RESUMO
The architecture of flowering plants exhibits both phenotypic diversity and plasticity, determined, in part, by the number and activity of axillary meristems and, in part, by the growth characteristics of the branches that develop from the axillary buds. The plasticity of shoot branching results from a combination of various intrinsic and genetic elements, such as number and position of nodes and type of growth phase, as well as environmental signals such as nutrient availability, light characteristics, and temperature (Napoli et al., 1998; Bennett and Leyser, 2006; Janssen et al., 2014; Teichmann and Muhr, 2015; Ueda and Yanagisawa, 2019). Axillary meristem initiation and axillary bud outgrowth are controlled by a complex and interconnected regulatory network. Although many of the genes and hormones that modulate branching patterns have been discovered and characterized through genetic and biochemical studies, there are still many gaps in our understanding of the control mechanisms at play. In this review, we will summarize our current knowledge of the control of axillary meristem initiation and outgrowth into a branch.
Assuntos
Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Magnoliopsida/genética , Reguladores de Crescimento de Plantas/metabolismo , Plasticidade Celular , Magnoliopsida/crescimento & desenvolvimento , Meristema/genética , Meristema/crescimento & desenvolvimento , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimentoRESUMO
Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2N242I and DAD2F135A, having substitutions in the core α/ß hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.
Assuntos
Compostos Heterocíclicos com 3 Anéis/química , Lactonas/química , Petunia/química , Reguladores de Crescimento de Plantas/genética , Proteínas de Plantas/química , Proteínas F-Box/química , Proteínas F-Box/genética , Regulação da Expressão Gênica de Plantas/genética , Hidrolases/química , Hidrolases/genética , Petunia/genética , Reguladores de Crescimento de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica/genética , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/genética , Transdução de Sinais/genéticaRESUMO
Strigolactones (SLs) are multifunctional plant hormones regulating essential physiological processes affecting growth and development. In vascular plants, SLs are recognized by α/ß hydrolase-fold proteins from the D14/DAD2 (Dwarf14/Decreased Apical Dominance 2) family in the initial step of the signaling pathway. We have previously discovered that N-phenylanthranilic acid derivatives (e.g. tolfenamic acid) are potent antagonists of SL receptors, prompting us to design quinazolinone and quinazolinedione derivatives (QADs and QADDs, respectively) as second-generation antagonists. Initial in silico docking studies suggested that these compounds would bind to DAD2, the petunia SL receptor, with higher affinity than the first-generation compounds. However, only one of the QADs/QADDs tested in in vitro assays acted as a competitive antagonist of SL receptors, with reduced affinity and potency compared with its N-phenylanthranilic acid 'parent'. X-ray crystal structure analysis revealed that the binding mode of the active QADD inside DAD2's cavity was not that predicted in silico, highlighting a novel inhibition mechanism for SL receptors. Despite a â¼10-fold difference in potency in vitro, the QADD and tolfenamic acid had comparable activity in planta, suggesting that the QADD compensates for lower potency with increased bioavailability. Altogether, our results establish this QADD as a novel lead compound towards the development of potent and bioavailable antagonists of SL receptors.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Petunia , Quinazolinonas , Receptores de Superfície Celular , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografia por Raios X , Petunia/química , Petunia/genética , Petunia/metabolismo , Ligação Proteica , Quinazolinonas/síntese química , Quinazolinonas/química , Quinazolinonas/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
The strigolactone (SL) family of plant hormones regulates a broad range of physiological processes affecting plant growth and development and also plays essential roles in controlling interactions with parasitic weeds and symbiotic fungi. Recent progress elucidating details of SL biosynthesis, signaling, and transport offers many opportunities for discovering new plant-growth regulators via chemical interference. Here, using high-throughput screening and downstream biochemical assays, we identified N-phenylanthranilic acid derivatives as potent inhibitors of the SL receptors from petunia (DAD2), rice (OsD14), and Arabidopsis (AtD14). Crystal structures of DAD2 and OsD14 in complex with inhibitors further provided detailed insights into the inhibition mechanism, and in silico modeling of 19 other plant strigolactone receptors suggested that these compounds are active across a large range of plant species. Altogether, these results provide chemical tools for investigating SL signaling and further define a framework for structure-based approaches to design and validate optimized inhibitors of SL receptors for specific plant targets.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Modelos Moleculares , Oryza , Petunia , Receptores de Superfície Celular , ortoaminobenzoatos , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Simulação por Computador , Oryza/química , Oryza/genética , Oryza/metabolismo , Petunia/química , Petunia/genética , Petunia/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologiaRESUMO
Branching has a major influence on the overall shape and productivity of a plant. Strigolactones (SLs) have been identified as plant hormones that have a key role in suppressing the outgrowth of axillary meristems. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes are integral to the biosynthesis of SLs and are well characterized in annual plants, but their role in woody perennials is relatively unknown. We identified CCD7 and CCD8 orthologues from apple and demonstrated that MdCCD7 and MdCCD8 are able to complement the Arabidopsis branching mutants max3 and max4 respectively, indicating conserved function. RNAi lines of MdCCD7 show reduced gene expression and increased branching in apple. We performed reciprocal grafting experiments with combinations of MdCCD7 RNAi and wild-type 'Royal Gala' as rootstocks and scion. Unexpectedly, wild-type roots were unable to suppress branching in MdCCD7 RNAi scions. Another key finding was that MdCCD7 RNAi scions initiated phytomers at an increased rate relative to the wild type, resulting in a greater node number and primary shoot length. We suggest that localized SL biosynthesis in the shoot, rather than roots, controls axillary bud outgrowth and shoot growth rate in apple.
