KARRIKIN UP-REGULATED F-BOX 1 (KUF1) imposes negative feedback regulation of karrikin and KAI2 ligand metabolism in Arabidopsis thaliana.

SignificanceKarrikins are chemicals in smoke that stimulate regrowth of many plants after fire. However, karrikin responses are not limited to species from fire-prone environments and can affect growth after germination. Putatively, this is because karrikins mimic an unknown signal in plants, KAI2 ligand (KL). Karrikins likely require modification in plants to become bioactive. We identify a gene, KUF1, that appears to negatively regulate biosynthesis of KL and metabolism of a specific karrikin. KUF1 expression increases in response to karrikin or KL signaling, thus forming a negative feedback loop that limits further activation of the signaling pathway. This discovery will advance understanding of how karrikins are perceived and how smoke-activated germination evolved. It will also aid identification of the elusive KL.

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.

KARRIKIN INSENSITIVE2 regulates leaf development, root system architecture and arbuscular-mycorrhizal symbiosis in Brachypodium distachyon.

KARRIKIN INSENSITIVE2 (KAI2) is an α/ß-hydrolase required for plant responses to karrikins, which are abiotic butenolides that can influence seed germination and seedling growth. Although represented by four angiosperm species, loss-of-function kai2 mutants are phenotypically inconsistent and incompletely characterised, resulting in uncertainties about the core functions of KAI2 in plant development. Here we characterised the developmental functions of KAI2 in the grass Brachypodium distachyon using molecular, physiological and biochemical approaches. Bdkai2 mutants exhibit increased internode elongation and reduced leaf chlorophyll levels, but only a modest increase in water loss from detached leaves. Bdkai2 shows increased numbers of lateral roots and reduced root hair growth, and fails to support normal root colonisation by arbuscular-mycorrhizal (AM) fungi. The karrikins KAR1 and KAR2 , and the strigolactone (SL) analogue rac-GR24, each elicit overlapping but distinct changes to the shoot transcriptome via BdKAI2. Finally, we show that BdKAI2 exhibits a clear ligand preference for desmethyl butenolides and weak responses to methyl-substituted SL analogues such as GR24. Our findings suggest that KAI2 has multiple roles in shoot development, root system development and transcriptional regulation in grasses. Although KAI2-dependent AM symbiosis is likely conserved within monocots, the magnitude of the effect of KAI2 on water relations may vary across angiosperms.

Karrikin perception and signalling.

Karrikins (KARs) are a class of butenolide compounds found in smoke that were first identified as seed germination stimulants for fire-following species. Early studies of KARs classified the germination and postgermination responses of many plant species and investigated crosstalk with plant hormones that regulate germination. The discovery that Arabidopsis thaliana responds to KARs laid the foundation for identifying mutants with altered KAR responses. Genetic analysis of KAR signalling revealed an unexpected link to strigolactones (SLs), a class of carotenoid-derived plant hormones. Substantial progress has since been made towards understanding how KARs are perceived and regulate plant growth, in no small part due to advances in understanding SL perception. KAR and SL signalling systems are evolutionarily related and retain a high degree of similarity. There is strong evidence that KARs are natural analogues of an endogenous signal(s), KAI2 ligand (KL), which remains unknown. KAR/KL signalling regulates many developmental processes in plants including germination, seedling photomorphogenesis, and root and root hair growth. KAR/KL signalling also affects abiotic stress responses and arbuscular mycorrhizal symbiosis. Here, we summarise the current knowledge of KAR/KL signalling and discuss current controversies and unanswered questions in this field.

Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2.

Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.

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

Karrikins (KARs), smoke-derived butenolides, are perceived by the α/ß-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 GR24ent-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 KAR1, KAR2 and rac-GR24, while root system development responds only to KAR1. 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.

Open Source IIoT Solution for Gas Waste Monitoring in Smart Factory.

Rapid development of smart manufacturing techniques in recent years is influencing production facilities. Factories must both keep up with smart technologies as well as upskill their workforce to remain competitive. One of the recent concerns is how businesses can contribute to environmental sustainability and how to reduce operating costs. In this article authors present a method of measuring gas waste using Industrial Internet of Things (IIoT) sensors and open-source solutions utilised on a brownfield production asset. The article provides a result of an applied research initiative in a live manufacturing facility. The design followed the Reference Architectural Model for Industry 4.0 (RAMI 4.0) model to provide a coherent smart factory system. The presented solution's goal is to provide factory supervisors with information about gas waste which is generated during the production process. To achieve this an operational technology (OT) network was installed and Key Performance Indicators (KPIs) dashboards were designed. Based on the information provided by the system, the business can be more aware of the production environment and can improve its efficiency.

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.

Perception of karrikins by plants: a continuing enigma.

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.

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.

LATERAL BRANCHING OXIDOREDUCTASE acts in the final stages of strigolactone biosynthesis in Arabidopsis.

Strigolactones are a group of plant compounds of diverse but related chemical structures. They have similar bioactivity across a broad range of plant species, act to optimize plant growth and development, and promote soil microbe interactions. Carlactone, a common precursor to strigolactones, is produced by conserved enzymes found in a number of diverse species. Versions of the MORE AXILLARY GROWTH1 (MAX1) cytochrome P450 from rice and Arabidopsis thaliana make specific subsets of strigolactones from carlactone. However, the diversity of natural strigolactones suggests that additional enzymes are involved and remain to be discovered. Here, we use an innovative method that has revealed a missing enzyme involved in strigolactone metabolism. By using a transcriptomics approach involving a range of treatments that modify strigolactone biosynthesis gene expression coupled with reverse genetics, we identified LATERAL BRANCHING OXIDOREDUCTASE (LBO), a gene encoding an oxidoreductase-like enzyme of the 2-oxoglutarate and Fe(II)-dependent dioxygenase superfamily. Arabidopsis lbo mutants exhibited increased shoot branching, but the lbo mutation did not enhance the max mutant phenotype. Grafting indicated that LBO is required for a graft-transmissible signal that, in turn, requires a product of MAX1. Mutant lbo backgrounds showed reduced responses to carlactone, the substrate of MAX1, and methyl carlactonoate (MeCLA), a product downstream of MAX1. Furthermore, lbo mutants contained increased amounts of these compounds, and the LBO protein specifically converts MeCLA to an unidentified strigolactone-like compound. Thus, LBO function may be important in the later steps of strigolactone biosynthesis to inhibit shoot branching in Arabidopsis and other seed plants.

Karrikin-KAI2 signalling provides Arabidopsis seeds with tolerance to abiotic stress and inhibits germination under conditions unfavourable to seedling establishment.

The control of seed germination in response to environmental conditions is important for plant success. We investigated the role of the karrikin receptor KARRIKIN INSENSITIVE2 (KAI2) in the response of Arabidopsis seeds to osmotic stress, salinity and high temperature. Germination of the kai2 mutant was examined in response to NaCl, mannitol and elevated temperature. The effect of karrikin on germination of wild-type seeds, hypocotyl elongation and the expression of karrikin-responsive genes was also examined in response to such stresses. The kai2 seeds germinated less readily than wild-type seeds and germination was more sensitive to inhibition by abiotic stress. Karrikin-induced KAI2 signalling stimulated germination of wild-type seeds under favourable conditions, but, surprisingly, inhibited germination in the presence of osmolytes or at elevated temperature. By contrast, GA stimulated germination of wild-type seeds and mutants under all conditions. Karrikin induced expression of DLK2 and KUF1 genes and inhibited hypocotyl elongation independently of osmotic stress. Under mild osmotic stress, karrikin enhanced expression of DREB2A, WRKY33 and ERF5 genes, but not ABA signalling genes. Thus, the karrikin-KAI2 signalling system can protect against abiotic stress, first by providing stress tolerance, and second by inhibiting germination under conditions unfavourable to seedling establishment.

A Selaginella moellendorffii Ortholog of KARRIKIN INSENSITIVE2 Functions in Arabidopsis Development but Cannot Mediate Responses to Karrikins or Strigolactones.

In Arabidopsis thaliana, the α/ß-fold hydrolase KARRIKIN INSENSITIVE2 (KAI2) is essential for normal seed germination, seedling development, and leaf morphogenesis, as well as for responses to karrikins. KAI2 is a paralog of DWARF14 (D14), the proposed strigolactone receptor, but the evolutionary timing of functional divergence between the KAI2 and D14 clades has not been established. By swapping gene promoters, we show that Arabidopsis KAI2 and D14 proteins are functionally distinct. We show that the catalytic serine of KAI2 is essential for function in plants and for biochemical activity in vitro. We identified two KAI2 homologs from Selaginella moellendorffii and two from Marchantia polymorpha. One from each species could hydrolyze the strigolactone analog GR24 in vitro, but when tested for their ability to complement Arabidopsis d14 and kai2 mutants, neither of these homologs was effective. However, the second KAI2 homolog from S. moellendorffii was able to complement the seedling and leaf development phenotypes of Arabidopsis kai2. This homolog could not transduce signals from exogenous karrikins, strigolactone analogs, or carlactone, but its activity did depend on the conserved catalytic serine. We conclude that KAI2, and most likely the endogenous signal to which it responds, has been conserved since the divergence of lycophytes and angiosperm lineages, despite their major developmental and morphogenic differences.

Effect of thixotropic agents as additives on the mechanical properties of maxillofacial silicone elastomers.

STATEMENT OF PROBLEM: The incorporation of thixotropic agents to prevent slumping during the silicone packing procedure may alter some favorable mechanical properties of the silicone elastomers and could possibly influence the success of the prosthesis. PURPOSE: The purpose of this in vitro study was to evaluate the effect of adding thixotropic agents on the mechanical properties of 3 commonly used silicone elastomers. MATERIAL AND METHODS: Specimens of 3 maxillofacial silicones (M511, Z004; Technovent Ltd, and A2000; Factor II Inc) were prepared according to the manufacturers' instructions. Tear and tensile strength values and percentages of elongation and hardness were evaluated for each material with and without thixotropic agents. Data were analyzed using 1-way ANOVA and the Bonferroni post hoc test (α=.05). RESULTS: Results showed that the 3 types of silicone elastomers had significantly different (P<.001) tensile and tear strength and hardness values and percentages of elongation. Z004 silicone showed the highest tensile and tear strength followed by A2000 and M511, regardless of the addition of thixotropic agent. The addition of a thixotropic agent decreased the tear strength (P<.001) but not the tensile strength for all types of silicone. Percentage of elongation was the highest in M511 and the lowest in A2000 and was significantly higher (P<.001) for silicones with no added thixotropic agent. Hardness was highest in A2000 and lowest in M511. Adding a thixotropic agent decreased hardness significantly (P<.001) for Z004 and A2000 only. CONCLUSIONS: The incorporation of thixotropic agents into the 3 maxillofacial silicone elastomers used in this study reduced some favorable mechanical properties, particularly tear strength and percentage of elongation. Z004 showed superior mechanical properties among the 3 tested silicones.

Development of a 3D printable maxillofacial silicone: Part II. Optimization of moderator and thixotropic agent.

STATEMENT OF PROBLEM: Conventionally, maxillofacial prostheses are fabricated by hand carving the missing anatomic defect in wax and creating a mold into which pigmented silicone elastomer is placed. Digital technologies such as computer numerical control milling and 3-dimensional (3D) printing have been used to prepare molds, directly or indirectly, into which a biocompatible pigmented silicone elastomer can be placed. PURPOSE: The purpose of this in vitro study was to develop a silicone elastomer that could be 3D printed directly without a mold to create facial or body prostheses by varying its composition. MATERIAL AND METHODS: The room temperature vulcanizing silicone composition was divided into 2 components which were mixed 1:1 to initiate polymerization in the printer before printing began. Different types of moderators and thixotropic agents were used, and the base composition was varied to obtain 11 formulations. The specimens were printed and polymerized from these formulations and tested for tear and tensile strength and hardness. Ten readings of the specimens were recorded for tear and tensile strength and 6 for hardness. Results were analyzed using ANOVA (α=.05). Visual assessment of uncured printed specimens was undertaken for 5 formulations to assess any differences in their ability to hold their shape after printing. RESULTS: The tear and tensile strength of the 11 formulations with varying moderators, thixotropic agents, and base compositions were statistically similar to each other (P>.05). Five of 11 formulations were chosen for the visual assessment as they had sufficient thixotropic agent to avoid slumping while printing. The specimens showed varied slumping behavior until they polymerized. The filler content was increased in the selected formulation, and the tear and tensile strength of the formulation was increased to 6.138 kNm-1 and 3.836 MPa; these increases were comparable to those of commercial silicones currently used for the fabrication of facial prostheses. CONCLUSIONS: The optimum combination of mechanical properties implies the use of one of the formulations as a suitable material for the 3D printing of facial prostheses.

Stereospecificity in strigolactone biosynthesis and perception.

MAIN CONCLUSION: Plants produce strigolactones with different structures and different stereospecificities which provides the potential for diversity and flexibility of function. Strigolactones (SLs) typically comprise a tricyclic ABC ring system linked through an enol-ether bridge to a butenolide D-ring. The stereochemistry of the butenolide ring is conserved but two alternative configurations of the B-C ring junction leads to two families of SLs, exemplified by strigol and orobanchol. Further modifications lead to production of many different strigolactones within each family. The D-ring structure is established by a carotenoid cleavage dioxygenase producing a single stereoisomer of carlactone, the likely precursor of all SLs. Subsequent oxidation involves cytochrome P450 enzymes of the MAX1 family. In rice, MAX1 enzymes act stereospecifically to produce 4-deoxyorobanchol and orobanchol. Strigol- and orobanchol-type SLs have different activities in the control of seed germination and shoot branching, depending on plant species. This can partly be explained by different stereospecificity of SL receptors which includes the KAI2/HTL protein family in parasitic plants and the D14 protein functioning in shoot development. Many studies use chemically synthesised SL analogues such as GR24 which is prepared as a racemic mixture of two stereoisomers, one with the same stereo-configuration as strigol, and the other its enantiomer, which does not correspond to any known SL. In Arabidopsis, these two stereoisomers are preferentially perceived by AtD14 and KAI2, respectively, which activate different developmental pathways. Thus caution should be exercised in the use of SL racemic mixtures, while conversely the use of specific stereoisomers can provide powerful tools and yield critical information about receptors and signalling pathways in operation.

