RESUMO
This study reveals striking differences in the content and composition of hydrophilic and lipophilic compounds in blackcurrant buds (Ribes nigrum L., cv. Ben Klibreck) resulting from winter chill or chemical dormancy release following treatment with ERGER, a biostimulant used to promote uniform bud break. Buds exposed to high winter chill exhibited widespread shifts in metabolite profiles relative to buds that experience winter chill by growth under plastic. Specifically, extensive chilling resulted in significant reductions in storage lipids and phospholipids, and increases in galactolipids relative to buds that experienced lower chill. Similarly, buds exposed to greater chill exhibited higher levels of many amino acids and dipeptides, and nucleotides and nucleotide phosphates than those exposed to lower chilling hours. Low chill buds (IN) subjected to ERGER treatment exhibited shifts in metabolite profiles similar to those resembling high chill buds that were evident as soon as 3 days after treatment. We hypothesise that chilling induces a metabolic shift which primes bud outgrowth by mobilising lipophilic energy reserves, enhancing phosphate availability by switching from membrane phospholipids to galactolipids and enhancing the availability of free amino acids for de novo protein synthesis by increasing protein turnover. Our results additionally suggest that ERGER acts at least in part by priming metabolism for bud outgrowth. Finally, the metabolic differences presented highlight the potential for developing biochemical markers for dormancy status providing an alternative to time-consuming forcing experiments.
Assuntos
Temperatura Baixa , Ribes , Ribes/metabolismo , Ribes/crescimento & desenvolvimento , Ribes/química , Dormência de Plantas/efeitos dos fármacos , Metaboloma , Flores/crescimento & desenvolvimento , Flores/metabolismo , Aminoácidos/metabolismoRESUMO
Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop.
Assuntos
Genoma de Planta/genética , Genômica , Solanum tuberosum/genética , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Variação Genética , Haplótipos/genética , Heterozigoto , Homozigoto , Imunidade Inata , Endogamia , Anotação de Sequência Molecular , Dados de Sequência Molecular , Doenças das Plantas/genética , Ploidias , Solanum tuberosum/fisiologiaRESUMO
Plant organs are made from multiple cell types, and defining the expression level of a gene in any one cell or group of cells from a complex mixture is difficult. Dicotyledonous plants normally have three distinct layers of cells, L1, L2 and L3. Layer L1 is the single layer of cells making up the epidermis, layer L2 the single cell sub-epidermal layer and layer L3 constitutes the rest of the internal cells. Here we show how it is possible to harvest an organ and characterise the level of layer-specific expression by using a periclinal chimera that has its L1 layer from Solanum pennellii and its L2 and L3 layers from Solanum lycopersicum. This is possible by measuring the level of the frequency of species-specific transcripts. RNA-seq analysis enabled the genome-wide assessment of whether a gene is expressed in the L1 or L2/L3 layers. From 13 277 genes that are expressed in both the chimera and the parental lines and with at least one polymorphism between the parental alleles, we identified 382 genes that are preferentially expressed in L1 in contrast to 1159 genes in L2/L3. Gene ontology analysis shows that many genes preferentially expressed in L1 are involved in cutin and wax biosynthesis, whereas numerous genes that are preferentially expressed in L2/L3 tissue are associated with chloroplastic processes. These data indicate the use of such chimeras and provide detailed information on the level of layer-specific expression of genes.
