Download Role of aquaporins in leaf physiology

Journal of Experimental Botany, Vol. 60, No. 11, pp. 2971–2985, 2009
doi:10.1093/jxb/erp171 Advance Access publication 19 June, 2009
DARWIN REVIEW
Role of aquaporins in leaf physiology
Robert B. Heinen*, Qing Ye* and Francxois Chaumont†
Institut des Sciences de la Vie, Université catholique de Louvain, Croix du Sud 5-15, B-1348 Louvain-la-Neuve, Belgium
Received 2 February 2009; Revised 1 May 2009; Accepted 5 May 2009
Abstract
Playing a key role in plant growth and development, leaves need to be continuously supplied with water and carbon
dioxide to fulfil their photosynthetic function. On its way through the leaf from the xylem to the stomata, water can
either move through cell walls or pass from cell to cell to cross the different tissues. Although both pathways are
probably used to some degree, evidence is accumulating that living cells contribute substantially to the overall leaf
hydraulic conductance (Kleaf). Transcellular water flow is facilitated and regulated by water channels in the
membranes, named aquaporins (AQPs). This review addresses how AQP expression and activity effectively regulate
the leaf water balance in normal conditions and modify the cell membrane water permeability in response to
different environmental factors, such as irradiance, temperature, and water supply. The role of AQPs in leaf growth
and movement, and in CO2 transport is also discussed.
Key words: Aquaporin, carbon dioxide, growth, hydraulic conductance, leaf, water.
Introduction
Plant growth and development are dependent on essential
physiological processes taking place in the leaf, such as
photosynthesis and transpiration. To function properly, the
leaf needs to maintain a balanced water content. This is
especially important during evapotranspiration, during
which water is unavoidably lost at the leaf surface to allow
CO2 uptake. Before evaporation through the stomata, the
high amounts of water transported from the soil to the leaf
(long-distance transport) must cross several cell layers of
different tissues (radial water transport). As in the composite transport model in the root, water can take different
paths on its way through the leaf, moving through the
apoplast (cell walls) or from cell to cell (Steudle, 1994;
Steudle and Peterson, 1998). The latter route is composed of
the symplastic (through the plasmodesmata) and the transcellular (across cell membranes) paths. The contribution of
each pathway to the overall leaf hydraulic conductance
(Kleaf) is unclear. Due to its low resistance to water flux, the
apoplastic path is believed to be the main route during
transpiration (Sack and Holbrook, 2006). However, several
recent studies have attributed an important role to the cellto-cell path (Cochard et al., 2007; Kim and Steudle, 2007;
Ye et al., 2008). The presence of lignified or suberized cell
walls, which constitute apoplastic barriers, forces water to
cross cell membranes, as do the Casparian strips in the root
endodermis (Lersten, 1997; Fricke, 2002a; Hachez et al.,
2008). In addition to radial water flux, which is controlled
by evapotranspiration, water movement across the leaf cell
membranes is also important for water homeostasis, in
increasing cell volume, and in maintaining turgor during
expansion, in regulating the opening and closure of
stomata, and in controlling leaf movement.
Water movement through cell membranes is facilitated by
water channels, called aquaporins (AQPs). These proteins
belong to the major intrinsic protein (MIP) family, members of which are found in almost all living organisms (Agre
et al., 1998). AQPs have six membrane-spanning alpha
helices and cytoplasmic N- and C- termini. The cytosolic
* These two authors contributed equally to this work.
y
To whom correspondence should be addressed: E-mail: [email protected]
Abbreviations: AQP, aquaporin; CA, cold-acclimated; ER, endoplasmic reticulum; gs, stomatal conductance; gm, mesophyll conductance; gi, internal conductance;
Kleaf, leaf hydraulic conductance; Lp, cell hydraulic conductivity; NA, non-acclimated; NIP, Nodulin26-like intrinsic protein; MIP, major intrinsic protein; PCMBS,
p-chloromercuribenzenesulphonic acid; PIP, plasma membrane intrinsic protein; ROS, reactive oxygen species; SIP, small basic intrinsic protein; TIP, tonoplast
intrinsic protein; WT, wild type; XIP, X intrinsic protein.
ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2972 | Heinen et al.
loop (loop B) between the second and third transmembrane
domains and the extracellular loop (loop E) between the
fifth and sixth transmembrane domains form short helices
that are relatively hydrophobic and are inserted into the
membrane from opposite sides. These two loops contain
highly conserved Asn-Pro-Ala (NPA) motifs located in the
middle of the pore (Murata et al., 2000). While some AQPs
behave as ‘strict’ water channels, others can conduct a wide
range of non-polar solutes, such as urea or glycerol, and the
non-polar gas carbon dioxide, the polar gas ammonia, the
reactive oxygen species (ROS) hydrogen peroxide, and the
metalloids antimonite, arsenite, and silicon (Bienert et al.,
2008; Maurel et al., 2008). AQPs are particularly abundant
in higher plants. Based on DNA sequence similarities, they
are classified into five different subfamilies, namely PIPs
(plasma membrane intrinsic proteins), TIPs (tonoplast
intrinsic proteins), NIPs (nodulin26-like intrinsic proteins),
SIPs (small basic intrinsic proteins), and XIPs (X intrinsic
proteins) (Chaumont et al., 2001; Johanson et al., 2001;
Sakurai et al., 2005; Danielson and Johanson, 2008).
Although water transport across membrane pores had
been proposed previously (Dainty and Ginzburg, 1963;
House, 1974; Finkelstein, 1987), the discovery of the
molecular structure of AQPs and detailed studies of their
function revolutionized studies on plant water relations, at
least those involving membranes (Steudle and Henzler,
1995; Maurel, 1997; Kjellbom et al., 1999; Tyerman et al.,
1999; Steudle, 2001). Evidence is accumulating that AQPs
play an important role in plant hydraulic relations at the
cell, tissue, organ, and whole plant level. They facilitate the
rapid, passive exchange of water across cell membranes and
are responsible for up to 95% of the water permeability of
plasma membranes (Henzler and Steudle, 2004). In general,
there are three ways by which water exchange across cell
membranes is regulated by AQPs: (i) their level of
expression, i.e. AQP abundance; (ii) their trafficking, i.e.
after synthesis in the ER, AQPs have to move to different
cellular locations via the secretory pathway; and (iii)
channel gating, i.e. the open/closed status of AQPs.
Many recent reviews have summarized the impressive
amount of data on the molecular structure, function, and
regulation of plant AQPs (Chaumont et al., 2005; Hachez
et al., 2006a; Kaldenhoff and Fischer, 2006; Maurel, 2007;
Kaldenhoff et al., 2008; Maurel et al., 2008). In this work,
the focus is on plant leaf AQPs, as a number of recent
papers have highlighted the importance of AQPs in leaf
growth and function. Firstly, the expression data for leaf
AQPs from different plant species that provide clues about
their roles are summarized; secondly, the responses of leaf
AQPs to multiple environmental factors are described; and,
finally, the data on the involvement of AQPs in leaf growth,
leaf movement, and CO2 uptake are discussed.
