Ca2+ signaling in plant

LightCold stressHeat shockMechanical stresses(Touch, wind &Wounding)Pathogen invadingPhytohormones(Auxin, ABA, GA)Gravity

[Ca2+]cyt

AM, FM, DU

DiverseCellularResponses

PrimaryCa2+-SignalDecoder

CalmodulinsCDPKsOther CBPs

Amplitude (AM)

Duration (DU)

Frequency (FM)

Model of Ca2+/calmodulin-mediated network in plants

• Calcineurin is a Ca2+, CaM-dependent protein phosphatase that is highly conserved in eukaryotes from yeast to mammals. Calcineurin is composed of two subunits. - catalytic subunit, calcineurin A (CaM-binding domain)- regulatory subunit, calcineurin B

• CaM and CBL(calcineurin B–like) protein are small proteins that contain multipleCa2+ binding domains but lack other effector domains, such as the kinase domain in CDPKs.CBLs interact with target proteins and regulate their activity. CBLs are encoded by a multigene family of at least 10 members in Arabidopsis.Unlike CaMs, which interact with a large variety of target proteins, CBLs appear to interact with a single family of protein kinases.

• CBL-interacting protein kinase (CIPK) all contain a unique C-terminal region that is both required and sufficient for interaction with the AtCBL-type but not calmodulin-type Ca2+ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca2+,suggesting that increases in cellular Ca2+ concentrations may trigger the formation of AtCBL–kinase complexes in vivo.

Calcineurin, CBL proteins, CIPK

A schematic presentation of calcineurin to CBL–CIPK

• Calcineurin B(CNB)• Calmodulin (CaM)• Calcineurin A (CNA)• Calcineurin B-like protein (CBL)

AtCBL4

AtCBL5

AtCBL9

AtCBL1

AtCBL2

AtCBL3

AtCBL6

AtCBL7

AtCBL8

Phylogenetic tree of AtCBL proteins in Arabidopsis thaliana

Overall structure of the AtCBL2–AtCIPK14 complex

CBL1, CBL2, CBL3, and CBL9 interacted with CIPK6, CIPK16, and CIPK23

Regulatory features of CBL–CIPK interactions

• CBLs interact with CIPKs through the C-terminal non kinase domain, which contains a conserved region among different CIPK members.-hydrophobic interaction with CBL proteins

• Regulatory feature concerns the role of Ca2+ in the CBL–CIPK interaction. Initial analysis of interaction between CBL1 and CIPK1 showed a requirement for micromolar levels of Ca2+.

• Regulatory C-terminal domain of CIPKs include the motif for interacting with a group of type-2C protein phosphatases (PP2Cs).

• Activating CIPK kinase activity, certain structural features of CBLs also suggest that these Ca2+-sensors can change cellular localization of the CBL–CIPK complexes.

Regulation of ion homeostasis by the SOS pathway during salt stress

SOS : Salt overly sensitive

SOS2 : CIPK24

SOS3 : CBL4

Hypothetical model of alternative targeting of CIPK24/SOS2 to plasmamembrane (PM) or tonoplast by CBL4/SOS3 or CBL10.

Stress and ABA Induction of CBL1 Protein Expression Patterns of the CBL9 Gene

Hypothetical Model of CIPK3 Function

Abiotic stress signaling through the CBL-CIPK network

A working model of the Ca2+-dependent pathway for potassium (K)channel activation in low-K response

Signaling pathways that regulate the expression and activities of ion transporters to maintain a low cytoplasmic concentration of Na+ under salt stress

1. Calcineurin B-like proteins (CBLs) represent a unique family of plant calcium sensors that relay signals by interacting with a family of protein kinases (CIPKs).

2. SOS3, a Ca2+ sensor, transduces the signal downstream after activating and interacting with SOS2 protein kinase.

3. This SOS3-SOS2 complex activates the Na+/H+ antiporter activity of SOS1 thereby reestablish cellular ion homeostasis.

SOS : Salt Overly Sensitive gene

SOS 1 : Na+/H+ andtiporter of plasma membrane

SOS 2 : CBL-interacting protein kinases (CIPKs_24)

SOS 3 : Calcineurin B-like protein, (CBL4 _ calcium sensor like calmodulin)

Expression pattern of the CBL10 gene.

F : flower

S: stems

L : rosette leaves

R : roots

SH : shoots

SL : siliques

 The cbl10 mutant is hypersensitive to NaCl

175mM NaCl

 Mature cbl10 mutant plants are hypersensitive to NaCl

300mM NaCl

NaCl hypersensitivity of the cbl10 mutant is caused by defects in ion homeostasis.

NaCl hypersensitivity of the cbl10 mutant is caused by defects in ion homeostasis

CBL10 interacts with CIPK24

CBL10+

CIPK24GST CIPK CBL10

 CBL10 is localized to punctate structures and vacuolar membranes

 CBL10 co-localizes with endosomal and tonoplast marker proteins

ARA6CBL10

CBL10 ARA7

CBL10

CBL1

FM4-64

FM4-64

CBL10

CBL1

TPC1

TPC1

ARA6, ARA7 : endosomesFM4-64 : plasma membraneTPC1 : vacuolar membrane

 CBL10 interacts with CIPK24 at the tonoplast

CBL10::CIPK24

DAPI/FM4-64

TPC1

CBL1::CIPK24

 Hypothetical model of CBL10 function

Summary

1. The CBL protein CBL10 functions as a crucial regulator of salt tolerance in Arabidopsis.

2. Cbl10 mutant plants exhibited significant growth defects and showed hypersensitive cell

death in leaf tissues under high-salt conditions.

3. The Na+ content of the cbl10 mutant was significantly lower than in the wild type under

either normal or high-salt conditions, suggesting that CBL10 mediates a novel Ca2+- signaling pathway for salt tolerance.

4. The CBL10 protein physically interacts with the salt-tolerance factor CIPK24 (SOS2), and

CBL10-CIPK24 (SOS2) complex is associated with the vacuolar compartments that are

responsible for salt storage and detoxification in plant cells.

5. These findings suggest that CBL10 and CIPK24 (SOS2) constitute a novel salt-tolerance

pathway that regulates the sequestration/compartmentalization of Na+ in plant cells.

6. These results identified CIPK24 as a multi-functional protein kinase that regulates different

aspects of salt tolerance by interacting with distinct CBL calcium sensors.