Molecular Control of Fruit Ripening and Sensory Quality of Charentais Melon

Traditional Charentais melons have a typical climacteric behavior with ethylene playing a major role in the regulation of the ripening process. Genetic studies using climacteric and non-climacteric types of Cucumis melo demonstrated that the climacteric character is dominant and conferred by 2 duplicated loci only which are of great importance for the regulation of storability and sensory quality. Commercial varieties of Charentais melon with long shelf-life have been generated, some of them by crossing with a non-ripening Charentais genotype (Vauclusien). The introduction of the long shelf-life character resulted in undesirable loss of aroma volatiles production. The inhibition of ethylene synthesis by knocking-down ACC oxidase gene expression has been achieved in Charentais melon. It results is a strong inhibition of the synthesis of aroma volatiles while the accumulation of sugars is not affected or is even improved and the softening of the flesh is strongly affected but not abolished. It was also demonstrated that ethylene-inhibited fruit exhibited better resistance to chilling injury. Due to the importance of aroma volatiles in sensory quality and to the strong negative correlation between aroma production and ethylene synthesis, we have developed a research program aimed at isolating genes involved in the synthesis of volatile esters, compounds that are essential for the flavor of Cantaloupe melons. We report here on the recent advances in the field with special emphasis on the characterization of two families of genes encoding aldehyde reductases and alcohol acyl transferases

Characterization of Genes Involved in the Formation of Aroma Volatiles in "Charentais" Melon Fruit

Volatiles esters impart distinct characteristics to the fruit quality. "Charentais" cantaloupe melon (Cucumis melo "cantalupensis") is characterized by abundant sweetness and aromatic flavour. Plant alcohol acyl transferase (AAT) genes have been identified and shown to be involved in aromas production. Recently,two cDNAs (Cm-AAT1 and Cm-AAT2) putatively involved in the formation of aroma volatile esters have been isolated from melon fruit. Cm-AAT1 protein exhibit alcohol acyl transferase activity while no such activity could be detected for Cm-AAT2. Two new cDNAs (Cm-AAT3 and Cm-AAT4) have been isolated from melon fruit that showed 69% and 36% similarity, respectively, with Cm-AAT1. The percentage similarity over the whole amino acid sequence between them is 34%. Cm-AAT3 and Cm-AAT4 show the highest similarity to the tobacco Nt-HSR201 protein and a rose alcohol acyltransferase Rh-AAT1, respectively. All Cm-AATs genes, share three conserved regions common to the BAHD acyltransferase gene superfamily. Heterologous expression in yeast revealed that some of the encoded proteins have a wide range of specificity while others are specific to a narrow range of substrates

Mechanisms of Fruit Ripening: Retrospect and Prospects

This paper aims at giving an overview of the progress made during the last decades on the mechanisms of fruit ripening and to present the most recent trends and prospects for the future. Important steps forward will be presented (respiratory climacteric, ethylene biosynthesis and action, isolation of genes involved in the ripening process, biotechnological control of fruit ripening....) by showing how the judicious exploitation of the data published previously, the strategies, methodologies and plant material adopted have been crucial for the advancement of knowledge. Opportunities of co-operation between geneticists and post-harvest physiologists as well as new possibilities offered by genomics, proteomics and metabolomics for the understanding of the fruit ripening process and the development of sensory quality will be developed

Role of ethylene on various ripening pathways and on the development of sensory quality of Charentais cantaloupe melons

Charentais melons (Cucumis melo L., var cantalupensis Naud.) in which ethylene biosynthesis has been suppressed by an antisense ACC oxidase gene have been used to better understand the role of ethylene in the regulation of the ripening process of climacteric fruit and on the development of sensory qualities. We have shown that a number of biochemical and molecular processes associated with the ripening of climacteric fruit are ethylene-independent. In some cases, such as softening of the flesh, the same pathway comprises both ethylene-dependent and -independent components. The various ethylene-dependent events exhibit differential sensitivity to ethylene. The threshold level for degreening of the rind is 1 ppm, while 2.5 ppm are required to trigger the ethylene-dependent component of the softening process. The saturating level of ethylene for all these events is less than 5 ppm, which is by far lower than the internal ethylene concentrations found in the fruit at the climacteric peak (around 100 ppm). Detachment of the fruit influences the development of respiratory climacteric. Fruit remaining attached to the vine, although producing higher levels of ethylene, exhibit a reduced climacteric rise in respiration as compared to detached fruit. The response of antisense ACO fruit to exogenous ethylene in terms of respiration is higher in detached than in attached fruit. Ethylene-suppressed melons show a severe reduction of aroma volatiles production, particularly in ester production. In the biosynthetic pathway of aliphatic esters, the dehydrogenation of fatty acids and aldehydes appears to be ethylene-dependent. In contrast, alcohol acetylation comprises ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyl transferases. In terms of sensory quality, these data show that the extension of shelf-life through the inhibition of ethylene production has some beneficial effects on texture and sugar accumulation but is detrimental for the generation of aroma