Leaf AQP expression in normal conditions
In addition to gating and trafficking, expression is one of
the most important means of AQP regulation. As AQPs
constitute a large and highly divergent protein family in
plants (Chaumont et al., 2001; Johanson et al., 2001;
Sakurai et al., 2005; Danielson and Johanson, 2008), the
study of their expression level and localization is highly
relevant to a better understanding of their diverse physiological roles.
Organ-specific expression
Many studies comparing AQP expression in different plant
organs might suggest that, under normal conditions, AQPs
play a more important role in roots than leaves. Indeed,
many PIP and TIP homologues studied have been reported
to be expressed at lower levels in leaves than in roots
(Johansson et al., 1996; Biela et al., 1999; Chaumont et al.,
2000; Baiges et al., 2001; Smart et al., 2001; Zhang et al.,
2008). For instance, Zea mays ZmPIP1;1, ZmPIP1;2, and
ZmPIP2;5 transcript levels are higher in the roots compared
to the leaves (Chaumont et al., 2000). This was confirmed
using antibodies raised against amino-terminal peptides of
several ZmPIPs (Hachez et al., 2006a, 2008). The Western
blots showed higher amounts of ZmPIPs in roots than in
leaves of 11-d-old plantlets or the 6th leaf of adult plants
(Fig. 1). However, other studies have shown that some
AQPs are expressed at the same amount, more abundantly
or even exclusively in leaves compared to roots. A recent
characterization of tulip PIPs showed that TgPIP2;1 and
TgPIP2;2 are highly expressed in leaves, but only TgPIP2;2
transcripts are detected, at a low level, in roots (Azad et al.,
2008). In Arabidopsis thaliana, AtPIP2;6 transcript levels
measured by real-time PCR are higher in the aerial parts
than the roots, while the expression of the other PIPs is
either lower (AtPIP1;1, AtPIP2;2, AtPIP2;3) or not
significantly different (Jang et al., 2004). In rice (Oryza
sativa), the expression of most AQP genes quantified by
reverse transcription-PCR shows clear organ specificity. The
levels of mRNA for 14 AQP homologues are higher in leaf
blades than roots, while only six are expressed at higher
levels in roots than leaves (Sakurai et al., 2005, 2008). All
these data suggest that AQPs play important roles, not only
in root water relations but also in the physiology and
development of leaves. However, to understand their involvement in leaf water and solute flow better, detailed
knowledge of their expression at the cellular, rather than the
organ level is required.
Expression according to developmental stage
Various studies have shown that AQP expression in leaves
is developmentally regulated. While some are highly
expressed in young, elongating leaf tissues, others are
expressed preferentially in fully developed, matured leaves
(Sarda et al., 1997; Chaumont et al., 1998; Maestrini et al.,
2004; Wei et al., 2007; Hachez et al., 2008). For example, in
Z. mays leaves, the expression of all 13 ZmPIP genes except
ZmPIP2;7 can be detected by real-time RT-PCR, although
to different extents, and the expression of most isoforms
depends on the developmental stage, with expression being
Aquaporins in leaves | 2973
cell-to-cell pathway where regulation of AQPs could be of
great importance (Hachez et al., 2008). In addition, the high
expression of AQPs in the transition zone between elongating and matured maize leaf is thought to be due to the
development of the metaxylem (built-up of apoplastic
barriers by lignification/suberization) and increasing radial
water flow due to higher photosynthetic activity and
transpiration of the exposed leaf (Hachez et al., 2008).
Finally, the isoforms highly expressed in fully developed,
matured leaves probably play various roles in physiological
processes, such as phloem loading, xylem water exit,
stomatal aperture, gas transport (e.g. CO2), and leaf
movement.
Tissue-specific expression
Fig. 1. Maize plasma membrane AQPs are more abundant in
roots than in leaves. ZmPIP protein amounts in 11-d-old maize
root and shoot, and leaf 6 of adult plant. Microsomal membranes
were extracted and analysed by Western blotting using antibodies
against ZmPIP1 (general), ZmPIP1;2, ZmPIP2 (general), ZmPIP2;1/
2;2, ZmPIP2;5 and ZmPIP2;6 as described in Hachez et al. (2006a
and 2008). A colloidal blue stained SDS-PAGE gel was used as
a loading control. The position of the molecular mass markers is
indicated.
highest at the end of the elongation zone (Hachez et al.,
2008). In barley (Hordeum vulgare), HvPIP1;3 is highly
expressed in mature leaf tissue and HvPIP1;5 and
HvPIP1;6 in the elongation zone (Wei et al., 2007). These
developmental-specific expression patterns presumably reflect the physiological roles of the different isoforms.
Growth-related expression of some AQPs has been described in other organs, such as the hypocotyl or root, in
various species (Ludevid et al., 1992; Chaumont et al., 1998;
Higuchi et al., 1998; Eisenbarth and Weig, 2005). In
addition to cell wall yielding and solute supply, tissue
hydraulic conductance and, thus, AQP activity are thought
to be potential factors limiting cell expansion and growth
(Fricke, 2002a) (see also the section ‘AQPs and leaf
growth’). As the elongation zone of the barley and maize
leaf is always covered by the sheath of older leaves, cells of
this tissue are never exposed to a dry atmosphere. Thus
water movement within this tissue, which is mainly driven
by gradients of osmotic potential, could dominantly use the
To understand the role of individual AQP in leaves,
information on tissue-specific expression is essential. Translational fusion, in situ hybridization, and immunolocalization are the most common approaches presently used to
study the tissue-specific expression of a single isoform.
Table 1 presents an overview of the tissue localization of
AQPs in leaves of various plant species. The large variety of
sites at which they are found reflects the wide range of tasks
they have to perform. Indeed, AQP expression has been
described in all leaf tissues in which high transmembrane
water movement is required. However, since AQPs can
facilitate the movement of other solutes or gas, such as
CO2, their localization in specific cells might be related to
functions other than hydraulic control.
Plants have to face the dilemma of taking up CO2 while
unavoidably losing water vapour through their stomata.
