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REVIEW
The CBL–CIPK network mediates different signaling pathwaysin plants
Qinyang Yu • Lijia An • Wenli Li
Received: 13 August 2013 / Accepted: 8 September 2013
� Springer-Verlag Berlin Heidelberg 2013
Abstract The calcineurin B-like protein–CBL-interact-
ing protein kinase (CBL–CIPK) signaling pathway in
plants is a Ca2?-related pathway that responds strongly to
both abiotic and biotic environmental stimuli. The CBL–
CIPK system shows variety, specificity, and complexity in
response to different stresses, and the CBL–CIPK signaling
pathway is regulated by complex mechanisms in plant
cells. As a plant-specific Ca2? sensor relaying pathway, the
CBL–CIPK pathway has some crosstalk with other sig-
naling pathways. In addition, research has shown that there
is crosstalk between the CBL–CIPK pathway and the low-
K? response pathway, the ABA signaling pathway, the
nitrate sensing and signaling pathway, and others. In this
paper, we summarize and review research discoveries on
the CBL–CIPK network. We focus on the different modi-
fication and regulation mechanisms (phosphorylation and
dephosphorylation, dual lipid modification) of the CBL–
CIPK network, the expression patterns and functions of
CBL–CIPK network genes, the responses of this network to
abiotic stresses, and its crosstalk with other signaling
pathways. We also discuss the technical research methods
used to analyze the CBL–CIPK network and some of its
newly discovered functions in plants.
Keywords CBL–CIPK � Ca2? � Abiotic stress �Signaling pathway � ABA � Potassium
Abbreviations
CBL Calcineurin B-like protein
CIPK CBL-interacting protein kinase
CaM Calmodulin
CDPK Ca2?-dependent protein kinase
AMPK AMP-activated protein kinase
PPI Protein phosphatase interaction motif
SNF1 Sucrose non-fermenting 1
PP2C Class 2C protein phosphatase
ABI1/2/3/4/5 ABA-insensitive protein 1/2/3/4/5
PFPF/FISL motif Indicated amino acids conserved in
each motif
NtSar1 Secretion-associated Ras-related
protein 1 of Nicotiana tabacum
BFA Brefeldin A
ROS Reactive oxygen species
SOS Salt overly sensitive
HKT High-affinity K?-transporter
AKT Arabidopsis K? transporter
AHA2 Arabidopsis H? ATPase 2
EF-hand Elongation factor-hand
MAPK Mitogen-activated protein kinase
GA Gibberellin
ABA Abscisic acid
Rboh Respiratory burst oxidase homolog
PM Plasma membrane
ER Endoplasmic reticulum
Sub1A Submergene 1A
SnRK SNF1 (sucrose non-fermenting-1)-
related protein kinase
OST1 Open stomata 1
ABRE ABA-responsive element
Communicated by N. Stewart.
Q. Yu � L. An � W. Li (&)
School of Life Science and Biotechnology, Dalian University of
Technology, Linggong Road No. 2, Dalian, Liaoning, China
e-mail: [email protected]
Q. Yu
e-mail: [email protected]
L. An
e-mail: [email protected]
123
Plant Cell Rep
DOI 10.1007/s00299-013-1507-1
AREB ABRE-binding protein
LEA Late embryogenesis abundant
PYR Pyrabactin resistance
PYL PYR 1-like
TF Transcription factor
RCAR Regulatory component of ABA
receptor
Y2H Yeast two-hybrid method
SUS Mating-based split-ubiquitin system
Introduction
Plants have their own unique ways to respond to environ-
mental stimuli, and have evolved strategies to survive from
abiotic and biotic stresses because they cannot escape by
moving to places that are more favorable. During evolution,
plants have formed complete systems to perceive, trans-
duce, and respond to stresses at the molecular, cellular, and
physiological levels (Li et al. 2009). Calcium (Ca2?) is
widely accepted as a ubiquitous second messenger. It
functions in a multitude of physiological and developmental
processes in plants. The ‘‘Ca2? signature’’ or [Ca2?]cyt
refers to the temporal and spatial changes in the cytosolic
Ca2? concentration. In recent decades, much research has
focused on how [Ca2?]cyt is detected, and how it functions
in the complex signaling network in plant cells. Researchers
have identified Ca2? sensor proteins, including sensor relay
proteins and sensor responder proteins. In plants, the Ca2?
sensor relay proteins do not have kinase activity. They
interact with sensor responder proteins to modulate down-
stream reactions after binding to Ca2?. The sensor proteins
include calmodulin (CaM) and the plant-specific calcineu-
rin B-like protein (CBL). In contrast, Ca2? sensor responder
proteins have all the functions of a Ca2? sensor relay pro-
tein, but also have kinase activity. An example of a Ca2?
sensor responder is the Ca2?-dependent protein kinase
(CDPK). In the calcineurin B-like protein–CBL-interacting
protein kinase (CBL–CIPK) system, separate proteins are
responsible for the Ca2?-binding function and kinase
activity. This two-protein system indicates that there is
complex and dynamic regulation of Ca2? signaling path-
ways in plants. It has become increasingly apparent that
signaling networks resemble webs or scale-free networks.
Collectively, Ca2? signals are regarded as one of the central
hubs of plant signaling networks. In recent years, much has
been learned about how the [Ca2?]cyt is transmitted by the
CBL–CIPK complex, and about the crosstalk between the
CBL–CIPK network and other signaling pathways.
Comparative genomic analyses of CBL/CIPK genes in
plants have provided details about the function, complexity,
and conservation of the CBL/CIPK family, and about the
evolution of the CBL–CIPK signaling network in various
plants, from algae to woody plants. Recent studies identified
10 CBLs and 26 CIPKs from Arabidopsis (Kolukisaoglu
et al. 2004; Lyzenga et al. 2013), 10 CBLs and 31 CIPKs
from rice (Oryza sativa) (Albrecht et al. 2001; Chen et al.
2011; Piao et al. 2010), 10 CBLs and 27 CIPKs from poplar
(Populus trichocarpa) (Zhang et al. 2008; Yu et al. 2007), 8
CBLs and 43 CIPKs from maize (Chen et al. 2011), 8 CBLs
and 21 CIPKs from grape (Vitis vinifera) (Weinl and Kudla
2009), and 6 CBLs and 32 CIPK-type kinases from Sorghum
bicolor (Weinl and Kudla 2009). Genetic analyses showed
that species-specific duplication or deletion events con-
tributed to the numerical divergence of the CBL/CIPK
family. Segmental duplicated genes encode the most closely
conserved protein pairs, whereas proteins encoded by tan-
demly oriented genes show a lower degree of conservation.
Therefore, segmental duplications explain a greater pro-
portion of the current complexity of the CBL/CIPK gene
family (Kolukisaoglu et al. 2004). A dendrogram clearly
indicated a monophyletic origin of the CBL/CIPK family,
and separate branches represented proteins with different
functions. Even recently duplicated representatives may
fulfill different functions (Kolukisaoglu et al. 2004).
In this paper, we particularly focus on reviewing the
CBL–CIPK network in Arabidopsis. In particular, we
summarize the current knowledge about the functions and
mechanisms of the CBL–CIPK network, including cross-
talk with the SOS signaling pathway, the potassium sensing
and signaling pathway, the ABA signaling pathway, and
the nitrate sensing and signaling pathway.
Expression patterns and functions of CBL–CIPK
network
Since the late 1990s, the CBL–CIPK network has been
shown to be involved in responses to high salinity, osmotic
or drought stress, cold, wounding, pH, ABA, low K?,
nitrate, low oxygen, and other stresses (Luan et al. 2009;
Batistic and Kudla 2004; Li et al. 2009; Luan 2009). There
is crosstalk between the CBL–CIPK network and other
classical pathways such as the CDPK, the AMP-activated
protein kinase (AMPK), salt overly sensitive (SOS), and
reactive oxygen species (ROS) pathways. Inducing the
CBL–CIPK system in plants could enhance their tolerance
to simultaneously occurring abiotic stresses. This could be
a significant factor in yield enhancement (Thapa et al.
2011).
There is a large body of research on the expression
patterns and functions of the CBL–CIPK network in
Arabidopsis. AtCBL4/SOS3 was the first CBL protein
identified in Arabidopsis. It was shown to function
Plant Cell Rep
123
specifically in the SOS pathway, interacting with At-
CIPK24/SOS2 under salinity stress in roots (Liu 1998; Liu
and Zhu 1997; Qiu et al. 2002), while AtCBL10–At-
CIPK24/SOS2 were reported to regulate Na? homeostasis
in the vacuolar membrane in shoots and leaves (Kim et al.
2007). Recently, it was reported that AtCBL10 directly
interacts with AKT1 to regulate K? homeostasis without
binding to any AtCIPKs (Ren et al. 2013). AtCBL1, which
was first cloned and functionally identified in 1999, is
induced by drought, cold, and wounding (Kudla et al.
1999), but not ABA (Albrecht et al. 2003). AtCBL1
responds to glucose and gibberellin (GA) signals during
germination and seedling development (Li et al. 2013b).
AtCBL9 is extremely homologous to AtCBL1; however,
AtCBL9 acts as a negative regulator in ABA signaling and
is involved in ABA biosynthesis under stress (Pandey et al.
2004). AtCBL9–AtCIPK3 plays roles in negatively regu-
lating the ABA response during seed germination (Pandey
et al. 2008). The AtCBL1–AtCIPK1 complex is involved in
ABA-dependent stress responses, while the AtCBL9–At-
CIPK1 complex plays roles in ABA-independent stress
responses (D’Angelo et al. 2006). AtCBL1/9–AtCIPK23
regulate both K? uptake and NO3- uptake in plants (Xu
et al. 2006; Cheong et al. 2007; Hashimoto and Kudla
2011). AtCBL1 and AtCBL9 regulate the Arabidopsis
NADPH oxidase RbohF after interacting with AtCIPK26 in
the ROS pathway (Sagi and Fluhr 2006; Kimura et al.
2013; Drerup et al. 2013). AtCBL2 and AtCBL3 are not
expressed in response to abiotic stress, but their expres-
sions are induced by light (Nozawa et al. 2001). The vac-
uolar membrane-localized proteins AtCBL2 and AtCBL3
serve as molecular connections between Ca2? signaling
and V-ATPase, and function as key regulators to maintain
intracellular ion homeostasis (Tang et al. 2012). In addi-
tion, AtCBL2 acts as a key Ca2? sensor in response to
ABA signaling in the tonoplast (Batistic et al. 2012). At-
CBL3 interacts with AtCIPK9, the most similar homolog to
AtCIPK23, to regulate K? homeostasis (Liu et al. 2013).
The AtCBL4–AtCIPK6 complex can mediate AtAKT2
translocation from the endoplasmic reticulum (ER) mem-
brane to the plasma membrane (PM) to regulate AtAKT2
activity (Held et al. 2011). AtCIPK7 may bind to AtCBL1
and play a role in the cold response (Huang et al. 2011).
AtCIPK11 is strongly induced by drought, salt, ABA, and
sucrose stress, but not by cold. AtCBL2/SCaBP1–At-
CIPK11/PKS5 regulates the H?-ATPase AHA2 in the
Arabidopsis plasma membrane (Fuglsang et al. 2007).
AtCBL5 may function as a positive regulator of salinity or
osmotic stress responses in plants, but does not play a role
in the ABA regulation pathway (Cheong et al. 2010). The
functions of AtCBL6/AtCBL7/AtCBL8 are unclear at
present and require further investigation. AtCIPK8 posi-
tively regulates the low-affinity phase of the primary nitrate
response and is involved in glucose sensing (Hu et al.
2009). AtCIPK6 plays a role in development and salt
stress, and studies on the Atcipk6 mutant revealed that
AtCIPK6 also functions in auxin transport (Tripathi et al.
2009). In addition, AtCIPK6 is involved in the AtCBL1–
AtCIPK6–PP2CA pathway to regulate the activity of
AKT1 (Lan et al. 2011). AtCIPK15 serves as a global
negative regulator of ABA responses (Guo et al. 2002).
The functions of other AtCIPK proteins (AtCIPK2/4/5/10/
12/13/17/18/19/20/21/22/25) are still unclear, and require
further research.
The expression patterns and functions of the CBL–CIPK
network have been widely researched in other species,
including maize (Zea mays) (Tai et al. 2013; Wang et al.
2007), rice (Oryza sativa) (Yim et al. 2012; Lee et al. 2009;
Yang et al. 2008; Piao et al. 2010; Xiang et al. 2007;
Kurusu et al. 2010); soybean (Glycine max) (Li et al.
2012c), poplar (Populus trichocarpa) (Zhang et al. 2008;
Yu et al. 2007), Ammopiptanthus mongolicus (Chen et al.
2010), Populus euphratica (Li et al. 2012a; Zhang et al.
2013; Li et al. 2013a), sorghum (sorghum bicolor), Bras-
sica napus (Chen et al. 2012), cotton (Gossypium hirsutum
L.) (He et al. 2013), Cicer arietinum (Tripathi et al. 2009),
Solanum lycopersicum (Kabir 2009), apple (Malus
domestica) (Wang et al. 2012; Hu et al. 2012), Hordeum
brevisubulatum (Li et al. 2012b), and Brassica juncea
(Kushwaha et al. 2011).
Transgenic plants expressing the AmCBL1 gene from
the desert shrub A. mongolicus show increased tolerance to
multiple abiotic stresses (Chen et al. 2010). Tobacco
ectopically expressing C. arietinum CaCIPK6, a homolog
of NtCIPK6, shows a well-developed root system,
increased basipetal auxin transport, and hypersensitivity to
auxin (Tripathi et al. 2009). Zhao et al. (2009) reported that
the expression of ZmCIPK16 is regulated by polyethylene
glycol (PEG), NaCl, ABA, dehydration, heat, and drought
stress during the seedling stage. ZmCIPK16 is mainly
located in the nucleus and at the plasma membrane, while
ZmCBL3, ZmCBL4, and ZmCBL5 are able to recruit
ZmCIPK16 to the plasma membrane. Transgenic Arabi-
dopsis plants over-expressing BnCIPK6 from B. napus
show increased tolerance to high salinity and low phos-
phate, and ABA signaling is involved in these responses
(Chen et al. 2012). GmCBL1 is homologous to AtCBL1 and
over-expression of GmCBL1 enhances tolerance to salinity
and drought stress in Arabidopsis (Li et al. 2012c). PeC-
BL6 and PeCBL10 confer tolerance to multiple stresses in
transgenic triploid poplar (Li et al. 2012a). ZmCIPK1, 3, 8,
17 and 18 are differently induced by ABA, PEG, CaCl2 and
H2O2 under water stress (Tai et al. 2013). Over-expression
of GhCIPK6 greatly enhances tolerance to salt, drought,
and ABA stresses (He et al. 2013). The PeCBL1–Pe-
CIPK24/25/26 complex was reported to regulate Na?/K?
Plant Cell Rep
123
homeostasis in P. euphratica (Zhang et al. 2013).
HbCIPK2 is a positive regulator of salt and osmotic stress
responses, and was shown to control Na?/K? homeostasis
in H. brevisubulatum (Li et al. 2012b). The SlCIPK gene
responds to various abiotic stresses, such as salt, dehy-
dration, and wounding, and plays a role in ABA-mediated
Ca2? signaling (Kabir 2009).
Xiang et al. (2007) investigated the stress-responsive
expressions of 30 OsCIPKs in rice plants, and found that
more than two-thirds of OsCIPKs respond to at least one
stress factor among drought, salt, low temperature, and
ABA. Among the CIPK proteins in rice, OsCIPK03, Os-
CIPK12, and OsCIPK15 function as positive regulators of
cold, drought, and salt stress tolerance, respectively (Xiang
et al. 2007). Moreover, OsCIPK03 was shown to nega-
tively regulate salt stress tolerance in rice (Rao et al. 2011).
OsCIPK14 and 15 play an important role in the defense
signaling pathway triggered by microbes in cultured rice
cells (Kurusu et al. 2010). OsCIPK23 is induced by mul-
tiple abiotic stresses, especially drought stress, and Os-
CIPK23 is involved in the pollination response in rice
(Yang et al. 2008). OsCIPK31/OsCK1 is associated with
abiotic stress responses during the seed germination and
seedling stage, leading to the differential expression of
various stress-responsive genes (Piao et al. 2010). Os-
CIPK19 responds to light and nutrients (Ohba et al. 2000).
OsCIPK15, a Ramy3D regulator, is involved in tolerance to
O2-deficiency in rice (Lee et al. 2009). Ramy3D belongs to
a family of genes encoding a-amylases, and it plays a
significant role during anaerobic germination (Lasanthi-
Kudahettige et al. 2007). The submergene 1A (Sub1A)
gene is associated with the flooding-tolerant phenotype. A
recent study (Kudahettige et al. 2011) showed that Os-
CIPK15 plays a role in up-regulating Ramy3D and causing
rapid elongation in plants lacking the Sub1A gene, allowing
starch degradation under O2-deficiency stress. Further
research showed that hexokinase plays a role in regulating
OsCIPK15 in the sugar signaling pathway; this was shown
by using a hexokinase inhibitor to release glucose-depen-
dent OsCIPK15 suppression (Yim et al. 2012).
Unraveling the CBL–CIPK network in Arabidopsis,
rice, and other plants, especially in crops and fruits, will
pave the way for transgenic breeding strategies to produce
crops with greater stress tolerance.
Modification regulation mechanisms in the CBL–CIPK
network
The CBL protein shows significant similarities to calci-
neurin B (CNB) and neuronal calcium sensors (NCS) in
animals; hence its name, the calcineurin B-like protein (Liu
1998; Kudla et al. 1999). As Ca2? sensors, the main
structural characteristic of CBLs is that they harbor several,
usually four, EF-hand (elongation factor-hand) motifs.
CIPKs consist of an N-terminal kinase catalytic domain
and a C-terminal regulatory domain. The C-terminal
domain includes a highly conserved FISL motif (NAF
motif) and a conserved protein phosphatase interaction
(PPI) motif. Several recent reviews have covered the
structural features, activation and regulation mechanisms,
gene expression patterns, and detailed functions of the
early parts of the CBL–CIPK network, mainly related to
abiotic stress responses (Batistic and Kudla 2004, 2009; Li
et al. 2009; Weinl and Kudla 2009; Luan 2009). Crystal-
lization studies have provided much information about the
structural features of CBLs/CIPKs, how CBL proteins
interact with CIPK proteins, and how Ca2? binds to CBLs
and regulates CBL–CIPK activity (Nagae et al. 2003;
Sanchez-Barrena et al. 2005, 2007; Akaboshi et al. 2008).
Experimental evidence has shown that CIPKs are activated
by CBLs in a Ca2?-dependent manner (Kim 2012); how-
ever, further research is required to provide more details
about the complex regulation mechanism.
Post-translational modifications, including protein
phosphorylation and dephosphorylation, and lipid modifi-
cations, affect key aspects of protein function, such as their
subcellular localization, activity, and their stability to
interact with other proteins (Du et al. 2011; Lin et al. 2009;
Batistic et al. 2008). Phosphorylation of the conserved Ser
residue in the PFPF motif of the CBL protein is a common
regulatory mechanism in Arabidopsis. This phosphoryla-
tion event enhances the interaction between CBLs and
CIPKs (Du et al. 2011). However, the mechanism of
phosphorylation-dependent activation of CIPKs suggests
that CIPKs may be phosphorylated by CDPKs or MAPKs
(Kolukisaoglu et al. 2004), thus connecting with other
classical pathways.
The PPI motif is responsible for binding to protein
phosphatase 2C proteins (PP2Cs) (Ohta et al. 2003).
Sequence variations determine how CIPK interacts with
protein phosphatase ABA-insensitive 1 (ABI1) or ABA-
insensitive 2 (ABI2) proteins. One possible regulation
mechanism is that PP2Cs can regulate the activity of
CIPKs though phosphorylation/dephosphorylation or
through physiological interactions. PP2Cs also play an
important role in the ABA signaling pathway (Nakashima
and Yamaguchi-Shinozaki 2013), and possibly play roles
in crosstalk between the ABA signaling pathway and other
pathways.
To fulfill its function in location-dependent signal
transduction in plant cells, the CBL protein must be
modified by dual lipid modifications (N-myristoylation and
S-acylation) (Batistic et al. 2008). However, not all CBLs
harbor N-myristoylation motifs, which are related to sub-
cellular localization in plant cells. Surprisingly, AtCBL10
Plant Cell Rep
123
can also attach to the membrane via its hydrophobic
domain (Quan et al. 2007). Either the N-myristoylation
motif or the hydrophobic domain enhances the attachment
of CBLs to the membrane. Subcellular location analyses of
AtCBL2, AtCBL3, AtCBL6, AtCBL7 showed that they
can be located in the membrane, even though they lack
lipid modification motifs (Batistic et al. 2008).
Targeting of the AtCBL1 protein to the plasma mem-
brane occurs via a novel BFA- and NtSar1-independent
pathway, which does not involve ER-to-Golgi transport,
and does not depend on COPII vesicle formation (Batistic
et al. 2008). The targeting pathways of all investigated
CBL proteins, which determine the localization of their
interacting kinases, do not involve COPII-mediated vesicle
transport via the Golgi (Batistic et al. 2010). Recently, it
was shown that AtCBL2 can be targeted to the vacuolar
membrane via a BFA- and Wortmannin-insensitive trans-
port pathway. S-acylation of three cysteine residues in the
N-terminus is essential for this transport step, and the
N-terminal domain of 22 amino acids is required and suf-
ficient for vacuolar membrane targeting (Batistic et al.
2012).
CBL–CIPK network mediation of responses to abiotic
stresses
The CBL–CIPK network mediates responses to various
abiotic stresses, such as high salt, osmotic/drought, cold (Li
et al. 2009), wounding, flooding (Bailey-Serres and Voe-
senek 2010), high pH (Li et al. 2009), and low K?. The
CBL–CIPK complex post-translationally phosphorylates
downstream target proteins, such as transcriptional factors
(TFs) and ion channels, to respond to external environ-
mental stimuli and adapt to adverse stresses. To date,
research on the CBL–CIPK system has shown that it is
closely connected to ion channels related to the influx or
efflux of various ions, including Na?, K?, Ca2?, NO3-,
and H?. Since the first report of AtCBL1/9 interacting with
AtCIPK23 to regulate AtAKT1 (Xu et al. 2006), there has
been much interest in the regulation of low-K? responses.
In the last 7 years, many studies have focused on how the
CBL–CIPK network mediates the low-K? response; this
will be discussed in next section. In this section, we focus
on the SOS pathway.
The SOS pathway, the main pathway for maintaining
ion homeostasis in plant cells, was the first CBL–CIPK
pathway identified in plant cells (Liu 1998; Guo et al.
2001; Gong et al. 2002; Qiu et al. 2002). Subsequently,
SOS3 has been renamed AtCBL4; this name is now used
more commonly. There are three main sodium-transport-
ing proteins in plants: the HKT family of transporters
(Na?/K? symporters), the NHX family (Na?/H?
exchangers in the tonoplast), and SOS1 proteins (Na?/H?
antiporters in the plasma membrane) (Shi et al. 2000). The
key to enhance salt tolerance is to allow transport of Na?
back into the soil, to sequester Na? in the vacuole, or to
recycle Na? into old tissues (Luan et al. 2009). External
stresses activate the AtCBL4/SOS3–AtCIPK24/SOS2
complex to stimulate the Na?/H? exchange activity of
SOS1 (Zhu 2002), resulting in exclusion of excess Na?
(Qiu et al. 2002). It was suggested that tonoplast Na?/H?
NHX antiporters are activated by AtCIPK24/SOS2
through a mechanism related to the Ca2? sensor AtCBL10
to sequester intracellular extra Na? in the vacuole (Cheng
et al. 2004). The HKT family is a group of Na?-specific
transporters, although they were initially described as
high-affinity K? transporters (Horie et al. 2009; Gassman
et al. 1996). HKT transporters can be divided into two
subclasses; Class 1 shows a preference to transport Na?
over other cations, while Class 2 shows greater K? and
Na? permeability (Horie et al. 2009). The SOS pathway
appears to regulate AtHKT1 activity under salt stress,
mediating Na? entry into root cells in Arabidopsis (Ma-
hajan and Tuteja 2005; Uozumi et al. 2000; Zhu 2002).
Further research indicated that AtHKT1;1/AtHKT1, which
encodes a low-affinity Na? transporter, is the only HKT
gene in Arabidopsis (Uozumi et al. 2000). AtHKT1;1 was
found to localize at the PM of xylem parenchyma cells. Its
function is to load Na? into phloem cells for transport
downwards from the shoots to the roots, thus preventing
the overaccumulation of Na? in shoots under salinity
stress (Horie et al. 2009). Unknown Ca2? sensors inter-
acting with CIPK24 may mediate the vacuolar membrane-
localized H?/Ca2? antiporter CAX1 to control intracellu-
lar Ca2? homeostasis (Quan et al. 2007).
The SOS pathway has also been studied in other
species. In apple, MdCIPK6L expression is positively
induced by abiotic stresses. Over-expression of
MdCIPK6L confers tolerance to salt, drought, and cold
stresses in transgenic tomatoes. Its ectopic expression can
functionally complement the Arabidopsis sos2 mutant,
even though it is not homologous to AtCIPK24/SOS2
(Wang et al. 2012). MdSOS2 isolated from apple shows
the highest similarity to AtCIPK24/SOS2, and it posi-
tively responds to salt stress and functionally comple-
ments the Arabidopsis sos2 mutant (Hu et al. 2012). The
structural and functional analysis of BjSOS3 established
the SOS pathway in B. juncea (Kushwaha et al. 2011).
OsCBL4 is most homologous to AtCBL4/SOS3 in rice.
Like OsCBL7 and OsCBL8, it was able to functionally
complement the sos3-1 mutant in Arabidopsis, indicating
that has the same function as AtCBL4/SOS3 (Martinez-
Atienza et al. 2007). ZmCBL4 is most similar to Os-
CBL4 and it can also complement the sos3-1 mutant in
Arabidopsis (Wang et al. 2007).
Plant Cell Rep
123
Involvement of the CBL–CIPK network in potassium
sensing and signaling
Potassium, the most abundant cation in plants, plays crucial
roles in many physiological and biochemical reactions in
plant cells. During evolution, plants have evolved complete
systems to take up sufficient potassium to meet their needs
under adverse environmental conditions. There are three
types of K? channels in plants; the plant Shaker-like
family, TPK proteins (tandem-pore K? channels), and
plant Kir-like channels (Luan et al. 2009). The Shaker-like
K? channels family has nine members, which can be
grouped into four subfamilies according to their biophysi-
cal properties and structure. The first subfamily is the
Arabidopsis K? transporter (AKT) subfamily, which
includes AKT1, AKT2/3, AKT5, and AKT6/SPIK. The
second subfamily, the K? transporters of Arabidopsis
thaliana (KAT)-type transporters, includes KAT1, KAT2,
and KC1. The other two subfamilies are the stellar K?-
outward rectifying (SKOR), and guard cell outward recti-
fying K? (GORK) channels (Luan et al. 2009). SKOR and
GORK are outward rectifying channels and the others are
inward-rectifying channels. The AKT, SKOR, and GORK
channels contain an ankyrin repeat domain. This domain is
phosphorylated or dephosphorylated by certain CIPKs or
PP2Cs, respectively. Each K? channel has its specific
function, although they are all related to K? transport.
These channels play roles in plant growth, K? transloca-
tion, and K? uptake in different organs. AtKAT1 is an
inward-rectifying potassium channel that plays an impor-
tant role in stomatal movement, possibly related to ABA-
mediated stomatal closure. AtKAT1 is regulated by OST1/
SnRK2.6 (Anderson et al. 1992; Sato et al. 2009; Yunta
et al. 2011; Nakashima and Yamaguchi-Shinozaki 2013).
AtAKT1, the Shaker-like potassium channel Arabidopsis
K? transporter 1, was first cloned and expressed in 1992
(Sentenac et al. 1992). The expression patterns and phys-
iological functions of AtAKT1 were identified in 1996
(Lagarde et al. 1996). This transporter is the major player
in K? uptake, and together with AtHAK5 (a high-affinity
K? transporter), it mediates high-affinity K? absorption in
Arabidopsis roots (Rubio et al. 2008; Gierth et al. 2005).
Further research (Rubio et al. 2008) showed that AtHAK5
is functionally inhibited by NH4? in the growth solution
and that AtAKT1 mainly mediates K? absorption. These
findings indicated that there are also effects from other
ions.
In plant root cells, low-potassium stress is associated
with ethylene and ROS production, leading to Ca2? fluc-
tuations that are sensed by CBLs (Hernandez et al. 2012;
Shin and Schachtman 2004); however, further research is
required to clarify the exact mechanism of Ca2? sensing. In
plants, ROS are generated during normal metabolic
processes in the mitochondria, peroxisomes, and cyto-
plasm. Overproduction of ROS leads to oxidative damage
of lipids, proteins, and nucleic acids, especially under
abiotic stresses such as drought, low K?, high salt, etc.
(McCord 2000). Under low-K? stress, ethylene acts
upstream of ROS (Ho and Tsay 2010), which mainly
accumulate in the region just behind the elongation zone in
the root (Shin and Schachtman 2004). The main form of
ROS, H2O2, is a signaling molecule in plants and functions
in ABA-mediated stomatal closure (Pei et al. 2000). The
AtCBL1/AtCBL9–AtCIPK23 complex can directly acti-
vate the plasma membrane-localized potassium channel
AtAKT1, enhancing K? uptake under low-K? conditions
(Xu et al. 2006; Li et al. 2006). Stomatal closure plays an
important role not only in CO2 transport, but also in tran-
spiration. The movement of solutes across the guard cell
plasma membrane and the tonoplast, with K? as the major
cation, determines the rate of transpiration (water loss).
The regulation of AtAKT1 by AtCIPK23 may also occur in
the stomata, and negatively affects plant performance
under water stress conditions. In conclusion, AtAKT1
plays an important role not only in K? uptake, but also in
stomatal movement and in the response to water stress
(Nieves-Cordones et al. 2012).
Besides the specific interaction between AtAKT1 and
AtCIPK23, AtCIPK9, the closest homolog to AtCIPK23,
can also interact with AtCBL3 to regulate K?-homeostasis
under low-K? stress in Arabidopsis (Liu et al. 2013).
Similarly, AtCIPK6 and AtCIPK16 both activate AtAKT1
by interacting with certain AtCBLs (Luan 2009; Lee et al.
2007). However, the interaction affinity among these
CBLs, CIPKs, and AKT1 is quite different. In addition, the
AtCBL4–AtCIPK6 complex can mediate translocation of
AtAKT2 from the ER membrane to the PM to regulate
AtAKT2 activity (Held et al. 2011). In grapevine, VvK1.1
is an inward K? channel that belongs to the Shaker family;
it can functionally complement AtAKT1 (Cuellar et al.
2010; Cuellar et al. 2013). The poplar proteins PeKC1 or
PeKC2 can also complement the function of AKT1 in the
akt1 mutant. These results showed that these proteins are
involved in the CBL1–CIPK23 signal transduction path-
way and play an important role in the plant response to
low-K? stress (Zhang et al. 2010).
AtAKT1 is not only positively activated by phosphor-
ylation, but is also regulated by dephosphorylation via
certain PP2Cs (Lee et al. 2007). AIP1 (PP2C-type phos-
phatase AKT1-interacting PP2C1) significantly reduces
AtAKT1 activity. It negatively regulates the K? high-
affinity channel and counteracts the function of the At-
CBL1/AtCBL9–AtCIPK23 complex (Lee et al. 2007). A
recent study elucidated the details of the CBL–CIPK–
PP2C–AKT1 regulation system (Lan et al. 2011). Group A
of the PP2Cs family contains nine members; ABI1, ABI2,
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123
HAB1, HAB2, AHG1, AHG3/AtPP2CA, HAI1, HAI2, and
HAI3 (Hirayama and Shinozaki 2007). Lan et al. (2011)
showed that PP2Cs can interact with CIPKs via both the
protein phosphatase interacting motif (PPI) and the kinase
domain. In addition, specific CBLs can interact with PP2C
and inactivate it to recover AKT1 activity. For example,
PP2CA inactivates AtAKT1 by interacting with both the
PPI domain and the kinase domain of AtCIPK6 (Lan et al.
2011). All the evidence suggests that the CBL–CIPK–
PP2C–AKT1 pathway is under complex regulation. For
example, AtAKT1 activity can be regulated separately by
four different proteins; AtCIPK23, AIP1, AtKC1, and At-
CBL10 (Ren et al. 2013). There is still much to learn about
how protein–protein interactions affect the various com-
ponents of the CBL–CIPK network.
Crosstalk between the CBL–CIPK network
and the ABA signaling pathway
Abscisic acid is one of the most important phytohormones.
It plays roles in plant growth, development, seed germi-
nation, and responses to abiotic stresses, especially osmotic
stress (Nakashima and Yamaguchi-Shinozaki 2013). ABA
mediates the stomatal closure signaling pathway (Ma-
cRibbie 1998), and regulates the expressions of stress-
responsive genes, such as those encoding LEA proteins.
ABA induces the production of ROS, such as H2O2, by
NADPH oxidases such as Arabidopsis AtRbohD and At-
RbohF (Ma et al. 2012), resulting in specific spatial and
temporal changes in Ca2? concentrations. ABA-induced
H2O2 and the H2O2-activated Ca2? channels are important
mechanisms for ABA-induced stomatal closure (Pei et al.
2000). In addition, ROS can affect the ABA signaling
pathway by regulating ABI1 and ABI2 (Hirayama and
Shinozaki 2007). ABI1 and ABI2 are involved in regulat-
ing seed germination and stomatal movement, while ABI3/
4/5 play roles in regulating seed maturation, germination,
and seedling growth (Pandey et al. 2004). ABI3/4/5 posi-
tively regulate ABA signaling pathways and ABI1/2 are
negative regulators. Over-expression of any one of the
ABI3/4/5 proteins leads to ABA hypersensitivity.
Another ABA-mediated stomatal closure mechanism is
to decrease K? influx through inward channels and
increase K? efflux through outward channels in guard
cells. AtKAT1, an inward-rectifying potassium channel,
plays an important role in stomatal movements related to
ABA-mediated stomatal closure, and is regulated by OST1/
SnRK2.6 (Anderson et al. 1992; Sato et al. 2009; Yunta
et al. 2011; Nakashima and Yamaguchi-Shinozaki 2013).
In addition, AtAKT1 plays an important role in plant
transpiration. Atakt1 mutants show more efficient stomatal
closure than that of wild-type, and show greater tolerance
to water loss. AtCIPK23 plays a positive role in regulating
AtAKT1; thus, it negatively affects water status in plants
(Nieves-Cordones et al. 2012).
The ABA response is precisely regulated by specific
responsive elements and different TFs. Various aspects of
ABA responses and its signaling pathway including ABA
synthesis, transport, receptors, regulation mechanisms, and
their responses to abiotic stresses have been reviewed
elsewhere (Nakashima and Yamaguchi-Shinozaki 2013;
Hirayama and Shinozaki 2007). The ABA-responsive ele-
ment (ABRE) is the major cis-element in the ABA
response, and ABRE-binding proteins (AREB)/ABRE-
binding factor (ABF) transcription factors regulate ABRE-
dependent gene expression together with other TFs. The
AREB/ABFs encode bZIP-type TFs and belong to the
group A subfamily. SNF1-related protein kinases 2
(SnRK2s) are key positive regulators in ABA signaling,
and function by post-transcriptionally modifying (phos-
phorylating) related TFs. Group A 2C-type protein kinases
(PP2Cs) are negative regulators in ABA signaling path-
ways, and inactivate SnRK2s via physical interactions and
dephosphorylation (Nakashima and Yamaguchi-Shinozaki
2013). The SnRK2s induce rapid physiological changes
including stomatal closure. The PYR/PYL/RCAR ABA
receptors can disrupt the interaction between SnRK2s and
PP2Cs in the presence of ABA, leading to reactivation of
SnRK2s (Santiago et al. 2009).
Calcium may be a positive regulator in GA signaling but
a negative regulator in ABA signaling (Pandey et al. 2008).
Although ABA-dependent and ABA-independent pathways
operate simultaneously to regulate stress-responsive genes,
the ABA-dependent signaling pathway plays an essential
role in regulating osmotic stress-responsive genes (Chin-
nusamy et al. 2004). AtCBL9 acts as a negative regulator in
ABA signaling and is involved in ABA biosynthesis under
stress conditions (Pandey et al. 2004). Atcbl9 shows
hypersensitivity to ABA (Pandey et al. 2004). The
expression of AtCIPK3 is induced by cold, high salt,
wounding, drought, and ABA, and AtCIPK3 functions as a
negative regulator in ABA signaling during seed germi-
nation (Kim 2003). The AtCBL9–AtCIPK3 complex neg-
atively regulates the ABA response during seed
germination (Pandey et al. 2008). The AtCBL1–AtCIPK1
complex is involved in ABA-dependent stress responses,
while the AtCBL9–AtCIPK1 complex in involved in ABA-
independent stress responses (D’Angelo et al. 2006). At-
CBL1/9 also interact with AtCIPK26 to regulate the
Arabidopsis NADPH oxidase in the ROS pathway (Kimura
et al. 2013; Sagi and Fluhr 2006; Drerup et al. 2013). Yeast
two-hybrid screening, in vitro pull-down, and bimolecular
fluorescence complementation (BiFC) assays confirmed
that AtCIPK26 interacts with the RING-type E3 ligase and
Keep on Going (KEG), and that it is degraded by the
Plant Cell Rep
123
ubiquitin–proteasome system (Lyzenga et al. 2013). KEG
targets the group A bZIP protein ABA-insensitive 5 (ABI5)
for degradation and negatively regulates the ABA signaling
pathway (Stone et al. 2006; Liu and Stone 2010). At-
CIPK26 interacts with ABI1, ABI2, ABI5 in the ABA
signaling pathway and plays a positive role in ABA sig-
naling in seed germination (Lyzenga et al. 2013). At-
CIPK15, which interacts with ABI2, serves as a global
negative regulator of ABA responses during seed germi-
nation, seedling growth, stomatal closure, and gene
expression (Guo et al. 2002).
More detailed evidence is required to explore the
crosstalk between the ABA signaling pathway and the
CBL–CIPK signaling pathway.
CBL–CIPK network mediation of nitrate sensing
and signaling
In soil, nitrate is the major form of nitrogen available to
most land plants. It is essential for plant growth and
physiological reactions in plant cells (Bouguyon et al.
2012). In 2009, two groups reported breakthroughs con-
cerning nitrate sensing and signaling (Ho et al. 2009; Hu
et al. 2009). The mechanism of nitrate sensing and sig-
naling in plants has been reviewed elsewhere (Bouguyon
et al. 2012; Ho and Tsay 2010). Here, we focus on the
crosstalk between the CBL–CIPK network with NO3-
sensing and signaling by CHL1 in Arabidopsis.
There are four main transporters responsible for nitrate
acquisition from the soil; CHL1 (AtNRT1.1), AtNRT1.2,
AtNRT2.1, and AtNRT2.1. Among them, AtNRT2.1 and
AtNRT2.1 function as high-affinity nitrate transporters, and
AtNRT1.2 is involved in low-affinity nitrate uptake. CHL1
(AtNRT1.1) was shown to function as a dual-affinity
nitrate transporter using the Xenopus oocyte expression
system (Shi et al. 2000). A possible regulation mechanism
of CHL1 (AtNRT1.1) is phosphorylation (Liu and Tsay
2003). The nitrate uptake mode of CHL1 can be regulated
by phosphorylation at threonine residue 101 (T101); that is,
phosphorylated CHL1 functions as a high-affinity trans-
porter, while the dephosphorylated form functions as a
low-affinity nitrate transporter (Liu and Tsay 2003). A
recent study suggested that CHL1 (AtNRT1.1) also func-
tions as a nitrate sensor. Therefore, it is the first transceptor
(dual function transporter and receptor) described in higher
plants (Ho et al. 2009). CHL1 functions as a nitrate sensor
responsible for primary nitrate sensing in an uptake activ-
ity-independent manner (Ho et al. 2009).
AtCIPK23 may function to regulate CHL1 by phos-
phorylating T101. This was determined by a microarray
analysis of the chl1-5 mutant, and was further confirmed by
extensive genetic and biochemical approaches (Ho et al.
2009). The increased high-affinity phase of the primary
nitrate response resulted in a higher Km (lower affinity) in
the cipk23 mutants than in WT plants; this suggests that
AtCIPK23 negatively regulates the primary nitrate
response in low-nitrate conditions (Ho et al. 2009).
A differential transcriptomic study showed that At-
CIPK8 is involved in regulating the primary nitrate
response in the chl1-5 mutant. A series of experiments on
the Atcipk8 mutant indicated that AtCIPK8 is involved in
long-term nitrate-regulated root growth and positively
regulates the primary nitrate response (Hu et al. 2009).
However, the mechanism by which AtCIPK8 accurately
regulates CHL1 is still unclear and needs further investi-
gation (Hu et al. 2009).
Discussion and conclusion
The CBL–CIPK network is involved in responses to vari-
ous abiotic stresses, and has crosstalk with different sig-
naling pathways in plant cells, such as the SOS pathway,
the low-K? response pathway, the nitrate sensing and
signaling pathway, and the ABA signaling pathway. Novel
pathways connected to the CBL–CIPK network have been
discovered recently, including the GA signaling pathway
(Li et al. 2013b), the O2-deficiency pathway (Lee et al.
2009), the glucose signaling pathway (Hu et al. 2009; Yim
et al. 2012; Li et al. 2013b), and the ROS pathway (Drerup
et al. 2013; Sagi and Fluhr 2006; Kimura et al. 2013). For
all of these signaling pathways, their crosstalks appear to
connect with different functions of, and interplay among,
small signaling molecules including GA, ABA, Ca2?, and
H2O2. As more is learned about the CBL–CIPK network,
the unraveling of the crosstalk among different pathways
will provide more information about the physiological
responses of plants, including transpiration (e.g., stomatal
movement), seed germination, seedling growth, and min-
eral nutrient uptake, under stress conditions.
Because of the complexity of the CBL–CIPK network,
current and future research should include system-level
approaches (e.g., transcriptomic and proteomic analyses,
suppression subtractive hybridization), and modern exper-
imental and computational approaches (e.g., bioinformatics
analyses). Bioinformatics and -omics research tools will
also be useful to clarify the evolution, diversification, and
functions of the CBL–CIPK network. In further studies,
new biotechnological tools and methods should be used to
identify the targets of kinases, to conduct detailed analyses
of the protein–protein interactions to prove their interac-
tions/relationships (e.g., mcBiFC), and to measure and
observe experimental results. Combining mature molecular
and physiological research methods with new technologies
from other fields, such as computational biology, may
Plant Cell Rep
123
provide more advanced, rapid, and unequivocal ways to
unravel the CBL–CIPK network.
Grefen and Blatt (2012) discussed the limitations of the
yeast two-hybrid (Y2H) method for studying membrane
proteins, and indicated that the mating-based split-ubiquitin
system (SUS) in yeast offers several advantages over the
Y2H approach, especially for studying integral membrane
proteins and membrane-anchored proteins. Their study
indicated that AtCBL9 and AtCBL4 can directly interact
with AtAKT1 without binding to AtCIPK23. Recently,
AtCBL10 was shown to directly interact with AKT1 to
regulate K? homeostasis without binding to any AtCIPKs
(Ren et al. 2013). These findings have advanced our
understanding of the functions and regulation mechanisms
of Ca2? sensors.
Research on the CBL–CIPK network has mainly
focused on identifying the functions of its components, its
regulation mechanisms, and its crosstalk with different
signaling pathways. The main challenge for future research
is to clarify the interconnections among pathways and to
explore the synergistic functions of these diverse signaling
systems to unravel the complex network of regulatory
mechanisms in plant cells.
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