp5-1 loss-of-function
mutation resulted in reduced stomatal opening in response to
light, in addition to insensitivity to ABA-induced stomatal clo-
sure. Overall, these changes resulted in higher WUEi because
stomatal conductance and transpiration in mrp5-1 were approx-
imately 22% less than the wild type; net CO2 assimilation un-
der saturating intensities was not changed in mrp5-1 plants
compared to wild type (Klein et al., 2003). Stomatal closure
facilitated by ectopic induction of ABA biosynthesis also im-
proved WUE, apparently by reducing transpiration. The enzyme
9-cis-epoxycarotenoid dioxygenase (NCED) catalyzes the rate-
limiting enzyme in ABA biosynthesis. Overexpression of NCED
in Solanum lycopersicum led to increased ABA accumulation
and decreased stomatal conductance and transpiration, but did
not appreciably affect CO2 assimilation and biomass accumula-
tion, resulting in higher WUEB (Thompson et al., 2007). Over-
expressing NCED in Arabidopsis thaliana (Iuchi et al., 2001)
and Nicotiana plumbaginifolia also reduced transpirational wa-
ter loss, but CO2 assimilation or biomass accumulation was
not reported (Qin and Zeevaart, 2002). Overexpression of the
ABA-responsive element-binding protein 9 (ABP9) in Arabidop-
sis moderately increased ABA levels and resulted in increased
WUEi (Zhang et al., 2008b). These reports indicate WUE can be
improved by genetic manipulation of stomatal regulation that re-
sults in moderately reduced transpiration without inhibiting net
CO2 assimilation (Figure 2).
The Arabidopsis homeodomain (HD)-START transcription
factor 11 (HDG11) regulates stomatal density, which is associ-
ated with reduced transpiration and increased carbon assimila-
tion, thereby increasing WUE (Yu et al., 2008). Overexpression
of AtHDG11 in tobacco reduced stomatal density and improved
WUEB , indicating that the function of HDG11 is conserved in
Arabidopsis and tobacco and, perhaps in crop species. HDG11
 422 C. Y. YOO ET AL.
overexpressing tobacco plants also exhibited water-deficit stress
tolerance that was attributed to changes in root architecture, ac-
cumulation of ABA and proline, and increased oxidative stress
tolerance. HDG11 seems to enhance both drought tolerance and
improved WUE, at least partially due to reduced stomatal den-
sity. The Arabidopsis GT2-like 1 (GTL1) transcription factor
is a member of the GT-2 family that contains two separate, but
similar, trihelix DNA-binding domains (helix-loop-helix-loop-
helix) (Kuhn et al., 1993). The null mutation in AtGTL1 (gtl1)
reduces stomatal density and transpiration, without a propor-
tional reduction in biomass, which results in improved WUEB
(Yoo et al., 2008). Together, these results indicate that a small
reduction in stomatal density can reduce transpiration and im-
prove WUE (Yu et al., 2008; Yoo et al., 2008). Presumably,
lower stomatal densities caused by gtl1 and hdg11 result in a re-
duction of transpiration over the range where CO2 assimilation
is near saturation (Figure 2; A → B).
Other transcription factors (TFs) have been implicated in
the regulation of plant WUE. The Arabidopsis dehydration re-
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sponsive element-binding protein (DREB1A) regulates drought
tolerance (Shinozaki and Yamaguchi-Shinozaki, 2000). Stress-
induced expression of AtDREB1A in peanut, driven by the
RD29A promoter, reduced stomatal conductance and transpi-
ration, and increased WUEB under well-watered conditions
(Bhatnagar-Mathur et al., 2007). However, constitutive expres-
sion of DREB1A in Arabidopsis caused severe growth retarda-
tion (Liu et al., 1998). These contrasting examples highlight
the fact that regulated expression of genes (e.g. temporally or
spatially, hormonal or environmental induction, etc.) that confer
drought tolerance may result in higher WUE.