Assuntos
Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Quimera , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Solanum lycopersicum/citologia , Anotação de Sequência Molecular , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de RNARESUMO
Brassinosteroid (BR)-regulated growth and development in Arabidopsis depends on BRASSINOSTEROID INSENSITIVE 1 (BRI1), the BR receptor that is responsible for initiating the events of BR signalling. We analysed the temporal and spatial regulation of BRI1 expression using stable transgenic lines that carried BRI1 promoter:reporter fusions. In both seedlings and mature plants the tissues undergoing elongation or differentiation showed elevated BRI1 gene activity, and it could be demonstrated that in the hypocotyl this was accompanied by accumulation of the BRI1 transcript and its receptor protein product. In seedlings the BRI1 promoter was also found to be under diurnal regulation, determined primarily by light repression and a superimposed circadian control. To determine the functional importance of transcriptional regulation we complemented the severely BR insensitive bri1-101 mutant with a BRI1-luciferase fusion construct that was driven by promoters with contrasting specificities. Whereas the BRI1 promoter-driven transgene fully restored the wild phenotype, expression from the photosynthesis-associated CAB3 and the vasculature-specific SUC2 and ATHB8 promoters resulted in plants with varying morphogenic defects. Our results reveal complex differential regulation of BRI1 expression, and suggest that by influencing the distribution and abundance of the receptor this regulation can enhance or attenuate BR signalling.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brassinosteroides/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas Quinases/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano/genética , Ritmo Circadiano/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Germinação/genética , Germinação/efeitos da radiação , Glucuronidase/metabolismo , Luminescência , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , Fatores de TempoRESUMO
Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, play critical roles. Detailed mechanisms of BR signaling have been examined in Arabidopsis (Arabidopsis thaliana), but the role of BRI1 and BAK1 phosphorylation in crop plants is unknown. As a foundation for understanding the mechanism of BR signaling in tomato (Solanum lycopersicum), we used liquid chromatography-tandem mass spectrometry to identify multiple in vitro phosphorylation sites of the tomato BRI1 and BAK1 cytoplasmic domains. Kinase assays showed that both tomato BRI1 and BAK1 are active in autophosphorylation as well as transphosphorylation of each other and specific peptide substrates with a defined sequence motif. Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue threonine-1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. Furthermore, analysis of transgenic lines expressing full-length tomato BRI1-Flag constructs in the weak tomato bri1 allele, curl3(-abs1), demonstrated that threonine-1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of TGF-ß Receptor Interacting Protein (TRIP1), which was previously shown to be a BRI1-interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.
Assuntos
Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Solanum lycopersicum/enzimologia , Sítios de Ligação , Cinética , Fosforilação , Proteínas de Plantas/química , Proteínas Serina-Treonina Quinases/química , Transdução de SinaisRESUMO
Many of the biochemical conversions in plant steroid hormone biosynthesis are catalysed by cytochrome P450 enzymes (CYPs or P450s). A recent paper by Toshiyuki Ohnishi et al. (2006) indicates the role of CYP90C1 and CYP90D1 in the synthesis of the most bioactive plant steroid hormone, brassinolide. These results highlight the need for refining the brassinolide biosynthesis pathway.
Assuntos
Colestanóis/metabolismo , Reguladores de Crescimento de Plantas/biossíntese , Esteroides Heterocíclicos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Brassinosteroides , Colestanóis/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica de Plantas , Estrutura Molecular , Filogenia , Esteroides Heterocíclicos/químicaRESUMO
Brassinosteroids (BRs) are plant steroid hormones and, when applied exogenously, they induce physiological responses, including tolerance to heat shock (HS). How endogenous BR content and altered perception of BRs influence thermal tolerance is poorly understood. BR-induced thermotolerance in tomato seedlings with altered BR homeostasis was examined by assessing the survival, ion leakage and lipid peroxidation of seedlings from a BR-deficient mutant (extreme dwarf d(x)), a partially BR-insensitive mutant curl3(-abs) allele (curl3 altered brassinolide sensitivity) and a line overexpressing the Dwarf, BR-biosynthesis gene (35SD). We confirmed that treatment with 1 µM of epi-brassinolide (EBL) induces thermotolerance of wild type seedlings following a HS regime at 45 °C. The curl3(-abs) seedlings had the highest basal tolerance to heat, whereas the EBL-induced thermal tolerance of d(x) seedlings was greatest and responded to lower EBL concentrations. The d(x) and 35SD seedlings had similar thermal tolerance; however, they showed increased signs of oxidative stress. EBL reduced the induction of lipid peroxidation of seedlings after recovery from heat. Highest oxidative stress and peroxidase (POX) activity (EC 1.11.1.7) was in BR-deficient d(x) mutant seedlings. EBL was able of inducing POX activity but not other antioxidant enzymes; however, effects of HS on POX activity of seedlings were absent or less marked. Taking together, results indicate that thermal tolerance is independent of endogenous BR content, but HS-mediated oxidative stress depends on BR levels.
