Ca 2+ signaling in plant Light Cold stress Heat shock Mechanical stresses (Touch, wind & Wounding) Pathogen invading Phytohormones (Auxin, ABA, GA) Gravity [Ca 2+ ] cyt AM, FM, DU Diverse Cellular Responses Primary Ca 2+ -Signal Decoder Calmodulins CDPKs Other CBPs Amplitude (AM) Duration (DU) Frequency (FM)

Model of Ca 2+ /calmodulin-mediated network in plants

Calcineurin is a Ca 2+, 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 multiple Ca 2+ 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 Ca 2+ binding proteins from plants. Interactions between the kinases and AtCBLs require micromolar concentrations of Ca 2+, suggesting that increases in cellular Ca 2+ 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 Ca 2+ in the CBL–CIPK interaction. Initial analysis of interaction between CBL1 and CIPK1 showed a requirement for micromolar levels of Ca 2+. 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 Ca 2+ -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 plasma membrane (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 Ca 2+ -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 Ca 2+ 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 + CIPK24 GST CIPK CBL10

CBL10 is localized to punctate structures and vacuolar membranes

CBL10 co-localizes with endosomal and tonoplast marker proteins ARA6CBL10 ARA7 CBL10 CBL1 FM4-64 CBL10 CBL1 TPC1 ARA6, ARA7 : endosomes FM4-64 : plasma membrane TPC1 : 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 Ca 2+ - 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.