The Arabidopsis HARDY (HRD) gene, an AP2/ERF-like
transcription factor, positively regulates the production of mes-
ophyll cells and secondary and tertiary roots (Karaba et al.,
2007). A gain-of-function hardy mutation increased mesophyll
cell layers and secondary and tertiary roots, all of which were
linked to increased drought and salt tolerance in Arabidopsis.
HRD overexpression in rice increased WUEB and WUEi , due
to reduced transpiration and increased carbon assimilation and
biomass production (Karaba et al., 2007). Although the phys-
iological mechanisms through which HARDY regulates these
processes are not fully understood, reduced transpiration might
be beneficial to maintain leaf water content and decrease overall
water use, leading to drought tolerance and improved WUE.
The enhanced suberin (esb1) mutant also results in improved
WUEB through reduced stomatal aperture and transpiration,
with only minor effects on overall biomass (Baxter et al., 2009).
esb1 mutants have higher root suberin content than wild type
plants, which limits water and ion transport, resulting in re-
duced stomatal aperture, transpiration, and leaf accumulation
of ions such as Ca and Fe. Increased suberin may cause en-
hanced hydraulic resistance to water transport from root to
shoot, which may lead to leaf development result in lower wa-
ter potential than normal, leading to the decreased stomatal
aperture.
C. Determinants That Regulate Transpiration and May
Improve Water Use Efficiency
Forward and reverse genetic approaches have led to the iden-
tification of numerous determinants involved in the regulation
of transpiration (Nilson and Assmann, 2007). Candidate genes
for improving WUE are those involved in regulation of stomatal
density, stomatal opening or closure, and nighttime transpira-
tion. Since these genes were selected with the possibility of
improving WUE, any mutants having severe growth defects or
unreported biomass or leaf area are excluded from this review.
Genes regulating cuticular transpiration or affecting boundary
layer conductance are also not included because their impact on
transpiration by genetic regulation is not clearly understood.
1. Stomatal Density Determinants
There are numerous genes regulating stomatal development
processes, including entry divisions, amplifying divisions, spac-
ing divisions, differentiation, and cell cycle/division (see re-
views by Bergmann and Sack, 2007; Nadeau, 2009). The vast
majority of identified mutants exhibit extreme division and spac-
ing pattern phenotypes such as clusters of stomata (e.g., er,
yoda, and tmm) or few functional stomata (e.g., flp, fama, spch,
and mute) (Bergmann and Sack, 2007). It may not be possi-
ble to manipulate genes involved in fundamental developmental
processes without substantial negative effects on growth and
development. However, mutations in genes listed here cause
a marginal reduction in stomatal density and no reduction of
growth, implying that these genes may be appropriate candi-
dates for improving WUE.
The STOMATAL DENSITY AND DISTRIBUTION (SDD1)
locus encodes a subtilisin-like serine protease that is expressed
in stomatal precursor cells and regulates stomatal differenti-
ation and pattern formation (Berger and Altmann, 2000; von
Groll et al., 2002). SDD1 overexpression decreased stomatal
density by ∼40% due to repression of stomatal divisions and
arrest of meristemoids and guard mother cells (GMCs) (Büssis
et al., 2006). The sdd1-1 null mutation increased stomatal num-
ber 3-fold without the one-cell spacing rule (von Groll et al.,
2002). Taken together, these studies indicate that SDD1 is a
negative regulator of stomatal density. Leaf area or plant size
is unchanged in SDD1 overexpression lines. Stomatal conduc-
tance in SDD1 overexpression lines was decreased only under
high light conditions, but transpiration was not reported. In-
terestingly, stomatal aperture in SDD1 overexpression lines in-
creased by 40–47%, suggesting that plants may compensate for
a reduction in stomatal density by increasing stomatal aperture
(Büssis et al., 2006).
EPIDERMAL PATTERNING FACTOR 1 (EPF1) encodes
a small secretory peptide that is expressed in stomata and
stomatal precursor cells (Hara et al., 2007). Overexpression
of EPF1 represses asymmetric cell division, decreasing stom-
atal density relative to the expression level of EPF1. High
expression of EPF1 resulted in a complete repression of stomatal
 TRANSPIRATION TO IMPROVE CROP WATER USE 423

TABLE 4
Genes regulating transpiration or transpiration-related phenotypes.
