Functional Characterization of Four Olive Squalene Synthases with Respect to the Squalene Content of the Virgin Olive Oil.

The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

Distinct Physiological Roles of Three Phospholipid:Diacylglycerol Acyltransferase Genes in Olive Fruit with Respect to Oil Accumulation and the Response to Abiotic Stress.

Three different cDNA sequences, designated OepPDAT1-1, OepPDAT1-2, and OepPDAT2, encoding three phospholipid:diacylglycerol acyltransferases (PDAT) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis showed the distinctive features typical of the PDAT family and together with phylogenetic analysis indicated that they encode PDAT. Gene expression analysis in different olive tissues showed that transcript levels of these three PDAT genes are spatially and temporally regulated and suggested that, in addition to acyl-CoA:diacylglycerol acyltransferase, OePDAT1-1 may contribute to the biosynthesis of triacylglycerols in the seed, whereas OePDAT1-2 could be involved in the triacylglycerols content in the mesocarp and, therefore, in the olive oil. The relative contribution of PDAT and acyl-CoA:diacylglycerol acyltransferase enzymes to the triacylglycerols content in olive appears to be tissue-dependent. Furthermore, water regime, temperature, light, and wounding regulate PDAT genes at transcriptional level in the olive fruit mesocarp, indicating that PDAT could be involved in the response to abiotic stresses. Altogether, this study represents an advance in our knowledge on the regulation of oil accumulation in oil fruit.

Modification of 13-hydroperoxide lyase expression in olive affects plant growth and results in altered volatile profile.

The C6 aldehydes, alcohols, and the corresponding esters are the most important compounds of virgin olive oil aroma. These C6 volatile compounds are synthesized via the 13-hydroperoxide lyase (13-HPL) branch of the lipoxygenase pathway. In this investigation, a functional analysis of the olive (Olea europaea L.) 13-HPL gene by its overexpression and silencing in olive transgenic lines was carried out. With this aim, sense and RNAi constructs of the olive 13-HPL gene were generated and used for the transformation of embryogenic olive cultures. Leaves from overexpressing lines showed a slight increase in 13-HPL gene expression, whereas RNAi lines exhibited a strong decrease in their transcript levels. Quantification of 13-HPL activity in two overexpressing and two RNAi lines showed a positive correlation with levels of transcripts. Interestingly, RNAi lines showed a high decrease in the content of C6 volatiles linked to a strong increase of C5 volatile compounds, altering the volatile profile in the leaves. In addition, the silencing of the 13-HPL gene severely affected plant growth and development. This investigation demonstrates the role of the 13-HPL gene in the biogenesis of olive volatile compounds and constitutes a functional genomics study in olive related to virgin olive oil quality.

The Oleic/Linoleic Acid Ratio in Olive (Olea europaea L.) Fruit Mesocarp Is Mainly Controlled by OeFAD2-2 and OeFAD2-5 Genes Together With the Different Specificity of Extraplastidial Acyltransferase Enzymes.

Fatty acid composition of olive oil has an important effect on the oil quality to such an extent that oils with a high oleic and low linoleic acid contents are preferable from a nutritional and technological point of view. In the present work, we have first studied the diversity of the fatty acid composition in a set of eighty-nine olive cultivars from the Worldwide Olive Germplasm Bank of IFAPA Cordoba (WOGBC-IFAPA), and in a core collection (Core-36), which includes 28 olive cultivars from the previously mentioned set. Our results indicate that oleic and linoleic acid contents displayed the highest degree of variability of the different fatty acids present in the olive oil of the 89 cultivars under study. In addition, the independent study of the Core-36 revealed two olive cultivars, Klon-14 and Abou Kanani, with extremely low and high linoleic acid contents, respectively. Subsequently, these two cultivars were used to investigate the specific contribution of different fatty acid desaturases to the linoleic acid content of mesocarp tissue during olive fruit development and ripening. Fatty acid desaturase gene expression levels, together with lipid analysis, suggest that not only OeFAD2-2 and OeFAD2-5 but also the different specificities of extraplastidial acyltransferase enzymes are responsible for the variability of the oleic/linoleic acid ratio in olive cultivars. All this information allows for an advancement in the knowledge of the linoleic acid biosynthesis in different olive cultivars, which can impact olive breeding programs to improve olive oil quality.

