RESUMEN
Firmness, one of the major determinants of postharvest quality and shelf life of fruits is determined by the mechanical resistance imposed by the plant cell wall. Expansins (EXP) are involved in the non-hydrolytic metabolic disassembly of plant cell walls, particularly in processes where relaxation of the wall is necessary, such as fruit development and ripening. As many carbohydrate-associated proteins, expansins have a putative catalytic domain and a carbohydrate-binding module (CBM). Several strategies have been pursued to control the loss of fruit firmness during storage. Most of the approaches have been to suppress the expression of key enzymes involved in the cell wall metabolism, but this is the first time that a CBM was overexpressed in a fruit aimed to control cell wall degradation and fruit softening. We report the constitutive overexpression of the CBM of Solanum lycopersicum expansin 1 (CBM-SlExp1) in the cell wall of tomato plants, and its effects on plant and fruit phenotype. Overexpression of CBM-SlExp1 increased the mechanical resistance of leaves, whereas it did not modify plant growth and general phenotype. However, transgenic plants showed delayed softening and firmer fruits. In addition, fruits were less susceptible to Botrytis cinerea infection, and the "in vitro" growth of the fungus on media containing AIR from the pericarp of transgenic fruits was lower than controls. The possibility of overexpressing a CBM of a fruit-specific expansin to control cell wall degradation and fruit softening is discussed.
Asunto(s)
Botrytis/fisiología , Frutas/metabolismo , Receptores de Superficie Celular/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Metabolismo de los Hidratos de Carbono , Pared Celular/metabolismo , Clorofila/metabolismo , Susceptibilidad a Enfermedades , Frutas/genética , Frutas/crecimiento & desarrollo , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Fenotipo , Desarrollo de la Planta , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Receptores de Superficie Celular/biosíntesisRESUMEN
α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.
Asunto(s)
Fragaria/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Adsorción , Secuencia de Aminoácidos , Bioensayo , Cromatografía de Afinidad , Clonación Molecular , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/aislamiento & purificación , Replegamiento Proteico , Estabilidad Proteica , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , TemperaturaRESUMEN
The accumulation of anthocyanin pigments is one of the most important traits that turn strawberry fruit attractive to consumers. During ripening, strawberry fruit color development is associated to anthocyanin synthesis through the phenylpropanoid pathway. Phenylalanine ammonia-lyase (PAL) is a key enzyme in this pathway, having a determining role in strawberry fruit quality. In this work, we studied the level of anthocyanins during fruit ripening of two cultivars that differ in color development (Camarosa and Toyonoka). Toyonoka showed a lower anthocyanin accumulation that was limited to external fruit tissue, while Camarosa accumulated higher amount of anthocyanins in both internal and external sections. In addition, we cloned a full-length gene (FaPAL6) and analyzed its expression in different strawberry plant tissues. The expression of this gene is fruit specific, and increases during fruit ripening in both cultivars along with anthocyanin accumulation. The mRNA level of FaPAL6 was higher in Camarosa. PAL enzyme activity increased at similar rates in both cultivars at early ripening stages, but at the end of ripening PAL activity diminished in Toyonoka while it rose markedly in Camarosa. PAL activity was higher in internal fruit tissue, showing no correlation with anthocyanin level of the same section in both cultivars. The higher FaPAL6 expression and activity detected in Camarosa could be associated to the enhanced anthocyanin accumulation found in this cultivar.
Asunto(s)
Antocianinas/metabolismo , Fragaria/genética , Fragaria/metabolismo , Fenilanina Amoníaco-Liasa/metabolismo , Secuencia de Aminoácidos , Antocianinas/genética , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Fragaria/enzimología , Fragaria/crecimiento & desarrollo , Frutas/enzimología , Frutas/genética , Frutas/crecimiento & desarrollo , Frutas/metabolismo , Datos de Secuencia Molecular , Fenilanina Amoníaco-Liasa/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Fleshy fruit soften during ripening mainly as a consequence of solubilization and depolymerization of cell wall components. We have performed a comparative study of the polysaccharide content of fruit cell walls during final steps of development and ripening of three strawberry (Fragaria x ananassa Duch.) cultivars with different softening rates. The three chosen varieties showed very different firmness; Camarosa was the firmest, Toyonaka the softest, and Pajaro intermediate between them. Cell walls were extracted, quantified and fractioned by sequential extraction to obtain particular subclasses of cell wall polymers. Cell wall content diminished during the process in the three cultivars. Differences among cultivar cell wall contents were detected only in immature stages. The amount of water soluble polymers (WSP) increased in all cultivars from small green (SG) to white (W) stage, although from the W to 100% red (100%R) stage the WSP remained constant in Camarosa and Pajaro and decreased in Toyonaka. On the contrary, the hydrochloric acid-soluble pectins (HSP) decreased during ripening of all the cultivars analyzed. Camarosa had the largest amount of HSP, but there were no differences between Pajaro and Toyonaka. The amount of hemicellulosic polysaccharides and cellulose also decreased in the three cultivars. Camarosa had the highest amounts of both polysaccharides while Toyonaka had the lowest at immature stages, but there were no differences among cultivars at 100%R stage. WSP showed depolymerization only in Toyonaka cultivar, while HSP showed depolymerization in Pajaro and Toyonaka cultivars. A slight depolymerization was observed in hemicelluloses extracted from any of three cultivars.
Asunto(s)
Pared Celular/metabolismo , Fragaria/metabolismo , Pectinas/metabolismo , Celulosa/metabolismo , Cromatografía en Gel , Fragaria/fisiología , Ácido Clorhídrico , Peso Molecular , Pectinas/química , Polisacáridos/metabolismo , Solubilidad , AguaRESUMEN
Peroxidase (POX) from strawberry fruits was analyzed for its capacity to bleach chlorophyll. The partially purified enzyme preperation catalyzed the bleaching of chlorophylls and their derivatives in the presence of H(2)O(2) and phenolic compounds. The optimal reaction conditions were 35 degrees C, pH 5.2 and ionic strength equal to 0.2. The maximum activity was observed at 1 mM of H(2)O(2), while higher concentrations inhibited enzyme activity. Compounds with a high affinity to the heme group, radical scavengers and reducing agents, showed an inhibitory effect. Phenolic compounds such as umbelliferone, naringenin and p-substituted monophenols acted as cofactors. Instead, other phenolic compounds tested such as caffeic acid, catechin, ellagic acid, esculin and quercetin inhibited the activity of POX on chlorophylls. Phenolic compounds extracted from strawberry fruits showed an inhibitory effect on POX-chlorophyll bleaching activity, although this effect decreased markedly during ripening. POX showed higher affinity for compounds derived from chlorophyll a than from chlorophyll b, and the enzyme preferentially degraded chlorophyll derivatives with the Mg(2+) ion present and the phytol group removed. The POX-chlorophyll bleaching activity was found in all ripening stages from small green to ripe, the highest activity corresponding to large green fruits.
Asunto(s)
Clorofila/metabolismo , Peroxidasas/metabolismo , Rosaceae/enzimología , Concentración de Iones de Hidrógeno , Concentración Osmolar , Fenoles/metabolismo , Especificidad por SustratoRESUMEN
Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.