Gene Protein Modification Phenotype Ref
Stomatal opening – photoreceptor/signal transduction/channel
PHOT1 serine/threonine kinase double mutation ↓E, SA (1)
PHOT2
CRY1 flavoprotein double mutation ↓WL, SA DT (2)
CRY2
MYB60 R2R3 MYB TF null mutation ↓WL, SA DT (4)
MYB61 R2R3 MYB TF overexpression ↓gs , SA (5)
ROP2 Small G protein constitutive expression ↓SA (6)
KAT1 inward-rectifying K+ channel dominant negative ↓WL, SA (3)
Stomatal closure – ABA signaling/Ca2+ signaling
ABF3 ABRE-binding bZip TF overexpression ↓WL (7)
ABH1/CBP80 Cap-binding protein null mutation ↓gs , SA (8)
ABI1 Protein phosphatase 2C null mutation ↓WL, SA DT (9)
CBP20 Cap-binding protein null mutation ↓gs DT (10)
ABO1/ELO2 a subunit of holo-Elongator null mutation ↓WL DT, ↓ SD (11)
Downloaded By: [Purdue University] At: 14:51 19 January 2010

HAB1 Protein phosphatase 2C null mutation ↓WL, SA DT (9)

(12)
MYB44 R2R3 MYB TF overexpression ↓WL, SA DT (13)
RGS1 Regulator of G-protein signaling overexpression ↓WL, SA DT (14)
SDIR1 RING finger E3 ligase overexpression ↓WL, SA DT (15)
SNAC1 NAC TF overexpression ↓E, SA (16)
CBL1 CBL9 Calcineurin-B like protein double mutation ↓WL, SA DT (19)
CIPK23 CBL-interacting protein kinase null mutation ↓WL, SA DT (19)
CPK23 Calcium-dependent protein kinase null mutation ↓SA DT (20)
Stomatal regulation – ABA catabolism/biosynthesis
CYP707A3 Cytochrome P450 monooxygenase overexpression ↓E DT (17)
NCED3 9-cis-epoxycarotenoid deoxygenase overexpression ↓E (18)
Abbreviations: TF, transcription factor; gs , stomatal conductance; WL, water loss; SA, stomatal aperture; DT, drought tolerance; SD, stomatal
density.
(1) Kinoshita et al., 2001; (2) Mao et al., 2005; (3) Cominelli et al., 2005; (4) Liang et al., 2005; (5) Jeon et al., 2008; (6) Kwak et al., 2001;
(7) Kang et al., 2002; (8) Hugouvieux et al., 2002; (9) Saez et al., 2006; (10) Papp et al., 2004; (11) Chen et al., 2006a; (12) Saez et al., 2004;
(13) Jung et al., 2008; (14) Chen et al., 2006b; (15) Zhang et al., 2007b; (16) Hu et al., 2006; (17) Umezawa et al., 2006; (18) Iuchi et al., 2001;
(19) Cheong et al., 2007; (20) Ma and Wu, 2007.

development changes, but did result in severe growth defects,
while mild expression of EPF1 resulted in a 10–20% decrease
in stomatal density without any morphological defects (Hara et
al., 2007), again suggesting that a slight decrease in stomatal
density may improve WUE by sustaining net CO2 assimilation
and decreasing transpiration (Figure 2; A → B).
The α subunit of GTP-binding protein (GPA1) in Arabidopsis
modulates epidermal cell proliferation by regulating cell divi-
sion. A null mutation in AtGPA1 (gpa1) resulted in reduced cell
division but increased cell expansion, leading to similar or even
greater leaf size compared to the wild type (Ullah et al., 2001).
Stomatal density and index and water loss were all reduced in
gpa1 mutant lines (Zhang et al., 2008a). These reports suggest
that GPA1 is another target to decrease stomatal density and
improve WUE.