Assuntos
Adaptação Fisiológica/fisiologia , Brassinosteroides/metabolismo , Resposta ao Choque Térmico/fisiologia , Estresse Oxidativo , Solanum lycopersicum/metabolismo , Esteroides Heterocíclicos/metabolismo , Estresse Fisiológico , Alelos , Brassinosteroides/farmacologia , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Plantas , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Homeostase , Temperatura Alta , Peroxidação de Lipídeos , Solanum lycopersicum/efeitos dos fármacos , Solanum lycopersicum/genética , Mutação , Peroxidase/metabolismo , Fitosteróis/genética , Fitosteróis/metabolismo , Plântula , Esteroides Heterocíclicos/farmacologiaRESUMO
Brassinosteroids (BRs) are perceived by Brassinosteroid Insensitive 1 (BRI1), that encodes a leucine-rich repeat receptor kinase. Tomato BRI1 has previously been implicated in both systemin and BR signalling. The role of tomato BRI1 in BR signalling was confirmed, however it was found not to be essential for systemin/wound signalling. Tomato roots were shown to respond to systemin but this response varied according to the species and growth conditions. Overall the data indicates that mutants defective in tomato BRI1 are not defective in systemin-induced wound signalling and that systemin perception can occur via a non-BRI1 mechanism.
RESUMO
The tomato Leu-rich repeat receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) has been implicated in both peptide (systemin) and steroid (brassinosteroid [BR]) hormone perception. In an attempt to dissect these signaling pathways, we show that transgenic expression of BRI1 can restore the dwarf phenotype of the tomato curl3 (cu3) mutation. Confirmation that BRI1 is involved in BR signaling is highlighted by the lack of BR binding to microsomal fractions made from cu3 mutants and the restoration of BR responsiveness following transformation with BRI1. In addition, wound and systemin responses in the cu3 mutants are functional, as assayed by proteinase inhibitor gene induction and rapid alkalinization of culture medium. However, we observed BRI1-dependent root elongation in response to systemin in Solanum pimpinellifolium. In addition, ethylene perception is required for normal systemin responses in roots. These data taken together suggest that cu3 is not defective in systemin-induced wound signaling and that systemin perception can occur via a non-BRI1 mechanism.
Assuntos
Peptídeos/farmacologia , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais/efeitos dos fármacos , Solanum lycopersicum/metabolismo , Solanum/efeitos dos fármacos , Álcalis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Brassinosteroides , Colestanóis/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Solanum lycopersicum/genética , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Proteínas de Plantas/genética , Solanum/crescimento & desenvolvimento , Esteroides Heterocíclicos/farmacologia , Ativação TranscricionalRESUMO
Based on its compact habit, Micro-Tom, a dwarf cultivar of tomato (Solanum lycopersicum L.), has been proposed as a preferred variety to carry out molecular research in tomato. This cultivar, however, is poorly characterized. It is shown here that Micro-Tom has mutations in the SELF-PRUNING (SP) and DWARF (D) genes. In addition to this, it is also shown that Micro-Tom harbours at least two independently segregating resistance loci to the plant pathogen Cladosporium fulvum. The presence of the self-pruning mutation in Micro-Tom, that generates a determinate phenotype, was confirmed by crossing and sequence analysis. It was also found that Micro-Tom has a mutation in the DWARF gene (d) that leads to mis-splicing and production of at least two shorter mRNAs. The d mutation is predicted to generate truncated DWARF protein. The d sequence defect co-segregates with dark-green and rugose leaves, characteristics of brassinosteroid biosynthesis mutants. Micro-Tom also carries at least another mutation producing internode length reduction that affects plant height but not active gibberellin (GA) levels, which were similar in dwarf and tall Micro-TomxSeverianin segregants. GAs and brassinosteroids act synergistically in Micro-Tom, and the response to GA depends on brassinosteroids because the elongation of internodes was at least six times higher when GA(3) was applied simultaneously with brassinolide. A novel variety, Micro-0 that is fully susceptible to C. fulvum and almost as dwarf as Micro-Tom, has been generated from the cross of Cf0xMicro-Tom. This line represents a valuable resource for future analysis of Cf resistance genes through breeding or transformation.