Adapting to local or global changes, stomata are able to
open and close rapidly as a result of osmosis-driven water
movement between the guard cells and surrounding tissue
(Hetherington and Woodward, 2003; Roelfsema and
Hedrich, 2005). Guard cell-localized AQP expression has
been detected for A. thaliana PIP1;2, sunflower (Helianthus
annuus) TIP7 and TIP20, Nicotiana tabacum AQP1, Norway
spruce (Picea abies) TIP, spinach (Spinacia oleracea)
PIP1;1, broad bean (Vicia faba) PIP1, and maize ZmPIP1;2
and ZmPIP2;1/2;2 (Kaldenhoff et al., 1995; Sarda et al.,
1997; Oliviusson et al., 2001; Sun et al., 2001; Cui et al.,
2005; Fraysse et al., 2005; Hachez et al., 2008; Uehlein
et al., 2008). In maize, ZmPIP2;1/2;2 and ZmPIP1;2
expression is not only detected in guard cells, but also in
the adjacent subsidiary cells, a second cell type contributing
to the physiology of the stomatal aperture (Hachez et al.,
2008) (Fig. 2A, B). In sunflower, the increase in SunTIP7
transcript levels during stomatal closure suggests its involvement in this mechanism by increasing guard cell water
efflux (Sarda et al., 1997). Recently, immunological and
translational fusion approaches have shown that the CO2
diffusion-facilitating NtAQP1 is not only found in guard
and mesophyll cell plasma membranes, but also in the inner
chloroplast membranes, indicating that it plays a role in the
CO2 assimilation process (Uehlein et al., 2008) (see also the
section ‘Leaf AQPs and CO2 transport’). Detection of
2974 | Heinen et al.
Table 1. Aquaporin tissue localization in leaves of various plant species
Species
Isoform
Leaf localization
Techniquea
Reference
Arabidopsis thaliana
PIP1;2
PIP1
MOD (PIP1)
c-TIP
PIP1
PIP
TIP7
TIP20
PIP1;6
MipA (PIP1)
Guard cells, in/around vascular bundles
Plasmalemmasomes of mesophyll cells
Blade, hydatodes
Bundle sheath > mesophyll > vascular parenchyma
Bundle sheath > mesophyll > vascular parenchyma
Mesophyll
Guard cells
Guard cells
Epidermis and adjacent mesophyll
Vascular tissue > mesophyll
Phloem associated cells, mesophyll, minor veins
Companion cells, sieve elements, young xylem
vessels (low)
Seedlings: apical meristem (high), petiole and stem:
vascular tissues, glandular trichomes, subepidermal cells
Stem: cells of the central part of xylem vessels (high),
layer surrounding the central vascular structure
Young developping xylem vessels, cells between xylem
and phloem, phloem (low), mesophyll (low)
Developing xylem, xylem parenchyma, companion cells,
spongy parenchyma (especially around
stomatal cavities)
Plasma and inner chloroplast membrane of guard and
mesophyll cells
Mesophyll (high), vascular tissues (very low)
Mesophyll (high), bundle sheath and epidermis (low)
Mesophyll
Mesophyll, epidermis, parenchyma
Differentiating vascular tissues, guard cells,
bundle sheath
Trichomes, marginal meristem
Vascular bundles, periderm, subperiderm
Vascular bundles
Vascular bundles, periderm, subperiderm
Palisade and spongy parenchyma
Phloem sieve elements
Guard cells
Guard cells
Xylem parenchyma, companion cells, around phloem strands
Leaf primordia
Companion cells (high), xylem parenchyma (high), bundle
sheath, blade mesophyll (low in seedlings), midrib
parenchyma, guard cells, subsidiary cells
(only mature plants), epidermis (only mature plants),
apical meristem (low), leaf primordia
Companion cells (high), xylem parenchyma (high), bundle
sheath, blade mesophyll, midrib parenchyma, subsidiary
cells, guard cells, epidermis, apical meristem, leaf primordia
Companion cells (high), xylem parenchyma (high),
bundle sheath,
mesophyll, midrib parenchyma (low)
GUS, ISH, ISI
ISI
GUS
ISI
ISI
TPI
ISH
ISH
ISP
ISH
GUS (tobacco)
ISI
Kaldenhoff et al., 1995
Robinson et al., 1996
Dixit et al., 2001
Frangne et al., 2001
Frangne et al., 2001
Ohshima et al., 2001
Sarda et al., 1997
Sarda et al., 1997
Wei et al., 2007
Yamada et al., 1995
Yamada and Bohnert, 2000
Kirch et al., 2000
GUS (tobacco)
Yamada et al., 1997
ISI
Kirch et al., 2000
ISI
Kirch et al., 2000
ISI
Otto et al., 2000
ISI, GFP
Uehlein et al., 2008
ISI
ISI
ISH
ISH
ISH
Sakurai et al., 2008
Sakurai et al., 2008
Li et al., 2008
Montalvo-Hernandez et al., 2008
Oliviusson et al., 2001
GUS (tobacco)
TPI, ISI
TPI, ISI
TPI, ISI
ISI
ISI
ISI
ISH
ISH
ISH
ISI
Jones and Mullet, 1995
Suga et al., 2003
Suga et al., 2003
Suga et al., 2003
Karlsson et al., 2000
Fraysse et al., 2005
Fraysse et al., 2005
Sun et al., 2001, Cui et al., 2005
Barrieu et al., 1998
Chaumont et al., 1998
Hachez et al., 2008
ISI
Hachez et al., 2008
ISI
Hachez et al., 2008
Brassica campestris
Brassica napus
Graptopetalum paraguayense
Helianthus annuus
Hordeum vulgare
Mesembryanthemum
crystallinum
MipB
MipF (TIP)
Nicotiana tabacum
Oryza sativa
Phaseolus vulgaris (Carioca)
Picea abies
Pisum sativum
Raphanus sativus
Spinacia oleracea
Vicia faba
Zea mays
AQP1
PIP1
PIP2;1
PIP2;7
AQP1 (TIP)
TIP
Trg31 (PIP1)
PIP1
PIP2;1
TIP1
d-TIP
PIP1;2
PIP1;1
PIP1
TIP1
PIP1;2
PIP2;1/2;2
PIP2;5
a
Techniques: GFP, promoter::GFP fusion; GUS, promoter::GUS fusion; ISH, in situ hybridization; ISI, in situ immunolocalization; ISP, in situ
PCR; TPI, tissue print immunoblotting.
several AQPs in guard cells indicate that they contribute to
the regulation of their volume and turgor. However, the
way AQP activity is regulated by the environmental and
internal signals is still unknown.
Besides their important role in CO2 assimilation and
photosynthesis, mesophyll cells constitute an important
element in the pathway taken by water in the transpiration
stream. Whether water on its way to the substomatal cavity
Aquaporins in leaves | 2975
Fig. 2. Models of aquaporin-facilitated transmembrane water flow in Z. mays stomatal apparatus and mesophyll cells. (A) Schematic
view on a stoma during its opening process. Maize stomata are composed of two guard cells (GC, light green) surrounded by two
subsidiary cells (SC, light blue). During stomatal opening, solute accumulation in the guard cells decreases the osmotic potential. To reestablish the osmotic equilibrium, water follows the solutes into the guard cells inducing an increase in their volume and turgor pressure
which finally causes the pore aperture allowing gas/vapour exchanges. The water influx could be facilitated by a high expression and
activity of aquaporins in the guard cell plasma membrane but also in the surrounding subsidiary and epidermal (EP) cells. The latter cell
types could play an important role in supplying the water required by the guard cells. (B) Schematic view of a stoma during its closure.
During the closure, the decrease in water potential in the guard cells induces a rapid water efflux, which could also be facilitated by AQPs
in a similar way as during the opening process. (C) Schematic view of a cross-section through a maize leaf showing the radial water flow
during transpiration out of the bundle sheath towards the stomatal cavity. Water of the transpiration stream can follow either the
apoplastic or the cell-to-cell pathway. The transcellular component of the cell-to-cell path could be increased by the presence of AQPs.
Note that (i) AQP expression in mesophyll cells could also facilitate the CO2 uptake, which is not represented in this figure and (ii)
plasmodesmata, important for the symplastic pathway, have been omitted to avoid an overcharge of the figure. All these models are
deduced from immunocytochemistry data demonstrating the presence of ZmPIP proteins in the plasma membrane of these cells
(Hachez et al., 2008). BS, bundle sheath; EP, epidermis; GC, guard cell; MP, mesophyll; SC, subsidiary cell.
takes the apoplastic path or the cell-to-cell path is still
a matter of debate and probably differs according to the
species and conditions (Sack and Holbrook, 2006; Cochard
et al., 2007; Ye et al., 2008). An essential contribution of
AQPs to the latter pathway seems likely (Fig. 2C), given
their high expression in mesophyll cells of various plant
species (Yamada et al., 1995; Robinson et al., 1996;
Karlsson et al., 2000; Otto and Kaldenhoff, 2000; Yamada
and Bohnert, 2000; Frangne et al., 2001; Ohshima et al.,
2001; Hachez et al., 2008; Li et al., 2008; MontalvoHernandez et al., 2008; Sakurai et al., 2008; Uehlein et al.,
2008). It should be noted that, in many species, part of the
mesophyll volume is airspace with limited cell-to-cell
contact (Sack and Holbrook, 2006). Thus, it cannot be
excluded that AQPs in these cells may rather play a role in
the osmotic adjustment of the cell water relations in
response to changes of apoplastic water potential. NtAQP1
expression is particularly high in the mesophyll cells around
stomatal cavities, possibly facilitating gas exchange (CO2
and water) between the plant and the atmosphere (Otto and
Kaldenhoff, 2000; Uehlein et al., 2008). However, whether
water evaporates in the close vicinity of the substomatal
cavity or deeper in the mesophyll tissue is still not clear
(Boyer, 1985; Sack and Holbrook, 2006).
Before entering the mesophyll, water has to cross the
bundle sheath when flowing out of the vascular bundles.
Due to its highly compacted cells and their sometimes
lignified or suberized cell walls, the bundle sheath prevents
exposure of the vascular tissues to the air in the intercellular
spaces. In the case of the presence of apoplastic barriers, all
substances including water leaving or entering the vascular
bundle have to enter the symplast as in the root endodermis
(Canny, 1990; Hachez et al., 2008; Obrien and Kuo, 1975).
Water movement through these cell layers could therefore
be efficiently controlled and enhanced by AQPs (Fig. 3B).
This hypothesis is supported by the expression of several
AQPs in bundle sheath cells or cells surrounding the
vascular bundles, which has been reported for AtPIP1;2,
Brassica napus c-TIP and PIP1, common ice plant (Mesembryanthemum crystallinum) MipB, rice OsPIP1, PaTIP,
ZmPIP1;2, ZmPIP2;1/2;2, and ZmPIP2;5 (Kaldenhoff
et al., 1995; Kirch et al., 2000; Frangne et al., 2001;
Oliviusson et al., 2001; Hachez et al., 2008; Sakurai et al.,
2008). Once in these cells, the water stream can continue
2976 | Heinen et al.
Fig. 3. Models of aquaporin-facilitated transmembrane water flow in Z. mays bundle sheath, xylem parenchyma, and phloem
companion cells. (A) Cross-section through a midrib vascular bundle giving an overview of the location of the different sections shown in
(B), (C), and (D). (B) Schematic view of a cross-section showing the bundle sheath (BS, light yellow) and radial water efflux out of the
metaxylem (MX, blue). Before entering the mesophyll (MP), water has to cross the bundle sheath. The presence of apoplastic barriers
(lignified/suberized cell walls, dark yellow) similar to Casparian strips in the root endodermis forces the water to cross the plasma
membrane. Transmembrane water movement through the bundle sheath could therefore be efficiently controlled and enhanced by
AQPs. (C) Schematic view of a longitudinal section through the metaxylem (MX) showing a gas embolism (GE) in the xylem vessel called
cavitation. AQPs expressed in the xylem parenchyma (XP) could play an important role in the refilling process which is still not fully
understood. (D) Schematic view of a cross-section through a phloem sieve element and its associated companion cell showing the
potential implication of AQPs during apoplastic sugar loading. The C4 pathway of sucrose biosynthesis and phloem loading is
represented by purple arrows starting in mesophyll cells with the first CO2 fixation, followed by the Calvin cycle in the bundle sheath cells,
and finally leading to the sucrose synthesis (for a review, see Lunn and Furbank, 1999). On their way into the vascular bundle, the carbon
assimilates follow the symplastic path through the plasmodesmata. Since sieve elements have no symplastic contact with surrounding
cells except their associated companion cell (the few existing plasmodesmata are thought to be especially narrow or even sealed),
apoplastic phloem-loading species need sucrose transporters to charge the carbohydrates into the phloem against their concentration
gradient (Slewinski et al., 2009; Turgeon, 2006). The resulting decrease in the osmotic potential drives an equilibrating water influx. High
AQP expression in companion cells might increase the membrane water permeability needed during phloem loading. All these models
are deduced from immunocytochemistry data demonstrating the presence of ZmPIP proteins in the plasma membrane of these cells
(Hachez et al., 2008). BS, bundle sheath; CC, companion cell; GE, gas embolism; MP, mesophyll; MX, metaxylem; PP, phloem
parenchyma; SE, sieve element; XP, xylem parenchyma.
symplastically or transcellularly, but the contribution of
each pathway is unknown.
Interestingly, when AQP expression is detected in a species’ leaves, it is often present in tissues of the vascular
bundles (Table 1). For example, ZmTIP1, ZmPIP1;2,
ZmPIP2;1/2;2, ZmPIP2;5, and NtAQP1 are highly
expressed in xylem parenchyma (Barrieu et al., 1998; Otto
and Kaldenhoff, 2000; Hachez et al., 2008), suggesting their
involvement in water exit from the xylem vessels. Studies
also indicate a role of xylem parenchyma located AQPs in
repairing embolism (Holbrook and Zwieniecki, 1999;
Meinzer et al., 2001). The process involved in the refilling
of gas-filled vessels to maintain the transpiration stream
after cavitation is still unclear, but has been shown to be
faster than expected, suggesting the involvement of water
channels (Fig. 3C) (Meinzer et al., 2001). ZmTIP1,
ZmPIP1;2, ZmPIP2;1/2;2, ZmPIP2;5, and NtAQP1 have
also been shown to be highly abundant in phloem
companion cells and this might be related to phloem sap
loading (Fig. 3D). Moreover, McMipA and SoPIP1;2 are
expressed in phloem sieve elements (Kirch et al., 2000;
Fraysse et al., 2005). In species with apoplastic sugar
Aquaporins in leaves | 2977
loading, phloem sieve elements with their associated companion cells are symplastically isolated from the neighbouring tissues. Photoassimilates need to be taken from the
apoplast by a companion cell-localized sucrose-H+ symporter (Giaquinta, 1983; Rae et al., 2005; Slewinski et al.,
2009). The resulting decrease in the osmotic potential drives
an equilibrating water influx. High AQP expression in
companion cells might increase the membrane water
permeability needed during phloem loading, an idea
supported by inhibition of the process by exogenous
application of the sulphhydryl-modifying compound pchloromercuribenzenesulphonic acid (PCMBS) (Bel et al.,
1993). However, PCMBS is a non-specific inhibitor, affecting sucrose transporters, AQPs, and a wide range of other
proteins. Specific AQP inhibitors are highly needed to
further verify the role of AQPs in the above mentioned
processes.
AQP expression has also been detected in the epidermis/
periderm and trichomes (Jones and Mullet, 1995; Yamada
et al., 1997; Suga et al., 2003; Wei et al., 2007; Hachez et al.,
2008; Montalvo-Hernandez et al., 2008; Sakurai et al.,
2008). Finally, several AQPs are expressed in leaf primordial cells and fast dividing apical or marginal meristem cells
(Jones and Mullet, 1995; Yamada et al., 1997; Chaumont
et al., 1998; Hachez et al., 2008).
From all of these studies, it can be concluded that AQPs
are present in all kind of tissues. However, while some
isoforms are ubiquitously expressed under normal conditions, others are highly tissue-specific.
Effect of deregulation of plant AQP
expression on leaf hydraulic parameters
Altering the level of expression of a single AQP gene by
reverse genetics, such as over-expression, silencing, and/or
knockout, is an interesting strategy for elucidating the
physiological function of specific AQP genes. Alteration of
AQP expression by these approaches has been shown to
affect leaf cell water permeability and, in some cases, other
physiological parameters, such as water potential, water loss
rate, and stomatal conductance, indicating the importance
of AQP for leaf function. However it is difficult to
generalize phenotypes at tissue and plant levels as different
mechanisms are probably used by plants to compensate
lower water permeability (Hachez et al., 2006b). In Arabidopsis plants with reduced expression of PIP1s and/or
PIP2s, the hydraulic conductivity (Lp) of isolated leaf
protoplasts is markedly reduced (Kaldenhoff et al., 1998;
Martre et al., 2002). To compensate the reduced cell Lp,
plants increase their root mass by 2.5–5 times as compared
with wild type (WT) plants to ensure sufficient water supply
to the plant. Overexpression of lily AqpL1 (a PIP1 gene) in
tobacco greatly increases the osmotic water permeability of
leaf protoplasts and the water conductivity of leaf cells
(Ding et al., 2004). The transgenic plants consume more
water than WT plants, have a higher stomatal density in
their young leaves, and exhibit a higher stomatal aperture
induced by light. These data indicate that overexpression of
this AQP gene, by enhancing the cell-to-cell pathway of
water movement within the leaf, might act as an internal
factor to adapt the stomatal apparatus and increase the leaf
transpiration (Ding et al., 2004). Overexpression of B. napus
BnPIP1 in tobacco also results in a faster swelling of leaf
protoplasts in hypotonic medium (Yu et al., 2005). In the
case of AtPIP1;2 overexpression in tobacco plants, a significant increase in plant growth rate, leaf transpiration rate,
stomatal density, and photosynthetic efficiency is observed
under favourable growing conditions (Aharon et al., 2003).
In a drought-sensitive cultivar of rice, overexpression of
OsPIP1;3 under the control of a stress-inducible promoter
results in a higher leaf water potential and transpiration
rate, indicating that OsPIP1;3 may play a role in drought
resistance (Lian et al., 2004). Overexpression of barley
HvPIP2;1 in rice leads to faster water loss by the leaf which
may be due to an increased transpiration rate. Overexpressing lines also show an increased CO2 assimilation rate and
a faster growth rate, possibly resulting from an increased
internal CO2 conductance (see also the section ‘Leaf AQPs
and CO2 transport’). However, the plants are more sensitive
to water stress, a fact that may lead to the observed
anatomical adaptations of the leaves such as smaller
mesophyll cells and thicker leaf cell walls (Hanba et al.,
2004).
To be able to conclude about the function of a specific
AQP by modifying its expression in plant, it is important to
(i) characterize the transport specificity of the channel and
(ii) carefully analyse possible compensation mechanisms by
close homologues. For instance, expression of AQPs
isolated from cucumber and figleaf gourd in Arabidopsis
perturbs the expressions of endogenous AQPs, especially
when plants are subjected to water stresses (Jang et al.,
2007). It is therefore impossible to attribute the detected
phenotypes to the transgene or to the altered expression of
endogenous AQPs.
Responses of leaf AQPs to environmental
conditions
While many AQPs coexist in plants, most isoforms seem to
have their own specific characteristics which allow the plant
to develop and grow adequately in ever-changing external
conditions.
