Functional and physical interaction between srk2 e and abi1

Activation of SRK2E/OST1 by ABA & low humidity stressA, ABA dose response forSRK2E/OST1 activationB, ABA-dependent kinase activities in the srk2emutantC, ABA-dependent kinaseactivity was not detected inwild-type leavesD, ABA-inducible genes wereexpressed in wild-typeleavesE, activation of SRK2E/OST1by low humidity stress in leaftissuesF, kinase activities in the low humidity-stressedsrk2emutantG, ABA and OS activated SRK2E-GFP inArabidopsisT87 cells, Cold Stress NOT

Effect of abi and abamutations on the activation of SRK2E/OST1A, SRK2E-GFP proteins were overexpressedin abi1–1 andabi2–1 These results indicate that two similar PP2C, ABI1 andB, SRK2E-GFP proteins were overexpressedin theaba2–1 ABI2, have distinct roles in ABA signaling in terms ofC , water loss in abi1–1 and aba2–1mutant regulation of SRK2E/OST1seedlings

Structural analysis of the C-terminal region in SnRK2 proteinsA, comparison of theC-terminal amino acids inArabidopsisSnRK2sB, SRK2D-GFP, SRK2F-GFP,SRK2G-GFP, and SRK2I-GFP proteins were overexpressed in T87 cellsC, comparison of the C-terminal amino acids inthree SnRK2sArabidopsis SRK2E/OST1Fava bean AAPKRiceSAPK8

The effect of N and C terminus amino acids on the activation of SRK2E/OST1A, schematic representation of thedeletion constructs introduced intoT87 cellsFull-length ,N terminus-deleted, andtwo types of C terminus-deletedSRK2E were designated as2EWT-GFP ,2EΔN-GFP ,2EΔC1-GFP, and 2EΔ C2-GFP ,respectivelyB, kinase activity of truncated SRK2E/OST1 in T87 cellsEach construct was overexpressed asa GFP fusion protein and treatedwith ABA )50 M( and sorbitol )0.8M(for the indicated times, and their cell extracts were subjected toin-gel kinase assay

Functional analysis of truncated SRK2E in the complementation of the srk2ephenotypeA ,35S:: 2EWT-GFP srk2eand35S::2EΔC1-GFP srk2eseedlings under low humiditystressRed triangles indicate wiltyleaves observed in the35S:: 2EΔ C1-GFP srk2eplantsB, water loss in eachcomplementation lineC, effects of ABA on the water loss in35S::2EWT-GFP srk2e and35S::2EC1-GFP srk2eplantsD, 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 assayA, estimation of partners wouldinteract with SRK2E/OST1Interactions betweenSRK2E/OST1 and eachsignaling factor, includingABI1, ABI2, AtRac1, andGPA1, were determined bygrowth assay on mediumlacking Leu, Trp, and His inthe presence of 20mM 3-ATB , effect of the abi1–1mutationon binding to SRK2E/OST1Degree of binding to SRK2E/OST1 was comparedbetween ABI1 and Abi1–1by growth assay SRK2E/OST1 was used as the bait protein

ABI1 physically binds to the C terminus of SRK2E/OST1A, constructs used for theidentification of the ABI1 bindingdomain on SRK2E/OST1 ,)Full-length )2EWT ,)C terminus deleted )(2E-(1–317 ,)C terminus )(2E-(319 –357C terminus of SRK2G/OSKL8 )2G-(311–353(( were used as the preyproteinsB, identification of the ABI1 bindingdomain in SRK2E/OST1Interactions between ABI1 and each construct presented inpanel Awere determined by growth assayon medium lacking Leu, Trp, andHis in the presence of 20 mM 3-AT .ABI1 was used as the bait protein

A model to explain SRK2E/OST1 signaling in stomatal closureWhen plants sense low humidity stress, thesignal may integrate into two pathwaysABA produced by low humidity may act onDomain II throughPathway I )(step 3ABI1 binds to this region and may regulateSRK2E activity )(step 4 ABA may also activatePathway II. In thiscase, ABI1 and ABI2 may function as negativeregulators )(step 5On 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, 9and result in the complete closure of stomata

Conclusion The direct interaction between SRK2E/OST1 and ABI1through 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 ABAMembers of SnRK2 family may activate and be activated by different downstream and upstream factors

Functional and physical interaction between srk2 e and abi1

by Amer Wazwaz on Jun 01, 2012

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