No abstract

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 KAR 1 , 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 KAR 2. The α/β-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 highlyexpressed homologue is sufficient to confer enhanced responses to KAR 1 relative to KAR 2 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.

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.

Strigolactones and karrikins are butenolide molecules that regulate plant growth. They are perceived by the a/b-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.

Karrikins are small butenolide molecules with the capacity to promote germination and enhance seedling establishment. Generated abiotically from partial combustion of vegetation, karrikins are comparatively rare in the environment, but studying their mode of action has been most informative in revealing a new regulatory pathway for plant development that uses the karrikin perception machinery. Recent studies suggest that the karrikin receptor protein KAI2 and downstream transcriptional co-repressors in the SMXL family influence seed germination, seedling photomorphogenesis, root morphology, and responses to abiotic stress such as drought. Based on taxonomic distribution, this pathway is ubiquitous and likely to be evolutionarily ancient, originating prior to land plants. However, we still do not have a good grasp on how karrikins actually activate the receptor protein, and we have yet to discover the assumed endogenous ligand for KAI2 that karrikins are thought to mimic. This review covers recent progress in this field, as well as current gaps in our knowledge.

16Wildfires can encourage the establishment of invasive plants by releasing potent germination 17 stimulants, such as karrikins. Seed germination of Brassica tournefortii, a noxious weed of 18 Mediterranean climates, is strongly stimulated by KAR1, which is the most abundant karrikin 19 produced from burning vegetation. In contrast, the closely-related yet non-fire-associated 20 ephemeral Arabidopsis thaliana responds preferentially to the less abundant KAR2. The α/β-21 hydrolase KARRIKIN INSENSITIVE2 (KAI2) is the putative karrikin receptor identified in 22Arabidopsis. Here we show that B. tournefortii differentially expresses three KAI2 23 homologues, and the most highly-expressed homologue is sufficient to confer enhanced 24 responses to KAR 1 relative to KAR 2 when expressed in Arabidopsis. We further identify two 25 variant amino acid residues near the KAI2 active site that explain the ligand selectivity. Our 26 results suggest that duplication and diversification of KAI2 proteins could confer upon weedy 27 ephemerals differential responses to chemical cues produced by environmental disturbance, 28 including fire. (146 words) 29 by the presence of a methyl group on the butenolide ring in KAR1, which is absent in KAR2 38 ( Supplementary Fig. 1a). Invasive plant species that are responsive to karrikins could utilise 39 natural and human-induced fires to facilitate their establishment 5, 6 . 40 Brassica tournefortii (Brassicaceae; Sahara mustard) is native to northern Africa and the Middle 41East, but is an invasive weed that blights many ecosystems with a Mediterranean climate and 42 chaparral-type vegetation that are prone to wildfires in North America, Australia and South 43 Africa. B. tournefortii seed can persist in the soil for many seasons, undergoing wet-dry cycling 44 that can influence dormancy and contribute to boom-bust cycles that outcompete native 45 3 species 7, 8 . B. tournefortii plants may radically alter fire frequency and intensity by influencing 46 fuel profiles 9, 10 , further exacerbating the impact of fire on susceptible native ecosystems. In 47 addition, seed of B. tournefortii is particularly responsive to smoke-derived karrikins, and 48 shows a positive germination response to KAR1 in the nanomolar range 11 . Accordingly, B. 49 tournefortii is particularly well positioned to invade areas disturbed by fire events 12, 13 . 50The putative karrikin receptor KARRIKIN INSENSITIVE 2 (KAI2) was identified in Arabidopsis 51 thaliana, a weedy ephemeral that originated in Eurasia but is now widely distributed 52 throughout the northern hemisphere 14, 15, 16 . Arabidopsis is not known to colonise fire-prone 53 habitats, but nevertheless seeds germinate in response to karrikins in the micromolar range 17 . 54Unlike most smoke-responsive species that respond more readily to KAR1 18, 19 , Arabidopsis 55 responds preferentially to the less abundant analogue KAR2 17 . KAI2 is an evolutionarily ancient 56 α/β-hydrolase and a paralogue of DWARF14 (D14), the receptor for strigolactones 20, 21 . 57 ...