1. The document discusses the activation of the SnRK2 protein SRK2E/OST1 by abscisic acid (ABA) and low humidity stress. 2. It examines the roles of the phosphatases ABI1 and ABI2 in regulating SRK2E/OST1 under ABA signaling and in response to water loss. 3. The C-terminal domain of SRK2E/OST1 is found to physically interact with ABI1, and this interaction plays a critical role in controlling stomatal closure in response to ABA and low humidity stress.

1. Activation of SRK2E/OST1 by ABA & low humidity stress
A, ABA dose response for
SRK2E/OST1 activation
B, ABA-dependent kinase
activities in the srk2emutant
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-stressedsrk2e
mutant
G, ABA and OS activated
SRK2E-GFP inArabidopsis
T87 cells, Cold Stress NOT

2. Effect of abi and abamutations on the activation of SRK2E/OST1
A, SRK2E-GFP proteins were overexpressed
in abi1–1 andabi2–1 These results indicate that
two similar PP2C, ABI1 and
B, SRK2E-GFP proteins were overexpressed
in theaba2–1 ABI2, have distinct roles in
ABA signaling in terms of
C , water loss in abi1–1 and aba2–1mutant regulation of SRK2E/OST1
seedlings

3. Structural analysis of the C-terminal region in SnRK2 proteins
A, comparison of the
C-terminal amino acids in
ArabidopsisSnRK2s
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
RiceSAPK8

4. 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 as2EWT-
GFP ,2EΔN-GFP ,2EΔC1-GFP, and
2EΔ 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

5. Functional analysis of truncated SRK2E in the
complementation of the srk2ephenotype
A ,35S:: 2EWT-GFP srk2eand
35S::2EΔC1-GFP srk2e
seedlings under low humidity
stress
Red triangles indicate wilty
leaves observed in the
35S:: 2EΔ C1-GFP srk2e
plants
B, water loss in each
complementation line
C, effects of ABA on the water
loss in35S::2EWT-GFP srk2e
and35S::2EC1-GFP srk2e
plants
D, kinase activity in C terminus-
deleted SRK2E )2EΔC1(
under low humidity stress

6. 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 abi1–1mutation
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

7. 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 inpanel 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

8. 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 throughPathway I )(step 3
ABI1 binds to this region and may regulate
SRK2E activity )(step 4
ABA may also activatePathway II. In this
case, ABI1 and ABI2 may function as negative
regulators )(step 5
On the other hand, the ABA-independent
pathway specifically acts onDomain I
throughPathway III )step 7( also regulates
)SRK2E activity )step 8
)These three pathways converge )steps 5, 9
and result in the complete closure of stomata

9. Conclusion
The 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