RESUMEN
Plant rhamnogalacturonan lyases (RGLyases) cleave the backbone of rhamnogalacturonan I (RGI), the "hairy" pectin and polymer of the disaccharide rhamnose (Rha)-galacturonic acid (GalA) with arabinan, galactan or arabinogalactan side chains. It has been suggested that RGLyases could participate in remodeling cell walls during fruit softening, but clear evidence has not been reported. To investigate the role of RGLyases in strawberry softening, a genome-wide analysis of RGLyase genes in the genus Fragaria was performed. Seventeen genes encoding RGLyases with functional domains were identified in Fragaria × ananassa. FaRGLyase1 was the most expressed in the ripe receptacle of cv. Chandler. Transgenic strawberry plants expressing an RNAi sequence of FaRGLyase1 were obtained. Three transgenic lines yielded ripe fruits firmer than controls without other fruit quality parameters being significantly affected. The highest increase in firmness achieved was close to 32%. Cell walls were isolated from ripe fruits of two selected lines. The amount of water-soluble and chelated pectins was higher in transgenic lines than in the control. A carbohydrate microarray study showed a higher abundance of RGI epitopes in pectin fractions and in the cellulose-enriched fraction obtained from transgenic lines. Sixty-seven genes were differentially expressed in transgenic ripe fruits when compared with controls. These genes were involved in various physiological processes, including cell wall remodeling, ion homeostasis, lipid metabolism, protein degradation, stress response, and defense. The transcriptomic changes observed in FaRGLyase1 plants suggest that senescence was delayed in transgenic fruits.
Asunto(s)
Fragaria , Fragaria/metabolismo , Frutas/genética , Frutas/metabolismo , Ramnogalacturonanos/metabolismo , Pectinas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Pared Celular/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Firmness is one of the most important fruit quality traits in strawberries. The postharvest shelf life of this soft fruit is highly limited by the loss of firmness, where cell wall disassembly plays an important role. Previous studies demonstrated that the polygalacturonase FaPG1 has a key role in remodelling pectins during strawberry softening. In this study, FaPG1 knockout strawberry plants have been generated using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Ten independent lines, cv. "Chandler", were obtained, and all of them were successfully edited as determined by PCR amplification and T7 endonuclease assay. The targeted mutagenesis insertion and deletion rates were analyzed using targeted deep sequencing. The percentage of edited sequences varied from 47% up to almost 100%, being higher than 95% for seven of the selected lines. Phenotypic analyses showed that 7 out of the eight lines analyzed produced fruits significantly firmer than the control, ranging from 33 to 70% increase in firmness. There was a positive relationship between the degree of FaPG1 editing and the rise in fruit firmness. Minor changes were observed in other fruit quality traits, such as colour, soluble solids, titratable acidity or anthocyanin content. Edited fruits showed a reduced softening rate during postharvest, displayed a reduced transpirational water loss, and were less damaged by Botrytis cinerea inoculation. The analysis of four potential off-target sites revealed no mutation events. In conclusion, editing the FaPG1 gene using the CRISPR/Cas9 system is an efficient method for improving strawberry fruit firmness and shelf life.
RESUMEN
In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.
Asunto(s)
Fragaria , Proantocianidinas , Antocianinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Flavonoides/metabolismo , Proantocianidinas/metabolismo , Flavonoles/metabolismo , Frutas/genética , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fragaria/genética , Fragaria/metabolismoRESUMEN
Volatile compounds produced during ripening of strawberry are key determinants of fruit quality and consumer preference. Strawberry volatiles are largely esters which are synthesized by alcohol acyltransferases (AATs) and degraded by carboxylesterases (CXEs). Although CXE activity can have a marked influence on volatile contents in ripe strawberry fruits, CXE function and regulation in them are poorly known. Here, we report the biochemical and functional characterization of the fruit receptacle-specific and ripening-related carboxylesterase FanCXE1. The expression of the corresponding gene was found to be antagonistically regulated by auxins and abscisic acid, key hormones that regulate fruit growth and ripening in strawberry. In vitro, FanCXE1 was able to hydrolyze artificial ester substrates similar to those produced by ripe strawberry fruits. Transient suppression of the FanCXE1 gene by RNAi resulted in an increase of important volatile esters such as methyl hexanoate, methyl butanoate and ethyl hexanoate as well as a decrease of the alcohols hexenol and linanool. The results of this work enhance our understanding of the molecular basis for volatile syntheses and facilitate production of better flavored strawberry fruits by introduction of the relevant alleles into common cultivars.
RESUMEN
BACKGROUND: Strawberry ripening involves a number of irreversible biochemical reactions that cause sensory changes through accumulation of sugars, acids and other compounds responsible for fruit color and flavor. The process, which is strongly dependent on methylation marks in other fruits such as tomatoes and oranges, is highly controlled and coordinated in strawberry. RESULTS: Repeated injections of the hypomethylating compound 5-azacytidine (AZA) into green and unripe Fragaria × ananassa receptacles fully arrested the ripening of the fruit. The process, however, was reversible since treated fruit parts reached full maturity within a few days after AZA treatment was stopped. Transcriptomic analyses showed that key genes responsible for the biosynthesis of anthocyanins, phenylpropanoids, and hormones such as abscisic acid (ABA) were affected by the AZA treatment. In fact, AZA downregulated genes associated with ABA biosynthetic genes but upregulated genes associated with its degradation. AZA treatment additionally downregulated a number of essential transcription factors associated with the regulation and control of ripening. Metabolic analyses revealed a marked imbalance in hormone levels, with treated parts accumulating auxins, gibberellins and ABA degradation products, as well as metabolites associated with unripe fruits. CONCLUSIONS: AZA completely halted strawberry ripening by altering the hormone balance, and the expression of genes involves in hormone biosynthesis and degradation processes. These results contradict those previously obtained in other climacteric and fleshly fruits, where AZA led to premature ripening. In any case, our results suggests that the strawberry ripening process is governed by methylation marks.
Asunto(s)
Fragaria , Ácido Abscísico/metabolismo , Antocianinas/metabolismo , Azacitidina/farmacología , Fragaria/metabolismo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Hormonas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Under climate change, the spread of pests and pathogens into new environments has a dramatic effect on crop protection control. Strawberry (Fragaria spp.) is one the most profitable crops of the Rosaceae family worldwide, but more than 50 different genera of pathogens affect this species. Therefore, accelerating the improvement of fruit quality and pathogen resistance in strawberry represents an important objective for breeding and reducing the usage of pesticides. New genome sequencing data and bioinformatics tools has provided important resources to expand the use of synthetic biology-assisted intragenesis strategies as a powerful tool to accelerate genetic gains in strawberry. In this paper, we took advantage of these innovative approaches to create four RNAi intragenic silencing cassettes by combining specific strawberry new promoters and pathogen defense-related candidate DNA sequences to increase strawberry fruit quality and resistance by silencing their corresponding endogenous genes, mainly during fruit ripening stages, thus avoiding any unwanted effect on plant growth and development. Using a fruit transient assay, GUS expression was detected by the two synthetic FvAAT2 and FvDOF2 promoters, both by histochemical assay and qPCR analysis of GUS transcript levels, thus ensuring the ability of the same to drive the expression of the silencing cassettes in this strawberry tissue. The approaches described here represent valuable new tools for the rapid development of improved strawberry lines.
RESUMEN
To disentangle the role of polygalacturonase (PG) genes in strawberry softening, the two PG genes most expressed in ripe receptacles, FaPG1 and FaPG2, were down-regulated. Transgenic ripe fruits were firmer than those of the wild type when PG genes were silenced individually. Simultaneous silencing of both PG genes by transgene stacking did not result in an additional increase in firmness. Cell walls from ripe fruits were characterized by a carbohydrate microarray. Higher signals of homogalacturonan and rhamnogalacturonan I pectin epitopes in polysaccharide fractions tightly bound to the cell wall were observed in the transgenic genotypes, suggesting a lower pectin solubilization. At the transcriptomic level, the suppression of FaPG1 or FaPG2 alone induced few transcriptomic changes in the ripe receptacle, but the amount of differentially expressed genes increased notably when both genes were silenced. Many genes encoding cell wall-modifying enzymes were down-regulated. The expression of a putative high affinity potassium transporter was induced in all transgenic genotypes, indicating that cell wall weakening and loss of cell turgor could be linked. These results suggest that, besides the disassembly of pectins tightly linked to the cell wall, PGs could play other roles in strawberry softening, such as the release of oligogalacturonides exerting a positive feedback in softening.
Asunto(s)
Fragaria , Pared Celular/metabolismo , Fragaria/genética , Fragaria/metabolismo , Frutas/genética , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Pectinas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Poligalacturonasa/genética , Poligalacturonasa/metabolismoRESUMEN
BACKGROUND: In soft fruits, the differential expression of many genes during development and ripening is responsible for changing their organoleptic properties. In strawberry fruit, although some genes involved in the metabolic regulation of the ripening process have been functionally characterized, some of the most studied genes correspond to transcription factors. High throughput transcriptomics analyses performed in strawberry red receptacle (Fragaria x ananassa) allowed us to identify a ripening-related gene that codes an atypical HLH (FaPRE1) with high sequence homology with the PACLOBUTRAZOL RESISTANCE (PRE) genes. PRE genes are atypical bHLH proteins characterized by the lack of a DNA-binding domain and whose function has been linked to the regulation of cell elongation processes. RESULTS: FaPRE1 sequence analysis indicates that this gene belongs to the subfamily of atypical bHLHs that also includes ILI-1 from rice, SlPRE2 from tomato and AtPRE1 from Arabidopsis, which are involved in transcriptional regulatory processes as repressors, through the blockage by heterodimerization of bHLH transcription factors. FaPRE1 presented a transcriptional model characteristic of a ripening-related gene with receptacle-specific expression, being repressed by auxins and activated by abscisic acid (ABA). However, its expression was not affected by gibberellic acid (GA3). On the other hand, the transitory silencing of FaPRE1 transcription by agroinfiltration in receptacle produced the down-regulation of a group of genes related to the ripening process while inducing the transcription of genes involved in receptacle growth and development. CONCLUSIONS: In summary, this work presents for the first time experimental data that support an important novel function for the atypical HLH FaPRE1 during the strawberry fruit ripening. We hypothesize that FaPRE1 modulates antagonistically the transcription of genes related to both receptacle growth and ripening. Thus, FaPRE1 would repress the expression of receptacle growth promoting genes in the ripened receptacle, while it would activate the expression of those genes related to the receptacle ripening process.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Fragaria/fisiología , Proteínas de Plantas/fisiología , Factores de Transcripción/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Fragaria/efectos de los fármacos , Fragaria/genética , Fragaria/crecimiento & desarrollo , Frutas/genética , Frutas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Desarrollo de la Planta/genética , Reguladores del Crecimiento de las Plantas/fisiología , Proteínas de Plantas/genética , Factores de Transcripción/genética , Triazoles/farmacologíaRESUMEN
NAC proteins are a family of transcription factors which have a variety of important regulatory roles in plants. They present a very well conserved group of NAC subdomains in the N-terminal region and a highly variable domain at the C-terminus. Currently, knowledge concerning NAC family in the strawberry plant remains very limited. In this work, we analyzed the NAC family of Fragaria vesca, and a total of 112 NAC proteins were identified after we curated the annotations from the version 4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes) and described its physicochemical and genetic features. A microarray transcriptomic analysis showed six of them expressed during the development and ripening of the Fragaria x ananassa fruit. Their expression patterns were studied in fruit (receptacle and achenes) in different stages of development and in vegetative tissues. Also, the expression level under different hormonal treatments (auxins, ABA) and drought stress was investigated. In addition, they were clustered with other NAC transcription factor with known function related to growth and development, senescence, fruit ripening, stress response, and secondary cell wall and vascular development. Our results indicate that these six strawberry NAC proteins could play different important regulatory roles in the process of development and ripening of the fruit, providing the basis for further functional studies and the selection for NAC candidates suitable for biotechnological applications.
