María Alejandra Moya-León

Increased accumulation of anthocyanins in Fragaria chiloensis fruits by transient suppression of FcMYB1 gene.

Anthocyanins and proanthocyanidins (PAs), flavonoid-derived metabolites with different physiological roles, are produced by plants in a coordinated manner during fruit development by the action of transcription factors (TFs). These regulatory proteins have either an activating or repressing effect over structural genes from the biosynthetic pathway under their control. FaMYB1, a TF belonging to the R2R3-MYB family and isolated from commercial strawberry fruit (Fragaria×ananassa), was reported as a transcriptional repressor and its heterologous over-expression in tobacco flowers suppressed flavonoid-derived compound accumulation. FcMYB1, an ortholog of FaMYB1 isolated from the white Chilean strawberry (Fragaria chiloensis ssp. chiloensis f. chiloensis), showed higher transcript levels in white (F. chiloensis) than in red (F.×ananassa cv. Camarosa) fruits. In order to assess its contribution to the discolored phenotype in F. chiloensis, FcMYB1 was transiently down-regulated in planta using an RNAi-based approach. Quantitative real-time PCR on FcMYB1 down-regulated fruits resulted an up-regulation of anthocyanidin synthase (ANS) and a strong repression of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) transcript accumulation. In addition, these fruits showed increased concentrations of anthocyanins and undetectable levels of flavan 3-ols. Altogether, these results indicate a role for FcMYB1 in regulation of the branching-point of the anthocyanin/PA biosynthesis determining the discolored phenotype of the white Chilean strawberry fruit.

Comparison of transcriptional profiles of flavonoid genes and anthocyanin contents during fruit development of two botanical forms of Fragaria chiloensis ssp. chiloensis

Difference in fruit pigmentation observed between two botanical forms of Fragaria chiloensis ssp. chiloensis (form chiloensis and form patagonica) was studied through transcriptional and chemical approaches. The proportion of different anthocyanins was demonstrated to be characteristic of each botanical form, with pelargonidin 3-glucoside being the most abundant in f. patagonica fruit and cyaniding 3-glucoside as the major one in f. chiloensis fruit. Partial gene sequences of the phenylpropanoid and flavonoid biosynthesis pathways were isolated from the native Chilean strawberry fruits, and used to design gene-specific primers in order to perform transcriptional analyses by qRT-PCR. These genes showed spatial, developmental, and genotypic associated patterns. The red fruit of f. patagonica exhibited higher transcript levels of anthocyanin-related genes and higher levels of anthocyanins compared to the barely pigmented fruit of f. chiloensis. The anthocyanin accumulation in F. chiloensis ssp. chiloensis fruits was concomitant with the particular progress of the transcriptional activity of genes involved in the biosynthesis of flavonoid pigments. The differences in anthocyanin contents, both in terms of type and quantity, between the two botanical forms of F. chiloensis ssp. chiloensis were coincident with the differential transcriptional patterns found in the anthocyanin-related genes.

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.).

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adhEU395433; Pa-pdcEU395434; and Pa-loxEU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.

VpAAT1, a gene encoding an alcohol acyltransferase, is involved in ester biosynthesis during ripening of mountain papaya fruit.

Mountain papaya ( Vasconcellea pubescens ) is a climacteric fruit that develops a strong and characteristic aroma during ripening. Esters are the main volatile compounds produced by the fruit, and most of them are dependent on ethylene. As esters are synthesized through alcohol acyltransferases (AAT), a full-length cDNA (VpAAT1) was isolated that displayed the characteristic motifs of most plant acyltransferases. The full-length cDNA sequence was cloned and expressed in yeasts, obtaining a functional enzyme with high AAT activity toward the formation of benzyl acetate. The transcript accumulation pattern provided by qPCR analysis showed that the VpAAT1 gene is expressed exclusively in fruit tissues and that a high level of transcripts is accumulated during ripening. The increase in VpAAT1 transcripts in fruit is coincident with the increase in AAT activity; transcript accumulation is induced by ethylene, and it is avoided by 1-methylcyclopropene (1-MCP) treatment. The data indicate that VpAAT1 is involved in aroma formation and that ethylene plays a major role in regulating its expression.

