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Calcium is an essential plant nutrient. It is required for various structural roles in the cell wall and membranes, it is a counter-cation for inorganic and organic anions in the vacuole, and the cytosolic Ca2+ concentration ([Ca2+]cyt) is an obligate intracellular messenger coordinating responses to numerous developmental cues and environmental challenges. This article provides an overview of the nutritional requirements of different plants for Ca, and how this impacts on natural flora and the Ca content of crops. It also reviews recent work on (a) the mechanisms of Ca2+ transport across cellular membranes, (b) understanding the origins and specificity of [Ca2+]cyt signals and (c) characterizing the cellular [Ca2+]cyt-sensors (such as calmodulin, calcineurin B-like proteins and calcium-dependent protein kinases) that allow plant cells to respond appropriately to [Ca2+]cyt signals.
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doi:10.1093/aob/mcg164, available online at www.aob.oupjournals.org
REVIEW ARTICLE
Calcium in Plants
PHILIP J. WHITE* and MARTIN R. BROADLEY
Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK
Received: 7 January 2003 Returned for revision: 26 March 2003 Accepted: 6 June 2003 Published electronically: 21 August 2003
Calcium is an essential plant nutrient. It is required for various structural roles in the cell wall and membranes,
it is a counter-cation for inorganic and organic anions in the vacuole, and the cytosolic Ca
2+
concentration
([Ca
2+
]
cyt
) is an obligate intracellular messenger coordinating responses to numerous developmental cues and
environmental challenges. This article provides an overview of the nutritional requirements of different plants
for Ca, and how this impacts on natural ¯ora and the Ca content of crops. It also reviews recent work on (a) the
mechanisms of Ca
2+
transport across cellular membranes, (b) understanding the origins and speci®city of
[Ca
2+
]
cyt
signals and (c) characterizing the cellular [Ca
2+
]
cyt
-sensors (such as calmodulin, calcineurin B-like
proteins and calcium-dependent protein kinases) that allow plant cells to respond appropriately to [Ca
2+
]
cyt
signals. ã 2003 Annals of Botany Company
Key words: Arabidopsis, ATPase, calcium (Ca
2+
), channel, cytosolic Ca
2+
, ecology, H
+
/Ca
2+
-antiport (CAX), kinase,
phylogeny, plasma membrane, root, vacuole.
INTRODUCTION
Calcium is an essential plant nutrient. As the divalent cation
(Ca
2+
), it is required for structural roles in the cell wall and
membranes, as a counter-cation for inorganic and organic
anions in the vacuole, and as an intracellular messenger in
the cytosol (Marschner, 1995). Calcium de®ciency is rare in
nature, but excessive Ca restricts plant communities on
calcareous soils. Calcium is taken up by roots from the soil
solution and delivered to the shoot via the xylem. It may
traverse the root either through the cytoplasm of cells linked
by plasmodesmata (the symplast) or through the spaces
between cells (the apoplast). The relative contributions of
the apoplastic and symplastic pathways to the delivery of Ca
to the xylem are unknown (White, 2001). However, the
movement of Ca through these pathways must be ®nely
balanced to allow root cells to signal using cytosolic Ca
2+
concentration ([Ca
2+
]
cyt
), control the rate of Ca delivery to
the xylem, and prevent the accumulation of toxic cations in
the shoot.
Calcium enters plant cells through Ca
2+
-permeable ion
channels in their plasma membranes (White, 2000). Since a
high [Ca
2+
]
cyt
is cytotoxic, a submicromolar [Ca
2+
]
cyt
is
maintained in unstimulated cells by Ca
2+
-ATPases and H
+
/
Ca
2+
-antiporters (Sze et al., 2000; Hirschi, 2001). These
enzymes remove cytosolic Ca
2+
to either the apoplast or the
lumen of intracellular organelles, such as the vacuole or
endoplasmic reticulum (ER). The rapid in¯ux of Ca
2+
through cation channels in the plasma membrane, tonoplast
and/or ER generates [Ca
2+
]
cyt
perturbations that initiate
cellular responses to a diverse range of developmental cues
and environmental challenges (White, 2000; Sanders et al.,
2002). Proteins that change conformation or catalytic
activity upon binding Ca
2+
, such as calmodulin (CaM),
calcineurin B-like proteins (CBLs) and Ca
2+
-dependent
protein kinases (CDPKs), allow the cellular perception and
transduction of the [Ca
2+
]
cyt
signal. These proteins are
termed `[Ca
2+
]
cyt
sensors'. It is speculated that cellular
responses to speci®c biotic and abiotic stimuli are encoded
by distinct [Ca
2+
]
cyt
perturbations and are transduced by
particular [Ca
2+
]
cyt
sensors. Much current work on Ca in
plants is dedicated to understanding the nature and
speci®city of [Ca
2+
]
cyt
signalling and response networks.
This article provides an overview of recent work on Ca in
plants. First, it discusses the Ca requirements of different
plant species, the mechanisms of Ca uptake and delivery to
the xylem, and the impact of these on natural ¯ora and the
Ca content of crops. It then highlights the insights made
possible by recent advances in electrophysiology, micro-
scopy and plant molecular biology that have enabled
researchers to characterize Ca
2+
transporters in cellular
membranes, begin to unravel the origins and speci®city of
[Ca
2+
]
cyt
signals, and identify the [Ca
2+
]
cyt
-sensors that
allow plant cells to respond to [Ca
2+
]
cyt
perturbations.
NUTRITION
The calcium requirements of plants
Plants growing with adequate Ca in their natural habitats
have shoot Ca concentrations between 0´1 and 5 % d. wt
(Marschner, 1995). These values re¯ect both Ca availability
in the environment and the contrasting Ca requirements of
different plant species. Calcium de®ciency is rare in nature,
but may occur on soils with low base saturation and/or high
levels of acidic deposition (McLaughlin and Wimmer,
1999). By contrast, several costly Ca-de®ciency disorders
occur in horticulture (Fig. 1; Shear, 1975). These generally
arise when suf®cient Ca is momentarily unavailable to
developing tissues. De®ciency symptoms are observed
* For correspondence. Fax 01789 470552, e-mail philip-j.white@
hri.ac.uk
Annals of Botany 92/4, ã Annals of Botany Company 2003; all rights reserved
Annals of Botany 92: 487±511, 2003
(a) in young expanding leaves, such as in `tipburn' of leafy
vegetables, (b) in enclosed tissues, such as in `brown heart'
of leafy vegetables or `black heart' of celery, or (c) in tissues
fed principally by the phloem rather than the xylem, such as
in `blossom end rot' of watermelon, pepper and tomato fruit,
`bitter pit' of apples and `empty pod' in peanut. They occur
because Ca cannot be mobilized from older tissues and
redistributed via the phloem. This forces the developing
tissues to rely on the immediate supply of Ca in the xylem,
which is dependent on transpiration. Transpiration is low in
young leaves, in enclosed tissues and in fruit. Other
physiological disorders, such as `cracking' in tomato,
cherry and apple fruit, occur in tissues lacking suf®cient
Ca upon hypo-osmotic shock (following increased humidity
or rainfall), presumably as a result of structural weaknesses
in cell walls. When excessive Ca is present in the
rhizosphere solution, plants may suffer Ca toxicity. This
may prevent the germination of seeds and reduce plant
growth rates (Fig. 2). In cultivated tomato, one symptom of
excess calcium is the development of tiny yellowish ¯ecks
or `gold spot' in the cell walls around the calyx and
shoulders of the fruit (Fig. 1). These ¯ecks are crystals of
calcium oxalate and their abundance is increased by high
humidity and high Ca fertilization (Bekreij et al., 1992).
Ecologists have classi®ed plant species into calcifuges,
which occur on acid soils with low Ca, and calcicoles, which
occur on calcareous soils. The Ca concentrations in
calcifuge and calcicole plants growing in their natural
habitats differ markedly. However, it is the ability to tolerate
excessive Al, Mn and Fe that largely determines the ¯ora of
acid soils, and an insensitivity to Fe- and P-de®ciencies that
determines the ¯ora of calcareous soils (Lee, 1999).
Nevertheless, calcifuges generally grow well at low Ca
2+
concentrations in the rhizosphere ([Ca
2+
]
ext
) and respond
little to increased [Ca
2+
]
ext
, which may even inhibit growth
(Fig. 2). Conversely, the mechanisms that enable calcicole
plants to maintain low [Ca
2+
]
cyt
in their natural habitat are
believed to restrict their growth at low [Ca
2+
]
ext
by inducing
Ca-de®ciency (Fig. 2; Lee, 1999). This is consistent with the
phenotype of plants overexpressing Ca
2+
-transporters that
remove Ca
2+
from the cytoplasm to the vacuole which show
Ca-de®ciency symptoms at low [Ca
2+
]
ext
(Hirschi, 2001).
Hence, the optimal [Ca
2+
]
ext
for a plant in hydroponics often
approximates the [Ca
2+
]
ext
of its natural habitat.
Distinct relationships between shoot Ca concentration
([Ca]
shoot
) and [Ca
2+
]
ext
have been attributed to contrasting
Ca `physiotypes' (Kinzel and Lechner, 1992). The ecology
of different Ca physiotypes is thought to re¯ect their ability
to utilize Ca as an osmoticum in primarily xerophytic,
calcareous environments. One characteristic of calcicole
plants, such as the Crassulaceae, Brassicaceae and
Fabaceae, is a high soluble Ca concentration. In these
F IG. 1. Calcium disorders in horticultural crops: (a) cracking in tomato fruit; (b) tipburn in lettuce; (c) calcium de®ciency in celery; (d) blossom end
rot in immature tomato fruit; (e) bitter pit in apples; (f) gold spot in tomato fruit with calcium oxalate crystals (inset). Photographs (A±E) are from the
HRI collection and (F) is courtesy of Lim Ho (HRI-Wellesbourne).
488 White and Broadley Ð Calcium in Plants
plants, which are also termed `calciotrophs' (Kinzel, 1982),
Ca accumulation is stimulated greatly by increasing
[Ca
2+
]
ext
. By contrast, calcifuges generally have a low
soluble Ca concentration. They include the `potassium
plants', such as the Apiales and Asterales, which are
characterized by high shoot K/Ca quotients, and the `oxalate
plants', which have high tissue oxalate concentrations.
Plants that accumulate oxalate can be subdivided into
species that contain soluble oxalate and those in which Ca-
oxalate is precipitated. Interestingly, the uptake of Ca does
not appear to increase with increasing [Ca
2+
]
ext
in plants
containing soluble oxalate, such as the Oxalidaceae (Kinzel
and Lechner, 1992). In plants that precipitate Ca-oxalate
such as the Caryophyllales families Caryophyllaceae,
Chenopodiaceae and Polygonaceae, there is a proportional
increase in both Ca and oxalate concentration with
increasing [Ca
2+
]
ext
(Libert and Franceschi, 1987; Kinzel
and Lechner, 1992).
Calcium uptake and movement to the shoot
Calcium is acquired from the soil solution by the root
system and translocated to the shoot via the xylem. The Ca
¯ux to the xylem is high, and a rate of 40 nmol Ca h
±1
g
±1
f.
wt root is not unreasonable in an actively growing plant
(White, 1998). The delivery of Ca to the xylem is restricted
to the extreme root tip and to regions in which lateral roots
are being initiated (Clarkson, 1993; White, 2001). In these
regions a contiguous, Casparian band between endodermal
cells is absent or disrupted, and/or the endodermal cells
surrounding the stele are unsuberized. The Casparian band
restricts the apoplastic movement of solutes (Clarkson,
1984, 1993; White, 2001) and suberization prevents Ca
2+
in¯ux to endodermal cells (Moore et al., 2002). These
observations suggest that Ca might reach the xylem solely
via the apoplast in regions where the Casparian band is
absent or disrupted, or circumvent the Casparian band by
entering the cytoplasm of unsuberized endodermal cells
when the Casparian band is present (Clarkson, 1984, 1993;
White 2001). These are referred to as the apoplastic and
symplastic pathways, respectively.
Each pathway of Ca movement across the root confers
distinct advantages and disadvantages. The apoplastic
pathway allows Ca to be delivered to the xylem without
impacting on the use of [Ca
2+
]
cyt
for intracellular signalling
(White, 1998). Intracellular signalling requires [Ca
2+
]
cyt
to
be maintained at submicromolar levels in the resting cell
and to increase rapidly in response to developmental cues or
environmental challenges. Since the Ca
2+
¯uxes required for
[Ca
2+
]
cyt
signalling are minute compared with those
required for adequate nutrition, both these requirements
for [Ca
2+
]
cyt
signalling might be compromised by high
nutritional Ca
2+
¯uxes through root cells (White, 2001).
