Carlos Sanz

Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance

Hydroperoxide lyases (HPLs) catalyze the cleavage of fatty acid hydroperoxides to aldehydes and oxoacids. These volatile aldehydes play a major role in forming the aroma of many plant fruits and flowers. In addition, they have antimicrobial activity in vitro and thus are thought to be involved in the plant defense response against pest and pathogen attack. An HPL activity present in potato leaves has been characterized and shown to cleave specifically 13-hydroperoxides of both linoleic and linolenic acids to yield hexanal and 3-hexenal, respectively, and 12-oxo-dodecenoic acid. A cDNA encoding this HPL has been isolated and used to monitor gene expression in healthy and mechanically damaged potato plants. HPL gene expression is subject to developmental control, being high in young leaves and attenuated in older ones, and it is induced weakly by wounding. HPL enzymatic activity, nevertheless, remains constant in leaves of different ages and also after wounding, suggesting that posttranscriptional mechanisms may regulate its activity levels. Antisense-mediated HPL depletion in transgenic potato plants has identified this enzyme as a major route of 13-fatty acid hydroperoxide degradation in the leaves. Although these transgenic plants have highly reduced levels of both hexanal and 3-hexenal, they show no phenotypic differences compared with wild-type ones, particularly in regard to the expression of wound-induced genes. However, aphids feeding on the HPL-depleted plants display approximately a two-fold increase in fecundity above those feeding on nontransformed plants, consistent with the hypothesis that HPL-derived products have a negative impact on aphid performance. Thus, HPL-catalyzed production of C6 aldehydes may be a key step of a built-in resistance mechanism of plants against some sucking insect pests.

Increasing ω-3 Desaturase Expression in Tomato Results in Altered Aroma Profile and Enhanced Resistance to Cold Stress

One of the drawbacks in improving the aroma properties of tomato (Solanum lycopersicum) fruit is the complexity of this organoleptic trait, with a great variety of volatiles contributing to determine specific quality features. It is well established that the oxylipins hexanal and (Z)-hex-3-enal, synthesized through the lipoxygenase pathway, are among the most important aroma compounds and impart in a correct proportion some of the unique fresh notes in tomato. Here, we confirm that all enzymes responsible for the synthesis of these C6 compounds are present and active in tomato fruit. Moreover, due to the low odor threshold of (Z)-hex-3-enal, small changes in the concentration of this compound could modify the properties of the tomato fruit aroma. To address this possibility, we have overexpressed the ω-3 fatty acid desaturases FAD3 and FAD7 that catalyze the conversion of linoleic acid (18:2) to linolenic acid (18:3), the precursor of hexenals and its derived alcohols. Transgenic OE-FAD tomato plants exhibit altered fatty acid composition, with an increase in the 18:3/18:2 ratio in leaves and fruits. These changes provoke a clear variation in the C6 content that results in a significant alteration of the (Z)-hex-3-enal/hexanal ratio that is particularly important in ripe OE-FAD3FAD7 fruits. In addition to this effect on tomato volatile profile, OE-FAD tomato plants are more tolerant to chilling. However, the different behaviors of OE-FAD plants underscore the existence of separate fatty acid fluxes to ensure plant survival under adverse conditions.

Changes in sugars, organic acids and amino acids in medlar (Mespilus germanica L.) during fruit development and maturation

Abstract The contents of sugars, organic acids, and amino acids (after acid hydrolysis) were determined during development and maturation of medlar (Mespilus germanica L.) fruit from 39 days after full bloom (DAF) until 2 weeks after the beginning of fruit drop (161 DAF). Fructose, glucose and sucrose were identified as the principal sugars and their levels varied remarkably during development. The fructose level increased continually through development reaching its maximum by 161 DAF (1200 mg/100 g fresh weight) while the increase of sucrose reached maximum at 131 DAF and had decreased at 161 DAF. After some fluctations at 69 DAF, glucose level remained high (686 mg/100 g fresh weight) at 161 DAF, when compared with Stage IV (131 DAF). While the level of malic acid increased continually, the ascorbic acid level decreased dramatically through fruit development; both acids reached their maximum and minimum levels at 161 DAF, i.e. 428 and 8.4 mg/100 g fresh weight, respectively. The total amino acid composition also changed in decreasing trend throughout development and remained low at 161 DAF. In the ripe fruit, glutamate and aspartate were the major amino compounds identified. These changes in the identified compounds can be related to the metabolic activity during fruit development and maturation.

