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Characterization and validation of olive FAD and SAD gene families: expression analysis in different tissues and during fruit development

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Abstract

Stearoyl-ACP desaturases (SADs) and fatty acid desaturases (FADs) play a critical role in plant lipid metabolism and also affect oil fatty acid composition introducing double bonds into the hydrocarbon chains to produce unsaturated fatty acids. In the present study, the genomic sequences of three SAD and three FAD candidate genes were characterized in olive and their expression was evaluated in different plant tissues. OeSAD genes corresponded to olive SAD1 and SAD2 and to a newly identified OeSAD4, sharing the conserved protein structure with other plant species. On the other hand, the full-length genomic sequences of two microsomal OeFAD genes (FAD2-1 and FAD2-2) and the plastidial FAD6, were released. When the level of expression was tested on different tissues of cv. Leccino, OeSAD1 and OeSAD2 were mainly expressed in the fruits, while OeFAD genes showed the lowest expression in this tissue. The mRNA profiling of all genes was directly studied in fruits of Leccino and Coratina cultivars during fruit development. In both genotypes, the expression level of OeSAD1 and OeSAD2 had the highest value during and after the pit-hardening period, when oil accumulation in fruit mesocarp is intensively increasing. Furthermore, the expression level of both OeFAD2 genes, which were the main candidates for oleic acid desaturation, were almost negligible during fruit ripening. These results have made possible to define candidate genes of the machinery regulation of fatty acid composition in olive oil, providing information on their sequence, gene structure and chromosomal location.

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Acknowledgements

This research was financed by the European Union’s Horizon 2020 Research and Innovation Program Marie Sklodowska-Curie—Before Project (Grant Agreement No 645595), with a partial contribution of CNR—Institute of Biosciences and Bioresources (IBBR), Consejo de Investigaciones Científicas y Técnicas (CONICET) and Instituto Nacional de Tecnología Agropecuaria (INTA).

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Authors and Affiliations

  1. Estación Experimental Agropecuaria San Juan, Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Juan, Argentina

    C. Contreras, P. Pierantozzi, L. Gentili, M. Tivani & M. Torres

  2. CNR — Institute of Biosciences and Bioresources (IBBR), Perugia, Italy

    R. Mariotti, S. Mousavi, L. Baldoni & N. Cultrera

  3. Department of Molecular Biology and Biochemistry, Science Faculty, University of Malaga, Malaga, Spain

    C. Guerrero

  4. Department of Biotechnology, Agricultural University of Athens, Athens, Greece

    L. Roka

  5. Instituto Multidisciplinario de Biología Vegetal, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de Córdoba, Córdoba, Argentina

    D. Maestri

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  1. C. Contreras
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  2. R. Mariotti
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  3. S. Mousavi
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  4. L. Baldoni
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Contributions

CC, RM, MTo, DM, LG, MT and LB conceived the study. CC, RM, SM, CG, LR, NC and PP provided the plant material and performed the mRNA synthesis. CC, RM, SM, NC, CG, LR and MTo performed the molecular and bioinformatics analyses. CC, RM, SM, LB, CG, PP, DM, LG, MT and MTo wrote the first draft of the manuscript. CC, RM, SM, LB, CG, PP, DM, LG, MT and MTo contributed to the writing and revised the manuscript. All the authors agreed on the final version of this work.

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Correspondence to R. Mariotti.

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Electronic supplementary material

Below is the link to the electronic supplementary material.Figure S1. a) Leccino cultivar tissues images from which RNA extraction was performed: roots, fruits (mesocarp at 165 DAF), flowers, young leaves, buds and cambium. b) Different stages of fruit development and ripening in cv. Leccino, referred as days after flowering (DAF).

Supplementary file1 (JPG 780 kb)

Figure S2. Simplified scheme of the olive oil biosynthesis pathway showing the fatty acid formation in the plastid, and the triacylglycerol assembly through the Kennedy pathway in the ER. The key genes coloured in green were studied in the present work

Supplementary file2 (JPG 717 kb)

Figure S3. Distance tree of FAD proteins encoded by the three studied genes after NCBI database blasting. The analysis involved 139 FAD amino acid sequences. All ambiguous positions were removed for each sequence pair. The accession number and the full name for each entry were left as reported in the NCBI database. The FAD protein sequences related to Olea europaea were highlighted with different shades of green, full for the sequences found in this work and empty for those previously published. Evolutionary analyses were conducted in MEGA7. The history was inferred using the Neighbor-Joining method. The optimal trees, after a bootstrap analysis running 10,000 times, are shown and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site

Supplementary file3 (JPG 10173 kb)

Figure S4. Distance tree of SAD proteins encoded by the three studied genes after NCBI database blasting. The analysis involved 107 SAD amino acid sequences. All ambiguous positions were removed for each sequence pair. The accession number and the full name for each entry were left as reported in the NCBI database. The protein sequences related to Olea europaea were highlighted with different shades of green, full for the sequences found in this work and empty for those previously published. Evolutionary analyses were conducted in MEGA7. The history was inferred using the Neighbor-Joining method. The optimal trees, after a bootstrap analysis running 10,000 times, are shown and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site

Supplementary file4 (JPG 4868 kb)

Figure S5. Relative expression levels of different genes belonging to the OeFAD and OeSAD families in fruit mesocarp at 165 DAF, flower, leaf, root, bud and cambium of cv. Leccino. Data are the average of two biological replicates and three technical replicates. Multiple comparison test was performed by Duncan test. Different letters correspond to significant level (p<0.05) of expression in the same gene family

Supplementary file5 (JPG 984 kb)

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Contreras, C., Mariotti, R., Mousavi, S. et al. Characterization and validation of olive FAD and SAD gene families: expression analysis in different tissues and during fruit development. Mol Biol Rep 47, 4345–4355 (2020). https://doi.org/10.1007/s11033-020-05554-9

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