Water stress
Water deficit, initially sensed by the roots, can affect the
regulation of water movement in the shoots. A number of
studies have shown that water stresses, including drought
and salinity, can alter leaf AQP expression and activity. In
the aerial part of Arabidopsis plants, drought stress induced
with 250 mM mannitol significantly alters the expression of
most PIP genes (Jang et al., 2004). For example, drought
treatment rapidly decreases levels of PIP1;5, PIP2;2,
PIP2;3, and PIP2;6 transcripts to one-tenth of the levels in
2978 | Heinen et al.
normal condition. The expression of PIP1;1 initially
increases, while that of PIP1;2, PIP2;7, and PIP2;8 remains
constant during the first 12 h of mannitol treatment, then
gradually decreases. Using a longer term (up to 12 d), but
gradual, drought stress, Alexandersson et al. (2005) showed
that PIP transcripts in Arabidopsis leaves are generally
down-regulated, except those for AtPIP1;4 and AtPIP2;5,
which are up-regulated. The different responses of AQP
expression (up/down-regulation or no change) to water
stresses suggest that AQP isoforms can be divided into
different groups which contribute differently to water transport and regulation, with some being stress-responsive
(Hachez et al., 2006b). Different responses of AQPs to
water stress were found in upland (drought-resistant) and
lowland (drought-sensitive) rice. PIP proteins increase
markedly in the roots of both types, but only in upland rice
leaves. OsPIP1;2, OsPIP1;3, OsPIP2;1, and OsPIP2;5
mRNA levels in roots and OsPIP1;2 and OsPIP1;3 mRNA
levels in leaves are significantly up-regulated in upland rice,
but their expression is unchanged or down-regulated in
lowland rice (Lian et al., 2006), indicating that AQPs
present in the same species, but in different cultivars, can
respond differently to water stress depending on their
tolerance to water deficits. Expression of the V. faba
VfPIP1 gene in Arabidopsis improves drought resistance in
the mutant plants, probably by promoting stomatal closure
under drought conditions, thus highlighting an important
role of AQPs when plants are subject to water stress (Cui
et al., 2008). Using double antisense Arabidopsis plants with
reduced amounts of both PIP1 and PIP2 proteins, Martre
et al. (2002) found that the leaf hydraulic conductance was
similar in the mutants and control plants. However, upon
water stress application, the mutant plants recovered their
hydraulic conductance and transpiration rates less rapidly
than the control plants and had a significantly lower leaf
water potential after rewatering. These data led the authors
to conclude that PIPs play an important role in the recovery
of Arabidopsis from water-deficient conditions. In the
heavy-metal accumulator Brassica juncea, BjPIP1 expression is up-regulated in leaves subjected to drought, salt, or
low temperature, or exposed to heavy metals (Zhang et al.,
2008). When overexpressed in tobacco plants, BjPIP1
enhances drought and cadmium resistance by decreasing
the transpiration rate and stomatal conductance, suggesting
that it might increase abiotic stress resistance by maintaining a reasonable water status in tobacco leaves (Zhang et al.,
2008). However, overexpression of AQPs is not always
beneficial to the plants. For instance, tobacco plants overexpressing AtPIP1;2 wilt more rapidly than control plants
under drought stress (Aharon et al., 2003).
Freezing and cold stress
Perennial plants are able to survive tough winters through
a phenomenon called cold acclimation. Low non-freezing or
subfreezing temperatures can increase the freezing tolerance
of plants by 3–5 C. Northern blot analysis and comparison
of expressed sequence tags from non-acclimated (NA) and
cold-acclimated (CA) Rhododendron catawbiense leaf tissues
showed a 10-fold down-regulation of RcPIP2;1 in CA
tissues (Wei et al., 2006). Similarly, overexpression of
RcPIP2s and Panax ginseng PIP1 in transgenic Arabidopsis
plants compromised their freezing tolerance and cold
acclimation ability, which is presumably due to their
decreased capacity to resist freeze desiccation (Peng et al.,
2007, 2008). The authors further demonstrated that all 13
endogenous PIPs except PIP2;5 are down-regulated in
wild-type Arabidopsis plants during cold acclimation, confirming previous data concerning the impact of different
abiotic stresses on Arabidopsis PIP transcription in the
aerial parts of 2-week-old seedlings (Jang et al., 2004).
Similar down-regulation of most rice PIP genes was
recorded when seedlings were chilled to 7 C, and their
expression recovered on return to 28 C. During recovery,
significantly higher expression of OsPIP1;1, OsPIP2;1, and
OsPIP2;7 was seen in shoots of a chilling tolerant variety
than in those from a chilling sensitive one (Yu et al., 2006).
All these data suggest that down-regulation of PIP transcripts during cold acclimation helps to prevent freezeinduced cellular dehydration, leading to increased freezing
tolerance. Moreover, rapid rehydration of the leaf after
cold/freeze stress might be mediated by AQPs. However,
these mechanisms might be species-dependent as, in wheat
leaves, a large increase in PIP transcript levels was seen
after cold acclimation (from 22 C to 4 C) (Herman et al.,
2006).
Responses of leaf AQPs to irradiance
In many plant species, the Kleaf can be increased several fold
by high irradiance. The response of the Kleaf to light is
rather fast, as shown in Quercus rubra, in which it increases
after only 30 min of high irradiance (1000–1200 lmol m 2
s 1) (Tyree et al., 2005). The increase in the Kleaf probably
involves AQPs, as it is inhibited by mercury compounds,
which are well-known AQP inhibitors. The lightinduced increase in the Kleaf in the burr oak is reduced by
HgCl2 treatment, and this effect can be reversed using 2mercaptoethanol (Voicu et al., 2008). Similar results were
obtained in the sunflower, in which the increase in the Kleaf
by light is sensitive to mercury inhibition (Nardini et al.,
2005). Using a cell pressure probe, Kim and Steudle (2007)
found, for the first time at the cellular level, that the
membrane water permeability of individual parenchyma
cells in the midrib of maize leaves increases by a factor of
3 after 30 min of illumination at intensities of 100—
200 lmol m 2 s 1. Interestingly, the same authors subsequently showed that higher light intensities (800 and 1800
lmol m 2 s 1) dramatically decrease the water permeability
of these leaf cells, possibly due to oxidative gating of AQPs
induced by high light (Kim and Steudle, 2009). All these
studies suggest that the effect of light on leaf hydraulic
properties is related to the regulation of AQPs. This idea is
further supported by the results of an analysis of the
expression of two PIP2 isoforms (JrPIP2;1 and JrPIP2;2) in
walnut (Juglans regia) leaves which correlates with the leaf
Aquaporins in leaves | 2979
hydraulic conductance, i.e. the increased Kleaf in response to
light is associated with marked accumulation of PIP2
proteins, while both the Kleaf and levels of PIP2 decrease
when the light is turned off (Cochard et al., 2007). This
effect of light on leaf AQPs is reflected by a fluctuating
expression pattern that follows the day/night cycle. In maize
leaves, most of the ZmPIP genes are more highly expressed
during the first hours of the light period than at the end of
the day or at night (Hachez et al., 2008). This correlates
with changes in the membrane water permeability measured
using a cell pressure probe (C Hachez, W Fricke, F
Chaumont, unpublished data). A diurnal oscillation in the
expression of NtAQP1 was also found in tobacco leaf
petioles, in which high expression is seen in the morning
and low expression in the evening, and this is consistent
with the diurnal changes in the osmotic water permeability
of protoplasts isolated from the same part of the leaf
(Siefritz et al., 2004). Similar results were obtained in motor
cells of Samanean samam, in which expression of the AQP
homologue gene SsAQP2 shows a diurnal rhythm concomitant with the diurnal regulation of water movement in these
cells (Moshelion et al., 2002). The regulation of AQP
expression and activity by irradiance seems important for
leaf hydraulics. However, the mechanism(s) by which light
modulates the Kleaf via AQP regulation is not yet fully
understood, and more precise investigations on the relationships between light and leaf AQPs are needed.