However, the Ca ¯ux to the xylem through the apoplastic
pathway is in¯uenced markedly by transpiration, which
could lead to vagaries in the amount of Ca supplied to the
shoot and the development of Ca disorders (Marschner,
1995; McLaughlin and Wimmer, 1999). Furthermore, the
apoplastic pathway is relatively non-selective between
divalent cations (White, 2001; White et al., 2002b), and
its presence could result in the accumulation of toxic solutes
in the shoot. By contrast, the symplastic pathway allows the
plant to control the rate and selectivity of Ca transport to the
shoot (Clarkson, 1993; White, 2001). It is thought that Ca
2+
enters the cytoplasm of endodermal cells through Ca
2+
-
permeable channels on the cortical side of the Casparian
band, and that Ca
2+
is pumped from the symplast by the
plasma membrane Ca
2+
-ATPases or Ca
2+
/H
+
-antiporters of
cells within the stele. By regulating the expression and
activity of these transporters, Ca could be delivered
selectively to the xylem at a rate consistent with the
requirements of the shoot.
Several lines of evidence suggest that both apoplastic and
symplastic pathways contribute to Ca delivery to the xylem.
First, since Ca is delivered to the xylem in regions of the
root where the Casparian band is fully developed and
F IG. 2. The relationships between Ca concentration in the soil solution
([Ca
2+
]
ext
) and (A) shoot dry weight and (B) shoot Ca content of two
calcifuge species Juncus squarrosus (®lled circles) and Nardus stricta
(®lled triangles), the mineral-tolerant Siegelingia decumbens (open
circles), and the calcicole species Origanum vulgare (®lled squares).
Plants were grown for approx. 4 weeks after germination in a quartz sand
to which was added a complete mineral solution containing various
concentrations of CaCl
2
. Data are taken from Jefferies and Willis (1964),
who observed (a) that Juncus squarrosus did not establish at [Ca
2+
]
ext
>
0´8 m
M and (b) that Origanum vulgare showed symptoms of Ca
de®ciency and was unable to survive at [Ca
2+
]
ext
less than about 0´8 mM.
White and Broadley Ð Calcium in Plants 489
apoplastic Ca transport is restricted, some Ca might bypass
the Casparian band through the cytoplasm of endodermal
cells (Clarkson, 1984, 1993; White, 2001). Secondly,
although Ca
2+
channels and Ca
2+
-ATPases are present and
thermodynamically capable of catalysing Ca
2+
in¯ux and
ef¯ux across the plasma membrane of root endodermal
cells, it has been calculated that there is insuf®cient ATP to
power (Flowers and Yeo, 1992) and insuf®cient proteins to
catalyse (White, 1998, 2001) the observed Ca
2+
¯uxes solely
through the symplast. Thirdly, if Ca reached the xylem
solely by a symplastic pathway, its accumulation in the
shoot would be expected to show the hallmarks of protein-
catalysed transport, which it does not. For example, both
Ca
2+
channels in the plasma membrane of root cells and
Ca
2+
-ATPases discriminate between divalent cations, but
there seems to be no discrimination between Ca
2+
,Ba
2+
and
Sr
2+
in their transport to the shoot (White, 2001).
Furthermore, there seems to be no competition, or inter-
actions, between Ca
2+
,Ba
2+
and Sr
2+
during their transport
to the shoot, and the accumulation of divalent cations in the
shoot is often linearly related to their concentrations in the
rhizosphere solution (White, 2001). Although the relative
contributions of the apoplastic and symplastic pathways to
the delivery of Ca to the xylem are unknown, it is likely that
a functional separation of apoplastic Ca
2+
¯uxes (for transfer
to the shoot) and symplastic Ca
2+
¯uxes (for cell signalling)
would enable the root to ful®l the demand of the shoot for
Ca without compromising intracellular [Ca
2+
]
cyt
signals.
The Ca concentration in xylem sap ([Ca]
xylem
)is
in¯uenced greatly by [Ca
2+
]
ext
, and [Ca]
xylem
between
300 m
M and 16´5 mM has been reported (White et al.,
1992; De Silva et al., 1998). The relative proportion of Ca
2+
to total Ca also varies, with organic acids, such as malate
and citrate, chelating Ca
2+
in the xylem sap (Marschner,
1995). When abundant Ca is present in the xylem sap, there
is a close relationship between Ca distribution to the shoot
and transpiration. Within the leaf, Ca follows the apoplastic
route of the transpiration stream and accumulates in either
the mesophyll cells, trichomes or epidermal cells adjacent to
guard cells, depending on the plant species (Karley et al.,
2000). Both the [Ca
2+
]
cyt
in guard cells and the closing of
stomata in detached epidermal strips are sensitive to
apoplastic Ca
2+
concentrations within the range of
[Ca
2+
]
xylem
(McAinsh et al., 1995) and the ability of some
calcicole species, such as Leontodon hispidus and
Centaurea scabiosa, to tolerate high [Ca
2+
]
ext
may be
related to their ability to accumulate Ca in their trichomes
(De Silva et al., 1996, 1998).
The phylogeny of shoot calcium concentration
When grown under identical conditions, the [Ca]
shoot
of
different plant species differs markedly (Figs 2 and 3 and
Table 1; Broadley et al., 2003a). A large proportion of this
variation can be attributed to the phylogenetic division
between eudicots and monocots (Thompson et al., 1997;
Broadley et al., 2003a). Eudicots generally have a higher
[Ca]
shoot
than monocots. Within the eudicots, orders within
both the rosid (Cucurbitales, Rosales, Malvales and
Brassicales) and asterid (Apiales, Asterales, Lamiales and
Solanales) clades have the highest [Ca]
shoot
. Within the
monocots, [Ca]
shoot
is higher in the non-commelinoid orders
(e.g. Asparagales) than in the commelinoid orders (e.g.
Poales). Phylogenetic differences in [Ca]
shoot
have not been
resolved at lower taxonomic levels, but it is noteworthy that
distinct Ca physiotypes (`calciotrophes', `potassium plants'
and `oxalate plants') occur in particular plant families
(Kinzel, 1982) and that traits such as the prevalence, shape
and tissue distributions of Ca oxalate crystals, are used as
taxonomic characters (e.g. Franceschi and Horner, 1980;
Kuo-Huang et al., 1994; Wu and Kuo-Huang, 1997; Prychid
and Rudall, 1999; Caddick et al., 2002).
Although the Ca physiotype of a plant determines its
ability to accumulate Ca in the shoot (Kinzel and Lechner,
1992; Broadley et al., 2003a), genotypic differences in the
activities of Ca
2+
transporters in root cell membranes
(Hirschi, 2001; White et al., 2002a) and/or in the relative
contributions of symplastic and apoplastic pathways to the
delivery of Ca to the xylem (White, 2001) will contribute
much to phylogenetic variation in [Ca]
shoot
. Historically,
[Ca]
shoot
has been correlated with the cation exchange
capacity (CEC) of plant roots (Fig. 3). The CEC is located in
the root apoplast, and is attributed to the free carboxyl
groups of galacturonic acids of cell wall pectins in the
middle lamella (Haynes, 1980; Sattelmacher, 2001). If the
pectin contents of shoot and root cell walls are similar, it is
unsurprising that the phylogenetic variation in CEC in
monocot roots parallels that in pectin content of shoot cell
walls (Jarvis et al., 1988). The pectin contents of shoot cell
walls are low in the Cyperaceae, Poaceae, Juncaceae and
Restionaceae families (all assigned to the Poales) and
intermediate in the commelinoid monocot families
Commelinaceae (Commelinales), Bromeliaceae (unas-
signed to order) and Typhaceae (Poales) and in the non-
commelinoid Pandanaceae (Pandanales) family. The pectin
content of shoot cell walls is similar to that of dicots in the
F IG. 3. The relationships between root CEC, derived from a literature
survey, and shoot calcium concentration, derived from both literature and
experimental data, for angiosperm orders (Table 1). Circles represent
shoot Ca content data from a literature survey. Filled circles and linear
regression represent orders with n > 3 species sampled. Triangles
represent shoot Ca content estimated in a phylogenetically balanced
experiment.
490 White and Broadley Ð Calcium in Plants
commelinoid monocot families Musaceae (Zingiberales)
and Sparganiaceae (Poales) and in the non-commelinoid
monocot families Velloziaceae (Pandanales), Alliaceae
Amaryllidaceae, Asphodelaceae, Iridaceae, Orchidaceae
(all Asparagales), Alismataceae, Araceae, Juncaginaceae,
Potamogetonaceae, Zosteraceae (all Alismatales) and
Liliaceae (Liliales).
In regions of the root where the Ca ¯ux to the xylem is
apoplastic, CEC may exert a direct effect on the transport of
Ca
2+
to the shoot. However, several reviews have asserted
that there is no direct connection between the capacity of
roots to bind cations in the free space and their active
(energy-dependent) accumulation (e.g. Haynes, 1980).
Nevertheless, ®xed negative charges, and charge screening,
can in¯uence both the absolute and relative concentrations
of cations in the apoplast, especially at low ionic activities.
Thus, root CEC could determine shoot cation content
indirectly by affecting the rate and selectivity of cation
uptake into the symplast in addition to cation transport
through the apoplast (Asher and Ozanne, 1961; Wacquant,
1977). The effects of CEC on cation transport may have
ecological implications. It has been suggested that, in
solutions of low ionic strength, plants with higher root CEC
compete for divalent cations more effectively, whereas
plants with lower root CEC compete for monovalent cations
more effectively (Smith and Wallace, 1956; Asher and
Ozanne, 1961).
Quantifying the phylogenetic impact on [Ca]
shoot
has
several uses. First, since [Ca]
shoot
correlates with ecological
traits (Kinzel, 1982; Thompson et al., 1997; Grime, 2001),
predictions of the responses of plant communities to
environmental change can be improved using this informa-
tion. Secondly, knowledge of phylogenetic variation in
[Ca]
shoot
can be used to predict the movement of Ca (and
chemically similar elements such as Sr, Mg and Ba; White,
2001; Broadley et al., 2003b) from soils to the shoots of
plant species whose transfer coef®cients are unknown and,
thereby, improve nutrient and contaminant cycling models
(Broadley et al., 2001). Thirdly, appreciation of phylo-
genetic in¯uences on [Ca]
shoot
can improve the delivery of
Ca to the human diet. Unsurprisingly, Ca-de®ciency
disorders have been observed in populations whose dietary
habits have changed from bean-rich to rice-rich sources of
food (Graham et al., 2001). Phylogenetic information can
help identify crops that are either predisposed to higher
[Ca]
shoot
or offer genetic potential for breeding.
TABLE 1. The mean relative shoot calcium concentration of angiosperm orders derived from both a literature survey and a
phylogenetically balanced experiment, and their mean relative root cation exchange capacity (CEC)
Literature Literature Experiment
Order
Root CEC
(meq 100 g
±1
d. wt root)
No.
spp.
Relative shoot
[Ca]
No.
spp.
Relative shoot
[Ca]
No.
spp.
Apiales 40´67 3 1´90 5 1´21 4
Aquifoliales 27´28 1 ± ± ± ±
Asparagales 27´08 6 1´22 4 1´10 13
Asterales 40´34 12 1´78 11 1´19 15
Brassicales 36´39 7 2´19 15 2´96 3
Caryophyllales 32´12 12 1´57 17 1´07 7
Cucurbitales 43´35 4 3´19 2 3´21 1
Dipsacales 35´01 1 ± ± 1´04 2
Ericales 22´54 2 ±0´01 1 1´04 1
Fabales 33´03 23 1´53 78 1´66 9
Gentianales 32´21 1 ± ± 1´36 4
Geraniales 28´41 4 1´53 2 ± ±
Lamiales 25´96 2 1´72 5 1´84 7
Laurales ±2´07 1 ± ± ± ±
Liliales 24´06 2 ± ± ± ±
Malpighiales 23´18 1 1´12 2 1´07 5
Malvales 34´91 1 3´04 2 2´50 3
Myrtales 26´21 1 0´70 1 1´13 6
Poales 14´94 50 0´37 48 0´43 18
Ranunculales 55´42 4 1´69 2 ± ±
Rosales 22´54 4 2´73 1 1´25 3
Sapidales 17´98 6 ± ± 1´29 4
Solanales 31´22 4 1´73 8 1´60 4
Vitaceae 15´67 2 ± ± ± ±
Data for shoot Ca concentration were taken from Broadley et al. (2003a) and for root CEC from the comparative studies cited by Asher and Ozanne
(1961), Heintz (1961), Crooke and Knight (1962), Fre
Â
jat et al. (1967), Snaydon and Bradshaw (1969) or Wacquant (1977).
All literature data sets were subjected to a residual maximum likelihood (REML) analysis, using a procedure described previously (Broadley et al.,
2001). This procedure adjusted for differences in between-study means to generate mean relative shoot Ca concentration (206 plant species in 18
orders from 244 studies) and mean relative root CEC (154 plant species from 93 studies).
Data are expressed as order means from n species sampled.
White and Broadley Ð Calcium in Plants 491
CALCIUM TRANSPORTERS IN CELLULAR
MEMBRANES
Ca
2+
ef¯ux from the cytosol: Ca
2+
-ATPases and H
+
/Ca
2+
-
antiporters
The removal of Ca
2+
from the cytosol against its electro-
chemical gradient to either the apoplast or to intracellular
organelles requires energized, `active' transport. This is
catalysed by Ca
2+
-ATPases and H
+
/Ca
2+
-antiporters (Fig. 4).
