Journal of Experimental Botany, Vol. 62, No. 7, pp. 2233–2250, 2011
doi:10.1093/jxb/err111
REVIEW PAPER
Calcium delivery and storage in plant leaves: exploring the
link with water flow
Matthew Gilliham, Maclin Dayod, Bradleigh J. Hocking, Bo Xu, Simon J. Conn, Brent N. Kaiser, Roger A. Leigh
and Stephen D. Tyerman*
Waite Research Institute, School of Agriculture, Food and Wine, University of Adelaide, PMB1, Glen Osmond, SA, 5064, Australia
* To whom correspondence should be addressed. E-mail: [email protected]
Received 9 December 2010; Revised 17 March 2011; Accepted 21 March 2011
Abstract
Calcium (Ca) is a unique macronutrient with diverse but fundamental physiological roles in plant structure and
signalling. In the majority of crops the largest proportion of long-distance calcium ion (Ca2+) transport through plant
tissues has been demonstrated to follow apoplastic pathways, although this paradigm is being increasingly
challenged. Similarly, under certain conditions, apoplastic pathways can dominate the proportion of water flow
through plants. Therefore, tissue Ca supply is often found to be tightly linked to transpiration. Once Ca is deposited
in vacuoles it is rarely redistributed, which results in highly transpiring organs amassing large concentrations of Ca
([Ca]). Meanwhile, the nutritional flow of Ca2+ must be regulated so it does not interfere with signalling events.
However, water flow through plants is itself regulated by Ca2+, both in the apoplast via effects on cell wall structure
and stomatal aperture, and within the symplast via Ca2+-mediated gating of aquaporins which regulates flow across
membranes. In this review, an integrated model of water and Ca2+ movement through plants is developed and how
this affects [Ca] distribution and water flow within tissues is discussed, with particular emphasis on the role of
aquaporins.
Key words: Aquaporins, calcium, calcium storage, leaf hydraulic conductance, leaf water flow, transpiration.
Introduction
Calcium (Ca) is an essential plant macronutrient with key
structural and signalling roles. Calcium ions (Ca2+) act as:
an osmoticum within vacuoles; a stabilizing element of
membranes; a strengthening agent in cell walls; and
a secondary messenger for a multitude of signals (White
and Broadley, 2003; McAinsh and Pittman, 2009; Dodd
et al., 2010). Within leaves, the current paradigm predicts
that Ca2+ moves via extracellular pathways and is separated
from water when water enters cells (Canny, 1993; Storey
and Leigh, 2004). It is known that aquaporins (AQPs)
facilitate the flow of water from the apoplast (extracellular
space) into the symplast (intracellular compartment) and
vice versa (Heinen et al., 2009). Furthermore, the cytosolic
Ca concentration ([Ca2+]cyt) has been implicated in regulat-
ing water flow through AQPs, although this has not yet
been related to any given physiological process (Gerbeau
et al., 2002; Alleva et al., 2006). However, the interactions
between Ca, the pathways of water flow, and AQP function
have not been considered together in detail. The importance
of investigating these links is particularly relevant as genetic
manipulation of water-use efficiency (WUE) and Ca biofortification are both active fields of research (Flexas et al.,
2009; Karley and White, 2009) and manipulation of either
entity in isolation may have a negative impact on the other
and eventually on the overall productivity of targeted food
crops (Dayod et al., 2010). For instance, regulation of water
flow by nitrate has been studied and has revealed a regulatory mechanism via gating of AQPs that may increase
Abbreviations: AQP, aquaporin; B, boron; Ca, calcium; Ca2+, calcium ion; CEC, cation exchange capacity; DFS, Donnan free space; ECM, extracellular matrix; ER,
endoplasmic reticulum; HGA, homogalacturonan; HPTS, 8-hydroxypyrene-1,3,6-trisulphonic acid, trisodium salt; PME, pectin methylesterase; RG,
rhamnoglacturonan-I; ROS, reactive oxygen species; UL, unstirred layer; WFS, water free space.
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2234 | Gilliham et al.
nitrate uptake by roots (Gorska et al., 2008a, b). These
findings have important implications for WUE since when
nitrogen is limiting for photosynthesis it may be advantageous to increase water use in order to increase nitrate
uptake (Cramer et al., 2009).
Although Ca2+ is considered a ubiquitous signalling
element it is not well appreciated that the storage of Ca
across plant tissues and cell types is heterogenous (Leigh
and Tomos, 1991; Karley et al., 2000a, b; Conn and
Gilliham, 2010). Recent reviews have focused on how
discrete expression patterns of Ca2+ transporters may
influence Ca2+ storage in plants (Conn and Gilliham, 2010;
Dayod et al., 2010). In these reviews it was acknowledged
that transpiration and water movement through plants can
influence Ca2+ storage, but the extent of the interdependency between water and Ca2+ transport was not fully
detailed. Therefore, in the present review the focus is on the
role of long-distance Ca2+ transport and water movement
as controlling factors for Ca2+ distribution in plants, and
the consequences this has for regulation of apoplastic
[Ca2+], water flow, and leaf function. For recent reviews
dealing exclusively with water transport in leaves, see Sack
and Holbrook (2006), Brodribb et al. (2010), and Heinen
et al. (2009). As most plant Ca is stored in leaves and root
to shoot transfer of Ca2+ has been elaborated at length
elsewhere (Clarkson, 1984; White, 2001; White and Broadley,
2003; Cholewa and Peterson, 2004; Karley and White,
2009), this review touches only briefly on the role of roots
and the processes that occur within them as a counterpoint
to those that occur in leaves. To conclude, a hypothesis is
presented on how the movement of water and Ca2+ are
linked in plant leaves.
Peterson, 2004; Dayod et al., 2010). The anatomy of leaves,
apoplastic barriers, distribution of plasmodesmata between
cells, and AQP densities on adjacent membranes will
determine the pathway of water flow and potentially the
distribution of apoplastically transported nutrients within
the leaves (Voicu and Zwiazek, 2010). From the double
exponential kinetics of rehydration of leaves it has been
hypothesized that there are at least three hydraulic designs,
where preferred water flow pathways from the xylem can
result in hydraulic compartmentation in the leaf (Zwieniecki
et al., 2007). Such hydraulic compartmentation will impact
upon the supply of apoplastically transported solutes, such
as Ca2+, to certain cells. This will be elaborated upon
further in the section ‘Does the pathway of water flow affect
distribution of Ca?’
In the roots, an apoplastic compartment can be formed
between the exodermis and the endodermis and another
internal to the endodermis (Fig. 1A). Similarly, the xylem of
Transport and storage of calcium in plants
Important features of the apoplast related to Ca and
water transport
Solute and water transport pathways within plants can be
broadly catagorized as symplastic (intracellular, via AQPs
and plasmodesmata) or apoplastic (extracellular); a transcellular pathway involving both apoplastic and symplastic
compartments has also been defined where solutes and
water cross multiple cellular membranes whilst traversing
plant tissue (Johansson et al., 2000). It is very difficult to
quantify contributions of each of these pathways, and
generally transcellular and symplastic pathways are considered together as the cell-to-cell pathway. The non-xylem
apoplast compartment within tissues has a relatively small
volume compared with the symplast and this can have
profound effects upon ion concentrations especially considering the interaction with water flow (e.g. Flowers et al.,
1991; Canny, 1995), as explained in detail later. The
predominant pathway of water (and Ca2+) flow through
plant tissues differs between species, organs, developmental
stage, and with environmental parameters (Johansson et al.,
2000; White, 2001; White and Broadley, 2003; Cholewa and
Fig. 1. Diagrammatic summary of water and solute flow in (A)
roots and (B) leaves. (B) Observed differences in vacuolar Ca2+
distributions (stippled) between eudicots and grasses, superimposed with the proposed differences in apoplastic sumps (solid
black) and likely pathways for Ca2+ diffusion (dashed lines). Sumps
are located at different sites in leaves of grasses and eudictoyledons. In grasses they occur in the intercellular spaces on the
inner side of bundle sheath parenchyma cells, while in eudicots
they are within the small tracheary elements surrounded by bundle
sheath cells. Water and Ca2+ flux diverge at the point where most
of the water enters the symplast. The relative amount of water that
enters the symplast will determine the diffusion gradient for Ca2+
and the degree of Ca2+ advection in the apoplast. Entry of water to
the symplast will depend on the activity of aquaporins.