Transcriptional Regulation of Stearoyl-Acyl Carrier Protein Desaturase Genes in Response to Abiotic Stresses Leads to Changes in the Unsaturated Fatty Acids Composition of Olive Mesocarp.

In higher plants, the stearoyl-acyl carrier protein desaturase (SAD) catalyzes the first desaturation step leading to oleic acid, which can be further desaturated to linoleic and α-linolenic acids. Therefore, SAD plays an essential role in determining the overall content of unsaturated fatty acids (UFA). We have investigated how SAD genes expression and UFA composition are regulated in olive (Olea europaea) mesocarp tissue from Picual and Arbequina cultivars in response to different abiotic stresses. The results showed that olive SAD genes are transcriptionally regulated by temperature, darkness and wounding. The increase in SAD genes expression levels observed in Picual mesocarp exposed to low temperature brought about a modification in the UFA content of microsomal membrane lipids. In addition, darkness caused the down-regulation of SAD genes transcripts, together with a decrease in the UFA content of chloroplast lipids. The differential role of olive SAD genes in the wounding response was also demonstrated. These data point out that different environmental stresses can modify the UFA composition of olive mesocarp through the transcriptional regulation of SAD genes, affecting olive oil quality.

Transcriptional Analysis of Stearoyl-Acyl Carrier Protein Desaturase Genes from Olive (Olea europaea) in Relation to the Oleic Acid Content of the Virgin Olive Oil.

The specific contribution of different stearoyl-ACP desaturase (SAD) genes to the oleic acid content in olive (Olea europaea) fruit has been studied. Toward that end, we isolated three distinct cDNA clones encoding three SAD isoforms from olive (cv. Picual), as revealed by sequence analysis. The expression levels of olive SAD genes were determined in different tissues from Picual and Arbequina cultivars, including developing mesocarp and seed, together with the unsaturated fatty acid content. Lipid and gene expression analyses indicate that OeSAD2 seems to be the main gene contributing to the oleic acid content of the olive fruit and, therefore, of the virgin olive oil. This conclusion was confirmed when the study was extended to Hojiblanca, Picudo, and Manzanilla cultivars. Furthermore, our data indicate that the olive microsomal oleate desaturase gene OeFAD2-2, but not OeSAD2, is responsible for the linoleic acid content in the virgin olive oil.

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues.

De novo assembly and functional annotation of the olive (Olea europaea) transcriptome.

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation.

Effect of different environmental stresses on the expression of oleate desaturase genes and fatty acid composition in olive fruit.

The regulation of microsomal and plastidial oleate desaturases by low and high temperature, darkness, and wounding was investigated. To this end, their gene expression levels and the fatty acid composition was determined in the mesocarp tissue of olive fruit from the Picual and Arbequina varieties subjected to the corresponding stress treatments. Firstly, a plastidial oleate desaturase from olive was cloned and its functional identity was confirmed by overexpression in Escherichia coli. The results showed that temperature and light regulate olive oleate desaturase genes at transcriptional level. However, no correlation between their expression levels and the linoleic acid content in microsomal and plastidial lipids was found. In addition, the involvement of microsomal but not plastidial oleate desaturases in the wounding response of olive fruit mesocarp is demonstrated. The fatty acid analysis revealed the appearance of palmitolinoleic acid only in microsomal lipids, reaching a maximum 3h after wounding.

Growth temperature control of the linoleic acid content in safflower (Carthamus tinctorius) seed oil.

The temperature and oxygen regulation of the microsomal oleate desaturase (FAD2) from safflower (Carthamus tinctorius L.) seeds was investigated. Heat-resistance profiles obtained in vivo and in vitro showed that the FAD2 enzyme maintained its maximal activity until 30 degrees C. A temperature increase from 10 to 40 degrees C caused a decrease of the FAD2 activity. However, when the temperature was decreased from 40 to 10 degrees C, no increase in the activity level was detected. The removal of hulls from safflower seeds followed by incubation in air did not change the FAD2 activity level, whereas incubation under nitrogen caused a strong decrease. Air replacement brought about the recovery of the initials levels. Oxygen concentrations less than 3% produced the inactivation of the enzyme. These data indicate that the higher thermal stability and the lower dependence on oxygen availability of the safflower FAD2 enzyme, compared with that of sunflower, could be the main factors to explain why the linoleate content of safflower seeds is more independent of growth temperature than that of sunflower seeds.