Inhibition of AQPs by reactive oxygen species
Among the many internal or external factors that can affect
AQP activities, hydrogen peroxide (H2O2) has been shown
to be an effective inhibitor of leaf AQPs. In Tradescantia
fluminesis, H2O2 perfusion via the leaf petiole results in
a drop in the water permeability of epidermal cells
measured with a cell pressure probe (Ye et al., 2008).
Similarly, H2O2 treatment following Fe2+ pretreatment
decreases the water permeability of parenchyma cells in
maize leaves by a factor of 30 (Kim and Steudle, 2008). The
effect could be overcome using an antioxidant, suggesting
that the inhibition of AQP activity is probably due to an
oxidative gating by ROS, such as *OH radicals, which can
be produced in the Fenton reaction (Ye and Steudle, 2006).
However, on the basis of the lack of effect of H2O2 on the
activity of individual AQPs expressed in Xenopus oocytes,
Boursiac et al. (2008) proposed that ROS do not gate AQPs
through a direct oxidative mechanism, but rather act
through a cell signalling mechanism and showed that H2O2
induces internalization of plasma membrane AQPs in
unidentified vesicles. This process could therefore decrease
the cell membrane water permeability. In addition, recent
studies showed that, instead of being inhibited by H2O2,
several AQPs isoforms are able to facilitate H2O2 transport
across the tonoplast and plasma membranes (Bienert et al.,
2006, 2007; Dynowski et al., 2008). The exact means by
which H2O2 regulates the cell membrane permeability is still
under investigation, but could involve different mechanisms, including direct oxidative gating, signal transduction
leading to the internalization of AQP proteins, or modification of their phosphorylation status, a post-translational
modification thought to regulate their gating (Johansson
et al., 1998; Tornroth-Horsefield et al., 2006; Van Wilder
et al., 2008). It has to be noted that ROS like H2O2 and
*OH are very aggressive and reactive chemicals. It is
possible that the inhibition of cell water permeability was
also due to indirect effects on AQPs such as lipid
peroxidation, which needs to be verified in future experiments. Nevertheless, responses of AQPs to ROS may
represent an efficient way to adjust water relations and cope
with different types of stresses from which plants may
suffer.
AQPs and leaf growth
Like other plant organs, leaves grow in size through the
irreversible expansion of cells, a process that requires the
continuous uptake of water. As mentioned above, AQPs are
expressed in leaf elongation zones and it is therefore
tempting to believe that they are involved in the growth
process (Fricke and Chaumont, 2007). Evidence that AQPs
are involved in leaf growth comes from cell water permeability measured from different zones of the leaf. Using
a pressure probe, Volkov et al. (2007) found that the water
permeability of barley epidermal cells in the leaf growing
elongation zone is higher than that of cells in the emerged
non-growing leaf zone. Similar results were obtained using
an osmotic swelling assay with protoplasts of mesophyll
cells sampled from the same zones (growing versus nongrowing) (Volkov et al., 2007). Barley HvPIP1;6, a functional water channel, was found to be specifically expressed
in the leaf epidermis and its expression was the highest in
the elongation zone, where it accounted for 85% of the
expression of known barley PIP1s, suggesting that it could
facilitate growth-associated water uptake into these cells
(Wei et al., 2007). However, both salinity (100 mM NaCl)
and nutrient source reduction (by removing the leaves
supplying nutrients to the newly emerged leaf), which were
shown previously to reduce the leaf elongation rate of the
growing barley leaf (Fricke, 2002b; Fricke and Peters, 2002;
Fricke et al., 2006), increase the expression of HvPIP1;6.
These authors proposed that, if the increase in HvPIP1;6
water channel activity assisted leaf growth under
stress conditions, it should occur primarily through posttranslational modifications, such as phosphorylation. Direct
evidence for the important role of AQPs in leaf growth was
provided by Uehlein et al. (2003). In tobacco, NtAQP1
overexpression increases leaf growth rate, an effect that can
be attributed to NtAQP1 acting as both a water channel
and a CO2 transporter (see also the section ‘Leaf AQPs and
CO2 transport’). The process of leaf cell expansion is
a complex process involving multiple cellular activities,
amongst which modification of the membrane permeability
through AQP regulation is probably one important factor.
It would be interesting to measure growth rate of plants
with altered expression of single AQP isoforms which were
2980 | Heinen et al.
shown to be active in cell membrane water transport, and in
parallel to determine hydraulic properties of these transgenic plants.
AQPs and leaf movement
It is known that the diurnal and circadian folding and
unfolding of tobacco leaves are determined by different
growth rates of the upper and lower leaf surfaces. Siefritz
et al. (2004) showed that NtAQP1 plays an essential role in
leaf movements in tobacco: (i) immunodetection experiments demonstrated that the peaks of the oscillating
expression pattern of NtAQP1 in leaf petioles coincide with
leaf unfolding; (ii) using an osmotic swelling assay on
protoplasts isolated from leaf petioles, it was shown that
the cell membrane water permeability is high during the
unfolding process (in the morning), and low in the evening;
and (iii) mutated tobacco plants with down-regulated
NtAQP1 expression show substantially lower cell water
permeability than the control plants, which eventually leads
to reduced leaf movement. In contrast to the irreversible
process of leaf growth controlled by cell elongation, leaf
movement can also be mediated by turgor-regulated reversible cell volume changes of motor organs called pulvini,
which were described by Darwin in one of his publications
more than a century ago (Darwin, 1880). This special organ
can co-ordinate rapid water exchange across the cell
membrane and induce swelling and shrinking of cells
located on opposite sides of the tissue, resulting in the
movement of leaves and leaflets. As mentioned previously,
the water permeability of S. saman protoplasts isolated
from pulvini motor cells was found to be regulated
diurnally (Moshelion et al., 2002). Functional analyses in
Xenopus oocytes showed that a PIP homologue gene
(SsAQP2) isolated from these cells encodes an efficient
water conducting channel (Moshelion et al., 2002). Interestingly, the SsAQP2 expression pattern shows diurnal
and circadian oscillation, with the highest mRNA levels in
the morning, probably to meet the requirement for high
osmotic water fluxes across the cell membrane when the
pulvini open. The contribution of the tonoplast AQP c-TIP
to the fast movements of leaves and leaflets in Mimosa
pudica was investigated (Fleurat-Lessard et al., 1997).