By removing Ca
2+
from the cytosol these enzymes perform
several important functions (Sze et al., 2000; Hirschi,
2001): (1) they maintain a low [Ca
2+
]
cyt
in the resting
(unstimulated) cell appropriate for cytoplasmic metabolism;
(2) they restore [Ca
2+
]
cyt
to resting levels following a
[Ca
2+
]
cyt
perturbation, thereby in¯uencing the magnitude,
kinetics and subcellular location of [Ca
2+
]
cyt
signals; (3)
they replenish intracellular and extracellular Ca
2+
stores for
subsequent [Ca
2+
]
cyt
signals and permit the generation of
local [Ca
2+
]
cyt
oscillations through their interplay with Ca
2+
channels (Klu
È
sener et al., 1995; Harper, 2001); (4) they
provide Ca
2+
in the ER for the secretory system to function
(Blatt, 2000b; Ritchie et al., 2002); (5) they remove divalent
cations, such as Mg
2+
,Mn
2+
,Ni
2+
or Zn
2+
, from the cytosol,
to support the specialized biochemistry of particular
organelles and to prevent mineral toxicities (Hirschi,
2001; Wu et al., 2002). The relative importance of Ca
2+
-
ATPases and H
+
/Ca
2+
-antiporters in each of these functions
is unknown. Hirschi (2001) suggested that the Ca
2+
-
ATPases, which have high af®nity (K
m
= 1±10 mM; Evans
and Williams, 1998) but low capacity for Ca
2+
transport, are
F IG. 4. Cartoon illustrating the subcellular location of Ca
2+
transporters in Arabidopsis thaliana based on Sze et al. (2000), Sanders et al. (2002) and
White et al. (2002). In the plasma membrane there are hyperpolarization-activated Ca
2+
channels (HACC, possibly encoded by annexin genes),
depolarization-activated Ca
2+
channels (DACC, one of which may be encoded by TPC1), Ca
2+
-permeable outward rectifying K
+
channels (KORC,
encoded by SKOR and GORK), voltage-insensitive cation channels (VICC, probably encoded by the CNGC and GLR genes) and Ca
2+
-ATPases (one
of which is ACA8). Biochemical and electrophysiological evidence indicates that IP
3
-receptors, IP
6
-receptors, cADPR-activated (ryanodine)-receptors
and two types of voltage-gated Ca
2+
channels (the depolarization-activated SV channels and the hyperpolarization-activated Ca
2+
channels) are present
in the tonoplast together with Ca
2+
-ATPases (including ACA4) and H
+
/Ca
2+
-antiporters encoded by the CAX genes. There is also biochemical and
electrophysiological evidence for the presence of NAADP-receptors, IP
3
-receptors, cADPR-activated (ryanodine)-receptors and depolarization-
activated Ca
2+
channels in the endoplasmic reticulum (ER), together with Ca
2+
-ATPases (including ECA1 and ACA2). The Ca
2+
-ATPase ACA1 is
located in the plastid inner membrane.
492 White and Broadley Ð Calcium in Plants
responsible for maintaining [Ca
2+
]
cyt
homeostasis in the
resting cell, whereas the H
+
/Ca
2+
-antiporters, which have
lower af®nities (K
m
= 10±15 mM) but high capacities for
Ca
2+
transport, are likely to remove Ca
2+
from the cytosol
during [Ca
2+
]
cyt
signals and thereby modulate [Ca
2+
]
cyt
perturbations. Consistent with this hypothesis is the observ-
ation that an H
+
/Ca
2+
-antiporter, but not the vacuolar Ca
2+
-
ATPase, resets [Ca
2+
]
cyt
in yeast following hypertonic shock
(Denis and Cyert, 2002). However, the Arabidopsis de-
etiolated 3 (det3) mutant, which has reduced tonoplast H
+
-
ATPase and (presumably) H
+
/Ca
2+
-antiporter activity, has a
constitutively high [Ca
2+
]
cyt
(Allen et al., 2000), suggesting
that H
+
/Ca
2+
-antiporters contribute to [Ca
2+
]
cyt
homeostasis,
and plants poisoned by the V-type H
+
-ATPase inhibitor
ba®lomycin show greater [Ca
2+
]
cyt
elevations in response to
hypo-osmotic shock (Takahashi et al., 1997).
Plant Ca
2+
ATPases belong to one of two major families
(Evans and Williams, 1998; Geisler et al., 2000; Sze et al.,
2000; Axelsen and Palmgren, 2001; Garciadeblas et al.,
2001). The ®rst family (the P-type ATPase IIA family) lacks
an N-terminal autoregulatory domain. Four members of this
family have been identi®ed in the arabidopsis genome
(termed AtECAs 1 to 4 by Axelsen and Palmgren, 2001).
They are likely to be present in the plasma membrane,
tonoplast and the ER/Golgi apparatus. In tomato, two
transcripts of a type IIA Ca
2+
-ATPase (LeLCA1) were
observed in phosphate-starved roots that correlated with two
distinct protein isoforms (120 and 116 kDa) located in the
plasma membrane and tonoplast of root cells, respectively
(Navarro-Avino et al., 1999). The second family of plant
Ca
2+
ATPases (the P-type ATPase IIB family) is character-
ized by an autoinhibitory N-terminal domain that contains a
binding site for Ca-CaM plus a serine-residue phosphoryl-
ation site. Their catalytic activity can be modulated by
[Ca
2+
]
cyt
either through activation upon binding CaM or by
inhibition following phosphorylation by Ca
2+
-dependent
protein kinases (CDPK; Hwang et al., 2000). Since CaM
binding-sites are generally quite diverse, each type-IIB
Ca
2+
-ATPase may have a different af®nity for CaM or may
bind a different CaM isoform. Ten members of the type-IIB
Ca
2+
-ATPase family have been identi®ed in the arabidopsis
genome (termed AtACAs 1, 2 and 4 and AtACAs 7 to 13 by
Axelsen and Palmgren, 2001). These reside on various
cellular membranes including the plasma membrane
(AtACA8), the tonoplast (AtACA4), ER (AtACA2) and
the plastid inner membrane (AtACA1). Several explan-
ations for the abundance of Ca
2+
-ATPase isoforms, and also
the presence of several isoforms on the same cellular
membrane (such as AtECA1 and AtACA2 in the root ER)
have been proposed (Geisler et al., 2000; Sze et al., 2000;
Axelsen and Palmgren, 2001). Such explanations typically
suggest that individual isoforms are functionally distinct
and specialized to speci®c cellular processes requiring
distinct spatial or temporal expression. They also imply a
requirement for CaM-independent and CaM-dependent
regulation of Ca
2+
-ATPase activities in the modulation of
[Ca
2+
]
cyt
perturbations during cell signalling. Multiple Ca
2+
-
ATPase genes are required to effect this because alternative
splicing events are rare in plants. Intriguingly, the expres-
sion of many Ca
2+
-ATPases is increased upon exposure to
high salinity or high [Ca
2+
]
ext
, and some Ca
2+
-ATPase genes
are expressed only under stress conditions (Geisler et al.,
2000; Garciadeblas et al., 2001). This may re¯ect a role in
maintaining [Ca
2+
]
cyt
homeostasis or in reducing Na
+
in¯ux
to the cytosol in saline environments.
The H
+
/Ca
2+
-antiporters present in the plasma membrane
and tonoplast have been characterized biochemically
(Evans and Williams, 1998; Sanders et al., 2002). These
have a lower af®nity for Ca
2+
than the Ca
2+
-ATPases, and
may also transport Mg
2+
. The stoichiometry of the dominant
H
+
/Ca
2+
-antiporter in the tonoplast is apparently 3H
+
/1Ca
2+
(Blackford et al., 1990). Eleven genes encoding putative H
+
/
Ca
2+
-antiporters (AtCAX) have been identi®ed in the
genome of Arabidopsis thaliana (Hirschi, 2001; Ma
È
ser
et al., 2001). The transporters AtCAX1, AtCAX2 and
AtCAX4 are located at the tonoplast (Hirschi, 2001; N.
Cheng et al., 2002, 2003). The AtCAX1 antiporter exhibits
both a high af®nity and high speci®city for Ca
2+
.By
contrast, the AtCAX2 transporter is a high-af®nity, high
capacity H
+
/heavy metal cation antiporter. The cation
speci®city of other AtCAXs is unknown, but may reside
in a speci®c stretch of nine amino acids termed the `Ca
2+
domain' (Shigaki et al., 2002). The increased transport
capacity of truncated versions of AtCAX1, AtCAX3 and
AtCAX4, and their relative inhibition by synthetic peptides
corresponding to these deletions, suggest that they are
subject to autoinhibition by their N-termini in an isoform-
speci®c manner (N. Cheng et al., 2002; Pittman et al.,
2002a, b). How, and whether, this regulatory mechanism
operates in vivo has yet to be revealed.
The AtCAXs have homologues in other plant species and
their physiological roles have been investigated using
transgenic plants (Hirschi, 2001). Transgenic tobacco
overexpressing AtCAX1 exhibits Ca-de®ciency disorders,
such as tipburn, metal-hypersensitivity and susceptibility to
chilling, that can be reversed by increasing Ca supply. Since
these plants have increased [Ca]
shoot
, Hirschi (2001) specu-
lated that such phenotypes resulted from depleted [Ca
2+
]
cyt
,
suggesting that the principal role of AtCAX1 was to
maintain [Ca
2+
]
cyt
homeostasis by removing excess cyto-
solic Ca
2+
to the vacuole, and noted that the expression of
AtCAX1 and AtCAX3 (but not AtCAX2 or AtCAX4) was
increased by raising Ca supply (Shigaki and Hirschi, 2000;
Hirschi, 2001; N. Cheng et al., 2002).
Ca
2+
in¯ux to the cytosol: calcium channels
Calcium-permeable channels have been found in all plant
membranes (Fig. 4). They have been classi®ed on the basis
of their voltage-dependence into depolarization-activated
(DACC), hyperpolarization-activated (HACC) and voltage-
independent (VICC) cation channels (White, 2000;
Miedema et al., 2001; Sanders et al., 2002). The presence
of diverse classes of Ca
2+
-permeable channels in a particular
membrane is thought to enable physiological ¯exibility.
The principal roles of Ca
2+
-permeable channels in the
plasma membrane appear to be in cell signalling, but they
may also contribute to nutritional Ca
2+
¯uxes in particular
cell types (White, 1998, 2000; Miedema et al., 2001).
Several types of DACCs have been observed in the plasma
White and Broadley Ð Calcium in Plants 493
membrane of plant cells (White, 1998, 2000). Although
each has distinct pharmacological and electrophysiological
properties, all are permeable to both monovalent and
divalent cations. They may therefore contribute to the
uptake of essential or toxic cations in addition to Ca
2+
. Most
DACCs activate signi®cantly at voltages more positive than
about ±150 to ±100 mV under physiological conditions
(White, 1998). The dominant DACCs in protoplasts from
arabidopsis tissues and carrot suspension cells appear to be
controlled by cytoskeletal interactions and stabilized by the
disruption of microtubules (Thion et al., 1996, 1998). It is
argued that DACCs transduce general stress-related signals
since plasma membrane depolarization is common to many
stimuli, occurs by many diverse mechanisms, and is likely
to increase [Ca
2+
]
cyt
throughout the cell periphery (White,
1998, 2000). However, speci®c roles for DACCs acting in
tandem with cytoskeletal rearrangements have been pro-
posed in the acclimation of chilling-resistant plants to low
temperatures (Mazars et al., 1997; White, 1998; Xiong et al.,
2002) and in the interactions of plants with microbes (White
et al., 2002a). The outward-rectifying K
+
channels
(KORCs) found in the plasma membrane of plant cells are
also Ca
2+
-permeable DACCs (White et al., 2002a). These
channels activate signi®cantly at voltages more positive
than about ±50 mV under most physiological conditions and
catalyse a large K
+
ef¯ux simultaneously with a small Ca
2+
in¯ux (White, 1997; Gaymard et al., 1998; De Boer, 1999;
Roberts and Snowman, 2000). The Ca
2+
in¯ux through
KORCs might increase [Ca
2+
]
cyt
to coordinate ion transport,
metabolism and gene expression. An elaborate model of
how negative feedback through [Ca
2+
]
cyt
might control the
loading of K
+
into the root xylem by KORCs has been
proposed by De Boer (1999).
Patch-clamp electrophysiological techniques have iden-
ti®ed HACCs in root cells (Kiegle et al., 2000a;Ve
Â
ry and
Davies, 2000; Foreman et al., 2003), onion epidermal cells
(Pickard and Ding, 1993), suspension-cultured tomato cells
(Blumwald et al., 1998), leaf mesophyll cells (Stoelzle et al.,
2003) and stomatal guard cells (Hamilton et al., 2000, 2001;
Pei et al., 2000; Murata et al., 2001; Perfus-Barbeoch et al.,
2002). These channels are permeable to many divalent
cations including Ba
2+
,Ca
2+
,Mg
2+
,Mn
2+
,Cd
2+
and Zn
2+
.