2. Stomatal Aperture Determinants
Forward and reverse genetic approaches have led to the iden-
tification of numerous determinants involved in the regulation
of stomatal opening and closing (Chaerle et al., 2005; Nilson
and Assmann, 2007; Shimazaki et al., 2007; Sirichandra et al.,
2009). Table 4 summarizes the modification of genes regulating
stomatal opening through mechanisms including photorecep-
tion, signal transduction pathways, activation of channels, and
stomatal closing through mechanisms including ABA signaling,
Ca2+ signaling, and ABA catabolism/biosynthesis.
PHOTOTROPIN 1 and 2 (PHOT1 and PHOT2) are ser-
ine/threonine protein kinases that function as blue-light recep-
tors for stomatal opening (Kinoshita et al., 2001). In the double
mutant phot1 phot2, stomata do not open in response to blue
light, which results in reduced transpiration (Kinoshita et al.,
 424 C. Y. YOO ET AL.
2001). Since phot1 phot2 grew similarly to wild type plants
under white light (70 µmol m−2 s−1 ) (Takemiya et al., 2005),
phot1 phot2 may improve WUE due to reduced transpiration by
inhibiting stomatal opening. CRYPTOCHROME 1 and 2 (CRY1
and CRY2) are also blue-light receptors, both of which encode
flavoproteins that are homologous to microbial DNA photolyase
(Ahmad and Cashmore, 1993; Lin et al., 1995; Mao et al., 2005).
Mutations in CRY1 and CRY2 exhibited reduced water loss in
detached leaves and reduced stomatal aperture in response to
blue light. The double mutation cry1 cry2 exhibited even lower
water loss and decreased stomatal aperture over either of the
single mutations (Mao et al., 2005). Any growth defect of cry1,
cry2, or cry1 cry2 was not reported. The existence of multiple
photoreceptors makes it feasible to control marginal reductions
in transpiration, which would allow for a similar net CO2 as-
similation and thus, an improvement in WUE.
AtMYB60 and AtMYB61 are members of the R2R3 MYB
transcription factor family in Arabidopsis. A T-DNA insertional
mutation in AtMYB60 (myb60) results in reduced stomatal aper-
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ture that is not due to increased ABA sensitivity. The myb60 mu-
tant showed reduced water loss in excised leaves and increased
drought tolerance (Cominelli et al., 2005). Morphological or de-
velopmental abnormalities were not observed. Overexpression
of AtMYB61 resulted in reduced stomatal aperture and stom-
atal conductance, independent of ABA signaling (Liang et al.,
2005). Reduced plant size or any observed growth defects were
not reported in these overexpression lines. AtROP2 encodes a
small G protein that functions as a negative regulator of light-
induced stomatal opening, potentially to prevent excessive water
loss (Jeon et al., 2008). Constitutive activation of ROP2 causes
a reduction in stomatal aperture, suggesting that transpiration
might be reduced by the activation of ROP2.
Repressing K+ in channels can decrease stomatal aperture by
depressing K+ accumulation necessary for stomatal opening.
A dominant negative mutation in KAT1 in Arabidopsis reduced
K+in current magnitudes, which inhibited stomatal opening and
transpirational water loss in detached leaves (Kwak et al., 2001).
Stomatal opening is not completely blocked in kat1 due to re-
dundant functional K+ in channels (Szyroki et al., 2001), which
may explain the similar plant size of kat1 to wild type. There-
fore, WUE is likely increased in kat1.
Since ABA is a key hormone that induces stomatal closure,
increased sensitivity to ABA signaling results in reduced tran-
spiration. ABA induces the expression of many genes having the
ABA-responsive element (ABRE) in their promoters. Transcrip-
tion factors that bind to ABREs have been identified in order
to understand the transcriptional network of ABA signaling.
