RESUMEN
Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes.
Asunto(s)
Etilenos/biosíntesis , Frutas/crecimiento & desarrollo , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Frutas/anatomía & histología , Solanum lycopersicum/anatomía & histología , Solanum lycopersicum/genética , Fenotipo , Proteínas de Plantas/genética , Plantas Modificadas GenéticamenteRESUMEN
The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Sitios de Unión , Inmunoprecipitación de Cromatina , Flavonoides/metabolismo , Perfilación de la Expresión Génica , Solanum lycopersicum/metabolismo , Proteínas de Dominio MADS/metabolismo , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ARNRESUMEN
The contamination of processed vegetable foods with genetically modified tomatoes was investigated by the use of qualitative PCR methods to detect the cauliflower mosaic virus 35S promoter (P35S) and the kanamycin resistance gene (NPTII). DNA fragments of P35S and NPTII were detected in vegetable juice samples, possibly due to contamination with the genomes of cauliflower mosaic virus infecting juice ingredients of Brassica species and soil bacteria, respectively. Therefore, to detect the transformation construct sequences of GM tomatoes, primer pairs were designed for qualitative PCR to specifically detect the border region between P35S and NPTII, and the border region between nopaline synthase gene promoter and NPTII. No amplification of the targeted sequences was observed using genomic DNA purified from the juice ingredients. The developed qualitative PCR method is considered to be a reliable tool to check contamination of products with GM tomatoes.
Asunto(s)
Secuencia de Bases/genética , ADN de Plantas/análisis , ADN de Plantas/genética , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Alimentos Modificados Genéticamente , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa/métodos , Solanum lycopersicum/genética , Caulimovirus/genética , Fragmentación del ADN , ADN Viral/análisis , Resistencia a la Kanamicina/genéticaRESUMEN
Tomato (Solanum lycopersicum) fruit cuticle has been extensively studied due to its effect on the biochemical and physiological properties of the fruit. To date, several tomato mutants defective in proper cuticle formation have been identified. To gain insight into tomato cuticle formation, we investigated one such mutant, sticky peel/light green (pe lg). We verified the responsible gene by fine mapping and obtained the same conclusion as a previous report. To elucidate the pleiotropic effects of cuticle deficiency caused by the cd2 mutation, CD2 suppression lines were constructed. As found in the pe lg mutant, the suppression lines showed enhanced water permeability and aberrant leaf and fruit cuticles. Water use efficiency of the suppression line was lower than that of the wild type. However, photosynthetic ability was not affected in the suppression line. Since these phenotypes are related to altered deposition of wax and cutin, other lipidic metabolites might be changed, too. To confirm this hypothesis, we conducted metabolite profiling. The metabolite profiling revealed that not only lipid but also sugar, flavonoid and glycoalkaloid metabolites in fruit were changed in the cd2 mutant. These results indicate that CD2 is essential both for normal cutin and wax deposition and for proper accumulation of specific metabolites in tomato fruit.
Asunto(s)
Frutas/genética , Mutación , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Alcaloides/metabolismo , Metabolismo de los Hidratos de Carbono/genética , Flavonoides/metabolismo , Frutas/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Regulación de la Expresión Génica de las Plantas , Metabolismo de los Lípidos/genética , Lípidos/análisis , Lípidos/química , Solanum lycopersicum/metabolismo , Lípidos de la Membrana/metabolismo , Metabolómica/métodos , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Agua/metabolismo , Ceras/metabolismoRESUMEN
The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.
Asunto(s)
Frutas/metabolismo , Proteínas de Dominio MADS/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Proteínas de Dominio MADS/genética , Proteínas de Plantas/genéticaRESUMEN
Abscission in plants is a crucial process used to shed organs such as leaves, flowers, and fruits when they are senescent, damaged, or mature. Abscission occurs at predetermined positions called abscission zones (AZs). Although the regulation of fruit abscission is essential for agriculture, the developmental mechanisms remain unclear. Here, we describe a novel transcription factor regulating the development of tomato (Solanum lycopersicum) pedicel AZs. We found that the development of tomato pedicel AZs requires the gene MACROCALYX (MC), which was previously identified as a sepal size regulator and encodes a MADS-box transcription factor. MC has significant sequence similarity to Arabidopsis (Arabidopsis thaliana) FRUITFULL, which is involved in the regulation of fruit dehiscent zone development. The MC protein interacted physically with another MADS-box protein, JOINTLESS, which is known as a regulator of fruit abscission; the resulting heterodimer acquired a specific DNA-binding activity. Transcriptome analyses of pedicels at the preabscission stage revealed that the expression of the genes involved in phytohormone-related functions, cell wall modifications, fatty acid metabolism, and transcription factors is regulated by MC and JOINTLESS. The regulated genes include homologs of Arabidopsis WUSCHEL, REGULATOR OF AXILLARY MERISTEMS, CUP-SHAPED COTYLEDON, and LATERAL SUPPRESSOR. These Arabidopsis genes encode well-characterized transcription factors regulating meristem maintenance, axillary meristem development, and boundary formation in plant tissues. The tomato homologs were specifically expressed in AZs but not in other pedicel tissues, suggesting that these transcription factors may play key roles in pedicel AZ development.
