RESUMEN
Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis "Old Blush" flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-ß-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.
Asunto(s)
Geraniltranstransferasa , Rosa , Geraniltranstransferasa/genética , Ácido Mevalónico/metabolismo , Rosa/metabolismo , Citosol/metabolismo , Filogenia , Terpenos/metabolismo , Flores/metabolismoRESUMEN
Nudix hydrolases are conserved enzymes ubiquitously present in all kingdoms of life. Recent research revealed that several Nudix hydrolases are involved in terpenoid metabolism in plants. In modern roses, RhNUDX1 is responsible for formation of geraniol, a major compound of rose scent. Nevertheless, this compound is produced by monoterpene synthases in many geraniol-producing plants. As a consequence, this raised the question about the origin of RhNUDX1 function and the NUDX1 gene evolution in Rosaceae, in wild roses or/and during the domestication process. Here, we showed that three distinct clades of NUDX1 emerged in the Rosoidae subfamily (Nudx1-1 to Nudx1-3 clades), and two subclades evolved in the Rosa genus (Nudx1-1a and Nudx1-1b subclades). We also showed that the Nudx1-1b subclade was more ancient than the Nudx1-1a subclade, and that the NUDX1-1a gene emerged by a trans-duplication of the more ancient NUDX1-1b gene. After the transposition, NUDX1-1a was cis-duplicated, leading to a gene dosage effect on the production of geraniol in different species. Furthermore, the NUDX1-1a appearance was accompanied by the evolution of its promoter, most likely from a Copia retrotransposon origin, leading to its petal-specific expression. Thus, our data strongly suggest that the unique function of NUDX1-1a in geraniol formation was evolved naturally in the genus Rosa before domestication.
Asunto(s)
Rosa , Rosaceae , Monoterpenos Acíclicos , Domesticación , Rosa/genética , Rosa/metabolismoRESUMEN
Roses use a non-canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1-2. In order to study the function of the RwNUDX1-2 protein, we analyzed the volatile profiles of an F1 progeny generated by crossing R. chinensis cv. 'Old Blush' with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1-2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E-farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1-2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E-farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1-2 gene co-localized with the QTL for E,E-farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.
Asunto(s)
Proteínas de Plantas/metabolismo , Pirofosfatasas/metabolismo , Rosa/metabolismo , Sesquiterpenos/metabolismo , Farnesol/metabolismo , Genes de Plantas/genética , Genes de Plantas/fisiología , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Pirofosfatasas/genética , Pirofosfatasas/fisiología , Sitios de Carácter Cuantitativo/genética , Rosa/genética , Alineación de Secuencia , Hidrolasas NudixRESUMEN
Floral scent is one of the most important characters in horticultural plants. Roses (Rosa spp.) have been cultivated for their scent since antiquity. However, probably by selecting for cultivars with long vase life, breeders have lost the fragrant character in many modern roses, especially the ones bred for the cut flower market. The genetic inheritance of scent characters has remained elusive so far. In-depth knowledge of this quantitative trait is thus very much needed to breed more fragrant commercial cultivars. Furthermore, rose hybrids harbor a composite genomic structure, which complexifies quantitative trait studies. To understand rose scent inheritance, we characterized a segregating population from two diploid cultivars, Rosa × hybrida cv H190 and Rosa wichurana, which have contrasting scent profiles. Several quantitative trait loci for the major volatile compounds in this progeny were identified. One among these loci contributing to the production of 2-phenylethanol, responsible for the characteristic odor of rose, was found to be colocalized with a candidate gene belonging to the 2-phenylethanol biosynthesis pathway: the PHENYLACETALDEHYDE SYNTHASE gene RhPAAS An in-depth allele-specific expression analysis in the progeny demonstrated that only one allele was highly expressed and was responsible for the production of 2-phenylethanol. Unexpectedly, its expression was found to start early during flower development, before the production of the volatile 2-phenylethanol, leading to the accumulation of glycosylated compounds in petals.
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Alcohol Feniletílico/metabolismo , Proteínas de Plantas/fisiología , Rosa/metabolismo , Alelos , Vías Biosintéticas , Flores/genética , Flores/metabolismo , Odorantes , Alcohol Feniletílico/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sitios de Carácter Cuantitativo , Rosa/genéticaRESUMEN
The large French research project GENIUS (2012-2019, https://www6.inra.genius-project_eng/ ) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.
