Increasing oil accumulation in olive mesocarp: Cloning and characterization of phospholipid: diacylglycerol acyltransferase (PDAT) genes from olive fruit

Motivation: To identify, characterize and study the regulation of genes and enzymes involved in the biosynthesis of triacylglycerols, in order to increase oil accumulation in olive mesocarp. In particular, to investigate the contribution of phospholipid:diacylglycerol acyltransferase (PDAT) genes (Dahlqvist et al., 2000).   Methods: Isolation of full-length cDNA clones by PCR, sequencing of amplified fragments, and sequence analysis using bioinformatic tools. Gene expression analysis using quantitative real-time PCR (qRT-PCR). Functional expression in a mutant of Saccharomyces cerevisiae (Sandager et al., 2002), using a yeast vector containing an inducible GAL1 promoter. Results: A cDNA sequence, designated OepPDAT2, encoding phospholipid:diacylglycerol acyltransferase (PDAT) has been isolated from olive. Gene expression analysis has been performed in Picual and Arbequina cultivars using different olive tissues and in olive mesocarp under different abiotic stresses such as draught, low and high temperature, darkness and wounding. Overexpression in yeast is being carried out in a quadruple mutant of S. cerevisiae that cannot synthesize triacylglycerols to confirm gene identity. Conclusions: Sequence analysis suggests that the OepPDAT2 gene encodes a PDAT enzyme. Gene expression analysis in olive seed and mesocarp tissues indicates that OePDAT2 participates in triacylglycerol synthesis during development and ripening of olive fruit, and it is transcriptionally regulated by different environmental factor

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

Mapping quantitative trait loci controlling fatty acid composition in olive

22 Páginas; 3 Tablas; 2 FigurasFatty acids are the main components of the olive oil and their composition has a critical influence on the oil quality. However, oil quality evaluation has not been frequently included in the selection of new bred cultivars. This can be due to the difficulties in analyzing oil quality in large set of genotypes and also to the long juvenile period of olive seedlings. Therefore, the identification of molecular markers associated to olive oil quality traits could facilitate their selection in breeding programs of this species. In the present work, the identification of the first QTLs for fatty acids on olive oil is reported. They have been located in a linkage map of a ‘Picual’ × ‘Arbequina’ progeny of the olive breeding program of Córdoba. Correlations among fatty acids are in agreement with previous reports of breeding progenies. QTLs found for oleic and linoleic acids explained 41.1 and 69.7% of the total variability, respectively, and were co-localized in the same linkage groups. In the same region, QTLs for monounsaturated, polyunsaturated and oleic/linoleic ratio were also identified. In other linkage groups, three QTLs for linolenic and one for palmitoleic acid were also located explaining 15.0–28.0% of the total variability. These results could be useful to increase the efficiency of breeding programs aimed at selecting new cultivars with high oleic acid content, and, therefore, with enhanced nutritional properties and oxidative stability of the olive oil.This work was partly supported by OLEAGEN Project funded by the Fundación Genoma España, Junta de Andalucía through Instituto de Investigación y Formación Agraria y Pesquera (IFAPA) and Corporación Tecnológica de Andalucía (CTA).Peer reviewe

Differential contribution of endoplasmic reticulum and chloroplast ω-3 fatty acid desaturase genes to the linolenic acid content of olive (Olea europaea) fruit

7 Figuras.-- 3 TablasLinolenic 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 oilaccumulating photosynthetic tissues.This work was supported by the Spanish Ministry of Science and Innovation [grants Nos. AGL2008-00258 and AGL2011–24442] and the JAE-Postdoctoral CSIC program [contract to M.L.H.

Specialized Functions of Olive FAD2 Gene Family Members Related to Fruit Development and the Abiotic Stress Response

2 Tablas.-- 7 FigurasThree different cDNA sequences, designated OepFAD2-3, OepFAD2-4 and OepFAD2-5, encoding three microsomal oleate desaturases (FAD2) have been isolated from olive (Olea europaea cv. Picual). Sequence analysis and functional expression in yeast of the corresponding cDNAs confirm that they encode microsomal oleate desaturases. Gene expression and lipid analysis indicate that these three genes are not involved in the linoleic acid present in seed lipids, while OeFAD2-5, together with OeFAD2-2, contributes mostly to the linoleic acid present in the mesocarp and, therefore, in the olive oil. Our results have also shown that olive FAD2-3, FAD2-4 and FAD2-5 gene expression is not only spatially and temporally regulated in olive fruit, but also is cultivar-dependent, as well as regulated by water regime, temperature, light and wounding. All these data suggest specialized physiological roles for the olive FAD2 gene family members with respect to both aspects of the biosynthesis of the linoleic acid, either present in storage lipids that constitute the olive oil or being part of membrane lipids, which are involved in the response to abiotic stresses, and highlight the differences on FAD2 gene regulation between oilseeds and oil fruits.This work was supported by the Spanish Ministry of Science, Innovation and Universities through Research Grants AGL2014-55300-R and AGL2017-87871-R (AEI/FEDER, UE)Peer reviewe