Asunto(s)
Fragaria/genética , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Proteínas de Plantas/genética , Factores de Transcripción/genética , Transcriptoma , Ácido Abscísico/farmacología , Mapeo Cromosómico , Sequías , Fragaria/efectos de los fármacos , Fragaria/crecimiento & desarrollo , Fragaria/metabolismo , Frutas/efectos de los fármacos , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Ácidos Indolacéticos/farmacología , Anotación de Secuencia Molecular , Familia de Multigenes , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismoRESUMEN
Only a few transcription factors have been described in the regulation of the strawberry (Fragaria x ananassa) fruit ripening process. Using a transcriptomic approach, we identified and functionally characterized FaDOF2, a DOF-type ripening-related transcription factor, which is hormonally regulated and specific to the receptacle, though high expression levels were also found in petals. The expression pattern of FaDOF2 correlated with eugenol content, a phenylpropanoid volatile, in both fruit receptacles and petals. When FaDOF2 expression was silenced in ripe strawberry receptacles, the expression of FaEOBII and FaEGS2, two key genes involved in eugenol production, were down-regulated. These fruits showed a concomitant decrease in eugenol content, which confirmed that FaDOF2 is a transcription factor that is involved in eugenol production in ripe fruit receptacles. By using the yeast two-hybrid system and bimolecular fluorescence complementation, we demonstrated that FaDOF2 interacts with FaEOBII, a previously reported regulator of eugenol production, which determines fine-tuning of the expression of key genes that are involved in eugenol production. These results provide evidence that FaDOF2 plays a subsidiary regulatory role with FaEOBII in the expression of genes encoding enzymes that control eugenol production. Taken together, our results provide new insights into the regulation of the volatile phenylpropanoid pathway in ripe strawberry receptacles.
Asunto(s)
Eugenol/metabolismo , Fragaria/metabolismo , Frutas/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Fragaria/genética , Fragaria/crecimiento & desarrollo , Frutas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Secuenciación de Nucleótidos de Alto Rendimiento , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Interferencia de ARN , Factores de Transcripción/genéticaRESUMEN
Strawberry is an ideal model for studying the molecular biology of the development and ripening of non-climacteric fruits. Hormonal regulation of gene expression along all these processes in strawberries is still to be fully elucidated. Although auxins and ABA have been pointed out as the major regulatory hormones, few high-throughput analyses have been carried out to date. The role for ethylene and gibberellins as regulatory hormones during the development and ripening of the strawberry fruit remain still elusive. By using a custom-made and high-quality oligo microarray platform done with over 32,000 probes including all of the genes actually described in the strawberry genome, we have analysed the expression of genes during the development and ripening in the receptacles of these fruits. We classify these genes into two major groups depending upon their temporal and developmental expression. First group are genes induced during the initial development stages. The second group encompasses genes induced during the final maturation and ripening processes. Each of these two groups has been also divided into four sub-groups according their pattern of hormonal regulation. By analyzing gene expression, we clearly show that auxins and ABA are the main and key hormones that combined or independently are responsible of the development and ripening process. Auxins are responsible for the receptacle fruit development and, at the same time¸ prevent ripening by repressing crucial genes. ABA regulates the expression of the vast majority of genes involved in the ripening. The main genes expressed under the control of these hormones are presented and their physiological rule discussed. We also conclude that ethylene and gibberellins do not seem to play a prominent role during these processes.
Asunto(s)
Fragaria/genética , Frutas/genética , Proteínas de Plantas/biosíntesis , Transcriptoma/genética , Ácido Abscísico/farmacología , Etilenos/farmacología , Fragaria/efectos de los fármacos , Fragaria/crecimiento & desarrollo , Frutas/efectos de los fármacos , Frutas/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/genética , Transcriptoma/efectos de los fármacosRESUMEN
Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.
RESUMEN
Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by ß-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative ß-galactosidase gene, FaßGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaßGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaßGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaßGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaßGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaßGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.