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Agricultura/tendencias , Sistemas CRISPR-Cas/genética , Productos Agrícolas/genética , Edición Génica/métodos , Animales , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Brachypodium/genética , Brachypodium/crecimiento & desarrollo , Bryopsida/genética , Bryopsida/crecimiento & desarrollo , Productos Agrícolas/crecimiento & desarrollo , Genoma de Planta/genética , Mutagénesis/genética , FenotipoRESUMEN
The shoot branching pattern is a determining phenotypic trait throughout plant development. During shoot branching, BRANCHED1 (BRC1) plays a master regulator role in bud outgrowth, and its transcript levels are regulated by various exogenous and endogenous factors. RhBRC1 (the homologous gene of BRC1 in Rosa hybrida) is a main branching regulator whose posttranscriptional regulation in response to sugar was investigated through its 3'UTR. Transformed Rosa calluses containing a construction composed of the CaMV35S promoter, the green fluorescent protein (GFP) reporter gene, and the 3'UTR of RhBRC1 (P35S:GFP::3'UTRRhBRC1) were obtained and treated with various combinations of sugars and with sugar metabolism effectors. The results showed a major role of the 3'UTR of RhBRC1 in response to sugars, involving glycolysis/the tricarboxylic acid cycle (TCA) and the oxidative pentose phosphate pathway (OPPP). In Rosa vegetative buds, sequence analysis of the RhBRC1 3'UTR identified six binding motifs specific to the Pumilio/FBF RNA-binding protein family (PUF) and probably involved in posttranscriptional regulation. RhPUF4 was highly expressed in the buds of decapitated plants and in response to sugar availability in in-vitro-cultured buds. RhPUF4 was found to be close to AtPUM2, which encodes an Arabidopsis PUF protein. In addition, sugar-dependent upregulation of RhPUF4 was also found in Rosa calluses. RhPUF4 expression was especially dependent on the OPPP, supporting its role in OPPP-dependent posttranscriptional regulation of RhBRC1. These findings indicate that the 3'UTR sequence could be an important target in the molecular regulatory network of RhBRC1 and pave the way for investigating new aspects of RhBRC1 regulation.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al ARN/genética , Rosa/genética , Factores de Transcripción/genética , Regiones no Traducidas 3'/genética , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Fenotipo , Proteínas de Plantas/genética , Rosa/metabolismo , Transducción de Señal/genética , Azúcares/metabolismoRESUMEN
FT/TFL1 family members have been known to be involved in the development and flowering in plants. In rose, RoKSN, a TFL1 homologue, is a key regulator of flowering, whose absence causes continuous flowering. Our objectives are to functionally validate RoKSN and to explore its mode of action in rose. We complemented Arabidopsis tfl1 mutants and ectopically expressed RoKSN in a continuous-flowering (CF) rose. Using different protein interaction techniques, we studied RoKSN interactions with RoFD and RoFT and possible competition. In Arabidopsis, RoKSN complemented the tfl1 mutant by rescuing late flowering and indeterminate growth. In CF roses, the ectopic expression of RoKSN led to the absence of flowering. Different branching patterns were observed and some transgenic plants had an increased number of leaflets per leaf. In these transgenic roses, floral activator transcripts decreased. Furthermore, RoKSN was able to interact both with RoFD and the floral activator, RoFT. Protein interaction experiments revealed that RoKSN and RoFT could compete with RoFD for repression and activation of blooming, respectively. We conclude that RoKSN is a floral repressor and is also involved in the vegetative development of rose. RoKSN forms a complex with RoFD and could compete with RoFT for repression of flowering.
Asunto(s)
Flores/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Proteínas Represoras/metabolismo , Rosa/crecimiento & desarrollo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Prueba de Complementación Genética , Inflorescencia/genética , Inflorescencia/crecimiento & desarrollo , Mutación/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Reproducción , Rosa/genéticaRESUMEN
Light and temperature are two environmental factors that deeply affect bud outgrowth. However, little is known about their impact on the bud burst gradient along a stem and their interactions with the molecular mechanisms of bud burst control. We investigated this question in two acrotonic rose cultivars. We demonstrated that the darkening of distal buds or exposure to cold (5 °C) prior to transfer to mild temperatures (20 °C) both repress acrotony, allowing the burst of quiescent medial and proximal buds. We sequenced the strigolactone pathway MAX-homologous genes in rose and studied their expression in buds and internodes along the stem. Only expressions of RwMAX1, RwMAX2 and RwMAX4 were detected. Darkening of the distal part of the shoot triggered a strong increase of RwMAX2 expression in darkened buds and bark-phloem samples, whereas it suppressed the acropetal gradient of the expression of RwMAX1 observed in stems fully exposed to light. Cold treatment induced an acropetal gradient of expression of RwMAX1 in internodes and of RwMAX2 in buds along the stem. Our results suggest that the bud burst gradient along the stem cannot be explained by a gradient of expression of RwMAX genes but rather by their local level of expression at each individual position.