Purification and characterization of an olive fruit β-glucosidase involved in the biosynthesis of virgin olive oil phenolics.

An olive beta-glucosidase was purified to apparent homogeneity from mature fruits ( Olea europaea cv. Picual) by selective extraction and successive anion exchange and hydrophobic interaction chromatographic procedures. The enzyme was shown to be a homodimer made up of two identical subunits of 65.4 kDa. Optimum activity was recorded at pH 5.5 and 45 degrees C. The enzyme was active on the main olive phenolic glycosides, with maximum activity toward oleuropein (100%), followed by ligstroside (65%) and demethyloleuropein (21%). The enzyme showed very low activity with apigenin and luteolin glucosides and was not active on verbascoside and rutin. Kinetic values show that olive beta-glucosidase is 200-fold more active against oleuropein than against the synthetic substrate p-nitrophenyl-beta-d-glucopyranoside (pNPG). According to its catalytic properties, the implication of the purified olive beta-glucosidase on the synthesis of virgin olive oil phenolics is discussed.

Contribution of olive seed to the phenolic profile and related quality parameters of virgin olive oil

BACKGROUND Conflicting results have been reported about the effect of fruit de-stoning on the virgin olive oil (VOO) phenolic profile. The aim of the present study was to determine whether olive seed plays any role in the synthesis of this oil phenolic fraction. RESULTS Increases of around 25% of total phenolic compounds were observed in oils obtained from de-stoned olive fruits in three main Spanish cultivars. To investigate the involvement of olive seed in determining the phenolic profile of VOO, whole intact olive fruits were added with up to 400% olive stones. Excellent regression coefficients were found in general for the decrease of total phenolic compounds and, particularly, of o-diphenolics in the resulting oils. On the other hand, it was found that olive seed contains a high level of peroxidase (POX) activity (72.4 U g(-1) FW), accounting for more than 98% of total POX activity in the whole fruit. This activity is able to modify VOO phenolics in vitro, similar to the effect of adding stones during VOO extraction. CONCLUSION Olive seed plays an important role in determining VOO phenolic profile during the process to obtain an oil that seems to be associated with a high level of POX activity. Copyright

Functional characterization of two 13-lipoxygenase genes from olive fruit in relation to the biosynthesis of volatile compounds of virgin olive oil

Two LOX cDNA clones, Oep1LOX2 and Oep2LOX2, have been isolated from olive ( Olea europaea cv. Picual). Both deduced amino acid sequences showed significant similarity to known plant LOX2, and they contain an N-terminal chloroplastic transit peptide. Genomic Southern blot analyses suggest that at least three copies of Oep1LOX2 and one copy of Oep2LOX2 should be present in the olive genome. Linolenic acid proved to be the preferred substrate for both olive recombinant LOXs, and analyses of reaction products revealed that both enzymes produce primarily 13-hydroperoxides from linoleic and linolenic acids. Expression levels of both genes were measured in the mesocarp and seeds during development and ripening of Picual and Arbequina olive fruit along with the level of volatile compounds in the corresponding virgin olive oils. Biochemical and gene expression data suggest a major involvement of the Oep2LOX2 gene in the biosynthesis of virgin olive oil aroma compounds.