Asunto(s)
Pared Celular/metabolismo , Regulación hacia Abajo , Fragaria/enzimología , Fragaria/genética , Frutas/enzimología , Galactosa/metabolismo , ARN sin Sentido/metabolismo , beta-Galactosidasa/genética , Carbohidratos/análisis , Pared Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Fragaria/efectos de los fármacos , Frutas/efectos de los fármacos , Frutas/genética , Frutas/crecimiento & desarrollo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Fenotipo , Filogenia , Reguladores del Crecimiento de las Plantas/farmacología , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Ácidos Urónicos/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Strawberry fruit (Fragaria × ananassa) is a soft fruit with high water content at ripe stage (more than 90% of its fresh weight). Aquaporins play an important role in plant water homeostasis, through the facilitation of water transport and solutes. We report the role played by FaNIP1;1 in the receptacle ripening process. The analysis by qRT-PCR of FaNIP1;1 showed that this gene is mainly expressed in fruit receptacle and has a ripening-related expression pattern that was accompanied by an increase in both the abscisic acid and water content of the receptacle throughout fruit ripening. Moreover, FaNIP1;1 was induced in situations of water deficit. Additionally, we show that FaNIP1;1 expression was positively regulated by abscisic acid and negatively regulated by auxins. The water transport capacity of FaNIP1;1 was determined by a stopped-flow spectroscopy in yeast over-expressing FaNIP1;1. Glycerol, H2O2 and boron transport were also demonstrated in yeast. On the other hand, GFP-FaNIP1;1 fusion protein was located in plasma membrane. In conclusion, FaNIP1;1 seems to play an important role increasing the plasma membrane permeability, that allows the water accumulation in the strawberry fruit receptacle throughout the ripening process.
Asunto(s)
Fragaria/crecimiento & desarrollo , Fragaria/genética , Frutas/crecimiento & desarrollo , Frutas/genética , Genes de Plantas , Proteínas de Plantas/genética , Ácido Abscísico/farmacología , Acuaporinas/química , Acuaporinas/genética , Acuaporinas/metabolismo , Transporte Biológico/efectos de los fármacos , Boro/metabolismo , Fragaria/efectos de los fármacos , Frutas/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glicerol/metabolismo , Peróxido de Hidrógeno/metabolismo , Ácidos Indolacéticos/farmacología , Manitol/farmacología , Presión Osmótica/efectos de los fármacos , Permeabilidad , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinación Genética/genética , Saccharomyces cerevisiae/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Agua/metabolismoRESUMEN
Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. In ripe strawberry (Fragaria × ananassa) fruit receptacles, eugenol is biosynthesized by eugenol synthase (FaEGS2). However, the transcriptional regulation of this process is still unknown. We have identified and functionally characterized an R2R3 MYB transcription factor (emission of benzenoid II [FaEOBII]) that seems to be the orthologous gene of PhEOBII from Petunia hybrida, which contributes to the regulation of eugenol biosynthesis in petals. The expression of FaEOBII was ripening related and fruit receptacle specific, although high expression values were also found in petals. This expression pattern of FaEOBII correlated with eugenol content in both fruit receptacle and petals. The expression of FaEOBII was repressed by auxins and activated by abscisic acid, in parallel to the ripening process. In ripe strawberry receptacles, where the expression of FaEOBII was silenced, the expression of cinnamyl alcohol dehydrogenase1 and FaEGS2, two structural genes involved in eugenol production, was down-regulated. A subsequent decrease in eugenol content in ripe receptacles was also observed, confirming the involvement of FaEOBII in eugenol metabolism. Additionally, the expression of FaEOBII was under the control of FaMYB10, another R2R3 MYB transcription factor that regulates the early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway. In parallel, the amount of eugenol in FaMYB10-silenced receptacles was also diminished. Taken together, these data indicate that FaEOBII plays a regulating role in the volatile phenylpropanoid pathway gene expression that gives rise to eugenol production in ripe strawberry receptacles.
Asunto(s)
Eugenol/metabolismo , Fragaria/metabolismo , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Flores/genética , Fragaria/efectos de los fármacos , Fragaria/genética , Frutas/efectos de los fármacos , Frutas/genética , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Genes de Plantas , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Vegetales/efectos de los fármacos , Células Vegetales/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Estirenos/metabolismo , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
This work characterized the role of the R2R3-MYB10 transcription factor (TF) in strawberry fruit ripening. The expression of this TF takes place mainly in the fruit receptacle and is repressed by auxins and activated by abscisic acid (ABA), in parallel to the ripening process. Anthocyanin was not produced when FaMYB10 expression was transiently silenced in fruit receptacles. An increase in FaMYB10 expression was observed in water-stressed fruits, which was accompanied by an increase in both ABA and anthocyanin content. High-throughput transcriptomic analyses performed in fruits with downregulated FaMYB10 expression indicated that this TF regulates the expression of most of the Early-regulated Biosynthesis Genes (EBGs) and the Late-regulated Biosynthesis Genes (LBGs) genes involved in anthocyanin production in ripened fruit receptacles. Besides, the expression of FaMYB10 was not regulated by FaMYB1 and vice versa. Taken together, all these data clearly indicate that the Fragaria × ananassa MYB10 TF plays a general regulatory role in the flavonoid/phenylpropanoid pathway during the ripening of strawberry.