Aroma Development during Ripening of Fragaria chiloensis Fruit and Participation of an Alcohol Acyltransferase (FcAAT1) Gene

Fragaria chiloensis is characterized for having great aroma and flavor properties. Using headspace-SPME different volatile compounds were identified and quantified during development and ripening of the fruit. The headspace was dominated by esters, butyl acetate, ethyl acetate, ethyl butanoate and ethyl hexanoate being the most abundant in fully ripe fruit. As esters are important for aroma and synthesized through alcohol acyltransferases (AAT), a full-length cDNA (FcAAT1) was isolated from F. chiloensis fruit which displayed the three motifs characteristic of most AATs. As the production of esters increased during ripening, a clear increment in FcAAT1 transcripts was observed in fruit tissue. A good correlation was found between AAT activity and the total content of esters, especially with acetates and hexanoates. Aroma-related esters displayed during ripening the same production profile as AAT activity. Therefore it can be suggested that the FcAAT1 gene may have a significant role in ester production of F. chiloensis fruit.

Characterization of two divergent cDNAs encoding xyloglucan endotransglycosylase/hydrolase (XTH) expressed in Fragaria chiloensis fruit.

Chilean strawberry (Fragaria chiloensis), the maternal progenitor of Fragaria×ananassa, has emerged as a new berry fruit with excellent organoleptic characteristics. The fast softening of strawberries is a limiting step for their commercialization. Fruit softening has been shown to be related to cell wall degradation. Several enzymatic activities related to this process have been isolated in strawberry fruit, however xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have not been identified or characterized so far. Two XTH genes were identified in an EST database of F. chiloensis fruit with high homology to other plant XTHs. We isolated the full-length cDNAs associated to these ESTs in F. chiloensis (Fc-XTH1, Fc-XTH2). Phylogenetic analysis suggests that both F. chiloensis XTH genes belong to distant phylogenetic groups of XTHs. Moreover, DNA gel-blot analysis indicates different genomic organization between the two genes. By means of Real Time qPCR analysis, gene expression profiles show a transcriptional profile of Fc-XTH1 transcripts congruent with a probable role during strawberry ripening, while that exhibited by Fc-XTH2 could be related with vegetative processes like leaf growth. On the other hand, immunodetection and enzyme activity assays allow the detection of XTH-related proteins and high xyloglucan transglycosylating (XETA) and degrading (XDA) activities at the turning stage. The data presented confirms the existence of two divergent XTH genes, and XET and XEH activities, in F. chiloensis fruit.

Structural characterization and substrate specificity of VpAAT1 protein related to ester biosynthesis in mountain papaya fruit

The aroma in fruits is an important attribute of quality that influences consumers acceptance. This attribute is a complex character determined by a set of low molecular weight volatile compounds. In mountain papaya fruit (Vasconcellea pubescens) the aroma is determined mainly by esters, which are produced through an esterification reaction catalyzed by the enzyme alcohol acyltransferase (AAT) that utilizes alcohols and acyl-CoAs as substrates. In order to understand the molecular mechanism involved in the production of esters in this fruit, an AAT gene which has been previously cloned and characterized from mountain papaya (VpAAT1) was expressed in yeasts, and the highest enzyme activity of the recombinant protein was obtained when the enzyme was tested for its ability to produce benzyl acetate. On the other hand, to gain insight the mechanism of action at the molecular level, a structural model for VpAAT1 protein was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The VpAAT1 structure consists of two domains connected by a large crossover loop, with a solvent channel in the center of the structure formed between the two domains. Residues H166 and D170, important for catalytic action, displayed their side chains towards the central cavity of the channel allowing their interaction with the substrates. The conformational interaction between the protein and several ligands was explored by molecular docking simulations, and the predictions obtained were tested through kinetic analysis. Kinetic results showed that the lowest K(M) values were obtained for acetyl-CoA and benzyl alcohol. In addition, the most favorable predicted substrate orientation was observed for benzyl alcohol and acetyl CoA, showing a perfect coincidence between kinetic studies and molecular docking analysis.

Development of aroma compounds and sensory quality of ‘Royal Gala’ apples during storage

Summary The effect of 1-MCP and CA storage on aroma production and consumer acceptance were investigated during storage of ‘Royal Gala’ apples. Fruit was harvested 132 d (Harvest 1) and 144 d (Harvest 2) after full bloom (DAFB), and stored under refrigerated air (RA; 0ºC; 90 – 95% RH), or in a controlled atmosphere (CA; 2.0 – 2.5 kPa CO2, 1.8 – 2.0 kPa O2), or treated at harvest with 625 nl l–1 1-methylcyclopropene (1-MCP) ) followed by RA storage. Ripening indices, aroma volatile production, and sensory quality were determined after 2, 4 and 6 months of storage, plus 7 d of simulated shelf-life. Butyl acetate and hexyl acetate were the most abundant esters produced by ‘Royal Gala’ apple, and hexanol, butanol, and 2-methyl butanol were the most abundant alcohols. Hexyl acetate, 2-methyl butyl acetate and butyl acetate were found to be major contributors to aroma. CA storage and 1-MCP-treatment reduced ethylene production, softening and acidity loss. Storage of apples under RA conditions gave the highest levels of volatiles, while CA and 1-MCP treatment depressed aromatic volatile production in the fruit. At harvest, volatile emission was higher in late-harvested apples than in fruit picked at commercial harvest. After RA storage, no improvement in volatile emission was observed in late-harvested fruit; however, under CA or 1-MCP treatment, late-harvested fruit retained higher odour values than fruit harvested earlier. There was a good correlation between volatile levels and aroma intensity perceived by taste panelists; similarly high correlations were found between ethylene levels and most of the odour-active compounds analysed. 1-MCP and CA-stored fruit were preferred by consumers in all trials, indicating that both treatments can ensure ‘Royal Gala’ apples maintain their preferred textural characteristics. Therefore, this technology could be used to retain the quality of ‘Royal’ Gala fruit, especially during long-term storage.

Structural analysis of the alcohol acyltransferase protein family from Cucumis melo shows that enzyme activity depends on an essential solvent channel

Alcohol acyltransferases (AAT) play a key role in ester biosynthesis. In Cucumis melo var. cantalupensis, AATs are encoded by a gene family of four members (CmAAT1–4). CmAAT1, CmAAT3 and CmAAT4 are capable of synthesizing esters, with CmAAT1 the most active. CmAAT2 is inactive and has an Ala268 residue instead of a threonine which is present in all other active AATs, although the role of this residue is still unclear. The present work aims to understand the molecular mechanism involved in ester biosynthesis in melon fruit and to clarify the importance of the Ala268 residue. First, structural models for each protein were built by comparative modelling methodology. Afterwards, conformational interaction between the protein and several ligands, alcohols and acyl‐CoAs was explored by molecular docking and molecular dynamics simulation. Structural analysis showed that CmAATs share a similar structure. Also, well‐defined solvent channels were described in the CmAATs except for CmAAT2 which does not have a proper channel and instead has a small pocket around Ala268. Residues of the catalytic HxxxD motif interact with substrates within the solvent channel, with Ser363 also important. Strong binding interaction energies were described for the best substrate couple of each CmAAT (hexyl‐, benzyl‐ and cinnamyl‐acetate for CmAAT1, 3 and 4 respectively). CmAAT1 and CmAAT2 protein surfaces share similar electrostatic potentials; nevertheless the entrance channels for the substrates differ in location and electrostatic character, suggesting that Ala268 might be responsible for that. This could partly explain the major differences in activity reported for these two enzymes.

Isolation of genes differentially expressed during development and ripening of Fragaria chiloensis fruit by suppression subtractive hybridization

Fragaria chiloensis, the native Chilean strawberry, is noted for its good fruit quality characters. However, it is a highly perishable fruit due to its rapid softening. With the aim to screen for genes differentially expressed during development and ripening of strawberry fruit, the subtractive suppressive hybridization (SSH) methodology was employed. Six libraries were generated contrasting transcripts from four different developmental stages. A set of 1807 genes was isolated and characterized. In our EST collection, approximately 90% of partial cDNAs showed significant similarity to proteins with known or unknown function registered in databases. Among them, proteins related to protein fate were identified in a large green fruit library and protein related with cellular transport, cell wall-related proteins, and transcription regulators were identified in a ripe fruit library. Thirteen genes were analyzed by qRT-PCR during development and ripening of the Chilean strawberry fruit. The information generated in this study provides new clues to aid the understanding of the ripening process in F. chiloensis fruit.