Asunto(s)
Frutas/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/metabolismo , Proteínas de Plantas/genética , Solanum lycopersicum/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Homeodominio/genética , Proteínas de Dominio MADS/genética , Proteínas de Plantas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
The RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, and its ripening inhibitor (rin) mutation yields non-ripening fruit. In this study, the molecular properties of RIN and the rin mutant protein were clarified. The results revealed that the RIN protein accumulates in ripening fruit specifically and is localized in the nucleus of the cell. In vitro studies revealed that RIN forms a stable homodimer that binds to MADS domain-specific DNA sites. Analysis of binding site selection experiments revealed that the consensus binding sites of RIN highly resemble those of the SEPALLATA (SEP) proteins, which are Arabidopsis MADS box proteins that control the identity of floral organs. RIN exhibited a transcription-activating function similar to that exhibited by the SEP proteins. These results indicate that RIN exhibits similar molecular functions to SEP proteins although they play distinctly different biological roles. In vivo assays revealed that RIN binds to the cis-element of LeACS2. Our results also revealed that the rin mutant protein accumulates in the mutant fruit and exhibits a DNA-binding activity similar to that exhibited by the wild-type protein, but has lost its transcription-activating function, which in turn would inhibit ripening in mutant fruit.
Asunto(s)
ADN de Plantas/metabolismo , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Transcripción Genética/fisiología , Transporte Activo de Núcleo Celular/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Frutas/citología , Solanum lycopersicum/citología , Datos de Secuencia Molecular , Mutación , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
We have previously shown a significant decrease in the ethylene production in tomato fruit from the RIN/rin genotype. In this present study, we evaluated the amount of 1-aminocyclopropane-1-carboxylic acid (ACC) and the gene expression and enzymatic activities of ACC synthase (ACS) and ACC oxidase (ACO) to find which type of regulation influenced this low ethylene production. The results suggest that the decreased ethylene production was due to transcriptional regulation of the ACS and ACO genes by the heterozygous effect of the rin gene.
Asunto(s)
Etilenos/biosíntesis , Frutas/metabolismo , Solanum lycopersicum/metabolismo , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos Cíclicos/metabolismo , Quimera , Frutas/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Liasas/genética , Liasas/metabolismo , Solanum lycopersicum/genética , MutaciónRESUMEN
The ripening inhibitor (rin) mutant tomato yields non-ripening fruit, and the rin hybrid fruit (RIN/rin) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the rin hybrid fruit than in the wild-type fruit. These allergenic proteins, which were beta-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the rin hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the rin hybrid fruit than to that of the wild fruit. These results suggest that the rin gene has the potential to regulate allergen accumulation in tomato fruit.
Asunto(s)
Antígenos de Plantas/genética , Antígenos de Plantas/inmunología , Hipersensibilidad a los Alimentos/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Solanum lycopersicum/efectos adversos , Quimera/genética , Expresión Génica , Genes de Plantas , Humanos , Inmunoglobulina E/sangre , Solanum lycopersicum/genética , Solanum lycopersicum/inmunología , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Poligalacturonasa/genética , Poligalacturonasa/inmunología , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/inmunologíaRESUMEN
Arabinogalactan proteins (AGPs) are plant proteoglycans that have been implicated in plant growth and development. The possible involvement of AGPs in the action of gibberellin (GA), a class of plant hormones, was examined by applying beta-glucosyl Yariv reagent (beta-Glc)3Y, a synthetic phenyl glycoside that interacts selectively with AGPs, to barley aleurone protoplasts. Gibberellin induces transcription and secretion of alpha-amylases in the protoplasts. Induction of alpha-amylase was clearly inhibited by (beta-Glc)3Y but not by (alpha-Gal)3Y, a negative control of the Yariv reagent that does not interact with AGPs. Transfection analysis, using an alpha-amylase promoter-GUS fusion gene in the protoplasts, indicated that the transcriptional activation of the alpha-amylase promoter was inhibited specifically by (beta-Glc)3Y. These observations are the first indication of an involvement of AGPs in a plant hormone function. The inhibitory effect of (beta-Glc)3Y was not observed when aleurone layers or half-seed grains were used. This result, together with the fact that protoplasts do not have cell walls, suggests that the AGPs that function in alpha-amylase induction reside at the plasma membrane. An aleurone-specific AGP was detected by reversed-phase HPLC, and supported the idea that an AGP may play an important role in aleurone-specific events. The possible mechanism of AGP function in gibberellin-induced alpha-amylase production is discussed.