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

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.

bHLH transcription factors that facilitate K⁺ uptake during stomatal opening are repressed by abscisic acid through phosphorylation.

Stomata open in response to light and close after exposure to abscisic acid (ABA). They regulate gas exchange between plants and the atmosphere, enabling plants to adapt to changing environmental conditions. ABA binding to receptors initiates a signaling cascade that involves protein phosphorylation. We show that ABA induced the phosphorylation of three basic helix-loop-helix (bHLH) transcription factors, called AKSs (ABA-responsive kinase substrates; AKS1, AKS2, and AKS3), in Arabidopsis guard cells. In their unphosphorylated state, AKSs facilitated stomatal opening through the transcription of genes encoding inwardly rectifying K⁺ channels. aks1aks2-1 double mutant plants showed decreases in light-induced stomatal opening, K⁺ accumulation in response to light, activity of inwardly rectifying K⁺ channels, and transcription of genes encoding major inwardly rectifying K⁺ channels without affecting ABA-mediated stomatal closure. Overexpression of potassium channel in Arabidopsis thaliana 1 (KAT1), which encodes a major inwardly rectifying K⁺ channel in guard cells, rescued the phenotype of aks1aks2-1 plants. AKS1 bound directly to the promoter of KAT1, an interaction that was attenuated after ABA-induced phosphorylation. The ABA agonist pyrabactin induced phosphorylation of AKSs. Our results demonstrate that the AKS family of bHLH transcription factors facilitates stomatal opening through the transcription of genes encoding inwardly rectifying K⁺ channels and that ABA suppresses the activity of these channels by triggering the phosphorylation of AKS family transcription factors.