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.
Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. We utilized the insertional, indexed, genome‐saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome‐wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat‐sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat‐induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we presented a high/medium‐confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high‐confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a high‐confidence Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.
High temperatures adversely affect crop and algal yields. Photosynthetic cells show varying physiological responses to different heat intensities. The specificity of genes required for thermotolerance at different temperatures in photosynthetic cells remains elusive. Additionally, organic carbon sources are present in nature for both plants and algae, but it is unclear how environmental availability of carbon sources interface with heat responses. We utilized the insertional mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii (CLiP) to perform genome-wide, quantitative, pooled screens under moderate (35oC) or acute (40oC) high temperatures with or without organic carbon. We screened for heat sensitive mutants based on growth rate quantification and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts/proteins in wildtype cultures and MapMan functional annotation data, we present a high-confidence list of 933 Chlamydomonas genes with putative roles in heat tolerance. These include genes with known roles in thermotolerance and those with little or no functional annotation, suggesting novel players in thermotolerance. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage model organisms, including crop species. Arabidopsis thaliana mutants deficient in the ortholog of a Chlamydomonas HTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.
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