They activate at voltages more negative than about ±100 to
±150 mV at physiological [Ca
2+
]
cyt
, but increasing [Ca
2+
]
cyt
shifts their activation potential to more positive voltages in
root hairs (Ve
Â
ry and Davies, 2000) or more negative
voltages in guard cells (Hamilton et al., 2000). At the apex
of root hairs and pollen tubes, and in cells of the root
elongation zone, the regulation of HACCs by [Ca
2+
]
cyt
may
provide a positive feedback system to generate and maintain
the elevated [Ca
2+
]
cyt
required for cell expansion (White,
1998, 2000; Ve
Â
ry and Davies, 2000; Miedema et al., 2001;
Demidchik et al., 2002a). Mechanosensitive HACCs may
orchestrate the changes in morphology induced by gravity,
touch or ¯exure in a similar manner (Pickard and Ding,
1993; Bibikova et al., 1999). Reactive oxygen species also
increase the activity of HACCs in root hairs (Foreman et al.,
2003), guard cells (Pei et al., 2000; Murata et al., 2001;
Klu
È
sener et al., 2002; Ko
È
hler et al., 2003) and suspension-
cultured cells (Lecourieux et al., 2002) through the activity
of NADPH oxidases. Oxidative bursts have been correlated
with elevated [Ca
2+
]
cyt
, the initiation of [Ca
2+
]
cyt
waves,
elongation growth, altered gene expression and the induc-
tion of antimicrobial activities. Elicitor-activated HACCs,
such as those activated by a heterotrimeric G-protein/
protein kinase cascade in suspension-cultured tomato cells,
are thought to raise [Ca
2+
]
cyt
to initiate cellular responses to
pathogens (Blumwald et al., 1998). In guard cells, HACCs
have a central role in closing stomata during water stress.
Increasing ABA concentration shifts the activation potential
of HACCs to more positive voltages, thereby promoting
their opening, and the subsequent entry of Ca
2+
not only
depolarizes the plasma membrane but also initiates the
[Ca
2+
]
cyt
-dependent events, including [Ca
2+
]
cyt
-dependent
Ca
2+
release (CICR) from intracellular stores, that lead to
stomatal closure (Blatt, 2000a, b; White, 2000; Murata et al.,
2001; Schroeder et al., 2001; Ko
È
hler and Blatt, 2002).
Many distinct VICCs are present in the plasma membrane
of plant cells, which differ in cation selectivity, voltage-
dependence and pharmacology (White et al., 2002a;
Demidchik et al., 2002b). Nevertheless, all VICCs appear
to be permeable to both monovalent and divalent cations
(Davenport and Tester, 2000; Demidchik and Tester, 2002;
Demidchik et al., 2002a, b) and are likely to provide a
weakly voltage-dependent Ca
2+
in¯ux to cells under
physiological ionic conditions (White and Davenport,
2002; Demidchik et al., 2002a). It has been suggested that
Ca
2+
in¯ux through VICCs, which are open at physiological
voltages and are generally insensitive to cytoplasmic
modulators, is required to balance the perpetual Ca
2+
ef¯ux
through Ca
2+
-ATPases and H
+
/Ca
2+
-antiporters to maintain
[Ca
2+
]
cyt
homeostasis in an unstimulated plant cell (White
and Davenport, 2002). This hypothesis is supported both by
the pharmacology of Ca
2+
in¯ux to plant root cells and the
linear dependence of their [Ca
2+
]
cyt
on cell membrane
potential (Demidchik et al., 2002a). Furthermore, VICCs
appear to be the only Ca
2+
-permeable channels open at the
resting potential of most plant cells.
There has been some recent speculation on the identity of
genes encoding Ca
2+
-permeable channels in the plasma
membranes of plant cells (Demidchik et al., 2002b;Ve
Â
ry
and Sentenac, 2002; White et al., 2002a). It has been
suggested (a) that homologues of the arabidopsis AtTPC1
gene (Furuichi et al., 2001) encode DACCs regulated by
[Ca
2+
]
cyt
,(b) that homologues of the arabidopsis AtSKOR
(Gaymard et al., 1998) and AtGORK (Ache et al., 2000)
genes encode KORCs, (c) that the annexin genes encode
HACCs and (d) that genes from the cyclic-nucleotide gated
channel (CNGC) and glutamate receptor (GLR) families
encode VICCs. Since CNGCs have a binding site for CaM
within a C-terminus binding site for cyclic nucleotide
monophosphates, CNGC could impose a `coincidence
hierarchy' for [Ca
2+
]
cyt
signalling by integrating the
response to two different signalling cascades acting simul-
taneously. The low-af®nity cation transporter in the plasma
membrane of wheat root cells (TaLCT1) could also
facilitate Ca
2+
uptake into plant cells (Clemens et al.,
1998), but it is not known whether this protein forms an ion
channel.
494 White and Broadley Ð Calcium in Plants
Several Ca
2+
-permeable channels co-reside in the tono-
plast (Allen and Sanders, 1997; White, 2000; Sanders et al.,
2002). At least three types of pharmacologically distinct
depolarization-activated Ca
2+
-permeable channels have
been observed, of which the SV type is the most common.
The SV channels are permeable to monovalent and divalent
cations and catalyse a small Ca
2+
in¯ux to the cytoplasm
under physiological conditions (Pottosin et al., 1997, 2001;
White, 2000). They are regulated by many effectors,
suggesting that they could be pivotal in effecting the
coincidence control of [Ca
2+
]
cyt
signalling (Sanders et al.,
1999, 2002; White, 2000). Increasing [Ca
2+
]
cyt
or decreas-
ing vacuolar Ca
2+
concentration ([Ca
2+
]
vac
) promotes their
opening at physiological trans-tonoplast voltages. Their
response to [Ca
2+
]
cyt
is sensitized both by CaM and by
cytosolic Mg
2+
. Cytoplasmic alkalinization or vacuolar
acidi®cation also promotes their opening under physio-
logical conditions. The SV channels are regulated by
[Ca
2+
]
cyt
-dependent phosphorylation at two sites. Phos-
phorylation at one site is inhibitory, whereas phosphoryl-
ation at the other site activates the channel. It has been
proposed that [Ca
2+
]
cyt
-dependent regulation of SV chan-
nels might prevent an excessive rise in [Ca
2+
]
cyt
or modulate
the kinetics of changes in [Ca
2+
]
cyt
(Sanders et al., 1999).
The activity of SV channels is also reduced by 14-3-3
proteins (van den Wijngaard et al., 2001). The genes(s)
encoding SV channels are unknown, but KCO1 has been
implicated in their formation, since SV-channel currents in
mesophyll protoplasts from the arabidopsis kco1 mutant are
smaller than those from wild-type plants (Schonknecht et al.,
2002). At least two types of pharmacologically distinct
hyperpolarization-activated, Ca
2+
-permeable channels
(HACC) have been reported in plant vacuoles (Allen and
Sanders, 1997; White, 2000). These open at trans-tonoplast
voltages within the physiological range (±20 to ±70 mV)
and catalyse Ca
2+
in¯ux to the cytoplasm. Both tonoplast
HACCs show spontaneous changes in their kinetic beha-
viour, which has been interpreted as resulting from inter-
actions with (hypothetical) regulatory ligands. One type of
tonoplast HACC is inhibited by a [Ca
2+
]
cyt
above 1 mM,
whereas the other type is insensitive to [Ca
2+
]
cyt
. The
opening of the second type of HACC is promoted by
increasing [Ca
2+
]
vac
and/or vacuolar alkalinization.
Several highly selective Ca
2+
channels activated by
cytosolic second messengers (IP
3
,IP
6
or cADPR) are also
present in the tonoplast (Allen and Sanders, 1997; White,
2000; Sanders et al., 2002). All mediate Ca
2+
in¯ux to the
cytoplasm. The IP
3
-dependent Ca
2+
channels are activated
half-maximally by IP
3
concentrations as low as 200 nM.
They open at physiological trans-tonoplast voltages, and
their opening is promoted by tonoplast hyperpolarization.
They may have a role in turgor regulation in response to salt
and hyper-osmotic stresses (Allen and Sanders, 1997;
Drùbak and Watkins, 2000; DeWald et al., 2001; Xiong
et al., 2002), in nastic movements (Kim et al., 1996), in
gravitropic movements of roots (Fasano et al., 2002) and
pulvini (Perera et al., 1999), in stomatal closure (Staxe
Â
n
et al., 1999; Blatt, 2000a, b;Nget al., 2001b; Schroeder
et al., 2001; Klu
È
sener et al., 2002) and in pollen tube
elongation (Malho
Â
et al., 1998; Rudd and Franklin-Tong,
2001). They may also have roles in pollen tube self-
incompatibility (Malho
Â
et al., 1998; Rudd and Franklin-
Tong, 2001) and in plant defence responses (Mitho
È
fer et al.,
1999; Sanders et al., 1999; Blume et al., 2000). The
cADPR-dependent Ca
2+
channels also open at physiological
trans-tonoplast voltages, but are inhibited by [Ca
2+
]
cyt
greater than 600 nM (Leckie et al., 1998). The pharmacol-
ogy of plant cADPR-dependent Ca
2+
channels resembles
that of the cADPR-activated, ryanodine-receptor channels
found in the endomembranes of animal cells (Allen and
Sanders, 1997). They are activated half-maximally by 20±
40 n
M cADPR, 20 nM ryanodine or millimolar concentra-
tions of caffeine. They are inhibited by micromolar
concentrations of ruthenium red and procaine. The
cADPR-dependent Ca
2+
channels have been implicated in
ABA-signalling pathways leading to cold acclimation,
desiccation tolerance (Wu et al., 1997) and stomatal closure
(Leckie et al., 1998; Klu
È
sener et al., 2002), in circadian
[Ca
2+
]
cyt
rhythms and in the activation of plant defence
responses (Durner et al., 1998; Klessig et al., 2000).
Evidence for Ca
2+
channels activated by sub-micromolar
concentrations of IP
6
and having a role in stomatal closure is
circumstantial but persuasive (Lemtiri-Chlieh et al., 2000).
A variety of voltage-dependent and ligand-activated Ca
2+
channels have also been revealed in the ER using bio-
chemical or electrophysiological techniques (White, 2000).
Depolarization-activated Ca
2+
channels were observed
when ER vesicles from the touch-sensitive tendrils of
Bryonia dioica (BCC1) (Klu
È
sener et al., 1995) or the root
tips of Lepidium sativum (LCC1) (Klu
È
sener and Weiler,
1999) were incorporated into planar lipid bilayers. Their
activities were modulated by the Ca
2+
gradient across the
ER membrane and enhanced by cytoplasmic acidi®cation.
An interesting model for the generation of oscillations in
[Ca
2+
]
cyt
through BCC1 has been proposed (Klu
È
sener et al.,
1995). At the resting membrane potential of the ER, which
is assumed to be close to zero, and with submicromolar Ca
2+
activities in the lumen of the ER, BCC1 is likely to be
closed. However, if the lumenal Ca
2+
activity is increased,
for example by the activity of an ER Ca
2+
-ATPase, BCC1
will open to release Ca
2+
into the cytoplasm. This reduces
the lumenal Ca
2+
and the channel recloses. This cycle will
generate transient local elevations of [Ca
2+
]
cyt
. The fre-
quency of these elevations could be determined by modu-
lation of BCC1, the Ca
2+
-ATPase or the Ca
2+
gradient across
the ER. In addition to these voltage-dependent Ca
2+
channels, biochemical studies indicate the presence of
Ca
2+
channels activated by cADPR (Navazio et al., 2001),
NAADP (nicotinic acid adenine dinucleotide phosphate)
(Navazio et al., 2000) and (possibly) IP
3
(Muir and Sanders,
1997). To date, no genes encoding ER Ca
2+
channels have
been identi®ed in plants, and there appear to be no plant
genes homologous to the IP
3
-receptors, cADPR-receptors,
NAADP-receptors or endomembrane voltage-gated
channels of animals.
A hyperpolarization-activated Ca
2+
-permeable channel
has been recorded in excised patches from the nuclear
envelope of red beet cells (Grygorczyk and Grygorczyk,
1998). This channel was unaffected by changes in [Ca
2+
]
cyt
,
but when the [Ca
2+
] of the perinuclear space (the lumen of
White and Broadley Ð Calcium in Plants 495
the nuclear envelope) exceeded about 1 mM the channel
opened at physiological voltages. Based on this observation,
a role for this channel in regulating Ca
2+
-dependent nuclear
processes was proposed (Grygorczyk and Grygorczyk,
1998). Interestingly, annexin-like proteins with the potential
to form Ca
2+
-permeable cation channels have been located
at the perinuclear membrane and in the nucleolus (Clark
et al., 1998; Kova
Â
cs et al., 1998; de Carvalho-Niebel et al.,
2002).
CYTOSOLIC CALCIUM SIGNALS
The evolution of [Ca
2+
]
cyt
signalling and the [Ca
2+
]
cyt
`signature'
The [Ca
2+
]
cyt
of plant cells increases in response to many
developmental cues and environmental challenges (Table 2).