ABRE-binding factor 3 (ABF3) encodes a bZIP transcription
factor and functions as a positive regulator of ABA signaling
(Kang et al., 2002). Overexpression of ABF3 caused hypersen-
sitive response to ABA treatment, which resulted in reduced
detached leaf water loss and drought tolerance. Mild growth
retardation was observed in transgenic plants, indicating that
the overexpression of ABF3 may reduce stomatal aperture to an
extent that limits CO2 uptake as well as transpiration. However,
overexpression of another ABRE-binding factor (ABF4) causes
severe growth retardation with ABA hypersensitivity (Kang et
al., 2002); thus, manipulation of ABF4 is likely not suitable for
improving WUE. ABA hypersensitive 1 (ABH1) and cap-binding
protein 20 (CBP20) encode subunits of the nuclear mRNA cap-
binding complex. Null mutations in ABH1 (Hugouvieux et al.,
2002) and CBP20 (Papp et al., 2004) resulted in ABA hyper-
sensitivity, leading to reduced stomatal conductance. A simi-
lar phenotype was observed in a null mutation in ABO1/ELO2
that encodes a subunit of holo-Elongator (Chen et al., 2006a).
Similarly, overexpression of RGS1 that encodes a regulator of
G-protein signaling (Chen et al., 2006b), MYB44 that encodes
a R2R3 MYB transcription factor (Jung et al., 2008), SNAC1
(Stress-responsive NAC 1) that encodes a member of the NAC
(NAM/ATAF/CUC) transcription factor family (Hu et al., 2006),
and SDIR1 that encodes a RING finger E3 ligase (Zhang et al.,
2007b) all lead to reduced stomatal conductance. Induced stom-
atal closure by enhancing ABA signaling is a useful modification
to reduce water loss and increase drought tolerance. However,
the application of these genes to improve WUE should be care-
fully examined due to the possibility of growth inhibition.
ABI1 (ABA-insensitive 1) and HAB1 (Hypersensitive to
ABA1) encode protein phosphatases 2C that function as neg-
ative regulators of ABA signaling (Gosti et al., 1999; Saez et
al., 2004). Null mutations in ABI1 (abi1-2 and abi1-3) or in
AtHAB1 (hab1-1) resulted in reduced water loss during water-
deficit stress. This phenotype was additive in the double mutants
hab1-1 abi1-2 and hab1-1 abi1-3 (Saez et al., 2006). These sin-
gle and double mutant plants were hypersensitive to ABA for
stomatal closure (Saez et al., 2006). These results suggest that
ABA signaling is finely controlled by a combination of many
genetic components.
Disruption of ABA catabolism by AtCYP707A3 (Umezawa
et al., 2006) or overexpression of ABA biosynthesis by NCED3
(Iuchi et al., 2001) resulted in ABA accumulation, which in-
duced stomatal closure, thereby reducing transpiration. How-
ever, shoot growth is also affected by ABA levels (Saab et al.,
1990). It is possible that the regulation of constitutive ABA ac-
cumulation to moderate levels would result in the reduction of
stomatal closure where carbon assimilation is less affected than
transpiration, leading to an increase in WUE. Increasing ABA
biosynthesis or reducing ABA catabolism with a water deficit
conditional promoter might also be a plausible alternative for
improving WUE with minimal impact on yield stability.
Ca2+ signaling mediates stomatal closure during drought
stress adaptation processes (Himmelbach et al., 2003). ABA in-
duces transient changes in cellular Ca2+ concentrations, which
are sufficient to initiate ABA responses, including stomatal clo-
sure (Luan, 2002). Calcineurin B-like proteins (CBL) are Ca2+
binding proteins that signal the CBL-interacting protein kinase
(CIPK) family (Luan, 2009). Double mutations in CBL1 and
 TRANSPIRATION TO IMPROVE CROP WATER USE
CBL9 caused additive effects on reduced water loss, stomatal
aperture, and drought tolerance (Cheong et al., 2007). A null
mutation in CIPK23, which functions downstream of CBL1 and
CBL9, also resulted in reduced water loss and stomatal aperture
(Cheong et al., 2007). A mutation in the calcium-dependent pro-
tein kinase 23 (CPK23) also exhibited a reduction in stomatal
aperture and improved drought tolerance (Ma and Wu, 2007).
Although biomass of these transgenic plants was not reported,
their morphological phenotypes based on photos are very sim-
ilar to the wild type, suggesting that genes involved in Ca2+
signaling may contribute to the regulation of WUE in plants.