Asunto(s)
Lactonas/metabolismo , Luz , Brotes de la Planta/crecimiento & desarrollo , Rosa/crecimiento & desarrollo , Rosa/genética , Transducción de Señal/genética , Temperatura , Mapeo Cromosómico , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Genes de Plantas/genética , Filogenia , Brotes de la Planta/genética , Brotes de la Planta/efectos de la radiación , Sitios de Carácter Cuantitativo/genética , Rosa/fisiología , Rosa/efectos de la radiación , Transducción de Señal/efectos de la radiaciónRESUMEN
The Quantitative Trait Loci (QTL) mapping of plant architecture is a critical step for understanding the genetic determinism of plant architecture. Previous studies adopted simple measurements, such as plant-height, stem-diameter and branching-intensity for QTL mapping of plant architecture. Many of these quantitative traits were generally correlated to each other, which give rise to statistical problem in the detection of QTL. We aim to test the applicability of kernel methods to phenotyping inflorescence architecture and its QTL mapping. We first test Kernel Principal Component Analysis (KPCA) and Support Vector Machines (SVM) over an artificial dataset of simulated inflorescences with different types of flower distribution, which is coded as a sequence of flower-number per node along a shoot. The ability of discriminating the different inflorescence types by SVM and KPCA is illustrated. We then apply the KPCA representation to the real dataset of rose inflorescence shoots (n=1460) obtained from a 98 F1 hybrid mapping population. We find kernel principal components with high heritability (>0.7), and the QTL analysis identifies a new QTL, which was not detected by a trait-by-trait analysis of simple architectural measurements. The main tools developed in this paper could be use to tackle the general problem of QTL mapping of complex (sequences, 3D structure, graphs) phenotypic traits.
Asunto(s)
Algoritmos , Rosa/anatomía & histología , Mapeo Cromosómico , Simulación por Computador , Cruzamientos Genéticos , Bases de Datos como Asunto , Inflorescencia/anatomía & histología , Patrón de Herencia/genética , Fenotipo , Análisis de Componente Principal , Sitios de Carácter Cuantitativo/genética , Estadísticas no Paramétricas , Máquina de Vectores de SoporteRESUMEN
This study aims to: (i) identify the Rosa S-locus controlling self-incompatibility (SI); (ii) test the genetic linkage of the S-locus with other loci controlling important ornamental traits, such as the continuous-flowering (CF) characteristic; (iii) identify the S-alleles (SC ) of old Chinese CF cultivars (e.g, Old Blush, Slater's Crimson China) and examine the changes in the frequency of cultivars with Sc through the history of breeding; (iv) identify wild species carrying the Sc-alleles to infer wild origins of CF cultivars. We identified a new S-RNase (SC2 ) of Rosa chinensis in a contig from a genome database that has not been integrated into one of the seven chromosomes yet. Genetic mapping indicated that SC2 is allelic to the previously-identified S-RNase (SC1 ) in chromosome 3. Pollination experiments with half-compatible pairs of roses confirmed that they are the pistil-determinant of SI. The segregation analysis of an F1 -population indicated genetic linkage between the S-locus and the floral repressor gene KSN. The non-functional allele ksn is responsible for the CF characteristic. A total of five S-alleles (SC1-5 ) were identified from old CF cultivars. The frequency of cultivars with SC dramatically increased after the introgression of ksn from Chinese to European cultivars and remains high (80%) in modern cultivars, suggesting that S-genotyping is helpful for effective breeding. Wild individuals carrying SC were found in Rosa multiflora (SC1 ), Rosa chinensis var. spontanea (SC3 ), and Rosa gigantea (SC2 , SC4 ), supporting the hypothesis of hybrid origins of CF cultivars and providing a new evidence for the involvement of Rosa multiflora.
