RESUMO
Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Hidrolases de Éster Carboxílico , Lactonas , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Lactonas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Cristalografia por Raios X , Reguladores de Crescimento de Plantas/metabolismo , Modelos Moleculares , Hidrólise , Conformação ProteicaRESUMO
Today, the use of artificial pesticides is questionable and the adaptation to global warming is a necessity. The promotion of favorable natural interactions in the rhizosphere offers interesting perspectives for changing the type of agriculture. Strigolactones (SLs), the latest class of phytohormones to be discovered, are also chemical mediators in the rhizosphere. We present in this review the diversity of natural SLs, their analogs, mimics, and probes essential for the biological studies of this class of compounds. Their biosynthesis and access by organic synthesis are highlighted especially concerning noncanonical SLs, the more recently discovered natural SLs. Organic synthesis of analogs, stable isotope-labeled standards, mimics, and probes are also reviewed here. In the last part, the knowledge about the SL perception is described as well as the different inhibitors of SL receptors that have been developed.
Assuntos
Lactonas , Reguladores de Crescimento de Plantas , Plantas , Lactonas/química , Lactonas/metabolismo , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/síntese química , Plantas/metabolismo , Plantas/químicaRESUMO
SUPPRESSOR OF MAX2 (SMAX)1-LIKE (SMXL) proteins are a plant-specific clade of type I HSP100/Clp-ATPases. SMXL genes are present in virtually all land plant genomes. However, they have mainly been studied in angiosperms. In Arabidopsis (Arabidopsis thaliana), 3 functional SMXL subclades have been identified: SMAX1/SMXL2, SMXL345, and SMXL678. Of these, 2 subclades ensure endogenous phytohormone signal transduction. SMAX1/SMXL2 proteins are involved in KAI2 ligand (KL) signaling, while SMXL678 proteins are involved in strigolactone (SL) signaling. Many questions remain regarding the mode of action of these proteins, as well as their ancestral roles. We addressed these questions by investigating the functions of the 4 SMXL genes in the moss Physcomitrium patens. We demonstrate that PpSMXL proteins are involved in the conserved ancestral MAX2-dependent KL signaling pathway and negatively regulate growth. However, PpSMXL proteins expressed in Arabidopsis cannot replace SMAX1 or SMXL2 function in KL signaling, whereas they can functionally replace SMXL4 and SMXL5 and restore root growth. Therefore, the molecular functions of SMXL proteins are conserved, but their interaction networks are not. Moreover, the PpSMXLC/D clade positively regulates SL signal transduction in P. patens. Overall, our data reveal that SMXL proteins in moss mediate crosstalk between the SL and KL signaling pathways.
Assuntos
Proteínas de Arabidopsis , Bryopsida , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Bryopsida/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Transdução de Sinais , Filogenia , Lactonas/metabolismoRESUMO
Under specific conditions, the germination of Arabidopsis thaliana is dependent on the activation of the KARRIKIN INSENSITIVE 2 (KAI2) signaling pathway by the KAI2-dependent perception of karrikin or the artificial strigolactone analogue, rac-GR24. To regulate the induction of germination, the KAI2 signaling pathway relies on MORE AXILLARY BRANCHED 2- (MAX2-)dependent ubiquitination and proteasomal degradation of the repressor protein SUPPRESSOR OF MAX2 1 (SMAX1). It is not yet known how the degradation of SMAX1 proteins eventually results in the regulation of seed germination, but it has been hypothesized that SMAX1-LIKE generally functions as transcriptional repressors through the recruitment of co-repressors TOPLESS (TPL) and TPL-related, which in turn interact with histone deacetylases. In this article, we show the involvement of histone deacetylases HDA6, HDA9, HDA19 and HDT1 in MAX2-dependent germination of Arabidopsis, and more specifically, that HDA6 is required for the induction of DWARF14-LIKE2 expression in response to rac-GR24 treatment.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Germinação , Proteínas de Arabidopsis/metabolismo , Lactonas/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Strigolactones (SLs) are plant hormones exuded in the rhizosphere with a signaling role for the development of arbuscular mycorrhizal (AM) fungi and as stimulants of seed germination of the parasitic weeds Orobanche, Phelipanche, and Striga, the most threatening weeds of major crops worldwide. Phelipanche ramosa is present mainly on rape, hemp, and tobacco in France. P. ramosa 2a preferentially attacks hemp, while P. ramosa 1 attacks rapeseed. The recently isolated cannalactone (14) from hemp root exudates has been characterized as a noncanonical SL that selectively stimulates the germination of P. ramosa 2a seeds in comparison with P. ramosa 1. In the present work, (-)-solanacol (5), a canonical orobanchol-type SL exuded by tobacco and tomato, was established to possess a remarkable selective germination stimulant activity for P. ramosa 2a seeds. Two cannalactone analogues, named (±)-SdL19 and (±)-SdL118, have been synthesized. They have an unsaturated acyclic carbon chain with a tertiary hydroxy group and a methyl or a cyclopropyl group instead of a cyclohexane A-ring, respectively. (±)-SdL analogues are able to selectively stimulate P. ramosa 2a, revealing that these minimal structural elements are key for this selective bioactivity. In addition, (±)-SdL19 is able to inhibit shoot branching in Pisum sativum and Arabidopsis thaliana and induces hyphal branching in the AM fungus Rhizophagus irregularis, like SLs.