3. Nighttime Transpiration Determinants
A significant genetic correlation between nighttime and
daytime stomatal conductance was shown in 12 Arabidopsis
thaliana accessions (Christman et al., 2008). However, there is
often only a weak correlation between daytime and nighttime
water loss in a large number of species (Mickelbart, unpub-
lished). Stomatal regulation at night does not have a direct
Downloaded By: [Purdue University] At: 14:51 19 January 2010
effect on daytime photosynthesis. This assumption is based
on observed daytime and nighttime stomatal conductance
in Arabidopsis near isogenic lines (NILs) and their parental
lines (Christman et al., 2008), and differential responses of
daytime and nighttime conductance to environmental stress
such as salinity (Christman et al., 2009b). Genetic variation for
nighttime water loss is known to exist among species (Caird
et al., 2007a, b; Snyder et al., 2003; Bucci et al., 2004; Daley
and Philips, 2006), and among genotypes within a species
(Christman et al., 2008; Caird et al., 2007b; Mickelbart,
unpublished). The only report of a specific gene related to
nighttime water loss is FRIGIDA (FRI), which is an activator
of the central floral repressor FLOWERING LOCUS C (FLC)
(Michaels and Amasino, 1999). The FRI-Col line with an active
FRI allele showed lower nighttime stomatal conductance than
the Col-0 Arabidopsis ecotype, but similar daytime stomatal
conductance (Christman et al., 2008). WUE of these genotypes
was not quantified. Despite growing evidence for genetic pro-
cesses unique to nighttime transpiration, common regulatory
mechanisms for daytime and nighttime transpiration cannot be
excluded (Caird et al., 2007a; Ludwig et al., 2006). Because the
physiological mechanisms that control nighttime transpiration
are still unclear, Identification of the genetic determinants for
nighttime leaf conductance may increase our understanding of
the relationship between nighttime transpiration and WUE.
IV. CONCLUSION
Analyses of natural variation, mutations, and transformed
plants in a number of species have led to a better understanding
of the physiological basis of WUE. We believe that efforts to
address global water shortages should focus on overall water-
saving strategies, not just on increasing drought tolerance in arid
regions, although increased WUE will likely result in increased
drought tolerance in certain situations. Natural variation in plant
425
WUE is often negatively correlated with transpiration, but not
with CO2 assimilation. This suggests that plants have physiolog-
ical mechanisms for improving WUE by reducing transpiration
that are under genetic control. Therefore, improving WUE can
be achieved by introgression of genetic determinants that regu-
late the physiological mechanisms affecting transpiration. The
major mechanisms that affect water loss include those anatom-
ical and physiological traits that control daytime and nighttime
transpiration, primarily through the stomata. While the cuticle
likely plays a role in reducing water loss from leaves, the signif-
icance of this water loss pathway is still unclear. Furthermore,
while leaf boundary layer resistance also affects the loss of wa-
ter from individual leaves, it is likely a minor factor in most
cropping situations.
There are other factors that may affect overall WUE that we
have not covered in this review, such as water absorption through
the epidermis, respiration, and root and leaf hydraulic conduc-
tance. Ultimately, any factor that affects the amount of carbon
sequestered as biomass or yield or the amount of water lost in
that process can affect WUE on a crop level, and all of these
physiological factors should be considered. We also recognize
that increases in individual leaf or even whole plant WUE will
not have large effects if flowering time is also considered when
breeding or engineering crops for improved water loss. How-
ever, our knowledge to date suggests that the primary means
of greatly affecting WUE may be through minor alterations of
transpiration, which will lead to large water savings over the
course of a season or cropping period.
ACKNOWLEDGMENTS
National Science Foundation awards MCB-0424850 and
IBN-0416773 supported research in the laboratory of PMH.
Purdue Agricultural Research Programs supported research in
the laboratory of MVM. We thank Robert Heath, Matthew
Jenks, Richard Meilan, Tom Sharkey, and Tom Sinclair for their
thoughtful reviews of this manuscript.
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