RESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Hidrolasas , Hidrolasas/genética , Bacillus subtilis , 4-Butirolactona , Lactonas/química , Percepción , Proteínas de Arabidopsis/genética , Reguladores del Crecimiento de las PlantasRESUMEN
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.
Asunto(s)
4-Butirolactona/análogos & derivados , Proteínas de Arabidopsis , Desarrollo de la Planta , Piranos , Furanos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Estrés Fisiológico , Proteínas de Arabidopsis/metabolismo , Lactonas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Reguladores del Crecimiento de las Plantas , Proteínas de Arabidopsis/genética , Hidrolasas , Arabidopsis/genética , Furanos , Piranos , Percepción , Lactonas/farmacologíaRESUMEN
Decades of intense herbicide use has led to resistance in weeds. Without innovative weed management practices and new herbicidal modes of action, the unabated rise of herbicide resistance will undoubtedly place further stress upon food security. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is the rate limiting enzyme of the eukaryotic mevalonate pathway successfully targeted by statins to treat hypercholesterolemia in humans. As HMGR inhibitors have been shown to be herbicidal, HMGR could represent a mode of action target for the development of herbicides. Here, we present the crystal structure of a HMGR from Arabidopsis thaliana (AtHMG1) which exhibits a wider active site than previously determined structures from different species. This plant conserved feature enables the rational design of specific HMGR inhibitors and we develop a tolerance trait through sequence analysis of fungal gene clusters. These results suggest HMGR to be a viable herbicide target modifiable to provide a tolerance trait.
Asunto(s)
Arabidopsis , Herbicidas , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Acilcoenzima A , Arabidopsis/metabolismo , Herbicidas/farmacología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Ácido MevalónicoRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Furanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Hidrolasas/genética , Piranos/farmacología , Arabidopsis/metabolismo , Plantones/genética , Plantones/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Brachypodium , Proteínas de Arabidopsis/fisiología , Brachypodium/genética , Furanos , Lactonas/farmacología , Reguladores del Crecimiento de las Plantas/farmacología , Hojas de la Planta/genética , Piranos , SimbiosisRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Lactonas , 4-Butirolactona/análogos & derivados , Proteínas de Arabidopsis/genética , Hidrolasas , Ligandos , Reguladores del Crecimiento de las PlantasRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/genética , Furanos/metabolismo , Hidrolasas/genética , Hipocótilo/crecimiento & desarrollo , Lotus/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Piranos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Furanos/química , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas/genética , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Hidrolasas/metabolismo , Hipocótilo/metabolismo , Lactonas/metabolismo , Ligandos , Lotus/genética , Análisis por Micromatrices , Filogenia , Reguladores del Crecimiento de las Plantas/química , Reguladores del Crecimiento de las Plantas/genética , Raíces de Plantas/metabolismo , Piranos/químicaRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Furanos/farmacología , Hidrolasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteolisis , Piranos/farmacología , Secuencias de Aminoácidos , Proteínas Portadoras/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Secuencia Conservada , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Hidrolasas/química , Lactonas/farmacología , Extractos Vegetales , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , Eliminación de Secuencia , Relación Estructura-Actividad , Nicotiana/efectos de los fármacosRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Brassica/metabolismo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Furanos/farmacología , Malezas/efectos de los fármacos , Malezas/metabolismo , Piranos/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis , Brassica/efectos de los fármacos , Brassica/genética , Dominio Catalítico , Incendios , Regulación de la Expresión Génica de las Plantas , Germinación/efectos de los fármacos , Germinación/fisiología , Hidrolasas/genética , Hidrolasas/metabolismo , Magnoliopsida , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantones , Semillas/efectos de los fármacos , Semillas/fisiología , Análisis de Secuencia de Proteína , Transcriptoma , Incendios ForestalesRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Furanos , Germinación , Hidrolasas , Lactonas , PercepciónRESUMEN
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.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hidrolasas/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolasas/genética , Hidrólisis , Señales de Poliadenilación de ARN 3' , Receptores de Superficie Celular/metabolismoRESUMEN
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.
Asunto(s)
Adaptación Fisiológica , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Furanos/metabolismo , Germinación , Hidrolasas/metabolismo , Piranos/metabolismo , Plantones/crecimiento & desarrollo , Semillas/fisiología , Estrés Fisiológico , Ácido Abscísico/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Furanos/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Germinación/efectos de los fármacos , Giberelinas/farmacología , Respuesta al Choque Térmico/efectos de los fármacos , Respuesta al Choque Térmico/genética , Calor , Modelos Biológicos , Mutación/genética , Nitratos/farmacología , Presión Osmótica/efectos de los fármacos , Piranos/farmacología , Plantones/efectos de los fármacos , Semillas/efectos de los fármacos , Semillas/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacosRESUMEN
Strigolactones are a structurally diverse class of plant hormones that control many aspects of shoot and root growth. Strigolactones are also exuded by plants into the rhizosphere, where they promote symbiotic interactions with arbuscular mycorrhizal fungi and germination of root parasitic plants in the Orobanchaceae family. Therefore, understanding how strigolactones are made, transported, and perceived may lead to agricultural innovations as well as a deeper knowledge of how plants function. Substantial progress has been made in these areas over the past decade. In this review, we focus on the molecular mechanisms, core developmental roles, and evolutionary history of strigolactone signaling. We also propose potential translational applications of strigolactone research to agriculture.
Asunto(s)
Evolución Biológica , Lactonas/metabolismo , Reguladores del Crecimiento de las Plantas/fisiología , Micorrizas/metabolismo , Micorrizas/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/parasitología , Plantas , Rizosfera , Transducción de SeñalRESUMEN
Karrikins are a family of compounds generated via the incomplete combustion of plant matter. Since their discovery as seed germination stimulants in 2004, a great deal has been learned about the chemistry and the biological mode of action of karrikins. Much interest and progress have stemmed from the structural similarity of karrikins to that of strigolactones - the shoot branching hormone. This review will provide a historical account of some of the more significant discoveries in this area of plant biology. It will discuss how the study of these abiotic signalling molecules, combined with advances in our understanding of strigolactones, has led us towards the discovery of new mechanisms that regulate plant growth and development.
RESUMEN
Karrikins are a small family of naturally occurring plant growth regulators present in the smoke and char produced from burning plant material in wildfires. They can stimulate germination of dormant seed and can influence seedling morphogenesis. Although Arabidopsis thaliana is not considered to be a smoke-responsive species, karrikins will stimulate seed germination under the appropriate circumstances and will cause repression of hypocotyl elongation in low light. This chapter describes how to conduct assays of the activity of karrikins on Arabidopsis seeds and seedlings. The methods presented can potentially be modified for use in a range of Arabidopsis genotypes or in other plant species.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Furanos/metabolismo , Germinación/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Piranos/metabolismo , Plantones/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Genotipo , Hipocótilo/metabolismo , Luz , Latencia en las Plantas/fisiología , Semillas/metabolismo , HumoRESUMEN
Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2). We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway.
RESUMEN
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.