Synthesis of volatile compounds of virgin olive oil is limited by the lipoxygenase activity load during the oil extraction process

The aim of this work was to determine whether the lipoxygenase (LOX) activity is a limiting factor for the biosynthesis of virgin olive oil (VOO) volatile compounds during the oil extraction process. For this purpose, LOX activity load was modified during this process using exogenous LOX activity and specific LOX inhibitors on olive cultivars producing oils with different volatile profiles (Arbequina and Picual). Experimental data suggest that LOX activity is a limiting factor for the synthesis of the oil volatile fraction, this limitation being significantly higher in Picual cultivar than in Arbequina, in line with the lowest content of volatile compounds in the oils obtained from the former. Moreover, there is evidence that this limitation of LOX activity takes place mostly during the milling step in the process of olive oil extraction.

Variability of virgin olive oil phenolic compounds in a segregating progeny from a single cross in Olea europaea L. and sensory and nutritional quality implications.

Virgin olive oil phenolic compounds are responsible for its nutritional and sensory quality. The synthesis of phenolic compounds occurs when enzymes and substrates meet as olive fruit is crushed during the industrial process to obtain the oil. The genetic variability of the major phenolic compounds of virgin olive oil was studied in a progeny of the cross of Picual x Arbequina olive cultivars (Olea europaea L.). They belong to four different groups: compounds that included tyrosol or hydroxytyrosol in their molecules, lignans, flavonoids, and phenolic acids. Data of phenolics in the oils showed that the progeny displayed a large degree of variability, widely transgressing the genitor levels. This high variability can be of interest on breeding programs. Thus, multivariate analysis allowed to identify genotypes within the progeny particularly interesting in terms of phenolic composition and deduced organoleptic and nutritional quality. The present study has demonstrated that it is possible to obtain enough degree of variability with a single cross of olive cultivars for compounds related to the nutritional and organoleptic properties of virgin olive oil.

Monitoring endogenous enzymes during olive fruit ripening and storage: Correlation with virgin olive oil phenolic profiles

The ability of olive endogenous enzymes β-glucosidase, polyphenol oxidase (PPO) and peroxidase (POX), to determine the phenolic profile of virgin olive oil was investigated. Olives used for oil production were stored for one month at 20 °C and 4 °C and their phenolic content and enzymatic activities were compared to those of ripening olive fruits. Phenolic and volatile profiles of the corresponding oils were also analysed. Oils obtained from fruits stored at 4 °C show similar characteristics to that of freshly harvested fruits. However, the oils obtained from fruits stored at 20 °C presented the lowest phenolic content. Concerning the enzymatic activities, results show that the β-glucosidase enzyme is the key enzyme responsible for the determination of virgin olive oil phenolic profile as the decrease in this enzyme activity after 3 weeks of storage at 20 °C was parallel to a dramatic decrease in the phenolic content of the oils.

Isolation, expression, and characterization of a 13-hydroperoxide lyase gene from olive fruit related to the biosynthesis of the main virgin olive oil aroma compounds.

A full-length cDNA clone (OepHPL) coding for hydroperoxide lyase was isolated from olive fruit ( Olea europaea cv. Picual). The deduced amino acid sequence shows significant similarity to known plant hydroperoxide lyases and contains a N-terminal sequence that displays structural features of a chloroplast transit peptide. Genomic Southern blot analysis indicates that at least one copy of OepHPL is present in the olive genome. The recombinant hydroperoxide lyase was specific for 13-hydroperoxide derivatives of linolenic and linoleic acids but did not use 9-hydroperoxy isomers as substrates. Analyses of reaction products revealed that this enzyme produces primarily (Z)-hex-3-enal, which partially isomerizes to (E)-hex-2-enal, from 13-hydroperoxylinolenic acid and hexanal from 13-hydroperoxylinoleic acid. Expression levels were measured in different tissues of Picual and Arbequina varieties, including mesocarp and seed during development and ripening of olive fruits. The involvement of this olive hydroperoxide lyase gene in the biosynthesis of virgin olive oil aroma compounds is discussed.