Evaluating the contribution of fruit photosynthesis toward the biogenesis of olive oil

Póster presentado en Olivebioteq 2018, the 6th International Conference on Olive Management and Olive Products, held in Seville, Spain, on 15th-19th October 2018.In olive, fruit growth is determined by the turgor and by the сarbon assimilated in photosynthesis. This carbon could come from photoassimilates produced in the leaves (heterotrophic) or produced in the fruit itself (autotrophic). The aim of this work was to evaluate whether the fruit photosynthesis actually represent a substantial contribution to oil synthesis in the fruit. The experiment was carried out in 10-year-old Arbequina olive trees under two irrigation strategies: full irrigation needs (FI) and regulated deficit irrigation (45RDI) applying 45% of the FI treatment. To analyze the origin of photoassimilates, we measured oil content and fatty acid composition in olive mesocarp from fruits in control branches, leafless branches which were also “girdled” (autotrophic), and branches whose fruits were bagged to avoid photosynthesis (heterotrophic). Samples were collected at three different stages of fruit development: immediately after stone lignification; when the olive fruit was completely green and when the olive fruit was yellowish. The 45RDI strategy caused an increase in olive oil content per fruit dry weight compared to the FI at the end of the experiment. However, in both irrigation treatments, autotrophic fruits decreased their oil content and accelerated the maturation process in four weeks. Interestingly, minor changes in fatty acid composition were detected among different treatments and water regimes. In particular, linolenic acid, a precursor of the phytohormone jasmonic acid, increased slightly, although significantly, in the autotrophic treatment.Peer reviewe

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

7 Figuras.-- 1 TablaThe 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.Research was supported by the Spanish Ministry of Economy and Competitiveness (Grant AGL2014-55300-R). F.P. was the recipient of a Ph.D. fellowship from the National Institute of Genetic Engineering and Biotechnology (Iran), and M.L.H. was the recipient of a contract from the JAE-Postdoctoral CSIC program (Spain)

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 3 h after wounding. © 2010 Elsevier Ltd. All rights reserved.This work was supported by Research Projects AGL2004-02060 from MEC (Spain) and P06-AGR-02151 from Junta de Andalucía (Spain).Peer Reviewe

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

16 Páginas.-- 8 FigurasThree 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.This work was funded by the Spanish Ministry of Science, Innovation, and Universities through Research Grant AGL2017-87871-R (AEI/FEDER, UE). Agrobioscience Ph.D. program at Scuola Superiore Sant’Anna granted the scholarship to SM.Peer reviewe

Carbon supply and water status regulate fatty acid and triacylglycerol biosynthesis at transcriptional level in the olive mesocarp

15 páginas.- 7 figuras.- referenciasThe relative contribution of carbon sources generated from leaves and fruits photosynthesis for triacylglycerol biosynthesis in the olive mesocarp and their interaction with water stress was investigated. With this aim, altered carbon source treatments were combined with different irrigation conditions. A higher decrease in mesocarp oil content was observed in fruits under girdled and defoliated shoot treatment compared to darkened fruit conditions, indicating that both leaf and fruit photosynthesis participate in carbon supply for oil biosynthesis being leaves the main source. The carbon supply and water status affected oil synthesis in the mesocarp, regulating the expression of DGAT and PDAT genes and implicating DGAT1-1, DGAT2, PDAT1-1, and PDAT1-2 as the principal genes responsible for triacylglycerol biosynthesis. A major role was indicated for DGAT2 and PDAT1-2 in well-watered conditions. Moreover, polyunsaturated fatty acid content together with FAD2-1, FAD2-2 and FAD7-1 expression levels were augmented in response to modified carbon supply in the olive mesocarp. Furthermore, water stress caused an increase in DGAT1-1, DGAT1-2, PDAT1-1, and FAD2-5 gene transcript levels. Overall, these data indicate that oil content and fatty acid composition in olive fruit mesocarp are regulated by carbon supply and water status, affecting the transcription of key genes in both metabolic pathways.This study was supported by Spanish Ministry of Science, Innovation and Universities through Research Grants AGL2015-71585-R and AGL2017-87871-R (AEI/FEDER, UE). A. Montero and A. Perez-Martin helped us with field measurements. A.P.-A. was the recipient of a contract from the FPI-CSIC program (Spain). We thank Internacional Olivarera, S.A.U. (Interoliva), for allowing us to make the experiments at Sanabria orchard.Peer reviewe