Assuntos
Arabidopsis , Micorrizas , Orobanchaceae , Orobanche , Striga , Germinação , Compostos Heterocíclicos com 3 Anéis , Lactonas/química , Lactonas/farmacologia , Raízes de Plantas/química , Plantas Daninhas , SementesRESUMO
Although the division of the pericycle cells initiates both lateral root development and root-derived callus formation, these developmental processes are affected differently in the strigolactone and karrikin/KARRIKIN INSENSITIVE 2 (KAI2) ligand signalling mutant more axillary growth 2 (max2). Whereas max2 produces more lateral roots than the wild type, it is defective in the regeneration of shoots from root explants. We suggest that the decreased shoot regeneration of max2 originates from delayed formation of callus primordium, yielding less callus material to regenerate shoots. Indeed, when incubated on callus-inducing medium, the pericycle cell division was reduced in max2 and the early gene expression varied when compared with the wild type, as determined by a transcriptomics analysis. Furthermore, the expression of the LATERAL ORGAN BOUNDARIES DOMAIN genes and of callus-induction genes was modified in correlation with the max2 phenotype, suggesting a role for MAX2 in the regulation of the interplay between cytokinin, auxin, and light signalling in callus initiation. Additionally, we found that the in vitro shoot regeneration phenotype of max2 might be caused by a defect in KAI2, rather than in DWARF14, signalling. Nevertheless, the shoot regeneration assays revealed that the strigolactone biosynthesis mutants max3 and max4 also play a minor role.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ligantes , Raízes de Plantas/metabolismo , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismoRESUMO
Strigolactones (SLs) are intriguing phytohormones that not only regulate plant development and architecture but also interact with other organisms in the rhizosphere as root parasitic plants (Striga, Orobanche, and Phelipanche) and arbuscular mycorrhizal fungi. Starting with a pioneering work in 2003 for the isolation and identification of the SL receptor in parasitic weeds, fluorescence labeling of analogs has proven a major strategy to gain knowledge in SL perception and signaling. Here, we present novel chemical tools for understanding the SL perception based on the enzymatic properties of SL receptors. We designed different profluorescent SL Guillaume Clavé (GC) probes and performed structure-activity relationship studies on pea, Arabidopsis thaliana, and Physcomitrium (formerly Physcomitrella) patens. The binding of the GC probes to PsD14/RMS3, AtD14, and OsD14 proteins was tested. We demonstrated that coumarin-based profluorescent probes were highly bioactive and well-adapted to dissect the enzymatic properties of SL receptors in pea and a resorufin profluorescent probe in moss, contrary to the commercially available fluorescein profluorescent probe, Yoshimulactone Green (YLG). These probes offer novel opportunities for the studies of SL in various plants.