Immunodetection experiments showed that this AQP is
almost exclusively present in the large central vacuoles. It
was proposed that c-TIP probably facilitates fast water
efflux from motor cells, leading to simultaneous turgor loss
and leaf movement. Another study revealed that the
interaction of two plasma membrane AQPs (MpPIP1;1 and
MpPIP2;1) from Mimosa pudica increases the oocyte
membrane water permeability (Temmei et al., 2005). It is
very likely that the interaction of these two proteins affects
their trafficking, as demonstrated in maize (Zelazny et al.,
2007). Furthermore, phosphorylation of a serine residue in
MpPIP1;1 leads to an increase in the co-operative effect of
the two isoforms on water channel activity (Temmei et al.,
2005). The role of these mechanisms in controlling the cell
turgor pressure of motor cells remains to be demonstrated.
Another example of leaf movement is the active trapping
closure of the Venus flytrap (Dionaea muscipula) to capture
insects. It is plausible that trap closure involves rapid water
fluxes in the leaf cells which are driven by the cell osmotic
pressure and are actively regulated by the plant (Brown,
1916; Forterre et al., 2005). Interestingly, AQP inhibitors
decrease the speed of trap closure (Volkov et al., 2008). All
these data indicate that AQPs represent important components involved in leaf movement in different species. The
elucidation of the rapid signalling events and the exact
molecular mechanisms leading to the regulation of water
channel activity and cell turgor modification will be the next
challenge.
Leaf AQPs and CO2 transport
CO2 assimilation is one of the most important physiological
functions of the leaf and is indispensable for the survival
and development of the plant. In plants, gas exchange
between the atmosphere and the site of carboxylation (CO2)
or the site of evaporation (H2O) has often been described by
Ohm’s law of serial resistances (Flexas et al., 2008; Sack and
Holbrook, 2006). After crossing the boundary layer and the
leaf surface via the stomata, atmospheric CO2 diffuses
through the substomatal cavity and intercellular spaces.
The cell wall, plasma membrane, and cytoplasm of the
mesophyll cells constitute the next barriers on its way to the
assimilation site in the chloroplasts. Diffusion through
stomata is known as stomatal conductance (gs), while that
between the internal cavities and the carboxylation site is
called mesophyll conductance (gm) or internal conductance
(gi) (for a review, see Flexas et al., 2008; Warren, 2008). Not
only the gs, but also the gi can be sufficiently small to limit
photosynthesis (Harley et al., 1992; Loreto et al., 1992;
Bernacchi et al., 2002). Relative limitation by gi can account
for up to 40% and be similar to that due to stomatal
conductance (Warren, 2008). Recent research has indicated
that AQPs could explain the rapid adaptation of the gi to
changes in environmental conditions. Using the non-specific
AQP blocker HgCl2, Terashima and Ono (2002) observed
a significant decrease in the gi in V. faba and Phaseolus
vulgaris leaves. Heterologous expression of human AQP1 in
Xenopus oocytes results in a significant increase in the CO2
permeability of the oocyte membrane (Nakhoul et al.,
1998). A few years later, tobacco NtAQP1 was shown to
increase the CO2 permeability of oocyte membranes in
a similar way (Uehlein et al., 2003). To monitor cellular
CO2 transport, 14C incorporation in homologous plant
systems was investigated using NtAQP1 silenced or overexpressing tobacco lines. While a lower NtAQP1 level
resulted in reduced incorporation, increased NtAQP1 expression led to higher incorporation rates compared with
controls. Moreover, net photosynthesis was reduced in the
antisense lines and increased in the overexpressing lines
(Uehlein et al., 2003). These results suggest that NtAQP1
also acts as a CO2 membrane-transport-facilitating protein
Aquaporins in leaves | 2981
in planta. The first evidence for a direct link between AQPs
and the gi was provided by Hanba et al. (2004). Overexpression of barley HvPIP2;1 in transgenic rice plants
increases the gi in leaves by 40% compared to wild-type
plants. However, the increase in internal CO2 conductance
is accompanied by anatomical (stomatal density, mesophyll
porosity, mesophyll cell wall thickness, etc) and physiological (Rubisco content) differences, which are known to have
an impact on CO2 assimilation and internal conductance
(Hanba et al., 2004). Such anatomical and physiological
alterations were not induced in NtAQP1 antisense or
overexpressing tobacco lines, in which differences in the gi
were again linked to AQP expression levels (Flexas et al.,
2007). In addition to being present in the plasma membrane, NtAQP1 was detected in the chloroplast inner
membrane by immunological and green fluorescent proteinfusion approaches (Uehlein et al., 2008). Moreover, the CO2
permeability, analysed by stopped flow spectrophotometry,
was significantly reduced in the chloroplast membrane
vesicles of the NtAQP1 silenced plants compared to the
wild type. All those studies suggest a direct involvement of
AQPs in the internal CO2 conductance gi via modulation of
the CO2 permeability of the cell membranes. However,
while these data obtained in plants seem very convincing,
the physiological relevance of AQP-facilitated transmembrane CO2 diffusion in mammalian cells remains a matter
of debate and the assumption that the biomembrane is an
effective barrier to CO2 diffusion has been called into question (Verkman, 2002). Recently, the influence of unstirred
layers was investigated by microelectrode measurements on
reconstituted planar lipid bilayers of different compositions
(Missner et al., 2008). The results showed that transmembrane CO2 permeability is not limited by the membrane
itself, but by unstirred layers.
highly dynamic, regulated by environmental cues and
affected by leaf developmental stages or anatomy (Sack
and Holbrook, 2006). Methodology will need to be developed to quantify and compare the contribution of AQPs
in the leaf water conductivity in various situations: for
example, within a species exposed to different irradiance or
drought conditions, in different plant species differing in
anatomy (venation and mesophyll). Plant lines deregulated
in AQP gene expression in specific leaf tissues or cells could
be very useful to this purpose, but careful characterization
of the compensation mechanisms including anatomical
modifications or alteration of endogenous AQP gene
expression will be required. Other physiological processes
that have to be explored are leaf elongation, embolism
recovery, and stomatal regulation. While the signal transduction pathway and mechanisms governing stomatal
aperture and closure are well documented, the exact role
and regulation of AQPs in these processes are still missing.
Finally, the cellular mechanisms regulating AQP trafficking
and gating and, therefore, their activity will need to be
taken into account in these studies to get an overall picture
of the role AQPs play in leaf physiology.
Acknowledgements
This work was supported by grants from the Belgian
National Fund for Scientific Research (FNRS), the Interuniversity Attraction Poles Programme—Belgian Science
Policy, and the ‘Communauté francxaise de Belgique—
Actions de Recherches Concertées’. QY was supported by
an individual Marie Curie European fellowship. RH is a
Research Fellow at the FNRS.
References
Conclusions
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evapotranspiration was only obtained recently. mRNA and
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important role of AQPs in different processes. Biophysical
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