Assuntos
Cladosporium/fisiologia , Giberelinas/fisiologia , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Esteroides/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Giberelinas/metabolismo , Solanum lycopersicum/microbiologia , Solanum lycopersicum/fisiologia , Dados de Sequência Molecular , Mutação , Fenótipo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/fisiologia , Splicing de RNA , Esteroides/metabolismoRESUMO
Plant steroid hormones, brassinosteroids (BRs), are essential for normal photomorphogenesis. However, the mechanism by which light controls physiological functions via BRs is not well understood. Using transgenic plants carrying promoter-luciferase reporter gene fusions, we show that in Arabidopsis (Arabidopsis thaliana) the BR-biosynthetic CPD and CYP85A2 genes are under diurnal regulation. The complex diurnal expression profile of CPD is determined by dual, light-dependent, and circadian control. The severely decreased expression level of CPD in phytochrome-deficient background and the red light-specific induction in wild-type plants suggest that light regulation of CPD is primarily mediated by phytochrome signaling. The diurnal rhythmicity of CPD expression is maintained in brassinosteroid insensitive 1 transgenic seedlings, indicating that its transcriptional control is independent of hormonal feedback regulation. Diurnal changes in the expression of CPD and CYP85A2 are accompanied by changes of the endogenous BR content during the day, leading to brassinolide accumulation at the middle of the light phase. We also show that CPD expression is repressed in extended darkness in a BR feedback-dependent manner. In the dark the level of the bioactive hormone did not increase; therefore, our data strongly suggest that light also influences the sensitivity of plants to BRs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ritmo Circadiano , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica de Plantas , Esteroide Hidroxilases/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Retroalimentação Fisiológica , Genes Reporter , Luz , Luciferases/genética , Luciferases/metabolismo , Fitocromo/genética , Fitocromo/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Esteroide Hidroxilases/metabolismoRESUMO
Brassinosteroids (BRs) are steroid hormones that are essential for plant growth and development. To gain insight into potential sites of BR synthesis, we studied promoter activities of the two Arabidopsis BR C-6 oxidase genes (CYP85A1 and CYP85A2) in transgenic plants carrying promoter fusions with the GUS, GFP or LUC reporter genes. BR-dependent feedback regulation of the GUS reporter constructs indicated that their expression corresponded to those of the native genes. Both the CYP85A1 and CYP85A2 promoters showed maximum activity during the first week following germination, particularly in the vascular tissues. Compared to CYP85A2, CYP85A1 expression was weaker and confined to the early stages of seedling development. Stronger CYP85A2 promoter activity was evident in both juvenile and adult plants. Comparison of the 5'-UTR and TATA box sequences of CYP85A1 and CYP85A2 revealed high homology, indicating a relatively recent gene duplication. We also found that transgenic Arabidopsis plants harbouring the tomato DWARF promoter-GUS fusion had similarities in the expression pattern to the Arabidopsis genes suggesting common transcriptional regulation of CYP85 genes in the two species.
Assuntos
Proteínas de Arabidopsis/genética , Colestanóis/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Esteroides Heterocíclicos/metabolismo , Região 5'-Flanqueadora/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Brassinosteroides , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histocitoquímica , Luciferases/genética , Luciferases/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Oxirredução , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Homologia de Sequência do Ácido Nucleico , Espectrometria de FluorescênciaRESUMO
Brassinosteroids are steroidal hormones essential for the growth and development of plants. Brassinolide, the most biologically active brassinosteroid, has a seven-membered lactone ring that is formed by a Baeyer-Villiger oxidation of its immediate precursor castasterone. Despite its potential key role in controlling plant development, brassinolide synthase has not been identified. Previous work has shown that the formation of castasterone from 6-deoxocastasterone is catalyzed by members of the CYP85A family of cytochrome P-450 monooxygenases. A null mutation in the tomato Dwarf (CYP85A1) gene, extreme dwarf (d(x)), causes severe dwarfism due to brassinosteroid deficiency, but the d(x) mutant still produces fruits. Here, we show that d(x) fruits contain brassinolide at a higher level than wild-type fruits and that a new CYP85A gene, CYP85A3, is preferentially expressed in tomato fruits. Tomato CYP85A3 catalyzed the Baeyer-Villiger oxidation to produce brassinolide from castasterone in yeast, in addition to the conversion of 6-deoxocastasterone to castasterone. We also show that Arabidopsis CYP85A2, which was initially characterized as castasterone synthase, also has brassinolide synthase activity. Exogenous application of castasterone and brassinolide to the Arabidopsis cyp85a1/cyp85a2 double mutant suggests that castasterone can function as an active brassinosteroid but that its conversion into brassinolide is necessary for normal vegetative development in Arabidopsis. We postulate that castasterone is the major active brassinosteroid during vegetative growth in tomato, whereas brassinolide may play an organ-specific role in fruit development in this species.
