Distinct triterpene synthases in the laticifers of Euphorbia lathyris.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

An Oleuropein ß-Glucosidase from Olive Fruit Is Involved in Determining the Phenolic Composition of Virgin Olive Oil.

Phenolic composition of virgin olive oil is determined by the enzymatic and/or chemical reactions that take place during olive fruit processing. Of these enzymes, ß-glucosidase activity plays a relevant role in the transformation of the phenolic glycosides present in the olive fruit, generating different secoiridoid derivatives. The main goal of the present study was to characterize olive fruit ß-glucosidase genes and enzymes responsible for the phenolic composition of virgin olive oil. To achieve that, we have isolated an olive ß-glucosidase gene from cultivar Picual (OepGLU), expressed in Nicotiana benthamiana leaves and purified its corresponding recombinant enzyme. Western blot analysis showed that recombinant OepGLU protein is detected by an antibody raised against the purified native olive mesocarp ß-glucosidase enzyme, and exhibits a deduced molecular mass of 65.0 kDa. The recombinant OepGLU enzyme showed activity on the major olive phenolic glycosides, with the highest levels with respect to oleuropein, followed by ligstroside and demethyloleuropein. In addition, expression analysis showed that olive GLU transcript level in olive fruit is spatially and temporally regulated in a cultivar-dependent manner. Furthermore, temperature, light and water regime regulate olive GLU gene expression in olive fruit mesocarp. All these data are consistent with the involvement of OepGLU enzyme in the formation of the major phenolic compounds present in virgin olive oil.

Assessment of volatile compound profiles and the deduced sensory significance of virgin olive oils from the progeny of Picual×Arbequina cultivars.

Volatile compounds are responsible for most of the sensory qualities of virgin olive oil and they are synthesized when enzymes and substrates come together as olive fruit is crushed during the industrial process to obtain the oil. Here we have studied the variability among the major volatile compounds in virgin olive oil prepared from the progeny of a cross of Picual and Arbequina olive cultivars (Olea europaea L.). The volatile compounds were isolated by SPME, and analyzed by HRGC-MS and HRGC-FID. Most of the volatile compounds found in the progeny's oil are produced by the enzymes in the so-called lipoxygenase pathway, and they may be clustered into different groups according to their chain length and polyunsaturated fatty acid origin (linoleic and linolenic acids). In addition, a group of compounds derived from amino acid metabolism and two terpenes also contributed significantly to the volatile fraction, some of which had significant odor values in most of the genotypes evaluated. The volatile compound content of the progeny was very varied, widely transgressing the progenitor levels, suggesting that in breeding programs it might be more effective to consider a larger number of individuals within the same cross than using different crosses with fewer individuals. Multivariate analysis allowed genotypes with particularly interesting volatile compositions to be identified and their flavor quality deduced.

Modulating oxidoreductase activity modifies the phenolic content of virgin olive oil.

The effect of modifying polyphenol oxidase (PPO) and peroxidase (POX) activity during the extraction of virgin olive oil has been assessed in terms of its influence on the phenolic profile of the oil produced. These enzymes were modified by adding exogenous enzyme or specific inhibitors during the milling and subsequent kneading step, studying the effect on specific phenolic compounds in the oils. PPO is the main enzyme involved in phenolic oxidation at the milling step whereas POX activity seems to be the main influence during the kneading step. The data obtained suggest it is possible to increase the nutritional and organoleptic quality of virgin olive oil by inhibiting these enzymes during olive fruit processing. Treatment with the PPO inhibitor tropolone produced a twofold increase in the phenolic fraction, which would therefore seem to be an interesting strategy to improve the nutritional and organoleptic properties of virgin olive oil.

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.

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.

Precursor uptake assays and metabolic analyses in isolated tomato fruit chromoplasts.

BACKGROUND: Carotenoids are the most widespread group of pigments found in nature. In addition to their role in the physiology of the plant, carotenoids also have nutritional relevance as their incorporation in the human diet provides health benefits. In non-photosynthetic tissues, carotenoids are synthesized and stored in specialized plastids called chromoplasts. At present very little is known about the origin of the metabolic precursors and cofactors required to sustain the high rate of carotenoid biosynthesis in these plastids. Recent proteomic data have revealed a number of biochemical and metabolic processes potentially operating in fruit chromoplasts. However, considering that chloroplast to chromoplast differentiation is a very rapid process during fruit ripening, there is the possibility that some of the proteins identified in the proteomic analysis could represent remnants no longer having a functional role in chromoplasts. Therefore, experimental validation is necessary to prove whether these predicted processes are actually operative in chromoplasts. RESULTS: A method has been established for high-yield purification of tomato fruit chromoplasts suitable for metabolic studies. Radiolabeled precursors were efficiently incorporated and further metabolized in isolated chromoplast. Analysis of labeled lipophilic compounds has revealed that lipid biosynthesis is a very efficient process in chromoplasts, while the relatively low incorporation levels found in carotenoids suggest that lipid production may represent a competing pathway for carotenoid biosynthesis. Malate and pyruvate are efficiently converted into acetyl-CoA, in agreement with the active operation of the malic enzyme and the pyruvate dehydrogenase complex in the chromoplast. Our results have also shown that isolated chromoplasts can actively sustain anabolic processes without the exogenous supply of ATP, thus suggesting that these organelles may generate this energetic cofactor in an autonomous way. CONCLUSIONS: We have set up a method for high yield purification of intact tomato fruit chromoplasts suitable for precursor uptake assays and metabolic analyses. Using targeted radiolabeled precursors we have been able to unravel novel biochemical and metabolic aspects related with carotenoid and lipid biosynthesis in tomato fruit chromoplasts. The reported chromoplast system could represent a valuable platform to address the validation and characterization of functional processes predicted from recent transcriptomic and proteomic data.

Factors limiting the synthesis of virgin olive oil volatile esters.

The aim of the present work was to establish the limiting factors affecting the biosynthesis of volatile esters present in virgin olive oil (VOO). Oil volatile fractions of the main Spanish olive cultivars, Arbequina and Picual, were analyzed. It was observed that acetate esters were the most abundant class of volatile esters in the oils, in concordance with the high content of acetyl-CoA found in olive fruit, and that the content of C6 alcohols is limited for the synthesis of volatile esters during the production of VOO. Thus, the increase of C6 alcohol availability during VOO production produced a significant increase of the corresponding ester in the oils in both cultivars at two different maturity stages. However, the increase of acetyl-CoA availability had no effect on the VOO volatile fraction. The low synthesis of these C6 alcohols seems not to be due to a shortage of precursors or cofactors for alcohol dehydrogenase (ADH) activity because their increase during VOO production had no effect on the C6 alcohol levels. The experimental findings are compatible with a deactivation of ADH activity during olive oil production in the cultivars under study. In this sense, a strong inhibition of olive ADH activity by compounds present in the different tissues of olive fruit has been observed.

Purification and characterization of an olive fruit beta-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.