Molecular and Genetic Regulation of Sensory Quality of Climacteric Fruit

The sensory quality of fruit has become a major criterion in making the purchasing decision by consumers. Breeding programs have mainly been directed, from the post-harvest stand point, towards improving shelf-life. Chance seedlings or mutants with improved agronomic traits and/or extended shelf-life have been used for introgressing the long shelf-life character and eventually improved sensory quality traits in commercial genotypes of apple, melon or tomato. Because the plant hormone ethylene plays a central role in both storability and ripening of climacteric fruit, the generation by biotechnology of ethylene-inhibited fruit has offered a powerful tool to better understand, at the molecular and genetic level, the interrelations between storability and sensory quality. In the melon, inhibition of ethylene synthesis results is a strong inhibition of the synthesis of aroma volatiles while the accumulation of sugars is not affected or is even improved. The softening of the flesh is strongly affected but not abolished. Mid or long shelf-life melons generated by classical breeding present the same behavior. The generation of recombinant inbred lines by crossing a typical climacteric melon (Cantaloupe Charentais of the cantalupensis group) with a non climacteric melon (PI161375 of the agrestis chinensis group) allowed to demonstrate that the climacteric character is conferred by 2 duplicated loci only, which are of great importance for the regulation of storability and sensory quality. 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 aroma volatiles. We will report on the recent advances in the field with special emphasis on the characterization of genes responsible for the synthesis of esters, a family of compounds crucial for the flavor of many fruit

Climacteric fruit ripening: Ethylene-dependent and independent regulation of ripening pathways in melon fruit

Cantaloupe melons have a typical climacteric behaviour with ethylene playing a major role in the regulation of the ripening process and affecting the ripening rate. Crossing of Cantaloupe Charentais melon with a non-climacteric melon indicated that the climacteric character is genetically dominant and conferred by two duplicated loci only. However, other experiments made by crossing two non-climacteric melons have generated climacteric fruit, indicating that different and complex genetic regulation exists for the climacteric character. Suppression of ethylene production by antisense ACC oxidase RNA in Charentais melon has shown that, while many ripening pathways were regulated by ethylene (synthesis of aroma volatiles, respiratory climacteric and degreening of the rind), some were ethylene-independent (initiation of climacteric, sugar accumulation, loss of acidity and coloration of the pulp). Softening of the flesh comprised both ethylene-dependent and independent components that were correlated with differential regulation of cell wall degrading genes. These results indicate that climacteric (ethylene-dependent) and non-climacteric (ethylene-independent) regulation coexist during climacteric fruit ripening. In addition, ethylenesuppressed melons allowed demonstrating that the various ethylene-dependent events exhibited differential sensitivity to ethylene and that ethylene was promoting sensitivity to chilling injury. Throughout this review, the data generated with melon are compared with those obtained with tomato and other fruit

Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wallrelated genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and b-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening

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

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

Transcriptomic Events Involved in Melon Mature-Fruit Abscission Comprise the Sequential Induction of Cell-Wall Degrading Genes Coupled to a Stimulation of Endo and Exocytosis

Background: Mature-fruit abscission (MFA) in fleshy-fruit is a genetically controlled process with mechanisms that, contrary to immature-fruit abscission, has not been fully characterized. Here, we use pyrosequencing to characterize the transcriptomes of melon abscission zone (AZ) at three stages during AZ-cell separation in order to understand MFA control at an early stage of AZ-activation. Principal Findings: The results show that by early induction of MFA, the melon AZ exhibits major gene induction, while by late induction of MFA, melon AZ shows major gene repression. Although some genes displayed similar regulation in both early and late induction of abscission, such as EXT1-EXT4, EGase1, IAA2, ERF1, AP2D15, FLC, MADS2, ERAF17, SAP5 and SCL13 genes, the majority had different expression patterns. This implies that time-specific events occur during MFA, and emphasizes the value of characterizing multiple time-specific abscission transcriptomes. Analysis of gene-expression from these AZs reveal that a sequential induction of cell-wall-degrading genes is associated with the upregulation of genes involved in endo and exocytosis, and a shift in plant-hormone metabolism and signaling genes during MFA. This is accompanied by transcriptional activity of small-GTPases and synthaxins together with tubulins, dynamins, V-type ATPases and kinesin-like proteins potentially involved in MFA signaling. Early events are potentially controlled by down-regulation of MADS-box, AP2/ERF and Aux/IAA transcription-factors, and up-regulation of homeobox, zinc finger, bZIP, and WRKY transcription-factors, while late events may be controlled by up-regulation of MYB transcription-factors. Significance: Overall, the data provide a comprehensive view on MFA in fleshy-fruit, identifying candidate genes and pathways associated with early induction of MFA. Our comprehensive gene-expression profile will be very useful for elucidating gene regulatory networks of the MFA in fleshy-fruit

Biotecnologia e genética pós-colheita: retrospectivas e perspectivas.

A genética e a biotecnologia são ferramentas essenciais na melhoria da qualidade sensorial e nutricional de produtos hortícolas e da sua conservação pós-colheita