Assuntos
Arabidopsis/enzimologia , Colestanóis/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Solanum lycopersicum/enzimologia , Esteroides Heterocíclicos/metabolismo , Arabidopsis/genética , Brassinosteroides , Catálise , Solanum lycopersicum/genética , Dados de Sequência Molecular , Filogenia , Extratos Vegetais/genética , Extratos Vegetais/metabolismoRESUMO
Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.
Assuntos
Frutas/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Esteroides/biossíntese , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica/fisiologia , Genes de Plantas/fisiologia , Solanum lycopersicum/genética , Solanum lycopersicum/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismoRESUMO
Plant steroid hormones, brassinosteroids (BRs), were originally isolated from extracts of pollen because of their growth-promoting properties and their potential use for enhancing crop production. Mutants in the biosynthesis, metabolism, and signaling of brassinolide (BL), the most bioactive BR, are important resources in helping to establish BRs' essential role in plant growth and development. The dark green and distinctive dwarf phenotype of BR-related mutants identified in pea, tomato, and rice highlights the importance of BRs in crops. These mutants are helping to elucidate both the conserved and the unique features of BR biosynthesis and signaling. Such insights are providing the key knowledge and understanding that will enable the development of strategies towards the production of crops with enhanced qualities.
RESUMO
Brassinosteroids (BRs) are growth-promoting plant steroid hormones, and in garden pea (Pisum sativum L.), the lka mutant is defective in BR perception. Here, we show that LKA encodes P. sativum BRI1 (PsBRI1), a homolog of BRI1, which is the Arabidopsis leucine-rich repeat receptor-like kinase/BR receptor. PsBRI1 was isolated by screening a pea cDNA library using Arabidopsis BRI1 cDNA as the probe. PsBRI1 is predicted to encode a 1188-amino-acid protein that has 78% similarity with Arabidopsis BRI1. Sequence analysis of PsBRI1 in the lka mutant led to the identification of a missense mutation that converts the highly conserved aspartic acid residue to asparagine, which is located in the leucine-rich repeat, just before the island domain that may bind BR or a BR-protein complex. The mutation identified in PsBRI1 co-segregated with the semi-erectoide lka phenotype. Transcript analysis of LKA/PsBRI1 indicates that it is ubiquitously expressed in pea and that the expression was downregulated by exogenous BR. The lka mutant was then utilized in further studies to analyze the independent actions of BR and gibberellin (GA) through the characterization of BR response on GA mutants and GA response on BR mutants.
Assuntos
Pisum sativum/genética , Proteínas Quinases/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , Sequência Conservada , Primers do DNA , Biblioteca Gênica , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Proteínas Quinases/química , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genéticaRESUMO
Brassinosteroids (BRs) are plant steroid hormones that are essential for normal plant development. To gain better understanding of the conservation of BR signaling, the partially BR-insensitive tomato mutant altered brassinolide sensitivity1 (abs1) was identified and found to be a weak allele at the curl3 (cu3) locus. BR content is increased in both of these mutants and is associated with increased expression of DWARF: The tomato homolog of the Arabidopsis Brassinosteroid Insensitive1 Leu-rich repeat (LRR) receptor-like kinase, named tBri1, was isolated using degenerate primers. Sequence analysis of tBRI1 in the mutants cu3 and abs1 revealed that cu3 is a nonsense mutant and that abs1 is a missense mutant. A comparison of BRI1 homolog sequences highlights conserved features of BRI1 sequences, with the LRRs in close proximity to the island domain showing more conservation than N-terminal LRRs. The most homologous sequences were found in the kinase and transmembrane regions. tBRI1 (SR160) also has been isolated as the putative receptor for systemin, a plant peptide hormone. This finding suggests a possible dual role for tBRI1 in steroid hormone and peptide hormone signaling.
Assuntos
Proteínas de Arabidopsis , Reguladores de Crescimento de Plantas/farmacologia , Proteínas Quinases/genética , Transdução de Sinais/genética , Solanum lycopersicum/genética , Alelos , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Brassinosteroides , Colestanóis/metabolismo , Clonagem Molecular , Códon sem Sentido , Solanum lycopersicum/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Peptídeos/metabolismo , Fenótipo , Fitosteróis/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Sequências Repetitivas de Aminoácidos/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esteroides Heterocíclicos/metabolismoRESUMO
Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90 genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1, CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 and CYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 and CYP85A2 are preferentially represented in shoots and CYP90C1, CYP90D1, and CYP85A1 mRNAs are more abundant in roots, whereas CYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/CPD transcript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis.