Activation of SRK2E/OST1 by ABA & low humidity stress

A, ABA dose response for SRK2E/OST1 activation

B, ABA-dependent kinase activities in the srk2e mutant

C, ABA-dependent kinase activity was not detected in wild-type leaves

D, ABA-inducible genes were expressed in wild-type leaves

E, activation of SRK2E/OST1 by low humidity stress in leaf tissues

F, kinase activities in the low humidity-stressed srk2e mutant

G, ABA and OS activated SRK2E-GFP in Arabidopsis T87 cells, Cold Stress NOT

Effect of abi and aba mutations on the activation of SRK2E/OST1

A, SRK2E-GFP proteins were overexpressed in abi1–1 and abi2–1

B, SRK2E-GFP proteins were overexpressed in the aba2–1

C, water loss in abi1–1 and aba2–1 mutant seedlings

These results indicate that two similar PP2C, ABI1 and ABI2, have distinct roles in ABA signaling in terms of regulation of SRK2E/OST1

Structural analysis of the C-terminal region in SnRK2 proteins

A, comparison of the C-terminal amino acids in

Arabidopsis SnRK2s

B, SRK2D-GFP, SRK2F-GFP, SRK2G-GFP, and SRK2I-GFP proteins were over expressed in T87 cells

C, comparison of the C-terminal amino acids in three SnRK2s

Arabidopsis SRK2E/OST1

Fava bean AAPK Rice SAPK8

The effect of N and C terminus amino acids on the activation of SRK2E/OST1

A, schematic representation of the deletion constructs introduced into T87 cells

Full-length, N terminus-deleted, and two types of C terminus-deleted SRK2E were designated as 2EWT-GFP, 2EΔN-GFP, 2EΔC1-GFP, and 2E Δ C2-GFP, respectively

B, kinase activity of truncated SRK2E/OST1 in T87 cells

Each construct was overexpressed as a GFP fusion protein and treated with ABA (50 M) and sorbitol (0.8M) for the indicated times, and their cell extracts were subjected to

in-gel kinase assay

Functional analysis of truncated SRK2E in the complementation of the srk2e phenotype

A, 35S::2EWT-GFP srk2e and35S::2EΔC1-GFP srk2e seedlings under low humidity stressRed triangles indicate wilty leaves observed in the 35S::2E Δ C1-GFP srk2e plants

B, water loss in each complementation line

C, effects of ABA on the water loss in 35S::2EWT-GFP srk2e and 35S::2EC1-GFP srk2e plants

D, kinase activity in C terminus-deleted SRK2E (2EΔC1) under low humidity stress

Physical interactions between SRK2E/OST1 and ABA signaling factors in the yeast two-hybrid assay

A, estimation of partners would interact with SRK2E/OST1

Interactions between SRK2E/OST1 and each signaling factor, including ABI1, ABI2, AtRac1, and GPA1, were determined by growth assay on medium lacking Leu, Trp, and His in the presence of 20mM 3-AT

B, effect of the abi1–1 mutation on binding to SRK2E/OST1

Degree of binding to SRK2E/ OST1 was compared between ABI1 and Abi1–1 by growth assay

SRK2E/OST1 was used as the bait

protein

ABI1 physically binds to the C terminus of SRK2E/OST1

A, constructs used for the identification of the ABI1 binding domain on SRK2E/OST1

Full-length (2EWT) ,C terminus deleted (2E-(1–317)) ,C terminus (2E-(319 –357)) ,

C terminus of SRK2G/OSKL8 (2G-(311–353)) were used as the prey proteins

B, identification of the ABI1 binding domain in SRK2E/OST1

Interactions between ABI1 and each construct presented in panel A were determined by growth assay on medium lacking Leu, Trp, and His in the presence of 20 mM 3-AT

ABI1 was used as the bait protein.

A model to explain SRK2E/OST1 signaling in stomatal closure

When plants sense low humidity stress, the signal may integrate into two pathways

ABA produced by low humidity may act on Domain II through Pathway I (step 3)

ABI1 binds to this region and may regulate SRK2E activity (step 4)

ABA may also activate Pathway II. In this

case, ABI1 and ABI2 may function as negative regulators (step 5)

On the other hand, the ABA-independent pathway specifically acts on Domain I through Pathway III (step 7) also regulates SRK2E activity (step 8)

These three pathways converge (steps 5, 9)

and result in the complete closure of stomata

ConclusionConclusionThe direct interaction between SRK2E/OST1 and ABI1

through Domain II plays a critical role in the control of stomatal closure

ABI1 and ABI2 may function as -ve regulators of ABA signaling

SRK2E without Domain II was activated by low humidity

Domain II is required for interaction with ABI1 and activation by ABA

Members of SnRK2 family may activate and be activated by different downstream and upstream

factors