Water and calcium interactions | 2235
the leaf vasculature is physically separated from the
apoplast surrounding the leaf mesophyll by bundle sheath
cells (Fig. 1B; Leigh and Tomos, 1991). Both the symplast
and apoplast can be further subdivided into spatially
distinct subcompartments by their local physical–chemical
properties or the presence of membranes with varying
degrees of permeability and selectivity. These can also vary
with plant development, time of day, or environmental
conditions (e.g. Vandeleur et al., 2009), and will impact
significantly upon water and Ca2+ flow through these
compartments. The movement of ions in the apoplast has
been described in terms of the apparent free space (Briggs
and Robertson, 1957) composed of the Donnan free space
(DFS; analogous to an ion exchange membrane containing
non-diffusable anionic charges within the cell wall) and the
water free space (WFS; the apoplastic space where ion
binding does not affect ion movement) (Grignon and
Sentenac, 1991; Sattelmacher, 2001), but a distinct physical
division between the DFS and WFS is unlikely (Sattelmacher,
2001). The movement of ions through the DFS and their
activity in the cell wall is determined by the concentration
of fixed anionic charges, the pH and ionic potential of the
medium, and the valency and size of the ion (Grignon and
Sentenac, 1991; Shomer et al., 2003; Kinraide, 2004;
Kinraide and Wang, 2010). This manifests itself in the case
of cations through a large variation in the cation exchange
capacity (CEC) of walls of different cell types from 0 to
1000 mM (Fritz, 2007).
The extracellular matrix (ECM) is the major structure of
the cell wall apoplast. It is a porous and diverse array of
cross-linked pectins, glycans, proteins, and uronic acids that
have been secreted from the adjacent cell (Burton et al.,
2010; Doblin et al., 2010). The structural composition of the
ECM is dynamic throughout the plant; the specific composition in any given location has a large influence on liquid
and gas exchange between the apoplast and the cell, as well
as apoplastic solute motility and binding of solutes into the
matrix itself. The primary cell wall of higher plants is
comprised of three main classes of glycans: the pectins
[arabinans, homogalacturonan (HGA) and rhamnogalacturonan (RG)], celluloses [predominantly (1,4)-linked b-Dglucan], and hemicelluloses (xyloglucan, arabinoxylan, and
mannan) (Cosgrove, 2005; Burton et al., 2010). The pectic
content of the cell wall largely determines its ionic binding
and electrical properties (DFS), with uronic acid groups
binding free cations and excluding anions from the matrix
(Grignon and Sentenac, 1991). The most significant associations with the fixed anionic charges formed by these acids
appear to involve Ca2+ and H+ (Fang et al., 2009). Calcium
ions can cross-link carboxyl groups of adjacent HGA chains
in the ECM, forming a stiff gel-like matrix (Grignon and
Sentenac, 1991; Cosgrove, 2005), and reduced apoplastic
Ca2+ below a threshold will solubilize pectins (Jarvis et al.,
1984; Virk and Cleland, 1988; Homblé et al., 1989). Boron
(B) forms cross-linkages between RG-II molecules
(Kobayashi et al., 1996) and it has also been shown that B
deficiency results in a number of physiological/metabolic
perturbations including a decrease in free Ca2+ within the
WFS (Goldbach and Wimmer, 2007). Whether this results
from complexation of the remaining B by Ca2+ (Turan
et al., 2009), increased pectin sequestering more Ca2+ into
cell walls (Yamauchi et al., 1986; Yu et al., 2002), or by
Ca2+ filling available RG-II-binding sites vacated by
B (Kobayashi et al., 1999; Turan et al., 2009) is still unclear.
On average, the pectic content of cereals compared with
many dicotyledonous plants is lower and this results in
a lower average CEC of cereal walls, which is thought to be
a contributing factor to the low Ca content of cereal tissues
(White and Broadley, 2003). The CEC of the cell wall will
also influence how apoplastic Ca2+ is transported and
stored within plant tissues.
While apoplastic [Ca2+] modifies the structure of the
ECM, conversely Ca2+ (and probably water) transport
through the apoplast are dictated by the structure of the
ECM. This can be seen with the numerous changes within
the ECM during growth, and under a variety of stress
conditions including dehydration, salinity, and cold stress,
affecting the mobility of Ca2+ and its affinity for the ECM
(Cosgrove, 2005). Liberman et al. (1999) showed using
mung bean hypocotyls that developmental changes in cell
wall glycans including longtitudinal de-esterification of
HGA impacted upon apoplastic [Ca2+]; older non-expanding
cells accumulated greater bound [Ca2+]apo, greater
demethyl-esterfied HGA, and higher PME (pectin methylesterase) activity (proteins that demethyl-esterify HGA)
than rapidly expanding cells. This was correlated with
a radial decrease in both cell wall thickness and [Ca2+]apo
from the epidermis to the cortical parenchyma (Liberman
et al., 1999), as also previously observed in flax hypocotyls
(Rihouey et al., 1995; Jauneau et al., 1997). A similar
correlation was noted in Arabidopsis thaliana hypocotyls
(Derbyshire et al., 2007a, b). Apoplastic [Ca2+]apo was
manipulated in Arabidopsis leaves by Conn et al. (2011a),
and it was observed that an increase in expression of PME
genes and the presence of low methyl-esterified HGA,
thicker cell walls, lower leaf extensibility, and lower growth
rate also correlated with a greater amount of Ca2+ in the
cell wall available to bind pectate. A lowering in [Ca2+]apo
resulted in the opposite; lower transcript abundance of
PME genes, a decrease in low methyl-esterified HGA,
decreased cell wall thickness, and increased cell wall
extensibility (Conn et al., 2011a). RG is known to be
developmentally regulated (Knox, 2008) and its accumulation increases prior to pollen formation, which coincides
with an increased sensitivity to B deficiency in wheat
(Rawson, 1996). It has also been shown that among 14 crop
plants (both monocots and dicots) there are species-specific
requirements for B that are strongly correlated with leaf B
concentration and RG accumulation (Hu et al., 1996). This
shows that the ECM and apoplastic ion levels are intimately
linked throughout development, and may be linked with
water movement as elaborated below.
In addition to the transport barrier represented by the
ECM, the apoplast contains unstirred layers (ULs)
(Grignon and Sentenac, 1991). These occur in proximity to
transport barriers where diffusion is disrupted by
2236 | Gilliham et al.
incomplete mixing (e.g. directly adjacent to the plasma
membrane or through pores of the ECM) (Grignon and
Sentenac, 1991; Sattelmacher, 2001; Evans et al., 2009).
Decreased diffusion across an UL can represent a significant
barrier to transport of apoplastic solutes and may allow
high concentrations of ions to occur at sites of concentrated
water uptake (Canny, 1995). Membrane transport processes
may act to set up significant concentration gradients across
ULs (e.g. H+ excretion). As such, the concentration of
solutes within the ECM does not necessarily reflect the
concentration adjacent to the plasma membrane especially
when the surface charge of the plasma membrane is taken
into account (Kinraide, 2004). Therefore, ULs and ion
exchange in the cell wall and on the cell membrane make it
difficult to determine native transport kinetics and ion concentrations adjacent to transport proteins experimentally.