This is considered essential for producing a physiological
response. It is thought that elevating [Ca
2+
]
cyt
is a primitive,
and universal, response to stress. Sanders et al. (1999)
observed that the low solubility product of Ca
2+
and
phosphate would have necessitated a [Ca
2+
]
cyt
lower than
the [Ca
2+
] of seawater to maintain energy metabolism. They
presumed that this required the early evolution of mechan-
isms to remove Ca
2+
from the cytoplasm, and noted that a
homeostatically maintained submicromolar [Ca
2+
]
cyt
would
have been ideal for the subsequent evolution of [Ca
2+
]
cyt
signalling systems, since it would confer sensitivity and
speed to any signal. Sanders et al. (1999) also noted that the
chemistry of Ca
2+
, which can coordinate six to eight
uncharged oxygen atoms, had fortuitously made possible
the evolution of proteins that change conformation upon
binding Ca
2+
, allowing the cellular perception and transduc-
tion of a [Ca
2+
]
cyt
signal.
To effect an appropriate physiological response to a
particular stimulus, it is thought that the [Ca
2+
]
cyt
perturb-
ation (termed the [Ca
2+
]
cyt
`signature') elicited by each
developmental cue or environmental challenge is unique.
This uniqueness is manifest in the sub-cellular location and/
or the kinetics or magnitude of the [Ca
2+
]
cyt
perturbation
(McAinsh and Hetherington, 1998; Trewavas, 1999; Rudd
and Franklin-Tong, 2001). An increase in [Ca
2+
]
cyt
is
effected by Ca
2+
in¯ux to the cytosol either from the
apoplast, across the plasma membrane, or from intracellular
stores such as the ER or vacuole. This Ca
2+
in¯ux is
mediated by Ca
2+
-permeable ion channels, and their type,
cellular location and abundance will in¯uence the spatial
characteristics of [Ca
2+
]
cyt
perturbations. Since the diffusion
of Ca
2+
within the cytoplasm is low (Clapham, 1995), and
the buffering of Ca
2+
in the cytoplasm is high (0´1 to 1 mM;
Malho
Â
et al., 1998; Trewavas, 1999), the opening of a Ca
2+
channel produces a local increase in [Ca
2+
]
cyt
that dissipates
rapidly after the channel has closed. The subcellular
localization of Ca
2+
channels is therefore critical for the
targeting of different cellular processes. In some circum-
stances, the proteins responding to the changes in [Ca
2+
]
cyt
must either be associated with the Ca
2+
channel itself or
tethered closely to the membrane. Thus, the opening of Ca
2+
channels in¯uences local biochemical processes.
To coordinate cellular responses, [Ca
2+
]
cyt
`waves' are
produced within the cytoplasm by the successive recruit-
ment of receptive Ca
2+
channels. Various authors have
speculated how these waves might be initiated and propa-
gated. Trewavas (1999) suggested that a local elevation of
[Ca
2+
]
cyt
might generate soluble second messengers, such as
IP
3
or cADPR, that diffuse through the cytoplasm to activate
a relay of spatially separated Ca
2+
-channels. This might
occur during the [Ca
2+
]
cyt
waves observed in the shank of
poppy pollen tubes during the self-incompatibility response
(Straatman et al., 2001; Rudd and Franklin-Tong, 2001) or
in plant cells responding to salt stress (Drùbak and Watkins,
2000). Antoine et al. (2001) suggested that the [Ca
2+
]
cyt
wave that crosses the egg following fertilization was the
result of the successive activation of (mechanosensitive)
Ca
2+
channels in the plasma membrane radiating from the
site of sperm entry, and Franklin-Tong et al. (2002)
suggested that repetitive Ca
2+
in¯ux across the plasma
membrane contributed to the [Ca
2+
]
cyt
waves that occurred
in the shank of poppy pollen tubes during the self-
incompatibility response. The spatial changes in elevated
[Ca
2+
]
cyt
that occur following the application of ABA to
guard cells, ®rst close to the plasma membrane and
subsequently adjacent to the vacuole (McAinsh et al.,
1992; Allen et al., 1999), are thought to re¯ect the
sequential opening of hyperpolarization-activated Ca
2+
channels at the plasma membrane and then second-
messenger activated Ca
2+
channels in the tonoplast
(Grabov and Blatt, 1998; White, 2000; Schroeder et al.,
2001). In addition to these subcellular [Ca
2+
]
cyt
waves,
`waves' of cells with high [Ca
2+
]
cyt
may also propagate
through plant tissues. These can be induced in root tissues
by mechanical stimulation (Legue
Â
et al., 1997; Fasano et al.,
2002) or saline shock (Moore et al., 2002), in cotyledons by
cold shock (Knight et al., 1993) and in leaves by chilling
plant roots brie¯y (Campbell et al., 1996). Electrical action
potentials, osmotic perturbations or chemical signals may
trigger these waves.
Although an elevated [Ca
2+
]
cyt
is necessary for signal
transduction, a prolonged increase in [Ca
2+
]
cyt
is lethal.
Indeed, sustained high [Ca
2+
]
cyt
is implicated in apoptosis,
both during normal development (e.g. in tissue patterning
and xylogenesis) and in hypersensitive responses to patho-
gens (Levine et al., 1996). To effect other responses,
[Ca
2+
]
cyt
perturbations must be either of low amplitude or
transient. Transient increases in [Ca
2+
]
cyt
can be single
(spike), double (biphasic) or multiple (oscillations). Unique
[Ca
2+
]
cyt
spikes can be generated by delaying the [Ca
2+
]
cyt
rise, or by altering the rate of change of [Ca
2+
]
cyt
, the
maximal [Ca
2+
]
cyt
reached or the duration [Ca
2+
]
cyt
is above
a certain threshold. Oscillations can differ in their [Ca
2+
]
cyt
amplitudes, periodicity or duration (Evans et al., 2001). The
production of [Ca
2+
]
cyt
waves allows speci®c spatiotem-
poral [Ca
2+
]
cyt
perturbations to be generated that may differ
in their cellular location, rate and extent of propagation and/
or their [Ca
2+
]
cyt
amplitude during propagation (Malho
Â
et al.,
1998). However, despite the seductive logic of a [Ca
2+
]
cyt
signature for each developmental cue or environmental
challenge, plant tissues are comprised of populations of
heterogeneous cells that may have contrasting abilities to
496 White and Broadley Ð Calcium in Plants
generate [Ca
2+
]
cyt
signatures. For example, (a) within the
root, the [Ca
2+
]
cyt
perturbations induced by mechanical
perturbation (Legue
Â
et al., 1997), salinity, osmotic stress,
cold shock or slow cooling (Kiegle et al., 2000b; Moore
et al., 2002) differ markedly between cell types, and (b)
shoot cells exhibit a biphasic [Ca
2+
]
cyt
perturbation during
anoxia, whereas only a slow increase in [Ca
2+
]
cyt
is observed
in root cells (Sedbrook et al., 1996; Plieth, 2001). Indeed,
even cells of the same type, such as the two guard cells of a
stomate, seldom generate an identical [Ca
2+
]
cyt
in response
to a de®ned stimulus (Allen et al., 1999). This heterogeneity
may arise from differences in cellular cytology, the
distribution of cytoplasmic Ca
2+
buffers and/or the activities
of Ca
2+
transporters.
The [Ca
2+
]
cyt
signature: temporal and spatial aspects
Several abiotic challenges result in an immediate, tran-
sient increase in [Ca
2+
]
cyt
that is restored to basal levels
within minutes (Table 2). Such challenges include mech-
anical perturbation (Knight et al., 1991, 1992; Haley et al.,
1995; Legue
Â
et al., 1997; Malho
Â
et al., 1998; van der Luit,
1999; Plieth, 2001; Fasano et al., 2002) and rapid cooling
for brief periods, termed `cold-shock' (Knight et al., 1991;
Plieth et al., 1999; van der Luit, 1999; Kiegle et al., 2000b;
Plieth, 2001). By contrast, sustained cooling below a
threshold temperature results in a biphasic response in
[Ca
2+
]
cyt
, in which an initial transient increase in [Ca
2+
]
cyt
is
followed by a more prolonged, second transient elevation of
[Ca
2+
]
cyt
(Plieth et al., 1999; Knight, 2000; Knight and
Knight, 2000; Moore et al., 2002). Heat shock, acute salt
(NaCl) stress, hyper-osmotic (mannitol) stress, annoxia and
exposure to oxidative stress also elicit an immediate,
transient increase in [Ca
2+
]
cyt
in plant cells, which is
followed by a more prolonged elevation of [Ca
2+
]
cyt
lasting
many minutes or hours (Table 2; McAinsh et al., 1996;
Sedbrook et al., 1996; Knight et al., 1997, 1998; Gong et al.,
1998; Clayton et al., 1999; Kiegle et al., 2000b; Knight,
2000; DeWald et al., 2001; Plieth, 2001; Lecourieux et al.,
2002; Moore et al., 2002). Biphasic [Ca
2+
]
cyt
perturbations
have been observed in plant cells in response to diverse
pathogens and elicitors, but the kinetics of these responses
varies greatly (Lecourieux et al., 2002). Rudd and Franklin-
Tong (2001) speculated that the slow generation of a
sustained [Ca
2+
]
cyt
elevation was a common feature of these
responses, and the sustained increase in [Ca
2+
]
cyt
alone was
subsequently correlated with the induction of defence
responses (Cessna and Low, 2001; Lecourieux et al., 2002).
Several developmental cues and environmental chal-
lenges produce [Ca
2+
]
cyt
oscillations (Table 2). The dur-
ation, periodicity and amplitude of [Ca
2+
]
cyt
oscillations
vary considerably, and their form is often dependent on the
strength and combination of speci®c stimuli (Felle, 1988;
McAinsh et al., 1995; Allen et al., 1999; Staxe
Â
n et al., 1999;
Evans et al., 2001). Periodicities may range from the
circadian oscillations observed in various plant cells
(Johnson et al., 1995; Wood et al., 2001), through the
slow oscillations observed in coleoptile cells following the
addition of auxin (periodicity »30 min; Felle, 1988) and in
guard cells either spontaneously or in response to stimuli
such as ABA, [Ca
2+
]
ext
, ozone, cold-shock and elicitors
(periodicity >3 min; Grabov and Blatt, 1998; Allen et al.,
1999, 2000; Staxe
Â
n et al., 1999; Evans et al., 2001; Ng et al.,
2001a; Klu
È
sener et al., 2002), to the rapid oscillations in
[Ca
2+
]
cyt
observed at the tip of growing pollen tubes
(periodicity 40±75 s; Holdaway-Clarke et al., 1997;
Malho
Â
et al., 1998, 2000; Messerli et al., 2000; Rudd and
Franklin-Tong, 2001) and in root hairs during nodulation
(periodicity 1±2 min; Wais et al., 2000; Walker et al., 2000;
Shaw and Long, 2003). It is noteworthy that oscillations can
be induced in guard cells by releasing caged Ca
2+
into the
cytosol (McAinsh et al., 1995) and oscillatory [Ca
2+
]
cyt
waves can be induced in pollen tubes by releasing caged
Ca
2+
or IP
3
into the cytosol (Malho
Â
and Trewavas, 1996;
Malho
Â
, 1998). Several hypotheses have been proposed for
the generation of [Ca
2+
]
cyt
oscillations. These include
models based on the successive emptying and re®lling of
®nite Ca
2+
stores (Klu
È
sener et al., 1995; Harper, 2001), on
the successive activation and deactivation of target Ca
2+
channels through co-incident signalling cascades (Sanders
et al., 1999; White, 2000) or by the stretching and relaxation
of a membrane (Holdaway-Clarke et al., 1997).
Different stimuli generate contrasting spatial [Ca
2+
]
cyt
perturbations by mobilizing Ca
2+
from different cellular
stores and/or by activating Ca
2+
channels in a restricted
location (Table 2; Sanders et al., 1999). For example, Ca
2+
in¯ux from the apoplast is implicated in elevating [Ca
2+
]
cyt
during brief cold-shock (White, 1998, 2000; Plieth et al.,
1999; Kiegle et al., 2000b; Knight, 2000; Plieth, 2001) and
oxidative stress (Knight et al., 1997; Clayton et al., 1999),
but release from internal stores contributes signi®cantly to
the [Ca
2+
]
cyt
elevations in response to prolonged cooling,
salinity, osmotic stresses or pathogens (Knight et al., 1996,
1997; Blume et al., 2000; Knight, 2000; Pauly et al., 2001;
Lecourieux et al., 2002; Moore et al., 2002) and dominates
the [Ca
2+
]
cyt
elevations in response to mechanical perturb-
ations (Haley et al., 1995; Legue
Â
et al., 1997) and anoxia
(Subbaiah et al., 1994, 1998). Van der Luit et al. (1999)
demonstrated that separate nuclear [Ca
2+
] and [Ca
2+
]
cyt
perturbations were initiated in tobacco seedlings in response
to wind and cold shock, respectively, and that each led to the
expression of a different CaM isoform. Pauly et al. (2001)
similarly proposed that different cytoplasmic and nuclear
signals were involved in discriminating between hypo- and
hyper-osmotic shock.
Graded [Ca
2+
]
cyt
responses to stimuli
Perturbations in [Ca
2+
]
cyt
often show a graded response
that is proportional to the strength of the stimulus. For
example, the frequency and amplitude of oscillations in the
[Ca
2+
]
cyt
of guard cells varies directly with the apoplastic
ABA (Staxe
Â
n et al., 1999) or sphingosine-1-phosphate (Ng
et al., 2001a) concentration, with corresponding effects on
the kinetics of stomatal closure. The form of the [Ca
2+
]
cyt
perturbations that occur during cooling are a complex
function of the rate of cooling, the duration of cooling, and
the magnitude of the temperature drop (Plieth et al., 1999).
Plant cells respond to a brief cold-shock with an immediate,
transient increase in [Ca
2+
]
cyt
that is proportional to the rate
White and Broadley Ð Calcium in Plants 497
TABLE 2. Examples of the developmental processes and responses to abiotic and biotic challenges initiated by a
perturbation in cytosolic Ca
2+
concentration ([Ca
2+
]
cyt
)
Developmental process or
environmental challenge
Characteristic [Ca
2+
]
cyt
perturbation
Stores releasing
Ca
2+
to cytosol
Selected references
Pollen tube elongation Oscillation of high apical
[Ca
2+
]
cyt
Apoplast and
internal
Malho
Â
and Trewavas, 1996; Holdaway-Clarke et al., 1997;
Malho
Â
et al., 1998; 2000; Messerli et al., 2000; Rudd and
Franklin-Tong, 2001
Pollen tube self-
incompatibility response
Intracellular [Ca
2+
]
cyt
wave
in shank
Apoplast
Internal (IP
3
-
dependent)
Rudd and Franklin-Tong, 2001; Straatman et al., 2001;
Franklin-Tong et al., 2002
Cell polarity after
fertilization
Intracellular [Ca
2+
]
cyt
wave
from sperm fusion site leading
to sustained [Ca
2+
]
cyt
elevation
Apoplast Antoine et al., 2001
Cell division Elevated [Ca
2+
]
cyt
Bush, 1995
Seed germination
(giberellins)
Slow rise in [Ca
2+
]
cyt
Bush, 1995; Anil and Sankara Rao, 2001
Apoptosis Slow, sustained [Ca
2+
]
cyt
elevation
Levine et al., 1996
Red light Elevated [Ca
2+
]
cyt
Apoplast Shacklock et al. 1992; Malho
Â
et al., 1998
Blue light Brief spike in [Ca
2+
]
cyt
(seconds)
Apoplast Malho
Â
et al., 1998; Baum et al., 1999
Circadian rhythms Circadian [Ca
2+
]
cyt
oscillation Johnson et al., 1995; Wood et al., 2001
Stomatal closure (ABA,
sphingosine-1-phosphate)
(1) Elevated [Ca
2+
]
cyt
at cell
periphery
(2) Elevated [Ca
2+
]
cyt
around vacuole
(3) Oscillations in [Ca
2+
]
cyt
(1) Apoplast
(2) Vacuole
(3) Apoplast
and internal
McAinsh et al., 1992; Allen et al., 1999, 2000; Blatt, 2000a, b;
White, 2000; Anil and Sankara Rao, 2001; Evans et al.. 2001;
Ng et al., 2001a, b; Schroeder et al., 2001; Klu
È
sener et al., 2002
CO
2
Elevated [Ca
2+
]
cyt
in guard
cells
Apoplast Webb et al., 1996
Increasing apoplastic
Ca
2+
Oscillations in [Ca
2+
]
cyt
of
guard cells
Apoplast McAinsh et al., 1995; Allen et al., 1999, 2000
Auxin responses (1) Slow, prolonged [Ca
2+
]
cyt
increase
Felle, 1988; Malho
Â
et al., 1998; Ng et al., 2001b; Plieth, 2001;
Plieth and Trewavas, 2002
(2) Oscillations in [Ca
2+
]
cyt
Xylem K
+
loading Elevated [Ca
2+
]
cyt
De Boer, 1999
Exocytosis Elevated [Ca
2+
]
cyt
Battey et al., 1999; Camacho and Malho
Â
, 2003
Root cell elongation Sustained [Ca
2+
]
cyt
elevation Apoplast Cramer and Jones, 1996; Demidchik et al., 2002a
Root hair elongation Sustained high apical
[Ca
2+
]
cyt
Apoplast Wymer et al., 1997; White, 1998; Bibikova et al., 1999
Inhibition of cyclosis Elevated [Ca
2+
]
cyt
Ayling and Clarkson, 1996
Nodulation (nod factors) Initial [Ca
2+
]
cyt
rise then
oscillations in [Ca
2+
]
cyt
Apoplast Ca
Â
rdenas et al., 2000; Wais et al., 2000; Walker et al., 2000;
Lhuissier et al., 2001; Shaw and Long, 2003
Senescence Sustained [Ca
2+
]
cyt
elevation Huang et al., 1997
UV-B Slow [Ca
2+
]
cyt
rise, elevated
[Ca
2+
]
cyt
sustained for
several minutes
Apoplast Frohnmeyer et al., 1999
Heat-shock Elevated [Ca
2+
]
cyt
sustained
for 15±30 min
Apoplast and
internal
(IP
3
-dependent)
Gong et al., 1998; Malho
Â
et al., 1998
Cold-shock (1) Single brief [Ca
2+
]
cyt
spike (seconds)
(2) Oscillations in [Ca
2+
]
cyt
(1) Apoplast Knight et al., 1991; Malho
Â
et al., 1998; White, 1998;
Plieth et al., 1999; van der Luit, 1999; Allen et al., 2000;
Kiegle et al., 2000b; Knight, 2000; Cessna et al., 2001; Plieth, 2001
Slow cooling Biphasic
(1) Brief [Ca
2+
]
cyt
spike
(seconds)
(2) Slow [Ca
2+
]
cyt
elevation
(minutes)
(1) Apoplast
(2) Apoplast
and internal
(IP
3
-dependent)
Knight et al., 1996; Plieth et al., 1999; Knight, 2000; Knight
and Knight, 2000; Moore et al., 2002
498 White and Broadley Ð Calcium in Plants
of cooling, whereas they respond to sustained cooling below
a threshold temperature with a prolonged biphasic [Ca
2+
]
cyt
perturbation (Table 2). The magnitude of the [Ca
2+
]
cyt
elevation induced in roots following their reorientation in a
gravitational ®eld is proportional to the angle of displace-
ment up to a maximum of 135°, which corresponds to the
optimal angle for amyloplasts to slide down the cell wall
(Plieth and Trewavas, 2002), and the [Ca
2+
]
cyt
perturbation
induced by mechanical stimulation is increased in propor-
tion to the stress and/or the time an organ is in motion
(Knight et al., 1992; Haley et al., 1995). Similarly, the
magnitude of the initial [Ca
2+
]
cyt
elevation induced in
tobacco suspension cells by hypo- or hyper-osmotic shock
depends upon the change in the osmolarity of the bathing
medium (Takahashi et al., 1997; Pauly et al., 2001), the
magnitude of the [Ca
2+
]
cyt
perturbation in response to
elicitors increases proportionally to the elicitor concentra-
tion applied (Mitho
È
fer et al., 1999; Blume et al., 2000;
Lecourieux et al., 2002) and the magnitude of the [Ca
2+
]
cyt
elevation induced by oxidative stress increases in proportion
to the concentrations of ozone, H
2
O
2
or methyl viologen
applied (Price et al., 1994; Levine et al., 1996; McAinsh
et al., 1996; Clayton et al., 1999; Lecourieux et al., 2002).
Explicit [Ca
2+
]
cyt
perturbations produce de®ned
physiological responses
Explicit [Ca
2+
]
cyt
signatures are responsible for the
activation of speci®c genes in animal cells (Dolmetsch
et al., 1997, 1998; Li et al., 1998). This is consistent with the
hypothesis that explicit [Ca
2+
]
cyt
signatures produce de®ned
physiological responses to speci®c developmental cues or
TABLE 2. Continued
Developmental process or
environmental challenge
Characteristic [Ca
2+
]
cyt
perturbation
Stores releasing
Ca
2+
to cytosol
Selected references
Oxidative stress
(paraquat,
superoxide, H
2
O
2
,
ozone)
(1) Brief [Ca
2+
]
cyt
spike
(2) Sustained [Ca
2+
]
cyt
elevation
(3) Oscillations in [Ca
2+
]
cyt
(1) Apoplast
(2) Apoplast
and internal
(IP
3
-dependent)
Price et al., 1994; Levine et al., 1996; McAinsh et al., 1996;
Knight et al., 1998; Malho
Â
et al., 1998; Clayton et al., 1999;
Allen et al., 2000; Kawano and Muto, 2000; Knight, 2000;
Klu
È
sener et al., 2002; Lecourieux et al., 2002
Anoxia Biphasic
(1) Slow spike (duration of
minutes)
(2) Sustained [Ca
2+
]
cyt
elevation (hours)
(1) Apoplast
(2) Internal
including
mitochondria
Subbaiah et al., 1994, 1998; Sedbrook et al., 1996;
Malho
Â
et al., 1998; Plieth, 2001
Drought/hyper-osmotic
stress (mannitol)
Biphasic
(1) Slow spike (duration of
minutes)
(2) Sustained [Ca
2+
]
cyt
elevation (hours)
Apoplast and
vacuole
Knight et al., 1997, 1998; Malho
Â
et al., 1998;
Kiegle et al., 2000b; Cessna et al., 2001; Pauly et al., 2001;
Plieth, 2001
Salinity (NaCl) Biphasic
Tissue [Ca
2+
]
cyt
wave
(1) Slow spike (duration of
minutes)
(2) Sustained [Ca
2+
]
cyt
elevation (hours)
(3) Reduced [Ca
2+
]
cyt
(days)
Apoplast and
vacuole
(IP
3
-dependent)
Knight et al., 1997; Kiegle et al., 2000b; Knight, 2000;
DeWald et al., 2001; Pauly et al., 2001; Moore et al., 2002;
Halperin et al., 2003
Hypo-osmotic stress Biphasic
(1) Small [Ca
2+
]
cyt
elevation
(2) Large [Ca
2+
]
cyt
elevation
(1) Apoplast
(2) Apoplast
and internal
(IP
3
-dependent)
Takahashi et al., 1997; Malho
Â
et al., 1998; Knight, 2000;
Cessna and Low, 2001; Cessna et al., 2001; Pauly et al., 2001;
Plieth, 2001
Mechanical stimulation
(motion, touch, wind)
Single brief [Ca
2+
]
cyt
spike
(seconds)
Tissue [Ca
2+
]
cyt
wave
Internal Knight et al., 1991, 1992; Haley et al., 1995; Legue et al., 1997;
Malho
Â
et al., 1998; van der Luit, 1999; Plieth, 2001;
Fasano et al., 2002
Aluminium stress Elevated [Ca
2+
]
cyt
Zhang and Rengel, 1999
Pathogens (elicitors) Biphasic
(1) Slow spike (duration of
minutes)
(2) Sustained [Ca
2+
]
cyt
elevation (hours)
(3) Oscillations in [Ca
2+
]
cyt
(1) Apoplast
(2) Apoplast
and internal
(IP
3
-dependent)
Knight et al., 1991; Malho
Â
et al., 1998; Mitho
È
fer et al., 1999;
Blume et al., 2000; Fellbrich et al., 2000; Grant et al., 2000;
Cessna and Low 2001; Cessna et al. 2001; Rudd and
Franklin-Tong, 2001; Klu
È
sener et al., 2002; Lecourieux et al., 2002
(the relative magnitude of
different phases varies with
elicitor identity)
The kinetics of the [Ca
2+
]
cyt
perturbations and the location of stores releasing Ca
2+
into the cytosol associated with each developmental process or
environmental challenge are indicated.
White and Broadley Ð Calcium in Plants 499
environmental challenges. However, there have been few
attempts to generate explicit [Ca
2+
]
cyt
perturbations arti®-
cially in plants. Although several experiments have demon-
strated that elevating [Ca
2+
]
cyt
using ionophores induces the
expression of Ca
2+
-dependent genes and physiological
responses to chilling and ABA (Monroy and Dhinsa,
1995; Sheen, 1996), the [Ca
2+
]
cyt
perturbations produced
by ionophores are unlikely to have the same form as the
[Ca
2+
]
cyt
perturbations elicited by these effectors. This
suggested to Plieth (2001) that the form of the [Ca
2+
]
cyt
signature was inconsequential for these responses. Indeed,
Plieth (2001) further argued that the notion of an explicit
[Ca
2+
]
cyt
signature entirely responsible for a de®ned
physiological response was fundamentally doubtful. He
cited these observations: (a) both osmotic stress and salt
stress induced similar [Ca
2+
]
cyt
perturbations, but result in
different levels of expression of the p5cs gene (Knight et al.,
1997), and (b) almost any [Ca
2+
]
cyt
perturbation could be
elicited in plant cells with an appropriate manipulation of
temperature (Plieth, 2001), to suggest that factors other
than, or in addition to, [Ca
2+
]
cyt
were involved in producing
an appropriate response to a particular challenge. Consistent
with this suggestion, [Ca
2+
]
cyt
transients have recently been
found to be necessary, but not suf®cient, for the expression
of ABA-inducible genes in plant cells (Webb et al., 2001).
Nevertheless, several biochemical and genetic conse-
quences have been attributed to speci®c [Ca
2+
]
cyt
signatures.
An insight into the [Ca
2+
]
cyt
signatures required for
speci®c cellular responses has been obtained by correlating
biochemical or genetic responses with particular character-
istics of [Ca
2+
]
cyt
perturbations common to diverse stimuli
or by dissecting [Ca
2+
]
cyt
perturbations using pharmaceut-
icals. Thus, Cessna and Low (2001) used pharmaceuticals to
alter the [Ca
2+
]
cyt
perturbations in response to hypo-osmotic
shock to demonstrate that only the Ca
2+
¯ux from internal
stores during the second transient increase in [Ca
2+
]
cyt
initiated an oxidative burst. They speculated that this was a
consequence of either a local elevation of [Ca
2+
]
cyt
and/or a
local distribution of the enzymes generating H
2
O
2
.
Similarly, Lecourieux et al. (2002) demonstrated that only
the second, sustained increase in [Ca
2+
]
cyt
elicited by
cryptogein was required for a hypersensitive response and
cell death. Blume et al. (2000) demonstrated that only a
sustained [Ca
2+
]
cyt
elevation induced phytoallexin synthesis
in response to elicitors, and Clayton et al. (1999) showed
that the induction of glutathione-S-transferase (GST) gene
expression by ozone relied only on the second, transient
[Ca
2+
]
cyt
elevation. Finally, the changes in [Ca
2+
]
cyt
per-
turbations following combinations of oxidative stress and
hyper-osmotic stress correlated well with the expression of
Ca
2+
-regulated osmotic stress induced genes (p5cs and
rab18), and the acquisition of osmotic stress tolerance
(Knight et al., 1998).
A direct demonstration that an explicit [Ca
2+
]
cyt
perturb-
ation was required to produce a de®ned physiological
response was performed recently on arabidopsis guard cells
(Allen et al., 2001). Arti®cially elevating [Ca
2+
]
cyt
in guard
cells through hyperpolarization (Grabov and Blatt, 1998;
Allen et al., 2000, 2001) or H
2
O
2
(Allen et al., 2000; Pei
et al., 2000) can induce stomatal closure. However,
although a single elevation of [Ca
2+
]
cyt
was suf®cient for
immediate stomatal closure, prolonged stomatal closure
could only be induced if the initial increase in [Ca
2+
]
cyt
was
followed by [Ca
2+
]
cyt
oscillations with a speci®c periodicity
(Allen et al., 2001). Interestingly, appropriate [Ca
2+
]
cyt
oscillations are induced in guard cells by ABA and stomates
of the ABA-insensitive mutant gca2, which shows aberrant
[Ca
2+
]
cyt
oscillations in response to ABA, can be induced to
close when appropriate [Ca
2+
]
cyt
are produced arti®cially
(Allen et al., 2001).
Habituation, `learning' and `memory'
Trewavas (1999) has likened the [Ca
2+
]
cyt
signalling
network to a basic cellular `memory'. He noted that, in
common with a neural network, [Ca
2+
]
cyt
signals have the
following properties: they may be (a) spatially structured,
through the occurrence and location of cellular components;
(b) subject to coincidence control, since certain elements are
the targets of multiple [Ca
2+
]
cyt
cascades and can process or
block speci®c signals arriving coincidentally; (c) synchron-
ized, for example by membrane depolarization or a sudden
increase in the concentration of a diffusable second
messenger; (d) modi®ed by exposure to speci®c challenges,
through changes in the abundance of cellular components
involved in [Ca
2+
]
cyt
signals; and (e) predisposed by
previous challenges to generate an appropriate [Ca
2+
]
cyt
signal in response to a current challenge. He suggested that
these prerequisites for cellular `learning' allow plant cells to
respond `intelligently' to the challenges they experience
through an innate phenotypic and physiological plasticity.
Previous sections have discussed the spatial distribution
of Ca
2+
-transporters within a plant cell and their modulation
by components of diverse intracellular signalling cascades
such as [Ca
2+
]
cyt
, cytoplasmic pH, reactive oxygen species,
kinases, phosphatases, cNMPs, IP
3
and cADPR. Such Ca
2+
transporters could be a point of convergence, and integra-
tion, of many developmental and environmental signals.
This phenomenon is termed `cross-talk' and might be a
mechanism whereby plants develop cross-tolerance to
various biotic and abiotic stresses (Bowler and Fluhr,
2000; Knight, 2000). Similarly many targets of [Ca
2+
]
cyt
signals are also regulated by components of diverse
signalling cascades (see below).
There is considerable evidence that [Ca
2+
]
cyt
signatures
are modi®ed by previous experience. A diminished [Ca
2+
]
cyt
elevation upon repetitive stimulation and/or a refractory
period during which an increase in [Ca
2+
]
cyt
cannot be
elicited by the same developmental cue or environmental
challenge is commonly observed. The magnitude of the
[Ca
2+
]
cyt
perturbation elicited by reorientation in a gravita-
tional ®eld (Plieth and Trewavas, 2002), touch (Legue
Â
et al.,
1997) or wind-induced motion (Knight et al., 1992)
becomes progressively smaller upon repeated stimulation
and a refractory period of several minutes is required before
a full response is observed again. Following one brief pulse
of phototropically active blue light, arabidopsis seedlings
will not respond maximally again for several hours (Baum
et al., 1999). A second exposure to an elicitor does not
in¯uence [Ca
2+
]
cyt
for several hours after its initial applic-
500 White and Broadley Ð Calcium in Plants
ation (Blume et al., 2000), and plant cells challenged with
H
2
O
2
fail to respond to H
2
O
2
again for several hours (Price
et al., 1994). An anoxic treatment reduces the magnitude of
the initial [Ca
2+
]
cyt
peak and delays the sustained elevation
of [Ca
2+
]
cyt
in response to a second anoxic treatment
(Sedbrook et al., 1996), and the magnitude of [Ca
2+
]
cyt
perturbations is diminished by continued chilling and
rewarming (Plieth et al., 1999). These observations may
indicate a desensitization of signalling cascades or a
depletion of stores releasing Ca
2+
to the cytosol. By
contrast, the magnitude of the second (vacuolar) [Ca
2+
]
cyt
elevation observed during slow cooling was increased
following a period of cold acclimation (Knight and
Knight, 2000), and a pretreatment with mannitol increased
the magnitude of the [Ca
2+
]
cyt
perturbations subsequently
induced by hyper-osmotic shock (Knight et al., 1998).
There is also evidence that the [Ca
2+
]
cyt
signatures
elicited by one environmental challenge can be modi®ed
by prior exposure to a contrasting one. For example, the
magnitude of the [Ca
2+
]
cyt
perturbations in response to
oxidative stress was reduced by a prior exposure to hyper-
osmotic stress (Knight et al., 1997; Knight, 2000), and the
magnitude of the [Ca
2+
]
cyt
perturbations in response to
hyper-osmotic stress was reduced by a prior exposure to
oxidative stress (Knight et al., 1998; Knight, 2000). These
observations may imply cross-talk between the signalling
cascades and/or Ca
2+
channels and stores responding to
these challenges. However, prior oxidative stress did not
affect the [Ca
2+
]
cyt
transient observed upon cold shock or
touch (Price et al., 1994; Knight et al., 1998), suggesting
that contrasting signalling cascades and/or Ca
2+
stores and
channels are recruited by these challenges. Similarly, during
the refractory period following heat shock, during which
additional heat shocks fail to elevate [Ca
2+
]
cyt
, [Ca
2+
]
cyt
can
be elevated by cold shock or mechanical perturbation (Gong
et al., 1998), and during the refractory period following
wind-induced motion, cells can still raise [Ca
2+
]
cyt
in
response to cold shock (Knight et al., 1992). The cross-
talk (and lack of cross-talk) between diverse signalling
cascades responding to each facet of the environment is
thought to optimize a plant's response to its surroundings.
RESPONDING TO CYTOSOLIC CALCIUM
SIGNALS
To respond appropriately to a speci®c [Ca
2+
]
cyt
perturbation,
a cell must activate a unique combination of Ca
2+
-binding
proteins. These [Ca
2+
]
cyt
sensors include CaMs, CaM-like
proteins, calcineurin B-like (CBL) proteins and Ca
2+
-
dependent protein kinases (CDPKs). Many of these proteins
bind Ca
2+
using a helix±loop±helix structure termed the `EF
hand', which binds a single Ca
2+
molecule with high af®nity
(Strynadka and James, 1989). Frequently, pairs of EF hands
interact through antiparallel b-sheets, which allows co-
operativity in Ca
2+
binding. When Ca
2+
binds to [Ca
2+
]
cyt
sensors their structural and/or enzymatic properties change
and their subsequent interactions with target proteins can
alter solute transport and enzymatic activities, cytoskeletal
orientation, protein phosphorylation cascades and gene
expression. It is believed that these changes result in stress
tolerance and/or a developmental switch. The form of the
physiological response is determined not only by the
[Ca
2+
]
cyt
perturbation itself, but also by the expression of
the [Ca
2+
]
cyt
sensors, their af®nities for both Ca
2+
and target
proteins, and the abundance and activity of the target
proteins. Since different cell types, and probably even
individuals of the same cell type, have contrasting tran-
script, protein and enzyme pro®les, a similar [Ca
2+
]
cyt
perturbation will result in individual responses, which may
contribute to phenotypic plasticity (Gilroy and Trewavas,
2001).
Calcium-binding proteins: CaM and CaM-related proteins
CaM is found in the apoplast and in the cytosol, ER and
nucleus of plant cells. Within the cytosol, the estimated CaM
concentration is 5 to 40 m
M (Zielinski, 1998). Calmodulin
has been implicated in Ca
2+
-dependent responses to light,
gravity, mechanical stress, phytohormones, pathogens,
osmotic stress, salinity, heavy metals, xenobiotics, annoxia,
oxidative stress, heat shock and chilling (Zielinski, 1998;
Snedden and Fromm, 2001; Reddy, 2001; Rudd and
Franklin-Tong, 2001; Fasano et al., 2002). Calmodulin is a
small (17 kDa), highly conserved, acidic protein with two
globular domains each containing two EF hands connected
by a ¯exible a-helical spacer (Zielinski, 1998; Reddy, 2001;
Snedden and Fromm, 2001; Luan et al., 2002). A Ca
2+
/CaM
complex generally interacts with target proteins, although
there are exceptions, such as the interaction of myosin-like
proteins with CaM alone (Reddy, 2001). The binding of Ca
2+
to CaM, which has a K
d
between 10
±7
and 10
±6
M, exposes a
hydrophobic surface on each globular domain. This enables
the Ca
2+
/CaM complex to wrap around its target protein and
bind to its CaM-binding site with an af®nity in the nanomolar
range through non-speci®c van der Waals interactions. The
co-location of two EF hands in each globular domain allows
Ca
2+
to bind cooperatively, which ensures that CaM
activation occurs over a narrow range of Ca
2+
concentra-
tions. The af®nity of CaM for Ca
2+
may be profoundly
in¯uenced by the presence of particular target proteins
(Zielinski, 1998). Calmodulins bind to many different
proteins implicated in diverse physiological processes
including cation transport (including [Ca
2+
]
cyt
homeostasis),
cytoskeletal rearrangements and cell division, phytohor-
mone and phospholipid signalling, disease resistance
(including the oxidative burst) and stress tolerance
(Zielinski, 1998; Reddy, 2001; Snedden and Fromm, 2001;
Luan et al., 2002; Reddy et al., 2002). Calmodulins can also
regulate gene expression by binding to speci®c transcription
factors (Szymanski et al., 1996; Reddy et al., 2000; Yang and
Poovaiah, 2000; Bouche
Â
et al., 2002).
Small gene families encode CaM isoforms in plants.
Many species possess several CaM genes encoding identical
proteins as well as other genes encoding divergent isoforms
(Zielinski, 1998; Snedden and Fromm, 2001). It is thought
that the presence of genes encoding identical CaMs may
re¯ect a need for diverse tissue-speci®c, developmental or
stress-induced expression, and the presence of genes
encoding different CaM isoforms may re¯ect a requirement
for speci®c interactions with target proteins (Reddy, 2001;
White and Broadley Ð Calcium in Plants 501
Snedden and Fromm, 2001). Indeed, it has been suggested
that particular CaM isoforms transduce speci®c environ-
mental or developmental signals. Consistent with this
hypothesis, it has been shown that environmental challenges
rapidly up-regulate the expression of different CaM iso-
forms. For example, a subset of CaM genes is up-regulated
by touch in various plants (Braam et al. 1997; Verma and
Upadhyaya, 1998; Zielinski, 1998), cold shock and wind
stimuli lead to the expression of different CaM isoforms
(van der Luit et al., 1999), and speci®c CaM genes are up-
regulated in response to wounding or pathogens (Heo et al.,
1999; Yamakawa et al., 2001), auxin or salinity (Botella and
Arteca, 1994). Many CaM isoforms show tissue-speci®c
and/or developmentally regulated expression (Gawienowski
et al. 1993; Takezawa et al., 1995; Yang et al., 1998;
Yamakawa et al., 2001; Duval et al., 2002), and CaM
isoforms can differentially regulate target enzymes or have
contrasting af®nities for Ca
2+
and CaM-binding peptides
(Liao et al., 1996; Liu et al., 1998; Reddy et al., 1999;
Ko
È
hler and Neuhaus, 2000; Lee et al., 2000; Duval et al.,
2002; Zielinski, 2002). Calmodulins can also be post-
translationally trimethylated, which in¯uences both their
stability and physiological activities (Zielinski, 1998). All
these properties are consistent with [Ca
2+
]
cyt
signatures
producing unique biochemical and physiological conse-
quences in cells expressing speci®c CaM isoforms and
target proteins. Interestingly, target proteins themselves
may have CaM-binding or non-binding isoforms (Snedden
and Fromm, 2001), which presumably allows for [Ca
2+
]
cyt
-
dependent and [Ca
2+
]
cyt
-independent regulatory cascades.