Asunto(s)
Cruzamientos Genéticos , Flavonoides/metabolismo , Fragaria/metabolismo , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Propanoles/metabolismo , Ácido Abscísico/farmacología , Antocianinas/metabolismo , Fragaria/efectos de los fármacos , Fragaria/genética , Fragaria/crecimiento & desarrollo , Frutas/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Silenciador del Gen , Ácidos Indolacéticos/farmacología , Metabolómica , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de Transcripción/metabolismoRESUMEN
Late blight caused by the plant pathogenic oomycete Phytophthora infestans is known as one of the most destructive potato diseases. Plant breeders tend to employ NB-LRR-based resistance for introducing genetically controlled late blight resistance in their breeding lines. However, P. infestans is able to rapidly escape this type of resistance, and hence, NB-LRR-based resistance in potato cultivars is often not durable. Previously, we identified a novel type of Phytophthora resistance in Arabidopsis. This resistance is mediated by the cell surface receptor LecRK-I.9, which belongs to the family of L-type lectin receptor kinases. In this study, we report that expression of the Arabidopsis LecRK-I.9 gene in potato and Nicotiana benthamiana results in significantly enhanced late blight resistance. Transcriptional profiling showed strong reduction in salicylic acid (SA)-mediated defence gene expression in LecRK-I.9 transgenic potato lines (TPLs). In contrast, transcripts of two protease inhibitor genes accumulated to extreme high levels, suggesting that LecRK-I.9-mediated late blight resistance is relying on a defence response that includes activation of protease inhibitors. These results demonstrate that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana and suggest that this novel type of LecRK-based resistance can be exploited in breeding strategies to improve durable late blight resistance in Solanaceous crops.
Asunto(s)
Arabidopsis/metabolismo , Phytophthora infestans/patogenicidad , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Solanum tuberosum/metabolismo , Solanum tuberosum/parasitología , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/prevención & control , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/microbiología , Solanum tuberosum/genéticaRESUMEN
Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate's suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1α and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.
Asunto(s)
Fragaria/genética , Fragaria/metabolismo , Fragaria/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell-cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.
Asunto(s)
Fragaria/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Poligalacturonasa/genética , Pared Celular/genética , Pared Celular/metabolismo , Pared Celular/ultraestructura , Cromatografía en Gel , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida , Fragaria/metabolismo , Fragaria/ultraestructura , Frutas/genética , Frutas/metabolismo , Frutas/ultraestructura , Silenciador del Gen , Microscopía Electrónica de Rastreo , Pectinas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/ultraestructura , Poligalacturonasa/metabolismoRESUMEN
Plant phenolics have drawn increasing attention due to their potential nutritional benefits. Although the basic reactions of the phenolics biosynthetic pathways in plants have been intensively analyzed, the regulation of their accumulation and flux through the pathway is not that well established. The aim of this study was to use a strawberry (Fragaria × ananassa) microarray to investigate gene expression patterns associated with the accumulation of phenylpropanoids, flavonoids, and anthocyanins in strawberry fruit. An examination of the transcriptome, coupled with metabolite profiling data from different commercial varieties, was undertaken to identify genes whose expression correlated with altered phenolics composition. Seventeen comparative microarray analyses revealed 15 genes that were differentially (more than 200-fold) expressed in phenolics-rich versus phenolics-poor varieties. The results were validated by heterologous expression of the peroxidase FaPRX27 gene, which showed the highest altered expression level (more than 900-fold). The encoded protein was functionally characterized and is assumed to be involved in lignin formation during strawberry fruit ripening. Quantitative trait locus analysis indicated that the genomic region of FaPRX27 is associated with the fruit color trait. Down-regulation of the CHALCONE SYNTHASE gene and concomitant induction of FaPRX27 expression diverted the flux from anthocyanins to lignin. The results highlight the competition of the different phenolics pathways for their common precursors. The list of the 15 candidates provides new genes that are likely to impact polyphenol accumulation in strawberry fruit and could be used to develop molecular markers to select phenolics-rich germplasm.