RESUMO
KAI2 proteins are plant α/ß hydrolase receptors which perceive smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). The number of functional KAI2 receptors varies among species and KAI2 gene duplication and sub-functionalization likely plays an adaptative role by altering specificity towards different KLs. Legumes represent one of the largest families of flowering plants and contain many agronomic crops. Prior to their diversification, KAI2 underwent duplication resulting in KAI2A and KAI2B. Here we demonstrate that Pisum sativum KAI2A and KAI2B are active receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a higher affinity for and hydrolyses a broader range of substrates including strigolactone-like stereoisomers. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical, structural, and mass spectra analyses of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, confirming its role as a bona fide enzyme. Our work uncovers the stereoselectivity of ligand perception and catalysis by diverged KAI2 receptors and proposes adaptive sensitivity to KAR/KL and strigolactones by KAI2B.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Catálise , Pisum sativum/genética , Pisum sativum/metabolismo , Percepção , Reguladores de Crescimento de Plantas/genéticaRESUMO
Strigolactones (SLs) are plant hormones and important signalling molecules required to promote arbuscular mycorrhizal (AM) symbiosis. While in plants an α/ß-hydrolase, DWARF14 (D14), was shown to act as a receptor that binds and cleaves SLs, the fungal receptor for SLs is unknown. Since AM fungi are currently not genetically tractable, in this study, we used the fungal pathogen Cryphonectria parasitica, for which gene deletion protocols exist, as a model, as we have previously shown that it responds to SLs. By means of computational, biochemical and genetic analyses, we identified a D14 structural homologue, CpD14. Molecular homology modelling and docking support the prediction that CpD14 interacts with and hydrolyses SLs. The recombinant CpD14 protein shows α/ß hydrolytic activity in vitro against the SLs synthetic analogue GR24; its enzymatic activity requires an intact Ser/His/Asp catalytic triad. CpD14 expression in the d14-1 loss-of-function Arabidopsis thaliana line did not rescue the plant mutant phenotype. However, gene inactivation by knockout homologous recombination reduced fungal sensitivity to SLs. These results indicate that CpD14 is involved in SLs responses in C. parasitica and strengthen the role of SLs as multifunctional molecules acting in plant-microbe interactions.
Assuntos
Ascomicetos , Proteínas de Plantas , Ascomicetos/metabolismo , Compostos Heterocíclicos com 3 Anéis , Lactonas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The synthetic strigolactone (SL) analog, rac-GR24, has been instrumental in studying the role of SLs as well as karrikins because it activates the receptors DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2) of their signaling pathways, respectively. Treatment with rac-GR24 modifies the root architecture at different levels, such as decreasing the lateral root density (LRD), while promoting root hair elongation or flavonol accumulation. Previously, we have shown that the flavonol biosynthesis is transcriptionally activated in the root by rac-GR24 treatment, but, thus far, the molecular players involved in that response have remained unknown. To get an in-depth insight into the changes that occur after the compound is perceived by the roots, we compared the root transcriptomes of the wild type and the more axillary growth2 (max2) mutant, affected in both SL and karrikin signaling pathways, with and without rac-GR24 treatment. Quantitative reverse transcription (qRT)-PCR, reporter line analysis and mutant phenotyping indicated that the flavonol response and the root hair elongation are controlled by the ELONGATED HYPOCOTYL 5 (HY5) and MYB12 transcription factors, but HY5, in contrast to MYB12, affects the LRD as well. Furthermore, we identified the transcription factors TARGET OF MONOPTEROS 5 (TMO5) and TMO5 LIKE1 as negative and the Mediator complex as positive regulators of the rac-GR24 effect on LRD. Altogether, hereby, we get closer toward understanding the molecular mechanisms that underlay the rac-GR24 responses in the root.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Flavonóis/genética , Flavonóis/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Organogênese Vegetal/genética , Transdução de SinaisRESUMO
Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.
Assuntos
Compostos Heterocíclicos com 3 Anéis/metabolismo , Hidrolases/genética , Isotiocianatos/metabolismo , Lactonas/metabolismo , Orobanchaceae/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Europa (Continente) , Hidrolases/química , Hidrolases/metabolismo , Orobanchaceae/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Daninhas/genética , Plantas Daninhas/metabolismo , Alinhamento de SequênciaRESUMO
In angiosperms, the α/ß hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (-)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (-)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.
Assuntos
Bryopsida/genética , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Orobanchaceae/fisiologia , Proteínas de Plantas/genética , Bryopsida/metabolismo , Bryopsida/parasitologia , Proteínas de Plantas/metabolismoRESUMO
Uncovering the basis of small-molecule hormone receptors' evolution is paramount to a complete understanding of how protein structure drives function. In plants, hormone receptors for strigolactones are well suited to evolutionary inquiries because closely related homologs have different ligand preferences. More importantly, because of facile plant transgenic systems, receptors can be swapped and quickly assessed functionally in vivo. Here, we show that only three mutations are required to turn the nonstrigolactone receptor, KAI2, into a receptor that recognizes the plant hormone strigolactone. This modified receptor still retains its native function to perceive KAI2 ligands. Our directed evolution studies indicate that only a few keystone mutations are required to increase receptor promiscuity of KAI2, which may have implications for strigolactone receptor evolution in parasitic plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Furanos/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Compostos Heterocíclicos com 3 Anéis/metabolismo , Hidrolases/metabolismo , Lactonas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Piranos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Hidrolases/genética , Mutação , Filogenia , Ligação ProteicaRESUMO
In this chapter, we will describe a method we set up to synthesize two profluorescent strigolactone (SL) mimic probes (GC240 and GC242) and the optimized protocols developed to study the enzymatic properties of various strigolactone receptors. The Arabidopsis AtD14 SL receptor is used here as a model for this purpose.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Bioensaio , Corantes Fluorescentes/síntese química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Ensaios de Triagem em Larga Escala , Medições Luminescentes , Reguladores de Crescimento de Plantas/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Transdução de SinaisRESUMO
The F-box protein MORE AXILLARY GROWTH 2 (MAX2) is a central component in the signaling cascade of strigolactones (SLs) as well as of the smoke-derived karrikins (KARs) and the so far unknown endogenous KAI2 ligand (KL). The two groups of molecules are involved in overlapping and unique developmental processes, and signal-specific outcomes are attributed to perception by the paralogous α/ß-hydrolases DWARF14 (D14) for SL and KARRIKIN INSENSITIVE 2/HYPOSENSITIVE TO LIGHT (KAI2/HTL) for KAR/KL. In addition, depending on which receptor is activated, specific members of the SUPPRESSOR OF MAX2 1 (SMAX1)-LIKE (SMXL) family control KAR/KL and SL responses. As proteins that function in the same signal transduction pathway often occur in large protein complexes, we aimed at discovering new players of the MAX2, D14, and KAI2 protein network by tandem affinity purification in Arabidopsis cell cultures. When using MAX2 as a bait, various proteins were copurified, among which were general components of the Skp1-Cullin-F-box complex and members of the CONSTITUTIVE PHOTOMORPHOGENIC 9 signalosome. Here, we report the identification of a novel interactor of MAX2, a type 5 serine/threonine protein phosphatase, designated PHYTOCHROME-ASSOCIATED PROTEIN PHOSPHATASE 5 (PAPP5). Quantitative affinity purification pointed at PAPP5 as being more present in KAI2 rather than in D14 protein complexes. In agreement, mutant analysis suggests that PAPP5 modulates KAR/KL-dependent seed germination under suboptimal conditions and seedling development. In addition, a phosphopeptide enrichment experiment revealed that PAPP5 might dephosphorylate MAX2 in vivo independently of the synthetic SL analog, rac-GR24. Together, by analyzing the protein complexes to which MAX2, D14, and KAI2 belong, we revealed a new MAX2 interactor, PAPP5, that might act through dephosphorylation of MAX2 to control mainly KAR/KL-related phenotypes and, hence, provide another link with the light pathway.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Germinação , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Nicotiana/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/genética , Furanos/metabolismo , Hidrolases/genética , Hipocótilo/crescimento & desenvolvimento , Lotus/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Piranos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Furanos/química , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/genética , Compostos Heterocíclicos com 3 Anéis/metabolismo , Hidrolases/metabolismo , Hipocótilo/metabolismo , Lactonas/metabolismo , Ligantes , Lotus/genética , Análise em Microsséries , Filogenia , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/genética , Raízes de Plantas/metabolismo , Piranos/químicaRESUMO
The arbuscular mycorrhizal symbiosis is a very common association between plant roots and soil fungi, which greatly contributes to plant nutrition. Root-exuded compounds known as strigolactones act as symbiotic signals stimulating the fungus prior to root colonization. Strigolactones also play an endogenous role in planta as phytohormones and contribute to the regulation of various developmental traits. Structure-activity relationship studies have revealed both similarities and differences between the structural features required for bioactivity in plants and arbuscular mycorrhizal fungi. In the latter case, bioassays usually measured a stimulation of hyphal branching on isolated fungi of the Gigaspora genus, grown in vitro. Here, we extended these investigations with a bioassay that evaluates the bioactivity of strigolactone analogs in a symbiotic situation and the use of the model mycorrhizal fungus Rhizophagus irregularis. Some general structural requirements for bioactivity reported previously for Gigaspora were confirmed. We also tested additional strigolactone analogs bearing modifications on the conserved methylbutenolide ring, a key element of strigolactone perception by plants. A strigolactone analog with an unmethylated butenolide ring could enhance the ability of R. irregularis to colonize host roots. Surprisingly, when applied to the isolated fungus in vitro, this compound stimulated germ tube elongation but inhibited hyphal branching. Therefore, this compound was able to act on the fungal and/or plant partner to facilitate initiation of the arbuscular mycorrhizal symbiosis, independently from hyphal branching and possibly from the strigolactone pathway.
Assuntos
Glomeromycota , Micorrizas , Hifas , Raízes de Plantas , SimbioseRESUMO
Strigolactone (SL) plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. GR24, a synthetic SL analog, is the worldwide reference compound used in all bioassays for investigating the role of SLs in plant development and in rhizospheric interactions. In 2012, the first characterization of the SL receptor reported the detection of an unknown compound after incubation of GR24 samples with the SL receptor. We reveal here the origin of this compound (P270), which comes from a by-product formed during GR24 chemical synthesis. We present the identification of this by-product, named contalactone. A proposed chemical pathway for its formation is provided as well as an evaluation of its bioactivity on pea, Arabidopsis, root parasitic plant seeds and AM fungi, characterizing it as a SL mimic. Quality of GR24 samples can be easily checked by carrying out microscale hydrolysis in a basic aqueous medium to easily detect P270 as indicator of the presence of the contalactone impurity. In all cases, before being used for bioassays, GR24 must be careful purified by preparative HPLC.
Assuntos
Arabidopsis/química , Compostos Heterocíclicos com 3 Anéis/análise , Lactonas/análise , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos , Compostos Heterocíclicos com 3 Anéis/síntese química , Lactonas/síntese química , Estrutura MolecularRESUMO
Strigolactones (SLs) are a family of terpenoid allelochemicals that were recognized as plant hormones only a decade ago. They influence a myriad of both above- and below-ground developmental processes, and are an important survival strategy for plants in nutrient-deprived soils. A rapidly emerging approach to gain knowledge on hormone signaling is the use of traceable analogs. A unique class of labeled SL analogs was constructed, in which the original tricyclic lactone moiety of natural SLs is replaced by a fluorescent cyanoisoindole ring system. Biological evaluation as parasitic seed germination stimulant and hypocotyl elongation repressor proved the potency of the cyanoisoindole strigolactone analogs (CISAs) to be comparable to the commonly accepted standard GR24. Additionally, via a SMXL6 protein degradation assay, we provided molecular evidence that the compounds elicit SL-like responses through the natural signaling cascade. All CISAs were shown to exhibit fluorescent properties, and the high quantum yield and Stokes shift of the pyrroloindole derivative CISA-7 also enabled in vivo visualization in plants. In contrast to the previously reported fluorescent analogs, CISA-7 displays a large similarity in shape and structure with natural SLs, which renders the analog a promising tracer to investigate the spatiotemporal distribution of SLs in plants and fungi.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Lactonas/química , Proteólise , Transdução de Sinais , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Corantes Fluorescentes , Germinação , Hipocótilo/genética , Hipocótilo/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Sementes/genética , Sementes/fisiologiaRESUMO
Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones.