pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier

Bongkrekic acid (BKA), isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In the present study, we tested the inhibitory effects of 17 bongkrekic acid analogues, derived from the intermediates obtained during its total synthesis, on the mitochondrial ATP/ATP carrier. Rough screening of these chemicals, done by measuring their inhibitory effects on the mitochondrial ATP synthesis, revealed that 4 of them, KH-1, 7, 16, and 17, had moderate inhibitory effects. Further characterization of the actions of these 4 analogues on mitochondrial function showed that KH-16 had moderate; KH-1 and KH-17, weak; and KH-7, negligible side effects of both permeabilization of the mitochondrial inner membrane and inhibition of the electron transport, indicating that only KH-7 had a specific inhibitory effect on the mitochondrial ADP/ATP carrier. Although the parental bongkrekic acid showed a strong pH dependency of its action, the inhibitory effect of KH-7 was almost insensitive to the pH of the reaction medium, indicating the importance of the 3 carboxyl groups of BKA for its pH- dependent action. A direct inhibitory effect of KH-7 on the mitochondrial ADP/ATP carrier was also clearly demonstrated

Lotus japonicus karrikin receptors display divergent ligand-binding specificities and organ-dependent redundancy

Author summary Plant hormone signaling is crucial for development and for adequate responses to biotic and abiotic environmental conditions. The most recently discovered plant hormone receptor KARRIKIN INSENSITVE 2 (KAI2), binds a small butenolide called karrikin that was discovered in smoke and induces germination of fire-following plants. Several lines of evidence suggest a yet elusive endogenous hormone, which acts as ligand for KAI2. Until its identification, synthetic karrikins or the strigolactone-like molecule GR24 are used to probe the karrikin signaling pathway. While the model plant Arabidopsis contains only one KAI2 gene, several copies are maintained in other species suggesting sub-functionalization. We report that genomes of species in the legume hologalegina clade encode two KAI2 versions. In Lotus japonicus, they diverge in their binding ability to synthetic ligands due to three amino acid changes in their binding pocket, of which two are conserved across legumes and one has independently occurred in several species across the angiosperm phylogeny. Surprisingly, L. japonicus hypocotyls react with developmental responses to two different karrikins (KAR(1), KAR(2)) and a synthetic strigolactone rac-GR24, while root development responds only to KAR(1). This shows that there is not only diversity in ligand-receptor relationships but possibly also organ-specific uptake or metabolism of divergent butenolide molecules. Karrikins (KARs), smoke-derived butenolides, are perceived by the alpha/beta-fold hydrolase KARRIKIN INSENSITIVE2 (KAI2) and thought to mimic endogenous, yet elusive plant hormones tentatively called KAI2-ligands (KLs). The sensitivity to different karrikin types as well as the number of KAI2 paralogs varies among plant species, suggesting diversification and co-evolution of ligand-receptor relationships. We found that the genomes of legumes, comprising a number of important crops with protein-rich, nutritious seed, contain two or more KAI2 copies. We uncover sub-functionalization of the two KAI2 versions in the model legume Lotus japonicus and demonstrate differences in their ability to bind the synthetic ligand GR24(ent-5DS) in vitro and in genetic assays with Lotus japonicus and the heterologous Arabidopsis thaliana background. These differences can be explained by the exchange of a widely conserved phenylalanine in the binding pocket of KAI2a with a tryptophan in KAI2b, which arose independently in KAI2 proteins of several unrelated angiosperms. Furthermore, two polymorphic residues in the binding pocket are conserved across a number of legumes and may contribute to ligand binding preferences. The diversification of KAI2 binding pockets suggests the occurrence of several different KLs acting in non-fire following plants, or an escape from possible antagonistic exogenous molecules. Unexpectedly, L. japonicus responds to diverse synthetic KAI2-ligands in an organ-specific manner. Hypocotyl growth responds to KAR(1), KAR(2) and rac-GR24, while root system development responds only to KAR(1). This differential responsiveness cannot be explained by receptor-ligand preferences alone, because LjKAI2a is sufficient for karrikin responses in the hypocotyl, while LjKAI2a and LjKAI2b operate redundantly in roots. Instead, it likely reflects differences between plant organs in their ability to transport or metabolise the synthetic KLs. Our findings provide new insights into the evolution and diversity of butenolide ligand-receptor relationships, and open novel research avenues into their ecological significance and the mechanisms controlling developmental responses to divergent KLs