Assuntos
Dioxigenases/genética , Lactonas/metabolismo , Malus/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Brotos de Planta/crescimento & desenvolvimento , Dioxigenases/metabolismo , Regulação da Expressão Gênica de Plantas , Malus/crescimento & desenvolvimento , Malus/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/genéticaRESUMO
Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal.
Assuntos
Meio Ambiente , Morfogênese , Petunia/crescimento & desenvolvimento , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Vias Biossintéticas/efeitos da radiação , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas , Luz , Dados de Sequência Molecular , Morfogênese/efeitos dos fármacos , Morfogênese/efeitos da radiação , Petunia/efeitos dos fármacos , Petunia/genética , Petunia/efeitos da radiação , Fósforo/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/efeitos da radiação , Caules de Planta/efeitos dos fármacos , Caules de Planta/genética , Caules de Planta/efeitos da radiação , Análise de Componente Principal , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/metabolismoRESUMO
Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha, which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) and MORE AXILLARY GROWTH 1 (MAX1). However, it retains some SL pathway components, including DWARF27 (D27) and CCD7. To help elucidate the function of these remaining components in M. polymorpha, knockout mutants were generated for MpD27-1, MpD27-2 and MpCCD7. Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1, Mpd27-2, and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED (ELI), and stress responses such as LATE EMBRYOGENESIS ABUNDANT (LEA) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS (ERFs) and SL and carotenoid related genes, such as TERPENE SYNTHASE (TS), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT). Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants.
RESUMO
The action of the petunia strigolactone (SL) hormone receptor DAD2 is dependent not only on its interaction with the PhMAX2A and PhD53A proteins, but also on its expression patterns within the plant. Previously, in a yeast-2-hybrid system, we showed that a series of a single and double amino acid mutants of DAD2 had altered interactions with these binding partners. In this study, we tested the mutants in two plant systems, Arabidopsis and petunia. Testing in Arabidopsis was enabled by creating a CRISPR-Cas9 knockout mutant of the Arabidopsis strigolactone receptor (AtD14). We produced SL receptor activity in both systems using wild type and mutant genes; however, the mutants had functions largely indistinguishable from those of the wild type. The expression of the wild type DAD2 from the CaMV 35S promoter in dad2 petunia produced plants neither quite like the dad2 mutant nor the V26 wild type. These plants had greater height and leaf size although branch number and the plant shape remained more like those of the mutant. These traits may be valuable in the context of a restricted area growing system such as controlled environment agriculture.
RESUMO
Differential scanning fluorimetry (DSF) is a method used for assessing the interaction of ligands with proteins. In most cases binding of a ligand to proteins tends to increase the melting temperature (Tm) of the protein involved. However, in the case of strigolactone receptors (e.g., D14, AtD14, DAD2, RMS3) from plants, the Tm tends to be reduced in the presence of strigolactones. This is likely due to increased flexibility of the receptors in the presence of hormone ligands.DSF experiments are simple, fast, amenable to high-throughput formats, and cost effective. They have therefore gained in popularity, including within the field of SL signaling. Typically in DSF the receptor protein is purified and incubated with the ligand (strigolactone, agonist, or antagonist) and a (fluorescent) reporter dye. The mixture is then placed in a quantitative PCR instrument and subjected to an increasing temperature gradient. Changes in fluorescence are recorded along the gradient, as the dye interacts with unfolded portions of the protein becoming accessible when the protein "melts". Differences in the temperature at which the protein unfolds in the absence and in the presence of the ligand are interpreted as indicating interactions between the ligand and the receptor.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fluorometria , Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Receptores de Superfície Celular/metabolismo , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala , Ligantes , Transdução de SinaisRESUMO
⢠CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants, including Arabidopsis, pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). ⢠Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8. ⢠Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence. ⢠The CCD pathway for control of branch development is conserved across a wide range of species, including kiwifruit, a woody perennial.
Assuntos
Actinidia , Arabidopsis/metabolismo , Senescência Celular , Dioxigenases/metabolismo , Genes de Plantas , Folhas de Planta/fisiologia , Caules de Planta , Actinidia/enzimologia , Actinidia/genética , Actinidia/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carotenoides/metabolismo , Dioxigenases/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Mutação , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , RNARESUMO
One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.
Assuntos
Morfogênese , Petunia/enzimologia , Petunia/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Transdução de Sinais , Biomassa , Segregação de Cromossomos/genética , Retroalimentação Fisiológica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação/genética , Tamanho do Órgão , Especificidade de Órgãos , Petunia/genética , Fenótipo , Proteínas de Plantas/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/enzimologia , Brotos de Planta/crescimento & desenvolvimento , Caules de Planta/enzimologia , Caules de Planta/genética , Interferência de RNA , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). RESULTS: The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. CONCLUSION: This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.
Assuntos
Actinidia/genética , Actinidia/fisiologia , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Frutas/crescimento & desenvolvimento , Pigmentação/genética , Paladar , Actinidia/crescimento & desenvolvimento , Actinidia/metabolismo , Adulto , Alérgenos/genética , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Criança , Códon , Sequência Consenso , Ésteres/metabolismo , Frutas/genética , Frutas/metabolismo , Genes de Plantas/genética , Marcadores Genéticos , Humanos , Repetições de Microssatélites , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Pigmentos Biológicos/biossíntese , Pigmentos Biológicos/genética , Polimorfismo de Nucleotídeo Único , Ácido Quínico/metabolismo , Análise de Sequência , Terpenos/metabolismoRESUMO
BACKGROUND: Apple fruit develop over a period of 150 days from anthesis to fully ripe. An array representing approximately 13000 genes (15726 oligonucleotides of 45-55 bases) designed from apple ESTs has been used to study gene expression over eight time points during fruit development. This analysis of gene expression lays the groundwork for a molecular understanding of fruit growth and development in apple. RESULTS: Using ANOVA analysis of the microarray data, 1955 genes showed significant changes in expression over this time course. Expression of genes is coordinated with four major patterns of expression observed: high in floral buds; high during cell division; high when starch levels and cell expansion rates peak; and high during ripening. Functional analysis associated cell cycle genes with early fruit development and three core cell cycle genes are significantly up-regulated in the early stages of fruit development. Starch metabolic genes were associated with changes in starch levels during fruit development. Comparison with microarrays of ethylene-treated apple fruit identified a group of ethylene induced genes also induced in normal fruit ripening. Comparison with fruit development microarrays in tomato has been used to identify 16 genes for which expression patterns are similar in apple and tomato and these genes may play fundamental roles in fruit development. The early phase of cell division and tissue specification that occurs in the first 35 days after pollination has been associated with up-regulation of a cluster of genes that includes core cell cycle genes. CONCLUSION: Gene expression in apple fruit is coordinated with specific developmental stages. The array results are reproducible and comparisons with experiments in other species has been used to identify genes that may play a fundamental role in fruit development.
Assuntos
Flores/crescimento & desenvolvimento , Frutas/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Malus/genética , Malus/metabolismo , Flores/genética , Flores/metabolismo , Frutas/genética , Frutas/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Malus/crescimento & desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Amido/metabolismo , Fatores de TempoRESUMO
In the past decade, enormous progress has been made in our understanding of the molecular and genetic control of meristem growth, maintenance and differentiation into plant organs. Several model plants have contributed to our current knowledge of meristem function. Research using Petunia has had a substantial share in this progress. Integration of information obtained from this species gives clues about the common and diverged pathways underlying the formation and functioning of plant meristems.
Assuntos
Meristema/fisiologia , Petunia/fisiologia , Arabidopsis/fisiologia , Petunia/classificação , Petunia/crescimento & desenvolvimento , Filogenia , Tulipa/fisiologiaRESUMO
Axillary meristems are formed in leaf axils and their growth into branches is a highly controlled process that is an important contributor to plant architecture. Here we discuss work that improves our understanding of the initiation and growth of axillary meristems. Recent results have implicated brassinosteroid signalling in the formation of axillary meristems. Our knowledge of axillary meristem outgrowth has also advanced, particularly in the areas of strigolactone signal production and perception, which have been shown to respond to environmental inputs. Auxins and cytokinins have also been linked to the control of axillary shoot development, revealing a complex network of signals that combine to regulate the outgrowth of an axillary meristem into a branch.
Assuntos
Meristema/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brassinosteroides/metabolismo , Citocininas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Lactonas/metabolismo , Meristema/genética , Meristema/metabolismo , Modelos Biológicos , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Brotos de Planta/genética , Brotos de Planta/metabolismoRESUMO
The signaling molecules strigolactone (SL) and karrikin are involved in seed germination, development of axillary meristems, senescence of leaves, and interactions with arbuscular mycorrhizal fungi. The signal transduction pathways for both SLs and karrikins require the same F-box protein (MAX2) and closely related α/ß hydrolase fold proteins (DAD2 and KAI2). The crystal structure of DAD2 has been solved revealing an α/ß hydrolase fold protein with an internal cavity capable of accommodating SLs. DAD2 responds to the SL analog GR24 by changing conformation and binding to MAX2 in a GR24 concentration-dependent manner. DAD2 can also catalyze hydrolysis of GR24. Structure activity relationships of analogs indicate that the butenolide ring common to both SLs and karrikins is essential for biological activity, but the remainder of the molecules can be significantly modified without loss of activity. The combination of data from the study of DAD2, KAI2, and chemical analogs of SLs and karrikins suggests a model for binding that requires nucleophilic attack by the active site serine of the hydrolase at the carbonyl atom of the butenolide ring. A conformational change occurs in the hydrolase that results in interaction with the F-box protein MAX2. Downstream signal transduction is then likely to occur via SCF (Skp-Cullin-F-box) complex-mediated ubiquitination of target proteins and their subsequent degradation. The role of the catalytic activity of the hydrolase is unclear but it may be integral in binding as well as possibly allowing the signal to be cleared from the receptor. The α/ß hydrolase fold family consists mostly of active enzymes, with a few notable exceptions. We suggest that DAD2 and KAI2 represent an intermediate stage where some catalytic activity is retained at the same time as a receptor role has evolved.
RESUMO
Strigolactones are a recently discovered class of plant hormone involved in branching, leaf senescence, root development, and plant-microbe interactions. They are carotenoid-derived lactones, synthesized in the roots and transported acropetally to modulate axillary bud outgrowth (i.e., branching). However, a receptor for strigolactones has not been identified. We have identified the DAD2 gene from petunia, an ortholog of the rice and Arabidopsis D14 genes, and present evidence for its roles in strigolactone perception and signaling. DAD2 acts in the strigolactone pathway, and the dad2 mutant is insensitive to the strigolactone analog GR24. The crystal structure of DAD2 reveals an α/ß hydrolase fold containing a canonical catalytic triad with a large internal cavity capable of accommodating strigolactones. In the presence of GR24 DAD2 interacts with PhMAX2A, a central component of strigolactone signaling, in a GR24 concentration-dependent manner. DAD2 can hydrolyze GR24, with mutants of the catalytic triad abolishing both this activity and the ability of DAD2 to interact with PhMAX2A. The hydrolysis products can neither stimulate the protein-protein interaction nor modulate branching. These observations suggest that DAD2 acts to bind the mobile strigolactone signal and then interacts with PhMAX2A during catalysis to initiate an SCF-mediated signal transduction pathway.
Assuntos
Hidrolases/metabolismo , Petunia/crescimento & desenvolvimento , Petunia/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Regulação da Expressão Gênica de Plantas , Hidrolases/química , Hidrolases/genética , Lactonas/metabolismo , Lactonas/farmacologia , Dados de Sequência Molecular , Petunia/genética , Reguladores de Crescimento de Plantas/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/embriologia , Raízes de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Transdução de SinaisRESUMO
Analysis of mutants with increased branching has revealed the strigolactone synthesis/perception pathway which regulates branching in plants. However, whether variation in this well conserved developmental signaling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologs of the ArabidopsisMAX1 and MAX2 genes to characterize their role in petunia architecture. A single ortholog of MAX1, PhMAX1 which encodes a cytochrome P450, was identified and was able to complement the max1 mutant of Arabidopsis. Petunia has two copies of the MAX2 gene, PhMAX2A and PhMAX2B which encode F-Box proteins. Differences in the transcript levels of these two MAX2-like genes suggest diverging functions. Unlike PhMAX2B, PhMAX2A mRNA levels change in leaves of differing age/position on the plant. Nonetheless, this gene functionally complements the Arabidopsismax2 mutant indicating that the biochemical activity of the PhMAX2A protein is not significantly different from MAX2. The expression of the petunia strigolactone pathway genes (PhCCD7, PhCCD8, PhMAX1, PhMAX2A, and PhMAX2B) was then further investigated throughout the development of wild-type petunia plants. Three of these genes showed changes in mRNA levels over a development series. Alterations to the expression patterns of these genes may influence the branching growth habit of plants by changing strigolactone production and/or sensitivity. These changes could allow both subtle and dramatic changes to branching within and between species.