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: biolwl@dlut.edu.cn

Q. Yu

e-mail: qinyangyu@gmail.com

L. An

e-mail: bioeng@dlut.edu.cn

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

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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?

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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

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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).

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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

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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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