Karrikin signalling: impacts on plant development and abiotic stress tolerance.

Plants rely upon a diverse range of metabolites to control growth and development, and to overcome stress that results from suboptimal conditions. Karrikins (KARs) are a class of butenolide compounds found in smoke that stimulate seed germination and regulate various developmental processes in plants. KARs are perceived via a plant α/ß-hydrolase called KARRIKIN INSENSITIVE2 (KAI2), which also functions as a receptor for a postulated phytohormone, provisionally termed KAI2 ligand (KL). Considered natural analogues of KL, KARs have been extensively studied for their effects on plant growth and their crosstalk with plant hormones. The perception and response pathway for KAR-KL signalling is closely related to that of strigolactones, another class of butenolides with numerous functions in regulating plant growth. KAR-KL signalling influences seed germination, seedling photomorphogenesis, root system architecture, abiotic stress responses, and arbuscular mycorrhizal symbiosis. Here, we summarize current knowledge of KAR-KL signalling, focusing on its role in plant development, its effects on stress tolerance, and its interaction with other signalling mechanisms.

Desmethyl butenolides are optimal ligands for karrikin receptor proteins.

Strigolactones and karrikins are butenolide molecules that regulate plant growth. They are perceived by the α/ß-hydrolase DWARF14 (D14) and its homologue KARRIKIN INSENSITIVE2 (KAI2), respectively. Plant-derived strigolactones have a butenolide ring with a methyl group that is essential for bioactivity. By contrast, karrikins are abiotic in origin, and the butenolide methyl group is nonessential. KAI2 is probably a receptor for an endogenous butenolide, but the identity of this compound remains unknown. Here we characterise the specificity of KAI2 towards differing butenolide ligands using genetic and biochemical approaches. We find that KAI2 proteins from multiple species are most sensitive to desmethyl butenolides that lack a methyl group. Desmethyl-GR24 and desmethyl-CN-debranone are active by KAI2 but not D14. They are more potent KAI2 agonists compared with their methyl-substituted reference compounds both in vitro and in plants. The preference of KAI2 for desmethyl butenolides is conserved in Selaginella moellendorffii and Marchantia polymorpha, suggesting that it is an ancient trait in land plant evolution. Our findings provide insight into the mechanistic basis for differential ligand perception by KAI2 and D14, and support the view that the endogenous substrates for KAI2 and D14 have distinct chemical structures and biosynthetic origins.

An allelic series at the KARRIKIN INSENSITIVE 2 locus of Arabidopsis thaliana decouples ligand hydrolysis and receptor degradation from downstream signalling.

Karrikins are butenolide compounds present in post-fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE 2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2-dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)-mutagenized populations of A. thaliana for mutants with kai2-like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2-10 encoded a D184N protein variant that was stable in planta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2-active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.

Perception of butenolides by Bacillus subtilis via the α/ß hydrolase RsbQ.

The regulation of behavioral and developmental decisions by small molecules is common to all domains of life. In plants, strigolactones and karrikins are butenolide growth regulators that influence several aspects of plant growth and development, as well as interactions with symbiotic fungi.1,2,3 DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are homologous enzyme-receptors that perceive strigolactones and karrikins, respectively, and that require hydrolase activity to effect signal transduction.4,5,6,7 RsbQ, a homolog of D14 and KAI2 from the gram-positive bacterium Bacillus subtilis, regulates growth responses to nutritional stress via the alternative transcription factor SigmaB (σB).8,9 However, the molecular function of RsbQ is unknown. Here, we show that RsbQ perceives butenolide compounds that are bioactive in plants. RsbQ is thermally destabilized by the synthetic strigolactone GR24 and its desmethyl butenolide equivalent dGR24. We show that, like D14 and KAI2, RsbQ is a functional butenolide hydrolase that undergoes covalent modification of the catalytic histidine residue. Exogenous application of both GR24 and dGR24 inhibited the endogenous signaling function of RsbQ in vivo, with dGR24 being 10-fold more potent. Application of dGR24 to B. subtilis phenocopied loss-of-function rsbQ mutations and led to a significant downregulation of σB-regulated transcripts. We also discovered that exogenous butenolides promoted the transition from planktonic to biofilm growth. Our results suggest that butenolides may serve as inter-kingdom signaling compounds between plants and bacteria to help shape rhizosphere communities.

Divergent receptor proteins confer responses to different karrikins in two ephemeral weeds.

Wildfires can encourage the establishment of invasive plants by releasing potent germination stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of Mediterranean climates, is strongly stimulated by KAR1, the archetypal karrikin produced from burning vegetation. In contrast, the closely-related yet non-fire-associated ephemeral Arabidopsis thaliana is unusual because it responds preferentially to KAR2. The α/ß-hydrolase KARRIKIN INSENSITIVE 2 (KAI2) is the putative karrikin receptor identified in Arabidopsis. Here we show that B. tournefortii expresses three KAI2 homologues, and the most highly-expressed homologue is sufficient to confer enhanced responses to KAR1 relative to KAR2 when expressed in Arabidopsis. We identify two amino acid residues near the KAI2 active site that explain the ligand selectivity, and show that this combination has arisen independently multiple times within dicots. Our results suggest that duplication and diversification of KAI2 proteins could confer differential responses to chemical cues produced by environmental disturbance, including fire.