Plants also possess `CAM-like' proteins. These have
between one and six EF hands and a limited homology to
CaM (de®ned arbitrarily as <75% identity with canonical
CaM isoforms; Reddy, 2001; Snedden and Fromm, 2001;
Luan et al., 2002; Zielinski, 2002). In arabidopsis, they
include: CaBP-22 (Ling and Zielinski, 1993), TCH2 and
TCH3 (Braam et al., 1997), AtCP1 (Jang et al., 1998),
centrins (Cordeiro et al., 1998), NADPH oxidases
(AtrbohA±F; Torres et al., 1998), homologues of the rice
ABA-inducible EFA27 protein (Frandsen et al., 1996) and
Ca
2+
-binding protein phosphatases such as ABI1 and ABI2
(Leung et al., 1997). These proteins have been implicated in
cellular responses to diverse environmental, developmental
and pathological challenges.
Calcium-binding proteins: calcineurin B-like proteins
Calcineurin B-like proteins (CBL) possess three EF hands
(Luan et al., 2002). In arabidopsis there are at least ten
AtCBL genes, including AtSOS3 (AtCBL4), which encodes a
Ca
2+
-sensor protein involved in salt tolerance (Liu and Zhu,
1998; Luan et al., 2002; Xiong et al., 2002). Many CBLs
have a conserved myristoylation site in their N-termini,
which allows membrane association. It has been proposed
that myristoylation of CBLs alters their cellular location and
intracellular interactions (Luan et al., 2002). It is thought
that particular CBLs transduce speci®c environmental or
developmental signals. Consistent with this hypothesis is
the induction of AtCBL1 expression by drought, salinity,
cold and wounding (Kudla et al., 1999; Piao et al., 2001)
and the accumulation of AtCBL1 and AtCBL2 transcripts in
leaves upon illumination (Nozawa et al., 2001). A family of
SNF1-like serine/threonine protein kinases (AtCIPK1
through AtCIPK25) has been identi®ed as Ca
2+
-dependent
targets for AtCBLs (Shi et al., 1999; Halfter et al., 2000;
Albrecht et al., 2001; Y. Guo et al., 2001; Luan et al., 2002).
The CIPKs interact with CBLs through a unique 24-amino-
acid domain termed the `NAF domain' (Albrecht et al.,
2001) and require divalent-cation cofactors for their activity
(Luan et al., 2002). Both AtCIPK1 and AtCIPK2 require
Mn
2+
as a cofactor and AtSOS2 (AtCIPK24) requires Mg
2+
.
Each CBL protein may interact with several CIPKs. For
example, AtCBL1 interacts with numerous AtCIPKs (Shi
et al., 1999) and AtSOS3 interacts with AtSOS2
(AtCIPK24) and at least seven other AtCIPKs (Halfter
et al., 2000; Y. Guo et al., 2001). Conversely, certain
AtCIPKs can interact with several AtCBLs (Shi et al., 1999;
Kim et al., 2000; Albrecht et al., 2001; Y. Guo et al., 2001).
Common interactions between AtCBLs and AtCIPKs may
allow cross-talk between signalling cascades, whereas
preferential associations between CBLs and CIPKs are
thought to underpin speci®c signalling cascades. In this
context it is noteworthy that the expression of CIPKs (in
addition to CBLs) can be tissue speci®c and/or regulated by
environmental stresses such as cold, drought, salinity,
wounding or nutrient starvation (Albrecht et al., 2001;
Y. Guo et al., 2001; Kim et al., 2003).
Calcium-binding proteins: calcium-dependent protein kinases
The activity of many protein kinases can respond to
[Ca
2+
]
cyt
signals directly. These can be placed in one of four
classes: Ca
2+
-dependent protein kinases (CDPKs), CDPK-
related proteins (CRKs), CaM-dependent protein kinases
(CaMKs) and chimeric Ca
2+
- and CaM-dependent protein
kinases (CCaMKs). McAinsh and Hetherington (1998)
proposed an interesting model for generating speci®c
physiological responses to [Ca
2+
]
cyt
perturbations through
the action of Ca
2+
-dependent and Ca
2+
-independent protein
kinases and phosphatases. In this model, speci®c [Ca
2+
]
cyt
perturbations in¯uence the degree of phosphorylation of a
target protein leading to contrasting or graded responses.
The CDPKs are ubiquitous in plants. There are at least 34
genes encoding CDPKs in the arabidopsis genome (Harmon
et al., 2001; S. H. Cheng et al., 2002) and similar numbers in
other plant species. They generally have four EF hands at
their C-terminus that bind Ca
2+
to activate their serine/
threonine kinase activity. They act as monomers and many
may be autoinhibited by autophosphorylation of a pseudo-
substrate domain (S. H. Cheng et al., 2002). Different
CDPKs have contrasting af®nities for Ca
2+
(Lee et al., 1998)
and the binding of Ca
2+
to some CDPKs is modulated by
lipids, interactions with 14-3-3 proteins or phosphorylation
(Reddy, 2001; S. H. Cheng et al., 2002; Sanders et al.,
2002). No CDPKs appear to be integral membrane proteins,
but many are associated with the cytoskeleton, nucleus,
plasma membrane and ER (Reddy, 2001; Sanders et al.,
2002). At least 24 arabidopsis CDPKs can potentially
undergo myristoylation and palmitoylation at their N-
502 White and Broadley Ð Calcium in Plants
termini, which may facilitate their association with mem-
branes (S. H. Cheng et al., 2002; Xiong et al., 2002).
The CDPKs are capable of converting [Ca
2+
]
cyt
signals
into biochemical and genetic consequences through the
phosphorylation of diverse target proteins including mem-
brane solute transporters (including the Ca
2+
-ATPase,
AtACA2), ion and water channels, NADPH oxidases,
enzymes involved in carbon and nitrogen metabolism,
cytoskeletal proteins, proteases and DNA-binding proteins
(Reddy, 2001; Rudd and Franklin-Tong, 2001; S. H. Cheng
et al., 2002; Sanders et al., 2002). They are implicated in
pollen development, control of the cell cycle, phytohormone
signal transduction, light-regulated gene expression, gravi-
tropism, thigmotropism, nodulation, cold acclimation,
salinity tolerance, drought tolerance and responses to
pathogens (Sheen, 1996; Saijo et al., 2000; Anil and
Sankara Rao, 2001; Reddy, 2001; Romeis et al., 2001; S. H.
Cheng et al., 2002; Xiong et al., 2002; Lee et al., 2003). It is
thought that the possession of many CDPKs with contrast-
ing Ca
2+
af®nities and target proteins allows plant cells to
respond appropriately to speci®c [Ca
2+
]
cyt
perturbations
(Anil and Sankara Rao, 2001; S. H. Cheng et al., 2002;
Sanders et al., 2002). In various plant species speci®c
CDPKs are induced by cold, drought, salinity, annoxia,
mechanical stress, wounding and pathogen elicitors (Urao
et al., 1994; Breviario et al., 1995; Monroy and Dhinsa,
1995; Botella et al., 1996; Ta
È
htiharju et al., 1997; Yoon
et al., 1999; Saijo et al., 2000; Anil and Sankara Rao, 2001;
Romeis et al., 2001; Chico et al., 2002; Lee et al., 2003). In
addition, the activities of CDPKs are affected by post-
translational modi®cations during development (Anil et al.,
2000) or in response to environmental challenges such as
wounding or pathogens (Romeis et al., 2001).
Other protein kinases responding to [Ca
2+
]
cyt
are less well
characterized than the CDPKs. There are at least seven CRKs
in the arabidopsis genome that are structurally similar to
CDPKs, but have degenerate or truncated EF hands that may
not be able to bind Ca
2+
(Reddy, 2001). Orthologues of these
are present in many plant species. Several CaMKs have also
been cloned from arabidopsis and other plants (Zhang and
Lu, 2003). Their kinase activity is activated by CaM-
dependent autophosphorylation and their catalytic activity is
also modulated by CaM. They are expressed highly in
rapidly growing cells and tissues of the root and ¯ower
(Zhang and Lu, 2003). Chimeric CCaMKs are expressed in
the anthers of several plant species, including lily and
tobacco (Liu et al., 1998), but none have been identi®ed in
arabidopsis (Zhang and Lu, 2003). The CCaMKs possess a
CaM-binding domain and three EF hands. They require only
Ca
2+
for autophosphorylation, but Ca
2+
and CaM for
substrate phosphorylation. Liu et al. (1998) demonstrated
that different CaM isoforms have contrasting effects on
substrate phosphorylation by lily and tobacco CCaMKs,
suggesting that these kinases could respond to speci®c
environmental or developmental challenges.
Calcium-binding proteins: proteins without EF hands
Several proteins lacking EF hands are also capable of
binding Ca
2+
(Reddy, 2001; Anil and Sankara Rao, 2001).
For example, the activity of phospholipase D (PLD), which
cleaves membrane phospholipids into a soluble head group
and phosphatidic acid, is regulated by [Ca
2+
]
cyt
through a
Ca
2+
/phospholipid binding-site termed the `C2 domain'
(Wang, 2001). Phospholipase D activity is implicated in
cellular responses to ethylene and ABA, a-amylase synthe-
sis in aleurone cells, stomatal closure, pathogen responses,
leaf senescence and drought tolerance (Ritchie et al., 2002).
Plants possess several PLD isoforms that differ in their
af®nity for Ca
2+
and their modulation by phosphoinositides,
free fatty acids and lysolipids (Wang, 2001). These
biochemical modulators of PLD activity are the substrates
or products of phospholipase C, which generates IP
3
and
diacylglycerol, phospholipase A
2
and diacylglycerol kinase,
both of which are regulated by CaM. It has therefore been
suggested that [Ca
2+
]
cyt
signalling cascades might coordin-
ate the activities of these diverse enzymes to effect speci®c
responses to contrasting environmental or developmental
stimuli (Wang, 2001).
In annexins, Ca
2+
is bound by the `endonexin fold',
which contains a characteristic GXGT-{38}-(D/E) motif
(Delmer and Potikha, 1997). This often appears only in
the ®rst quarter of plant annexins, although animal
annexins generally possess four such motifs. The
binding of Ca
2+
to annexins enables them to associate
with membranes to form cation-channels (Hofmann et al.,
2000; White et al., 2002a). In plants, annexins are
encoded by small gene families. Seven annexin genes
(AnnAt1-7) have been identi®ed in arabidopsis (Clark
et al., 2001; White et al., 2002a). Most annexin genes
are expressed throughout the plant, but annexins are
especially abundant in highly secretory cell types, where
they are located at the cell periphery and may have a
role in membrane fusion, membrane traf®cking and/or
secretion (Clark et al., 2001). The increased expression
of certain annexins upon speci®c developmental or
environmental challenges has linked them with Ca
2+
signals during root nodulation, pathogen attack, ABA
responses, fruit ripening and cold acclimation (White
et al., 2002a).
Other Ca
2+
-binding proteins include calreticulin, calse-
questrin, calnexin and BiP, which sequester Ca
2+
in the ER.
These proteins are implicated in Ca
2+
homeostasis, protein
folding and post-translational modi®cations (Crofts and
Denecke, 1998; Michalak et al., 1998). The expression of
calreticulin is increased in reproductive tissues. Calreticulin
possesses two Ca
2+
-binding domains. The ®rst is a proline-
rich, low-capacity/high-af®nity Ca
2+
-binding domain
(K
d
=1 mM; 1 mol Ca
2+
mol protein
±1
) that contains a highly
conserved stretch of amino acids (KPEDWD). The second is
a high-capacity/low-af®nity Ca
2+
-binding domain (K
d
=
2m
M; 25 mol Ca
2+
mol protein
±1
) comprising acidic amino
acid residues (generally glutamate and aspartate). Both
calreticulin and BiP, and a Ca
2+
-binding protein (PCP)
expressed in Brassica pistils and implicated in pollen±pistil
interactions (Furuyama and Dzelzkalns, 1999) also possess
high-capacity/low-af®nity Ca
2+
-binding domains. Calnexin
possesses a proline-rich low-capacity/high-af®nity Ca
2+
-
binding domain.
White and Broadley Ð Calcium in Plants 503
Calcium and CaM-binding transcription factors
Changes in [Ca
2+
]
cyt
may