A proteomics study of chilling injury in tomato fruit, a low-temperature stress-induced physiological disorder affecting fruit quality

Trabajo presentado a la conferencia "Molecular Basis of Plant Stress" celebrado en Bulgaria del 21 al 23 de septiembre de 2011.-- FP7 REGPOT Project "BioSupport".Tomato (Solanum lycopersicum L.) is the second most cultivated horticultural crop in the world in terms of harvested area and production, after the potato (FAOSTAT 2011, data from 2009). A main problem regarding its postharvest life is its sensitivity to low temperature stress during refrigerated storage, which constitutes a main obstacle for its long-term commercialization. This sensitivity is reflected in the physiopathy of chilling injury (CI), which negatively affects the final fruit quality. The main symptoms of CI-affected fruits are skin depressions, tissue decomposition and impaired ripening, which results in deficient flavour and aroma. Physiological and biochemical events involved in CI progress have been extensively described, but the precise molecular mechanisms that ultimately regulate the plant response to cold stress remain unclear. In order to investigate this response at molecular level in tomato fruit a proteomics strategy has undertaken. The proteome analysis provides a direct insight on the changes undergone by proteins, which are the major functional determinants of the cell machinery, in a certain biological situation such as low temperature stress. The protein expression profile of chilled tomato fruits has been compared with fruits stored at nonchilling temperature (control). The protein analysis has been performed by two-dimensional differential-in-gel-electrophoresis (2D-DIGE), and mass spectrometry of protein spots was applied to identify proteins differentially expressed. Comparative analysis revealed significant changes in abundance of 31 identified proteins between the proteomes of chilled and control fruits. Major modifications in the expression profile are related to those proteins specifically involved in stress (chaperonins and heat-shock proteins); cell wall biochemistry (hydrolytic enzymes), and carbohydrate metabolism (enzymes involved in glycolisis, tricarboxylic acids cycle, photosynthesis and sucrose biosynthesis).This work was supported by the Spanish Ministry of Science and Innovation (MICINN) through grant and PIE2009-40I080, and by the Council of Science and Technology from the Spanish Region of Murcia (Fundación SENECA) through grant no. 04553/GERM/06.Peer Reviewe

Effects of a pretreatment with nitric oxide (NO) free radical on the conservation of peach at room temperature

[SPA] El melocotón (Prunus persica L.) es un fruto de gran importancia económica en la agricultura del Levante español, pero su reducido periodo de vida poscosecha constituye una seria limitación para su comercialización. Este problema se debe a su rápida senescencia causada por una elevada producción etileno autocatalítico al iniciarse su maduración, rasgo propio de los frutos climatéricos como los de esta especie. Se ha tratado de superar este problema mediante la aplicación de compuestos antagonistas de la acción de esta hormona a los frutos inmediatamente tras su recolección. Uno de los compuestos más novedosos y prometedores de este tipo es el radical libre del oxido nítrico (NO). Se ha realizado un tratamiento de melocotón var. ‘Rojo Rito’ con 5 ppm de NO en una atmósfera anaeróbica confinada durante 4 horas a 20ºC. Posteriormente los frutos se almacenaron en una cámara a 20ºC tomándose muestras a los 3, 6, 10 y 14 días para su análisis. Frutos no tratados y almacenados en idénticas condiciones fueron utilizados como control de la experiencia. Mediante este tratamiento previo se ha podido observar una mejora manifiesta en la conservación de los frutos a temperatura ambiente. La producción de etileno y la tasa respiratoria de los frutos tratados con NO era menor que la de los controles, al igual que el porcentaje de salida de electrolitos, parámetro utilizado para estimar la pérdida de semipermeabilidad de las membranas celulares y por tanto su grado de desintegración, que es un claro síntoma de senescencia. Los frutos tratados también mantenían una firmeza significativamente superior a lo largo de la conservación. Con respecto a otros parámetros de calidad como el parámetro de color ‘H’, la acidez valorable y el contenido en sólidos solubles no presentaron diferencias significativas entre frutos tratados y no tratados. [ENG] Peach (Prunus persica L.) is a fruit of great economical importance in the agricultural sector of South and East areas of Spain. One of the most serious limitations for the commercial potential of this produce is its reduced period of postharvest shelf life. This is due to the rapid onset of senescence, caused by the autocatalytic ethylene production occurring during ripening, a typical characteristic of climacteric fruits such as it is this species. One strategy used to try to overcome this drawback is the application of antagonistic compounds of ethylene action on harvested fruits. One of the most novel and promising compounds of this type is the nitric oxide (NO) free radical. A treatment of 5 ppm NO during 4 hours at 20ºC in a confined anaerobic atmosphere on peach fruit cv. ‘Rojo Rito’ has been carried out. The treated fruits were stored at 20ºC during 3, 6, 10 and 14 days. Non-treated fruits stored in identical conditions were used as controls of the assay. An apparent improvement on the quality and physiology of conservation at room temperature of the NOtreated fruit was observed. Ethylene production and respiratory rate of fruits treated with NO were lower than controls, as well as percentage of ion leakage, a parameter used to estimate cell membrane semipermeability loss and therefore disruption of membrane integrity, which is a typical symptom of senescence. Also treated fruits have got significant higher pulp firmness values along the conservation period. With regard to other quality parameters such as ‘H’ colour parameter, titrable acidity and soluble solids content, they do not show significant differences between both types of samples, NO-treated fruits and untreated ones.Queremos expresar nuestro agradecimiento a la cooperativa agrícola “Finca La Caprichosa” sita en Cieza (Murcia), y al Sr. J. Molina por la provisión de frutos para la realización de esta experiencia. Este trabajo ha sido financiado por el proyecto CICYT (Ref. AGL2003-01457)

A PK–PD model of ketamine-induced high-frequency oscillations

Objective. Ketamine is a widely used drug with clinical and research applications, and also known to be used as a recreational drug. Ketamine produces conspicuous changes in the electrocorticographic (ECoG) signals observed both in humans and rodents. In rodents, the intracranial ECoG displays a high-frequency oscillation (HFO) which power is modulated nonlinearly by ketamine dose. Despite the widespread use of ketamine there is no model description of the relationship between the pharmacokinetic–pharmacodynamics (PK–PDs) of ketamine and the observed HFO power. Approach. In the present study, we developed a PK–PD model based on estimated ketamine concentration, its known pharmacological actions, and observed ECoG effects. The main pharmacological action of ketamine is antagonism of the NMDA receptor (NMDAR), which in rodents is accompanied by an HFO observed in the ECoG. At high doses, however, ketamine also acts at non-NMDAR sites, produces loss of consciousness, and the transient disappearance of the HFO. We propose a two-compartment PK model that represents the concentration of ketamine, and a PD model based in opposing effects of the NMDAR and non-NMDAR actions on the HFO power. Main results. We recorded ECoG from the cortex of rats after two doses of ketamine, and extracted the HFO power from the ECoG spectrograms. We fit the PK–PD model to the time course of the HFO power, and showed that the model reproduces the dose-dependent profile of the HFO power. The model provides good fits even in the presence of high variability in HFO power across animals. As expected, the model does not provide good fits to the HFO power after dosing the pure NMDAR antagonist MK-801. Significance. Our study provides a simple model to relate the observed electrophysiological effects of ketamine to its actions at the molecular level at different concentrations. This will improve the study of ketamine and rodent models of schizophrenia to better understand the wide and divergent range of effects that ketamine has.National Institutes of Health (U.S.) (Pioneer Award DP1-OD003646)Burroughs Wellcome Fund (Career Award at the Scientific Interface)National Institutes of Health (U.S.) (Grant 5R01MH061976)National Institutes of Health (U.S.) (New Innovator Award DP2-OD006454

Effectiveness of Tronador to Control Brittlebush in Buffelgrass Pastures at Central Sonora, México

Buffelgrass (Cenchrus ciliaris L.) is an introduced species which is planted in low productive semi-arid rangelands in northern Mexico to increase productivity and ranchers profit. Brittlebush (Encelia farinosa) is a native half size shrub of low forage value that invades buffelgrass stands. Buffelgrass pastures lose productivity as brittlebush densities increase. Once brittlebush infestations occur plant densities do not decline unless brush control practices are applied. Mechanical treatments are mainly used when grass seeding is needed. Prescribed burning and manual control as well as granular herbicides have resulted on good plant control. Most liquid herbicides are either not as effective for plant control or not economically feasibles. Tronador is a new released herbicide but no data is available for its use. This study was conducted to evaluate several doses of Tronador to control brittlebush infestations and measure buffelgrass forage responses

Two highly divergent alcohol dehydrogenases of melon exhibit fruit ripening-specific expression and distinct biochemical characteristics

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes (15% identity at the amino acid level) of Cantaloupe Charentais melon (Cucumis melo var. Cantalupensis) have been isolated. Cm-ADH1 belongs to the medium-chain zinc-binding type of ADHs and is highly similar to all ADH genes expressed in fruit isolated so far. Cm-ADH2 belongs to the short-chain type of ADHs. The two encoded proteins are enzymatically active upon expression in yeast. Cm-ADH1 has strong preference for NAPDH as a co-factor, whereas Cm-ADH2 preferentially uses NADH. Both Cm-ADH proteins are much more active as reductases with Kms 10–20 times lower for the conversion of aldehydes to alcohols than for the dehydrogenation of alcohols to aldehydes. They both show strong preference for aliphatic aldehydes but Cm-ADH1 is capable of reducing branched aldehydes such as 3-methylbutyraldehyde, whereas Cm-ADH2 cannot. Both Cm-ADH genes are expressed specifically in fruit and up-regulated during ripening. Gene expression as well as total ADH activity are strongly inhibited in antisense ACC oxidase melons and in melon fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. These data suggest that each of the Cm-ADH protein plays a specific role in the regulation of aroma biosynthesis in melon fruit

Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon