Click-to-lead design of a picomolar ABA receptor antagonist with potent activity in vivo

Abscisic acid (ABA) is a key plant hormone that mediates both plant biotic and abiotic stress responses and many other developmental processes. ABA receptor antagonists are useful for dissecting and manipulating ABA’s physiological roles in vivo. We set out to design antagonists that block receptor–PP2C interactions by modifying the agonist opabactin (OP), a synthetically accessible, high-affinity scaffold. Click chemistry was used to create an ∼4,000-member library of C4-diversified opabactin derivatives that were screened for receptor antagonism in vitro. This revealed a peptidotriazole motif shared among hits, which we optimized to yield antabactin (ANT), a pan-receptor antagonist. An X-ray crystal structure of an ANT–PYL10 complex (1.86 Å) reveals that ANT’s peptidotriazole headgroup is positioned to sterically block receptor–PP2C interactions in the 4′ tunnel and stabilizes a noncanonical closed-gate receptor conformer that partially opens to accommodate ANT binding. To facilitate binding-affinity studies using fluorescence polarization, we synthesized TAMRA–ANT. Equilibrium dissociation constants for TAMRA–ANT binding to Arabidopsis receptors range from ∼400 to 1,700 pM. ANT displays improved activity in vivo and disrupts ABA-mediated processes in multiple species. ANT is able to accelerate seed germination in Arabidopsis, tomato, and barley, suggesting that it could be useful as a germination stimulant in species where endogenous ABA signaling limits seed germination. Thus, click-based diversification of a synthetic agonist scaffold allowed us to rapidly develop a high-affinity probe of ABA–receptor function for dissecting and manipulating ABA signaling.

Initiation and amplification of SnRK2 activation in abscisic acid signaling

The phytohormone abscisic acid (ABA) is crucial for plant responses to environmental challenges. The SNF1-regulated protein kinase 2s (SnRK2s) are key components in ABA-receptor coupled core signaling, and are rapidly phosphorylated and activated by ABA. Recent studies have suggested that Raf-like protein kinases (RAFs) participate in ABA-triggered SnRK2 activation. In vitro kinase assays also suggest the existence of autophosphorylation of SnRK2s. Thus, how SnRK2 kinases are quickly activated during ABA signaling still needs to be clarified. Here, we show that both B2 and B3 RAFs directly phosphorylate SnRK2.6 in the kinase activation loop. This transphosphorylation by RAFs is essential for SnRK2 activation. The activated SnRK2s then intermolecularly trans-phosphorylate other SnRK2s that are not yet activated to amplify the response. High-order Arabidopsis mutants lacking multiple B2 and B3 RAFs show ABA hyposensitivity. Our findings reveal a unique initiation and amplification mechanism of SnRK2 activation in ABA signaling in higher plants.

SiCEP3, a C-terminally encoded peptides from Setaria italica, promotes ABA import and signaling pathway

C-terminally encoded peptides (CEPs) are small peptides, typically post-translationally modified, and highly conserved in many species. CEPs are known to play roles in inhibition of plant growth and regulation of development, but the mechanisms are not well understood. In this study, we searched for CEP peptides in foxtail millet (Setaria italica). The 14 peptides we identified are divided into two subfamilies. The transcripts of most SiCEPs were more abundant in roots than in other tissues. SiCEP3, SiCEP4, and SiCEP5 were also expressed at high levels in panicles. Moreover, expression of all SiCEPs was induced by biotic stress and phytohormones. SiCEP3 overexpression and application of biosynthetic SiCEP3 both inhibited the growth of seedlings. In the presence of ABA, growth inhibition and ABA content of seedlings increased with the concentration of SiCEP3. Transcripts encoding two ABA transporters and one ABA receptor were induced by SiCEP3, ABA, and the two in combination. Further analysis revealed that SiCEP3 promoted ABA transport via NRT1.2 and ABCG40. In addition, SiCEP3, ABA, or the combination inhibited the kinase activities of CEP receptors CEPR1/2. Taken together, our results indicated that the CEP–CEPR module mediates ABA signaling by regulating ABCG40, NRT1.2, and PYL4 in planta.HighlightSiCEP3, a C-terminally encoded peptide, can promote ABA import and signaling pathway by inhibiting the kinase activity of its receptors under abiotic stress in Setaria italica.

OsPP2C09 Is a Bifunctional Regulator in Both ABA-Dependent and Independent Abiotic Stress Signaling Pathways

Clade A Type 2C protein phosphatases (PP2CAs) negatively regulate abscisic acid (ABA) signaling and have diverse functions in plant development and in response to various stresses. In this study, we showed that overexpression of the rice ABA receptor OsPYL/RCAR3 reduces the growth retardation observed in plants exposed to osmotic stress. By contrast, overexpression of the OsPYL/RCAR3-interacting protein OsPP2C09 rendered plant growth more sensitive to osmotic stress. We tested whether OsPP2CAs activate an ABA-independent signaling cascade by transfecting rice protoplasts with luciferase reporters containing the drought-responsive element (DRE) or ABA-responsive element (ABRE). We observed that OsPP2CAs activated gene expression via the cis-acting drought-responsive element. In agreement with this observation, transcriptome analysis of plants overexpressing OsPP2C09 indicated that OsPP2C09 induces the expression of genes whose promoters contain DREs. Further analysis showed that OsPP2C09 interacts with DRE-binding (DREB) transcription factors and activates reporters containing DRE. We conclude that, through activating DRE-containing promoters, OsPP2C09 positively regulates the drought response regulon and activates an ABA-independent signaling pathway.