Development of a 3D printable maxillofacial silicone: Part I. Optimization of polydimethylsiloxane chains and cross-linker concentration.

STATEMENT OF PROBLEM: Conventionally, maxillofacial prostheses are fabricated by hand carving the missing anatomic defect in wax and creating a mold into which pigmented silicone elastomer is placed. Digital technologies such as computer numerical control (CNC) milling and 3-dimensional (3D) printing have been used to prepare molds directly or indirectly into which a biocompatible pigmented silicone elastomer is placed. PURPOSE: The purpose of this in vitro study was to develop a silicone elastomer by varying composition that could eventually be 3D printed directly without a mold to create facial/body prostheses. MATERIAL AND METHODS: The silicone was composed of polydimethylsiloxane (PDMS), filler, catalyst, and cross-linker. Four types of base silicone polymers were prepared with different PDMS molecular weight combinations with long, medium, and short chain length PDMS. The effect of the cross-linker (2.5% to 12.5%) content in these bases was assessed for the effect upon the mechanical properties of the elastomer. Ten readings were made for each formulation, and differences in the means were evaluated with a 2-way ANOVA (α=.05). RESULTS: Variations in silicone composition resulted in hardness from 6.8 to 28.5 durometer, tensile strength from 0.720 to 3.524 kNm-1 and tear strength from 0.954 to 8.484 MPa. Significant differences were observed among all formulations (P<.05). These formulations have mechanical properties comparable with the commercial silicones currently used for the fabrication of facial prostheses. The formulation with 5% cross-linker content and high content of long-chain PDMS chains with optimum mechanical properties was chosen for further development. CONCLUSIONS: The optimum combination of mechanical properties implies the use of one of these formulations for further evaluation in a 3D printer capable of actively mixing and extruding 2-component, room temperature vulcanization silicone.

The karrikin response system of Arabidopsis.

Arabidopsis thaliana provides a powerful means to investigate the mode of action of karrikins, compounds produced during wildfires that stimulate germination of seeds of fire-following taxa. These studies have revealed close parallels between karrikin signalling and strigolactone signalling. The two perception systems employ similar mechanisms involving closely related α/ß-fold hydrolases (KAI2 and AtD14) and a common F-box protein (MAX2). However, karrikins and strigolactones may be distinguished from each other and elicit different responses. The karrikin response requires a newly discovered protein (SMAX1), a homologue of rice protein D53 that is required for the strigolactone response. Mutants defective in the response to karrikins have seeds with increased dormancy, altered seedling photomorphogenesis and modified leaf shape. As the karrikin and strigolactone response mechanisms are so similar, it is speculated that the endogenous signalling compound for the KAI2 system may be a specific strigolactone. However, new results show that the proposed endogenous signalling compound is not produced by the known strigolactone biosynthesis pathway via carlactone. Structural studies of KAI2 protein and its interaction with karrikins and strigolactone analogues provide some insight into possible protein-ligand interactions, but are hampered by lack of knowledge of the endogenous ligand. The KAI2 system appears to be present throughout angiosperms, implying a fundamentally important function in plant biology.

Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis.

Karrikins are butenolides derived from burnt vegetation that stimulate seed germination and enhance seedling responses to light. Strigolactones are endogenous butenolide hormones that regulate shoot and root architecture, and stimulate the branching of arbuscular mycorrhizal fungi. Thus, karrikins and strigolactones are structurally similar but physiologically distinct plant growth regulators. In Arabidopsis thaliana, responses to both classes of butenolides require the F-box protein MAX2, but it remains unclear how discrete responses to karrikins and strigolactones are achieved. In rice, the DWARF14 protein is required for strigolactone-dependent inhibition of shoot branching. Here, we show that the Arabidopsis DWARF14 orthologue, AtD14, is also necessary for normal strigolactone responses in seedlings and adult plants. However, the AtD14 paralogue KARRIKIN INSENSITIVE 2 (KAI2) is specifically required for responses to karrikins, and not to strigolactones. Phylogenetic analysis indicates that KAI2 is ancestral and that AtD14 functional specialisation has evolved subsequently. Atd14 and kai2 mutants exhibit distinct subsets of max2 phenotypes, and expression patterns of AtD14 and KAI2 are consistent with the capacity to respond to either strigolactones or karrikins at different stages of plant development. We propose that AtD14 and KAI2 define a class of proteins that permit the separate regulation of karrikin and strigolactone signalling by MAX2. Our results support the existence of an endogenous, butenolide-based signalling mechanism that is distinct from the strigolactone pathway, providing a molecular basis for the adaptive response of plants to smoke.