RESUMEN
The pattern of development of the inflorescence is an important characteristic in ornamental plants, where the economic value is in the flower. The genetic determinism of inflorescence architecture is poorly understood, especially in woody perennial plants with long life cycles. Our objective was to study the genetic determinism of this characteristic in rose. The genetic architectures of 10 traits associated with the developmental timing and architecture of the inflorescence, and with flower production were investigated in a F(1) diploid garden rose population, based on intensive measurements of phenological and morphological traits in a field. There were substantial genetic variations in inflorescence development traits, with broad-sense heritabilities ranging from 0.82 to 0.93. Genotypic correlations were significant for most (87%) pairs of traits, suggesting either pleiotropy or tight linkage among loci. However, non-significant and low correlations between some pairs of traits revealed two independent developmental pathways controlling inflorescence architecture: (1) the production of inflorescence nodes increased the number of branches and the production of flowers; (2) internode elongation connected with frequent branching increased the number of branches and the production of flowers. QTL mapping identified six common QTL regions (cQTL) for inflorescence developmental traits. A QTL for flowering time and many inflorescence traits were mapped to the same cQTL. Several candidate genes that are known to control inflorescence developmental traits and gibberellin signaling in Arabidopsis thaliana were mapped in rose. Rose orthologues of FLOWERING LOCUS T (RoFT), TERMINAL FLOWER 1 (RoKSN), SPINDLY (RoSPINDLY), DELLA (RoDELLA), and SLEEPY (RoSLEEPY) co-localized with cQTL for relevant traits. This is the first report on the genetic basis of complex inflorescence developmental traits in rose.
Asunto(s)
Inflorescencia/anatomía & histología , Inflorescencia/genética , Sitios de Carácter Cuantitativo/genética , Rosa/anatomía & histología , Rosa/genética , Mapeo Cromosómico , Cruzamientos Genéticos , Ligamiento Genético , Variación Genética , Genoma de Planta/genética , Genotipo , Análisis de los Mínimos Cuadrados , Carácter Cuantitativo Heredable , Factores de TiempoRESUMEN
We have constructed the first integrated consensus map (ICM) for rose, based on the information of four diploid populations and more than 1,000 initial markers. The single population maps are linked via 59 bridge markers, on average 8.4 per linkage group (LG). The integrated map comprises 597 markers, 206 of which are sequence-based, distributed over a length of 530 cM on seven LGs. By using a larger effective population size and therefore higher marker density, the marker order in the ICM is more reliable than in the single population maps. This is supported by a more even marker distribution and a decrease in gap sizes in the consensus map as compared to the single population maps. This unified map establishes a standard nomenclature for rose LGs, and presents the location of important ornamental traits, such as self-incompatibility, black spot resistance (Rdr1), scent production and recurrent blooming. In total, the consensus map includes locations for 10 phenotypic single loci, QTLs for 7 different traits and 51 ESTs or gene-based molecular markers. This consensus map combines for the first time the information for traits with high relevance for rose variety development. It will serve as a tool for selective breeding and marker assisted selection. It will benefit future efforts of the rose community to sequence the whole rose genome and will be useful for synteny studies in the Rosaceae family and especially in the section Rosoideae.
Asunto(s)
Mapeo Cromosómico/métodos , Diploidia , Rosa/genética , Ligamiento Genético , Genotipo , Fenotipo , Carácter Cuantitativo HeredableRESUMEN
Metabolic profile is a key component of fruit quality, which is a challenge to study due to great compound diversity, especially in species with high nutritional value. This study presents optimized analytical methods for metabolic profiling in the fruits of three Solanaceae species: Lycium barbarum, Lycium chinense and Solanumlycopersicum. It includes the most important chemical classes involved in nutrition and taste, i.e., carotenoids, phenolic compounds and primary compounds. Emphasis has been placed on the systematic achievement of good extraction yields, sample stability, and high response linearity using common LC-ESI-TQ-MS and GC-EI-MS apparatuses. A set of 13 carotenoids, 46 phenolic compounds and 67 primary compounds were profiled in fruit samples. Chemometrics revealed metabolic markers discriminating Lycium and Solanum fruits but also Lycium barbarum and Lycium chinense fruits and the effect of the crop environment. Typical tomato markers were found to be lycopene, carotene, glutamate and GABA, while lycibarbarphenylpropanoids and zeaxanthin esters characterized goji (Lycium spp.) fruits. Among the compounds discriminating the Lycium species, reported here for the first time to our knowledge, chlorogenic acids, asparagine and quinic acid were more abundant in Lycium chinense, whereas Lycium barbarum accumulated more lycibarbarphenylpropanoids A-B, coumaric acid, fructose and glucose.
RESUMEN
Exhaustive studies on flowering control in annual plants have provided a framework for exploring this process in other plant species, especially in perennials for which little molecular data are currently available. Rose is a woody perennial plant with a particular flowering strategy--recurrent blooming, which is controlled by a recessive locus (RB). Gibberellins (GA) inhibit flowering only in non-recurrent roses. Moreover, the GA content varies during the flowering process and between recurrent and non-recurrent rose. Only a few rose genes potentially involved in flowering have been described, i.e. homologues of ABC model genes and floral genes from EST screening. In this study, we gained new information on the molecular basis of rose flowering: date of flowering and recurrent blooming. Based on a candidate gene strategy, we isolated genes that have similarities with genes known to be involved in floral control in Arabidopsis (GA pathway, floral repressors and integrators). Candidate genes were mapped on a segregating population, gene expression was studied in different organs and transcript abundance was monitored in growing shoot apices. Twenty-five genes were studied. RoFT, RoAP1 and RoLFY are proposed to be good floral markers. RoSPY and RB co-localized in our segregating population. GA metabolism genes were found to be regulated during floral transition. Furthermore, GA signalling genes were differentially regulated between a non-recurrent rose and its recurrent mutant. We propose that flowering gene networks are conserved between Arabidopsis and rose. The GA pathway appears to be a key regulator of flowering in rose. We postulate that GA metabolism is involved in floral initiation and GA signalling might be responsible for the recurrent flowering character.
Asunto(s)
Flores/genética , Flores/fisiología , Genes de Plantas , Giberelinas/metabolismo , Rosa/crecimiento & desarrollo , Rosa/genética , Arabidopsis/genética , Mapeo Cromosómico , Recolección de Datos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Familia de Multigenes/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta.
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Monoterpenos/metabolismo , Odorantes , Plastidios/enzimología , Pirofosfatasas/biosíntesis , Rosa/enzimología , Terpenos/metabolismo , Compuestos Orgánicos Volátiles/metabolismo , Monoterpenos Acíclicos , Datos de Secuencia Molecular , Pirofosfatasas/genética , Rosa/genética , Transcriptoma , Hidrolasas NudixRESUMEN
Knowledge of the flowering process - an important trait in ornamental plants such as roses - is necessary for efficient control of flowering. This study was carried out to develop and characterize new resources to gain further insight into floral control in rose. We studied floral initiation in a nonrecurrent blooming rose (hybrid of Rosa wichurana) and a recurrent blooming rose (Rosa hybrida Black Baccara. In Black Baccara, floral initiation took place rapidly after bud burst, whereas in the greenhouse R. wichurana remained vegetative. During floral initiation, the apex enlarged and domed quickly and concomitantly. This is the first description of this transition between the vegetative and floral bud stages in rose. From these vegetative and pre-floral tissues, two cDNA libraries were constructed and 5,000 ESTs sequenced. By collecting our ESTs and those available in public databases, we developed a comprehensive database representing approximately 5,000 unique sequences after clustering. By screening this database for candidate genes involved in the flowering process, we identified 13 genes potentially involved in gibberellic acid signalling, photoperiod pathways, and floral development. Based on expression data, we put forward different hypotheses on the control of flowering in rose (photoperiod control and involvement of gibberellins) relative to what is already known in Arabidopsis.
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Flores/crecimiento & desarrollo , Flores/genética , Rosa/genética , Clonación Molecular , Cruzamientos Genéticos , Etiquetas de Secuencia Expresada , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Genes del Desarrollo , Genes de Plantas , Marcadores Genéticos/fisiología , Filogenia , Rosa/crecimiento & desarrollo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Two Bordetella bronchiseptica mutants, lacking the adenylate cyclase (Cya) or both Cya and pertactin (Prn), were compared with their parental strain NL1013 in their abilities to colonize the nose of neonate piglets and to induce local and systemic antibody responses against filamentous hemagglutinin (FHA) after intranasal (i.n.) inoculation. The number of bacteria recovered and the duration of infection in the nasal secretions were greater for the wild-type parent strain than for the Cya-deficient mutant, indicating that Cya plays an important role during B. bronchiseptica colonization of the nasal cavity. The double mutant did not colonize the nasal cavity and was less able to adhere to epithelial cells in vitro than the other two strains, supporting the hypothesis that Prn plays a major role in cell adhesion. In piglets inoculated with the wild type strain, anti-FHA IgM was found in the nasal secretions one week after inoculation, followed two weeks later by anti-FHA IgA; their presence was concomitant with decreases in bacterial counts. Anti-FHA IgG appeared at six weeks after infection in the serum. In contrast, i.n. inoculation with either mutant failed to induce a nasal secretory antibody response but did induce an earlier and higher IgM response in the serum than inoculation with the wild type strain. However, only the Cya-deficient mutant was able to prime the piglets for the development of a secondary nasal IgM and serum IgG response to FHA after intranasal inoculation with the wild type B. bronchiseptica.