Long-distance transport of calcium is predominantly
apoplastic
Ca present in soil as a divalent cation (Ca2+) enters the root
apoplast along with mass flow of water (Barber, 1995) and
follows apoplastic or symplastic pathways to the xylem
where exchange between xylem parenchyma and the xylem
sap will alter the ion concentrations in ascending sap
(White, 1998, 2001; White and Broadley, 2003). The
casparian band surrounding the root endodermis can limit
the apoplastic passage of solutes across the endodermis into
the xylem. Therefore, movement of Ca2+ to xylem must use
a symplastic pathway when an impermeable casparian band
is present (Fig. 1Ai, ii; Clarkson, 1993; White, 2001). In this
case, the apoplastic Ca2+ is taken up by cells on the cortical
side of the endodermis through Ca2+-permeable channels
(White, 2001). Subsequently, Ca2+ is actively effluxed across
the plasma membrane from the symplast by Ca2+ transporters (e.g. Ca2+-ATPases) in the stele. However, the
overall Ca2+ flux into the xylem appears to be greater than
if purely protein mediated (Clarkson, 1984; White, 2001;
Cholewa and Peterson, 2004). White (2001) proposed that
Ca might also traverse the symplast as Ca chelates in order
to maintain low [Ca2+]cyt.
The apoplastic or symplastic pathways have distinct
characteristics. The Ca2+ apoplastic flux is significantly
dependent on the transpiration rate, but the pathway is
relatively non-selective for divalent cations (White, 1998;
White, 2001; White and Broadley, 2003). Root endodermal/
exodermal bypass flow to the xylem, via the discontinuities
in the apoplastic barriers surrounding lateral roots, can
allow significant amounts of Na+ to enter the shoot, and
cultivar differences have been observed in salt tolerance
related to the degree of bypass flow in rice (Faiyue et al.,
2010a, b), but this has not been observed in grapevines
(Gong et al., 2010). It is possible that this may also affect
Ca2+ transfer to the shoot. Interestingly, enhanced suberization of root observed in the Arabidopsis knockout line
esb1 was linked with decreased daytime transpiration and
a 50% decrease in shoot [Ca], presumably from the
enhanced suberin barrier restricting water and solute
movement through the root apoplast (Baxter et al., 2009).
The symplastic pathway is more selective and controls Ca2+
transport into the xylem depending on the demand for Ca2+
in the shoot (Clarkson, 1993; White, 1998, 2001; White and
Broadley, 2003). It is likely that the proportion of Ca2+
transported via the symplast increases as the flux of Ca2+ to
the shoot decreases. This may occur following increased
suberization of the endodermis, low Ca supply, or low
transpiration (Clarkson, 1984; Baxter et al., 2009).
The proportional contribution of apoplastic and symplastic pathways to Ca delivery into xylem is unclear in
most plants but appears to be dependent on the species
(Cholewa and Peterson, 2004; Hayter and Peterson, 2004;
Baxter et al., 2009; Conn and Gilliham, 2010). Likewise
with water, there appear to be large differences between
species in the degree to which water bypasses the endodermal symplast and flows radially to the xylem predominantly
via the apoplast (Bramley et al., 2009, 2010). The presence
of such a barrier to apoplastic flow of water and solutes
could give a certain level of control over root-to-shoot
transfer of solutes through the symplastic loading and
unloading steps, as well as the possibility of diversion to
the vacuole for storage (Fig. 1A). Chemical differences in
suberin and the amount present on a surface area basis may
also exert some control on selectivity (Schreiber et al., 2005)
Within both the root and shoot the degree of secondary
wall deposits can vary between apoplastic subcompartments. For instance, maize roots grown in aeroponics have
an exodermis that alters solute flow across the root, but it is
absent in hydroponically grown maize even following
treatment with abscisic acid (ABA; Freundl et al., 1998,
2000; Hose et al., 2001). Similarly the degree of suberization
within the axial walls of leaf bundle sheath cells (parenchymatous and C4 Kranz) is reported to vary between species,
and for C4 grasses to correlate with the type of C4
photosynthesis (Hattersley and Browning, 1981). Whereas
no casparian bands have been detected in Arabidopsis
thaliana leaves under any conditions (MD, unpublished
results), they can be routinely detected in leaves of grasses,
cereals, and many dicots (Hattersley and Browning, 1981;
Evert et al., 1996; Lersten, 1997). Although there often
appear to be discontinuities in both root and shoot apoplastic barriers that allow apoplastic transport of solutes
and water (Evert et al., 1985), the presence of casparian
bands will still influence the route taken through leaves by
a large proportion of water and solute.
Calcium delivery to shoots is linked to the transpiration
rate
Ca deficiencies are often manifested in tissues that have low
relative rates of transpiration compared with other parts of
the plant, which clearly highlights the role of transpiration
in supply of Ca2+ and the low rate of symplastic transport
of Ca2+ within most tissues. This is discussed in greater
detail in Dayod et al. (2010) with particular reference to the
nutritional value of plants as food crops. Experiments show
that root pressure and recycled phloem water (Munch
Water and calcium interactions | 2237
water) are capable of delivering sufficient quantities of most
nutrients to the shoot, but not Ca which requires high rates
of transpiration (Tanner and Beevers, 2001). However, even
when the leaf transpiration rate is high, Ca deficiency can
also occur in adjacent lowly transpiring organs on the same
plant, for example fruits (Dayod et al., 2010). Presumably,
this is a consequence of insufficient Ca delivery to these
tissues because when the transpiration rate of these regions
is increased then the severity of Ca deficiency symptoms
such as blossom end rot or tipburn is reduced (Chang and
Miller, 2004; Frantz et al., 2004). Thus, under some circumstances (e.g. high growth rates and/or impeded transpiration
in folded leaves), it appears that long-distance symplastic
transport is unable to deliver sufficient Ca2+ to tissues that
have low transpiration, especially when competing against
tissues with higher transpiration rates.
Lower transpiration results in lower Ca content of plant
tissues, therefore climate change which is predicted to
reduce transpiration through the effects of elevated atmospheric CO2 and increased frequency of drought and salinity
is also likely to decrease plant Ca content (MartinezBallesta et al., 2010). A meta-analysis of experiments
regarding nutrient concentrations under high CO2 showed
that Ca declined by ;7.5% in leaves (Loladze, 2002). The
decline in foliar Ca was similar to that of other cation
macronutrients (Loladze, 2002). Ca deficiency symptoms
are observed in lettuce when grown under high light and
CO2 (Frantz et al., 2004), and macronutrient deficiencies
occur in strawberry grown at high CO2 (Keutgen et al.,
1997). This reduced nutrient content of plants relative to
carbon content will therefore have a negative impact on
plant quality and human nutrition (Dayod et al., 2010).
Homeostasis of calcium concentration—ubiquitous or
heterogeneous?
The [Ca2+]cyt is tightly controlled. When the cell is ‘resting’,
[Ca2+]cyt is ;100 nM but can be >1 lM during signalling
events (White and Broadley, 2003; McAinsh and Pittman,
2009; Dodd et al., 2010). During normal physiological
events, [Ca2+]cyt elevations are generally not prolonged and
instead oscillate in order to maintain cell viability (McAinsh
and Pittman, 2009). Signalling events, via changes in [Ca2+],
also occur in other compartments such as the nucleus
(McAinsh and Pittman, 2009). However, it is not known
whether there is tight homeostatic control of [Ca2+] in other
compartments, or if Ca storage within organelles [vacuole,
endoplasmic reticulum (ER), chloroplast, and mitochondrion] or the apoplast exists to service [Ca2+]cyt homeostasis.
Within the WFS [Ca2+] is generally in the micromolar range
(Sattelmacher, 2001; Fritz, 2007) and must be maintained
below ;500 lM in order to prevent stomatal closure
(DeSilva et al., 1996).
There is a constant and significant electrochemical
gradient for the passive uptake of Ca2+ into the cytoplasm
from the apoplast, and [Ca2+]cyt is responsive to [Ca2+]apo.
Elevations in [Ca2+]apo have been used in experiments to
manipulate [Ca2+]cyt and stomatal movements (Allen et al.,
2001), but the extent to which [Ca2+]apo is regulated or even
sensed is not fully understood. The identification of an
‘extracellular’ Ca2+ sensor, the CAS protein, will be a useful
tool in exploring this phenomenon further (Han et al.,
2003). Mutant guard cells devoid of this chloroplastlocalized protein do not, unlike wild-type stomata, raise
[Ca2+]cyt or close the stomatal pore in response to an
elevation in extracellular [Ca2+] to 5 mM (Weinl et al.,
2008). Further examples of apoplastic Ca2+ ‘sensors’ or
modulators include pectin, calmodulin, annexins, AQPs,
and ATP (Hepler and Winslip, 2010). Therefore, it is
possible that different cells respond differently to extracellular calcium dependent upon its complement of intra- and
extracellular Ca2+ sensors. How this may affect the flow of
water and [Ca2+] through plant tissues is further elaborated
below (see ‘Does the pathway of water flow affect distribution of Ca?’).
The leaf vacuole is widely considered to be the major Ca
store in plant tissues but, unlike the cytoplasm, the [Ca2+]vac
varies considerably between cell types (Karley et al., 2001a;
White and Broadley, 2003; Conn and Gilliham, 2010; Conn
et al., 2011a, b). Within roots, where overall Ca content is
low, [Ca]vac is <10 mM unless precipitated with oxalate,
whereas in some leaf cells it can reach >150 mM (Fricke
et al., 1995; Storey et al., 2003; White and Broadley, 2003;
Storey and Leigh, 2004). Furthermore, Ca2+ is preferentially
secreted into vacuoles of specific cell types in leaves (Leigh
and Storey, 1993; Karley et al., 2001a; Storey and Leigh,
2004; Kerton et al., 2009; Conn and Gilliham, 2010; Conn
et al., 2011a, b). In cereals, total [Ca]vac is significantly
higher in epidermal cells (up to 150 mM in ‘interstomatal’
cells; Fricke et al., 1995) than in mesophyll vacuoles
(<10 mM), whereas in most eudicots studied [Ca]vac is low
in epidermal and bundle sheath cells (<10 mM) but
>60 mM in the mesophyll (Karley et al., 2000a, b; Conn
and Gilliham, 2010; Conn et al., 2011a, b). In addition,
point source stores of Ca within the form of oxalate are
frequently observed in crystal-containing idoblasts within
the roots and shoots of many ‘oxalate plants’ (Franceschi
and Nakata, 2005).
Experimental manipulations where plant tissue is exposed
to high [Ca2+] through the transpiration stream, or using
vacuum infiltration, maintain this distribution of Ca (Storey
and Leigh, 2004), perhaps indicating that it is the transport
properties of the cells which determine where Ca is stored.
However, such manipulations affect the water flow through
tissues and circumvent the influence that water flow
normally has because it imposes high [Ca2+]apo by diffusion
or force (MD, unpublished results). Once Ca2+ is deposited
in vacuoles it is not redistributed because, with the
exception of the small amounts involved in signalling, most
intracellular Ca2+ is relatively immobile (White and Broadley,
2003) and there is limited evidence for symplastic movement
or redistribution of Ca2+ through organelles such as the ER.
Therefore, Ca2+ feeding experiments are likely to report
faithfully the cell types that have the potential to accumulate high [Ca] within leaves, although the distribution seen
in non-fed leaves will ultimately be dependent upon the
2238 | Gilliham et al.
routes that Ca2+ and water take through the leaf (Kerton
et al., 2009).
In Arabidopsis, a vacuolar-localized Ca2+/H+ exchanger
CAX1 was expressed preferentially in the leaf mesophyll
and found to be necessary for a significant proportion of
mesophyll-specific vacuolar storage of calcium (Conn et al.,
2011a). The calcium-accumulating ability of mesophyll cells
was reduced in plants lacking expression of CAX1 but
resulted in an increase in [Ca2+]apo, with some dramatic
effects upon leaf physiology (Conn et al., 2011a). First,
expression of cell wall synthesis and modifying genes was
perturbed, which ultimately resulted in thicker and less
extensible cell walls and, secondly, high [Ca2+]apo reduced
stomatal apertures which reduced transpiration and CO2
assimilation. The net effect of greater [Ca2+]apo was that
plants grew more slowly, implicating mesophyll cell vacuolar compartmentation as a key control point for the Carelated physiology of leaves. It was also apparent that Ca2+
is likely to contribute significantly to the osmotic potential
of mesophyll cells and, when mesophyll [Ca2+]vac was
reduced, mesophyll-specific accumulation of magnesium
(Mg2+) substituted for the osmotic role of Ca2+ (Conn
et al., 2011b). In serpentine or low Ca2+ growth conditions,
MRS2-1 and MRS2-5, which encode vacuolar-localized
Mg2+ transporters, were both up-regulated and appear to
play a key role in maintaining optimal growth (Conn et al.,
2011b). Such studies emphasize the importance of the
vacuole in co-ordinating fluxes across the plasma
membrane.
The pathway of water flow in leaves and the
impact upon Ca distribution
The pathway of water flow is dynamic
Water is usually initially taken up from the soil through the
plant root system and transported to the shoot via the
xylem. However, during leaf development water may also
be transported by the phloem (Schmalstig and Geiger,
1985). Water moves down water potential gradients where
component osmotic potential gradients require a semipermeable membrane for flow to occur. Expansion growth
requires water uptake, with most of the volume increase
accounted for by vacuolar expansion (Schmalstig and
Geiger, 1985). Growing tissues therefore must develop
water potential gradients and water must be conducted to
growing tissues. Hydraulic conductivity can in some cases
become rate limiting to growth (Tang and Boyer, 2002;
Boyer and Silk, 2004). Plant cells alter the water potential
gradient by changing the osmolyte content, or the rate of
volume increase relative to solute increase in expansion
growth, in order to favour an influx or efflux of water
(Sharp et al., 1990; Kaldenhoff and Eckert, 1999; Johansson
et al., 2000). Such osmotic adjustment can maintain turgor
to varying degrees (Jones and Turner, 1980; Westgate and
Steudle, 1985; Tyerman et al., 1989). Changes in osmolyte
and water content of the apoplast and symplast will
potentially have an impact upon both where Ca2+ is stored
and the flow of water through leaves.
Cells adjacent to xylem vessels in leaves may have an
important role in facilitating water flow between the
apoplastic and symplastic compartments (Frangne et al.,
2001; Heinen et al., 2009). It has been shown for leaves of
some species that apoplastic water movement from the
xylem is essentially blocked (Fitzgerald and Allaway, 1991),
and water flow can be entirely cell to cell to the epidermis
(Ye et al., 2008; Nardini et al., 2010a), while in others the
apoplastic pathway seems to predominate (Voicu et al.,
2009). This is similar to the variation observed in roots.
(Bramley et al., 2009). The bundle sheath in leaves may
have suberin lamellae and/or apoplastic barriers on radial
walls, thereby decreasing the apoplastic flow of water
(Lersten, 1997), and in other cases bundle sheath extensions
can allow high connectivity to the epidermis and thence
various degrees of connectivity to the mesophyll (Voicu and
Zwiazek, 2010). The preferred pathway of water flow within
leaf tissue may involve a combination of pathways at some
point depending on leaf developmental stage (Evert et al.,
1985; Voicu and Zwiazek, 2010). It would be useful to
examine species displaying these differences for differences
in Ca compartmentation in leaves as this may show
interesting correlations, but at present this has not been
done. However, it is known that many cereals have vein
extensions that connect vascular bundles directly to the
epidermis and this correlates with the cell types in which
most Ca is stored (Karley et al., 2001b). Interestingly and in
contrast to the current paradigm, Metzner et al. (2010)
observed, using cryo-SIMS (secondary ion mass spectrometry) on Phaseolus vulgaris leaves, that the unloading of
Ca2+ from the xylem did not follow an apoplastic pathway
but it was rapidly taken up into xylem parenchyma cells. It
was speculated that this may be an artefact due to the
loading of tracer through an excised stem causing the
opening of Ca2+ channels in cells surrounding the xylem.
For rice there is evidence using membrane-impermeant dyes
for endocytosis as a mechanism for unloading from the
xylem into xylem parenchyma cells where they abut pit
membranes (Botha et al., 2008). These observations clearly
highlight the need to re-examine the route of Ca2+ unloading from the xylem.
Does the pathway of water flow affect distribution of
Ca?
Calcium uptake and distribution at the whole-plant level is
influenced by water flow to transpiring organs and by the
relative rate of Ca2+ uptake along the transport pathway
(McLaughlin and Wimmer, 1999). In leaves, the arrangement of xylem has been suggested to have a direct effect on
water and solute transport (Roth-Nebelsick et al., 2001;
Loeffe et al., 2007). Water transported via the xylem is
proposed to be rapidly taken into the symplast of leaves by
flow across the plasma membrane of cells adjacent to veins
(Canny, 1990). This flow of water into the symplast would,
conceptually, increase the concentration of the less
Water and calcium interactions | 2239
membrane-permeable solutes within the apoplast and consequently create local ‘sumps’ (Canny, 1990). Furthermore,
once separated from water, mineral cations in the apoplast
would move at much slower rates than would occur solely
in water (Canny, 1993). Yet, only Ca2+ fulfils the prediction
fully (Canny, 1993), which implies that Ca2+ transport
within the leaf is not primarily by mass flow (Atkinson
et al., 1992) but it still occurs via an extracellular pathway
(Clarkson, 1984; Canny, 1993). This was also suggested by
the experiments of Storey and Leigh (2004) who found that
preventing cuticular transpiration from the astomatous
adaxial epidermis of citrus leaves did not affect Ca2+
accumulation in the palisade mesophyll underlying this
surface. Uncoupling of Ca2+ and water flows from the
xylem has been reported in a number of monocot and dicot
species (Atkinson et al., 1992; Kerton et al., 2009), which is
likely to be due to binding of Ca2+ to the xylem walls and
its movement along an exchange column whose length and
total capacity increase relative to cell and plant growth
(Clarkson, 1984). The site of water and Ca2+ unloading of
the xylem would therefore affect local [Ca2+] in the apoplast
and availability for cells to accumulate Ca2+.
In general, [Ca2+] within cereal leaf vacuoles will be lower
than that of most dicots (Conn and Gilliham, 2010).
However, in some cereal epidermal cells (those adjacent to
guard cells), the vacuolar [Ca2+] far exceeds that in
mesophyll cells of most dicots. This has been explained in
terms of both supply of solutes and the need for excretion
of apoplastic Ca away from sensitive sites, for example
stomatal guard cells (Karley et al., 2001a). The potential
volume for excretion of Ca in cereals away from the
apoplast is less than in dicots as the volume of the
mesophyll vacuole is greater on a whole-leaf basis. For
instance, the proportion of epidermal and mesophyll cells
within a barley leaf is estimated to be 27% and 42%, whilst
in spinach it is 3% and 58%, and in Arabidopsis, 13% and
60%, respectively (Pyke et al., 1991; Winter et al., 1993,
1994). The vein extensions within cereal leaves mean the
epidermis will be preferentially exposed to Ca2+; furthermore, the Ca will be drawn to stomatal guard cells through
mass flow of water in the transpiration stream. It is likely,
therefore, that epidermal cells will accumulate greater
amounts of vacuolar Ca as they are exposed to more
apoplastic Ca2+, and it is likely that these cells preferentially
express certain transporters that confer the capacity preferentially to sequester Ca2+ into their vacuoles (Conn and
Gilliham, 2010). In contrast, phosphor imaging of excised
coriander leaves fed 4 mM Ca2+ labelled with 45Ca showed
an unequal distribution of Ca2+ (mainly at the centre of the
leaf) despite uniform water loss through the leaf surface
detected by thermal imaging (Kerton et al., 2009). However,
rapid movement of recently imported Ca2+ to the leaf
margin was observed when supplied with 40 mM Ca2+
(Kerton et al., 2009). In coriander leaves, mesophyll cells in
the centre of the leaf are exposed to higher apoplastic Ca2+,
perhaps due to the pattern of the xylem networks, and they
thus accumulate more Ca2+ in their vacuoles (Kerton et al.,
2009). For a schematic of how these two water flow
pathways may result in the observed Ca distributions see
Fig. 1B.
There are numerous examples of interactions between
water flow and Ca2+ in the literature. Foremost is the effect
of apoplastic Ca2+ on stomatal aperture (Ruiz et al., 1993;
DeSilva et al., 1996; Webb et al., 2001; Yang et al., 2006).
As water flow through the plant is dominated by stomatal
conductance, stomatal aperture is the most important factor
in drawing water through the plant. It is known that
[Ca2+]apo adjacent to the guard cell will regulate stomatal
aperture, for example in response to low temperature stress
(Wilkinson et al., 2001), and it has been proposed that this
may involve an interaction with AQPs in the guard cells
(Yang et al., 2006). There has also been a report of a 4-fold
difference in sensitivity to Ca2+ between the adaxial and
abaxial guard cells of Vicia faba (Wang et al., 1998), which
may have implications for delivery of Ca2+ to different sides
of the leaf. However, it is clear that hydraulic conductance
through the leaf can be a substantial fraction of whole-plant
hydraulic conductance (Tsuda and Tyree, 2000), and as
such the way in which Ca2+ interacts with this internal
conductance will influence the delivery of Ca2+ to different
cell types including the guard cells.
AQPs are responsible for finely regulating water flows
across plant membranes (Kjellbom et al., 1999; Tyerman
et al., 1999) and therefore may be responsible for regulating
flow pathways in the leaf. Sumps are located at different
sites in leaves of grasses and eudictoyledons. In grasses they
occur in the intercellular spaces on the inner side of bundle
sheath parenchyma cells, while in eudicots they are within
the small tracheary elements surrounded by bundle sheath
cells (Fig. 1B). Bundle sheath cells have been shown to have
high expression of plasma membrane intrinsic protein (PIP)
and tonoplast intrinsic protein (TIP) AQPs in Brassica
napus (Frangne et al., 2001).
The role of AQPs in regulating the pathway
of water flow in leaves
Plant aquaporins
AQPs are water-conducting major intrinsic proteins (MIPs)
that facilitate the flow of water across biomembranes
(Quigley et al., 2001; Tyerman et al., 2002). It is generally
accepted that AQPs are key factors in regulating cell-to-cell
water flow either as components of the flow pathway
(Chaumont et al., 2005; Hachez et al., 2006; Kaldenhoff
and Fischer, 2006; Maurel, 2007) or potentially as sensors
(Hill et al., 2004). They appear to account for the major
portion of the hydraulic conductivity of the plasma
membrane and tonoplast (Maurel et al., 1997; Tyerman
et al., 1999, 2002; Maurel, 2007), but not always the major
proportion of the tissue hydraulic conductivity (Bramley
et al., 2009; Voicu et al., 2009). The role of AQPs in flow of
water through leaves has been comprehensively reviewed
recently by Heinen et al. (2009), but the interaction with
Ca2+ transport and distribution through leaves was not
considered.
2240 | Gilliham et al.
The MIP superfamily in plants had been originally
divided into four subfamilies: the PIPs, TIPs, nodulin-like
intrinsic proteins (NIPs), and the small basic intrinsic
proteins (SIPs) (Zardoya and Villalba, 2001; Johanson and
Gustavsson, 2002; Quigley et al., 2002; Forrest and Bhave,
2007). Two new subfamilies have been identified in the moss
Physcomitrella patens: the GlypF-like intrinsic proteins
(GIPs) (Gustavsson et al., 2005) and the hybrid intrinsic
proteins (HIPs) (Danielson and Johanson, 2008). Another
subfamily called the X intrinsic proteins (XIPs) has been
identified, with members in a number of dicotyledonous
plants including tomato (Sade et al., 2009) and grapevine
(Danielson and Johanson, 2008). Some MIPs may facilitate
the transport of small, uncharged molecules, such as
ammonia (Tyerman et al., 2002; Jahn et al., 2004; Beitz
et al., 2006), hydrogen peroxide (Henzler and Steudle,
2000), glycerol (Biela et al., 1999; Holm et al., 2004; Bietz
et al., 2006), urea (Liu et al., 2003; Holm et al., 2004; Beitz
et al., 2006), silicate (Chiba et al., 2009), B (Dordas et al.,
2000; Fitzpatrick and Reid, 2009; Schnurbusch et al., 2010),
and CO2 (Uehlein et al., 2003, 2008; Hanba et al., 2004)
across specific membranes. In addition to their role in
permeation of other solutes, the large family of AQPs in
plants may indicate that water transport within plant tissues
is complex and tightly regulated to allow them to adapt to
changes in environmental or developmental conditions
(Vandeleur et al., 2005; Kaldenhoff et al., 2008). It has been
shown that tissue-specific expression patterns of numerous
AQP homologues and their regulation in plants are affected
by environmental factors such as day/night cycles, water
stress, or pathogens (Gerbeau et al., 2002), which suggest
that plants have a capacity to regulate the uptake and loss,
or relocation, of water within plant cells and tissues.
Tissue versus cellular water flow
AQP-mediated water flow is thought to be a predominant
mechanism used by plants to control their cellular and
tissue flow (Maurel, 2007), although some plants have
apparently high AQP activity at the cell level with no
indication that they regulate flow across the tissue, for
example roots of some plant species under certain conditions (Bramley et al., 2009). On the other hand, their role
in regulation of flow across tissues has been indicated by
correlations between AQP expression and tissue/organ
hydraulic conductivity (Cochard et al., 2007; Lovisolo
et al., 2007; Parent et al., 2009; Vandeleur et al., 2009;
Postaire et al., 2010; Sade et al., 2010) and reversible effects
of inhibitors or conditions that normally inhibit AQP
activity (De Boer and Volkov, 2003; Volkov et al., 2007;
Bramley et al., 2010; Sadok and Sinclair, 2010; Voicu and
Zwiazek, 2010).
In leaves there is evidence of dynamic adjustment in
hydraulic conductivity that may indicate that AQPs control
the proportion of water flow in the cell-to-cell pathway after
water has exited xylem vessels. However, the total hydraulic
conductivity of a leaf includes a vascular (major and minor
veins) and non-vascular component (after water has exited
the xylem). The contribution of the non-vascular pathway
relative to the vascular pathway has been estimated in
Helianthus annuus L leaves, where it was 58% in the light
period and 72% in the dark period (Nardini et al., 2005).
Leaf hydraulic conductivity and aquaporins
Increased leaf hydraulic conductivity in response to increased irradiance occurs to varying degrees between species
(Tyree et al., 2005; Scoffoni et al., 2008), and the involvement of AQPs has been suggested from correlations
with expression of PIP AQPs (Cochard et al., 2007) or from
the inhibitory effects of mercurials (Nardini et al., 2005).
Interestingly, there is a larger response to light in heterobaric species (bundle sheath extensions between the upper
and lower epidermis limiting lateral gas diffusion within the
leaf) compared with homobaric species (lateral gas diffusion
not limited) (Scoffoni et al., 2008). From the hypothesis on
the hydraulic compartmentation within the leaf (Zwieniecki
et al., 2007; see above), this difference might be related to
heterobaric species having a greater total leaf cell volume
fed by the cell-to-cell pathway from the xylem. However,
Voicu et al. (2009) examined the expected change in the
water transport pathway (apoplastic versus cell-to-cell) by
examining the exudation of an apoplastic dye [8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS)] from leaves of oak
(Quercus macrocarpa Michx.) infused with the dye in
combination with mercurials. They found no evidence of
a change in pathway, or any changes in expression of four
PIP transcripts in the short term, which correlated with
changes in leaf hydraulic conductivity, suggesting that no
such relationship exists or that the experimental system was
not appropriate. A similar study on Populus tremuloides
Michx leaves, also using perfusion of HPTS dye as an
apoplastic tracer, indicated that most water flow occurred
via the apoplastic pathway, yet there was a strong effect of
leaf metabolic status on the leaf hydraulic conductivity
(Voicu et al., 2008, 2009). Light may increase the hydraulic
conductivity of the vascular pathway through increases in
the K+ concentration in the xylem, which may increase the
conductance of pit membranes through an ionic effect on
the pectin hydrogel (Nardini et al., 2010b; Zwieniecki et al.,
2001). In some species, Ca2+ has been shown to depress this
response to K+ (Nardini et al., 2007) and further illustrates
the link between Ca2+ and hydraulic conductivity.
Regulation of water flow in leaves in response to altered
transpiration has been investigated by altering atmospheric
humidity (Sack and Holbrook, 2006). Whole-plant hydraulic conductivity (normalized to leaf surface area) of
Arabidopsis increased when plants were exposed to low
humidity (implying higher transpiration) (Levin et al.,
2007). It might be expected that leaf cell turgor would
decrease at high transpiration rates (Kim and Steudle, 2007;
Zimmermann et al., 2008) and there appears to be a positive
linear correlation between leaf cell turgor and leaf hydraulic
conductivity (Brodribb and Holbrook, 2006). Thus increased transpiration might be expected to decrease leaf
hydraulic conductivity. However, these observations are not
Water and calcium interactions | 2241
in accord with those of Levin et al. (2007), but in this study
it was not known what proportion of the increase in wholeplant hydraulic conductivity in response to low humidity
could be in the leaves or the roots. Interestingly roots
showed increases in AQP transcripts following a low
humidity treatment (Levin et al., 2009). Vandeleur et al.
(2009) also found a positive linear correlation between
transpiration and root hydraulic conductivity. Taken together these observations may indicate an opposite response
between roots and leaves to transpiration rate, which would
tend to decrease the amplitude of changes in stem water
potential with fluctuating transpiration rates. For this to
occur it might also be expected that AQPs are differentially
regulated in roots and leaves when transpiration varies.
The effect of changes in AQP expression has also been
examined using transgenic approaches. A reverse genetic
approach has demonstrated the importance of AtPIP1;2 in
regulation of water flow in Arabidopsis rosettes (Postaire
et al., 2010). Interestingly, Arabidopsis showed an increase
in rosette conductance in continuous darkness, in contrast
to other plants, and AQP inhibitors had a greater effect
under these conditions. The same PIP AQP also contributes
to a substantial portion of the pressure-induced flow
through roots. A PIP isolated from lily and overexpressed
in tobacco increased water permeability of both leaf
protoplasts and intact leaf cells (Ding et al., 2004). Overexpression of OsPIP2;7, normally located in mesophyll
cells, in two independent rice lines resulted in higher
transpiration (Li et al., 2008). However, at the same time
transcripts of two other PIP2 isoforms increased in these
transgenic plants, so it is uncertain whether the increase in
transpiration was a result of overexpression of the specific
isoform or the combined pleiotropic effects. Regardless, it
would be interesting to examine Ca accumulation in these
overexpressing rice lines.
Leaf cells and aquaporins
At the cell level there is evidence for the involvement of
AQPs in regulating water flow in leaves (Johansson et al.,
1996; Morillon and Chrispeels, 2001; Kim and Steudle,
2007; Cohen et al., 2011). Maize midrib cells show an
increase in hydraulic conductivity in moderate light
(650 lmol m 2 s 1), but a reduction in turgor caused by
transpiration compensated for this effect (Kim and Steudle,
2007). At higher light intensities (800–1600 lmol m 2 s 1)
and under experimental conditions that prevented a drop in
turgor, cell hydraulic conductivity was reduced and reactive
oxygen species (ROS) inhibition of AQPs was implicated
(Kim and Steudle, 2009). It is interesting to note that the
leaf was perfused with CaCl2 in these experiments and it
would be interesting to explore the possibility that Ca2+
may have had a role in changing AQP activity. In spinach
leaves, SoPIP2;1 (PM28a) AQP is constitutively expressed
and shows reduced phosphorylation with decreased water
potential (and lower turgor) (Johansson et al., 1996).
In vitro the phosphorylation is Ca2+ dependent, and in
Xenopus laevis oocytes phosphorylation of SoPIP2;1
increases water permeability (Johansson et al., 1998). Thus
reduction of turgor or leaf water potential would tend to
decrease cell-to-cell flow and increase the proportion of
apoplastic flow if SoPIP2;1 accounts for most of the plasma
membrane water flow. These results indicate an optimum in
light intensity for water transport, and superimposed on this
is an inhibitory effect of water deficit, indicating a tight
regulation mediated by AQPs and involvement of Ca2+.
High transpiration rates (low humidity treatment) decreased the water permeability of isolated leaf protoplasts
(which have zero turgor), and conversely low transpiration
(high humidity or ABA treatment) increased protoplast
water permeability in Arabidopsis (Morillon and Chrispeels,
2001). This appeared to be independent of a direct effect of
ABA, since ABA-insensitive mutants and ABA-deficient
mutants both showed an increase in protoplast water
permeability at high humidity. However, the transpiration
transduction mechanism may have elements in common
with the ABA transduction mechanism (Morillon and
Chrispeels, 2001). An increase in PIP expression in leaves
under low transpiration caused by a variety of treatments
was also observed for P. vulgaris (Aroca et al., 2006). These
results have been interpreted as demonstrating a greater
proportion of apoplastic flow (lower cell-to-cell water transport) in leaves under high transpiration, and a smaller
proportion of apoplastic flow (higher cell-to-cell water
transport) under low transpiration, analogous to the
situation that is proposed to occur in roots purely in
response to changes in the predominant driving force
(hydrostatic versus osmotic) (Steudle and Peterson, 1998).
In absolute terms this would result in much greater changes
in apoplastic flow with changes in transpiration than would
occur if the proportion of flow in transcellular and apoplastic pathways remained the same, or if the effect on cell
water permeability was the other way around. On the other
hand, it would tend to even out the changes in flow via the
cell-to-cell pathway. It remains to be seen what effect this
would have on the apoplastic Ca concentration.
There is evidence from the location of AQPs in specific
leaf cells and genetic evidence that indicates AQP involvement in water flow through leaves. In Arabidopsis, expression of AtPIP1 in mesophyll cells has been reported
(Robinson et al., 1996). In tobacco, NtAQP1 was found to
accumulate in spongy parenchyma cells, with the highest concentration around substomatal cavities (Otto and Kaldenhoff,
2000). Rice OsPIP1 and OsPIP2;1 were reported to be
localized mainly in mesophyll cells of leaf blades (Sakurai
et al., 2008) as was OsPIP2;7 (Li et al., 2008). In addition,
VfPIP1 isolated from the leaf epidermis of V. faba is
localized in the plasma membrane (Cui et el., 2008). Also,
all maize PIPs are expressed in leaves, except for ZmPIP2;7,
and the diurnal expression correlates with protoplast water
permeability from different parts of the leaves (Hachez
et al., 2008). Immunolocalization further indicates that PIPs
are most probably involved in cell-to-cell water flow from
the vascular tissue (Hachez et al., 2008). A barley PIP
(HvPIP1;6), which incidentally seems to increase water
permeability (at the protein level) in response to ABA, is
2242 | Gilliham et al.
linked with growth-associated water uptake in the expansion zone of barley leaves (Wei et al., 2007). In olive (Olea
europaea) there is a positive correlation between the
expression of PIP AQPs and the hydraulic conductivity of
leaves of dwarfing and vigorous root stock varieties
(Lovisolo et al., 2007). It may be assumed that different
AQP homologues are localized in specific cell membranes
presumably to play distinct roles in the regulation of water
flow and/or CO2 diffusion within those particular cells.
Calcium regulation of AQPs
In Arabidopsis, water transport in purified plasma membrane vesicles was inhibited by Ca2+, with half-inhibition
(Pf) at 50–100 lM free Ca2+, indicating a Ca2+-induced
down-regulation of plant cell hydraulic conductivity
(Gerbeau et al., 2002). There appears to be a complex
interaction of different cations having direct or indirect
effects on the AQP-mediated water transport activity as
exhibited in Capsicum annuum plants (Cabanero et al., 2004;
Cabanero and Carvajal, 2007). Under saline conditions,
a positive effect of Ca2+ on AQPs in melon plants has been
reported (Carvajal et al., 2000). These effects may be due to
plasma membrane stabilization by Ca2+, activation of
a protein kinase that regulates PIP AQPs (Johansson et al.,
1996), or direct gating of the AQPs (Chaumont et al., 2005;
Alleva et al., 2006; Hedfalk et al., 2006).
Alleva et al. (2006) showed that Ca2+ has a clear effect on
AQP activity in plasma membrane vesicles from Beta
vulgaris storage root, with two distinct ranges of sensitivity
to [Ca2+]cyt (between pCa 8 and pCa 4) (Fig. 2). Also shown
in Fig. 2 are other observations of AQP sensitivity to Ca
from the literature. They suggested that since the normal
[Ca2+]cyt sits between these ranges, it allows for the
possibility of changes in Ca2+ to finely regulate AQP
Fig. 2. Effects of free cytosolic Ca2+ concentration on AQPs in
plant plasma membrane vesicles. The lines are dose–responses
curves for: Beta vulgaris storage root (square symbols solid line;
Alleva et al., 2006); AtPIP2;1 in proteoliposomes (dashed line taken
from Verdoucq et al., 2008); and plasma membrane vesicles from
Arabidopsis suspension cells (dotted line from Gerbeau et al.,
2002).
activity. Using inside-out and right-side out vesicles, Alleva
et al. (2006) showed that AQP activity is likely to be
dependent on the concentration of Ca2+ in the symplast,
rather than the apoplast. Since these experiments were
carried out on isolated plasma membrane vesicles, it may
imply a direct effect of Ca2+ on the cytosolically localized
residues of the AQP proteins; however, membrane-bound
Ca2+-dependent protein kinases and phosphatases cannot
be ruled out. It has been suggested that AQPs in plant
membranes may undergo Ca2+-dependent phosphorylation,
which can increase their water channel activity (Maurel
et al., 1995; Johansson et al., 1998; Johanson and Gustavsson,
2002). Structural analysis of SoPIP2;1 AQP in the open and
closed configuration indicates that direct Ca2+ binding is
involved in AQP gating (Hedfalk et al., 2006; TornrothHorsefield et al., 2006). Evidence for a direct role for Ca
and other divalent binding involving specific residues in
AtPIP2;1 has been obtained by reconstitution of the purified
protein into liposomes (Verdoucq et al., 2008; Fig. 2). The
water permeability was 50% inhibited by 4.2 lM Ca2+
which was higher than that observed on native plasma
membrane vesicles by Alleva et al. (2006). Also in contrast
to the Alleva study was the lack of any apparent cooperativity in the gating by Ca2+ at very low concentrations. These differences may indicate a more complex
situation in the native membrane that may involve other
proteins interacting with multiple AQP isoforms.
A proposed model for calcium distribution
and water flow within leaves
It has been demonstrated that both the rate and the
concentration at which Ca2+ in the transpiration stream is
delivered to leaves are likely to be affected by the pathway
of water flow to and through leaves. The rate of water
movement through leaves will be dominated by stomatal
conductance, but the route that water takes through the leaf
is likely to be affected by interactions between water and
Ca2+ transport through the apoplast and across cells that lie
between the xylem and the sites of evaporation. It is
probable that symplastic flow of Ca2+ is minimal in leaves,
as demonstrated by Ca deficiency symptoms in organs with
low transpiration. Therefore, the movement of Ca2+ relies
upon the rate of water movement and the Ca2+-binding
capacity (absorption–desorption) of the intervening cells,
but at the same time Ca2+ can affect water transport.
A model proposing the regulation of water flow by Ca2+
within and around leaf cells is presented in Fig. 3. The role
of stomatal conductance in the model has not been incorporated at this stage as interest was focused on the
internal hydraulic conductance and pathways in the leaf,
not the overall rate. Furthermore, uncoupling of leaf
hydraulic conductivity from stomatal closure has recently
been shown. A decrease in leaf hydraulic conductivity has
been correlated with a reduction in the activity of AQPs
solely in the bundle sheath cells by ABA, not the mesophyll,
a mechanism that is independent of stomatal closure
Water and calcium interactions | 2243
Fig. 3. Proposed calcium distribution and pathway of water flow through leaves. Left panel, low transpiration and low [Ca2+]apo; right
panel, high transpiration and high [Ca2+]apo. (1A) When [Ca2+]apo is low during low transpiration, water may preferentially enter and pass
through cells as [Ca2+]cyt is low and AQPs are predominantly in the open state; water may also pass directly across the membrane (black
dashed arrow). (1B) At the same time, Ca2+ is convected to the cell membrane with water where it is either taken up into the cell by
specific Ca2+ channels or (1C) accumulates around the cell in the WFS (blue dotted arrow). (2) When there are high rates of delivery of
Ca2+ to the leaf, e.g. high transpiration with abundant Ca2+ in the growth medium (White and Broadley, 2003), this may also initiate
Ca2+-induced Ca2+ release and will increase the [Ca2+]cyt in those cells controlling entry of water to the symplast. (3) Elevated [Ca2+]cyt
shuts AQPs, reduces the hydraulic conductivity of the leaf (Levin et al., 2007), and diverts water flow around the cell via the apoplast,
which may prevent excessive build up of [Ca2+]apo. (4) This diversion advects apoplastic Ca2+ along specific apoplastic routes of water
flow that are different in eudicot and grass leaves (Fig. 1B). This directs the Ca2+ to mesophyll cells in eudicots and to epidermal cells in
grasses for storage. Note that for both eudicots and grasses the bundle sheath cells, which are proposed to be important sites of water
entry to the symplast, do not accumulate Ca2+ (see Fig. 1B; Conn and Gilliham, 2010). (5) [Ca2+]cyt will decrease through transport of
Ca2+ into the vacuole or back into the apoplast, Ca2+ inhibition of AQPs will be relieved, and water entry into the cell wall increases. This
may result in oscillations in hydraulic conductivity that match those in stomatal aperture and turgor (Li et al., 2004; Zimmermann et al.,
2010). During long periods of high transpiration there is also likely to be transcriptional control of AQP activity (Morillon and Chrispeels,
2001; Aroca et al., 2006; Postaire et al., 2010). However, when the rate of xylem delivery of Ca2+ falls, when transpiration decreases,
symplastic flow of water will again predominate (1A).
(Cohen et al., 2011). However, the identity of the responsive
AQPs and the mechanism of down-regulation is as yet
unknown—but may well be Ca2+ related as ABA induces
Ca2+ entry into cells associated with the vasculature
(Gilliham and Tester, 2005). This has further implications
for the model as when the ABA concentration in the xylem
increases in conditions of stress, then water flow into the
leaf is decreased and [Ca2+]apo increases, which will further
affect AQP activity and close stomata. It is also likely that
patchiness of stomatal opening will lead to differences in the
transpiration draw of Ca2+, which may be interesting to
investigate with respect to the dynamics of apoplastic [Ca2+]
in a leaf.
The following model is proposed (Fig. 3), where the
degree of water entry into the symplast is responsive to
[Ca2+]apo. Increases in [Ca2+]apo will increase [Ca2+]cyt; these
changes in [Ca2+]cyt are likely to oscillate to maintain cell
viability (Allen et al., 2001). Stomatal aperture also
oscillates, is sensitive to [Ca2+]apo, and is proposed to be
regulated too by AQP sensitivity to [Ca2+]cyt (Li et al.,
2004); oscillations in turgor also occur with a similar period
(Zimmermann et al., 2010). A common factor in all these is
[Ca2+]apo, and therefore the delivery of apoplastic Ca2+ and
Ca2+ storage in the leaf could underpin all these phenomenon and potentially oscillations in hydraulic conductivity.
This model predicts that cells that accumulate high
[Ca2+]vac have a reduced capacity to flush Ca2+ from the
surrounding apoplast and have AQPs that may be desensitized to elevated [Ca2+]cyt. The net effect of these processes
would be a regulation of [Ca2+]apo and the distribution of
[Ca2+]vac in different cell types that may partially reflect the
pathway of water flow. For instance, the higher capacity of
mesophyll cells compared with epidermal cells of Arabidopsis
to store Ca in their vacuoles, by virtue of the higher
expression of the Ca2+/H+ antiporter on the tonoplast
membrane (CAX1), correlates with these cells being
exposed to Ca2+ upon unloading from the xylem apoplast
(Conn et al., 2011a). Alternatively, in plants that accumulate Ca in the epidermis, water flow may deliver Ca2+
initially around these cells (Fig. 1B). Increasing the rate of
movement and dispersal of Ca2+ through the apoplast by
closure of AQPs decreases the dependency of the distribution on diffusion (some 100–1000 times slower in the
apoplast than in water) and increases the effective membrane surface area over which Ca2+ can be accumulated
from the transpiration stream by cells that have the capacity
to store Ca2+ in their vacuoles. Ultimately a dynamic
equilibrium will be established or regular oscillations will
occur that balance the relative flow of water via the cell-tocell pathway and apoplastic pathway depending on the
2244 | Gilliham et al.
[Ca2+]apo, and the capacity of the cells to sequester Ca2+ in
the vacuole. The model has been simplified for clarity. In
reality, the interaction mechanism among the entities may
be more complex and involve other signalling molecules,
including ABA delivered in the xylem.
Bramley H, Turner NC, Turner DW, Tyerman SD. 2009. Roles of
morphology, anatomy, and aquaporins in determining contrasting
hydraulic behavior of roots. Plant Physiology 150, 348–364.
Acknowledgements
Briggs GE, Robertson RN. 1957. Apparent free space. Annual
Review of Plant Physiology and Plant Molecular Biology 8, 11–30.
Work in our Laboratory is funded by the Australian
Research Council and by the Waite Bequest of the
University of Adelaide. We thank Sue Tyerman for rigorous
editing of the manuscript.
Brodribb TJ, Holbrook NM. 2006. Declining hydraulic efficiency as
transpiring leaves desiccate: two types of response. Plant, Cell and
Environment 29, 2205–2215.
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