Reconstitution of Abscisic Acid Signaling from the Receptor to DNA via bHLH Transcription Factors

Abstract

ORCID IDs: 0000-0002-9406-4093 (Y.T.); 0000-0001-6450-8506 (K.S.).The plant hormone abscisic acid (ABA) confers drought tolerance in plants through stomatal closure and regulation of gene expression. The complex consisting of the ABA receptor PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR), type 2C protein phosphatase (PP2C), and SNF1-related protein kinase 2 (SnRK2) has a key role in ABA signaling. Basic helix-loop-helix (bHLH) transcriptional activator ABA-RESPONSIVE KINASE SUBSTRATE1 (AKS1, also known as FBH3) is released from DNA by phosphorylation-induced monomerization in response to ABA in guard cells. Here we reconstituted the release of AKS1 from DNA via the ABA signaling core complex in vitro. We first obtained evidence to confirm that AKS1 is an endogenous substrate for SnRK2s. Phosphorylation of AKS1 and activation of SnRK2 showed the same time course in response to ABA in guard cells. AKS1 was bound to SnRK2.6 in vivo. Three ABA-responsive SnRK2s (SnRK2.2/ SRK2D, SnRK2.3/SRK2I, and SnRK2.6/SRK2E/OST1) phosphorylated AKS1 in vitro, and the phosphorylation was eliminated by the triple mutation of SnRK2s in plants. We reconstituted the AKS1 phosphorylation in vitro via the signaling complex containing the ABA receptor PYR1, a PP2C, HYPERSENSITIVE TO ABA1 (HAB1), and a protein kinase, SnRK2.6 in response to ABA. We further reconstituted the release of AKS1 from the target gene of POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1 (KAT1) via the complex in response to ABA. These results demonstrate that AKS1 provides a link between the signaling complex and ABA-responsive genes and furnish evidence for a minimal signaling mechanism from ABA perception to DNA.

Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook–Like10 phosphorylation required for stress growth regulation

“…These included the [pSP] motif, consistent with phosphorylation by MPKs and other proline-directed kinases (10) as well as variations of the [RxxpS] motif that can be targeted by SnRKs and Calcium-Dependent Protein Kinases (CPKs) (22) and serine surrounded by acidic residues (E or D) that may be recognized by casein kinase II (23). Consistent with these enriched motifs, five of the putative HAI1 target proteins are also putative SnRK2 substrates (Dataset S5) (6,7,24). Also consistent with enrichment of the [RxxpS] motif, a phosphopeptide from CPK9 was more abundant in hai1-2 (Dataset S2), suggesting that HAI1 may affect some [RxxpS] sites via regulation of CPKs as well as SnRK2s.…”

Phosphoproteomics of Arabidopsis Highly ABA-Induced1 identifies AT-Hook Like10 phosphorylation required for stress growth regulation

“…S2 (22) and serine surrounded by acidic residues (E or D) which may be recognized by Casein kinase II (23). Consistent with these enriched motifs, five of the putative HAI1 target proteins are also putative SnRK2 substrates (Dataset S5) (6,7,24). Also consistent with enrichment of the [RxxpS] motif, a phosphopeptide from CPK9 was more abundant in hai1-2 (Dataset S2), suggesting that HAI1 may affect some [RxxpS] sites via regulation of CPKs as well as SnRK2s.…”

Abscisic acid-independent stomatal CO 2 signal transduction pathway and convergence of CO 2 and ABA signaling downstream of OST1 kinase

“…Then, GCPs were harvested by centrifugation at 13,000 × g for 1 min and lysed by the addition of SDS/PAGE sample loading buffer. Proteins were separated by SDS/PAGE using gels containing 0.5 mg/mL histone type III-S, and SnRK2 kinase activity was detected by in-gel kinase assays (35,50).…”

“…The ABA-responsive kinase activity is most likely due to the OST1/SnRK2.6 protein kinase because the corresponding kinase activity was not detected in ost1-3 mutant guard cells ( Fig. 5A) (8,50). Longer exposure of in-gel kinase 32 P signals showed a weak background kinase activity in the range expected for OST1/SnRK2 protein kinases that was, however, enhanced only by ABA but not by 900 ppm CO 2 or NaHCO 3 exposure (Fig.…”