The fruit size of a cultivated olive tree is consistently larger than its corresponding wild relatives because fruit size is one of the main traits associated with olive tree domestication. Additionally, large fruit size is one of the main objectives of modern olive breeding programs. However, as the long juvenile period is one main hindrance in classic breeding approaches, obtaining genetic markers associated with this trait is a highly desirable tool. For this reason, GWAS analysis of both genetic markers and the genes associated with fruit size determination, measured as fruit weight, was herein carried out in 50 genotypes, of which 40 corresponded to cultivated and 10 to wild olive trees. As a result, 113 genetic markers were identified, which showed a very high statistically significant correlation with fruit weight variability, p < 10⁻¹⁰. These genetic markers corresponded to 39 clusters of genes in linkage disequilibrium. The analysis of a segregating progeny of the cross of “Frantoio” and “Picual” cultivars allowed us to confirm 10 of the 18 analyzed clusters. The annotation of the genes in each cluster and the expression pattern of the samples taken throughout fruit development by RNAseq enabled us to suggest that some studied genes are involved in olive fruit weight determination.

The olive fruit is a drupe whose development and ripening takes several months from flowering to full maturation. During this period, several biochemical and physiological changes occur that affect the skin color, texture, composition, and size of the mesocarp. The final result is a fruit rich in fatty acids, phenolic compounds, tocopherols, pigments, sterols, terpenoids, and other compounds of nutritional interest. In this work, a transcriptomic analysis was performed using flowers (T0) and mesocarp tissue at seven different stages during olive fruit development and ripening (T1–T7) of the ‘Picual’ cultivar. A total of 1755 genes overexpressed at any time with respect to the flowering stage were further analyzed. These genes were grouped into eight clusters based on their expression profile. The gene enrichment analysis revealed the most relevant biological process of every cluster. Highlighting the important role of hormones at very early stages of fruit development (T1, Cluster 1), whereas genes involved in fatty acid biosynthesis were relevant throughout the fruit developmental process. Hence, genes coding for different fatty acid desaturase (SAD, FAD2, FAD3, FAD4, FAD5, FAD6, and FAD7) enzymes received special attention. In particular, 26 genes coding for different fatty acid desaturase enzymes were identified in the ‘Picual’ genome, contributing to the improvement of the genome annotation. The expression pattern of these genes during fruit development corroborated their role in determining fatty acid composition.

Olive fruit weight is a crucial trait to consider in olive breeding programs due to its impact on final yield and its relevance for mechanical harvesting and fruit processing. Although environmental conditions influence this trait, fruit weight is primarily determined by genetic factors and exhibits a high degree of heritability in breeding progenies. Despite several studies identifying potential markers associated with fruit weight, these markers have not been validated. In this study, we analyzed 40 genetic markers linked to fruit weight using a dataset comprising 73 cultivars (including 33 newly sequenced varieties) and 10 wild olives with a wide range of phenotypic characteristics, spanning from very light (0.41 g) to very heavy fruits (8.57 g). By examining the phenotype distribution for each genotype of the newly sequenced varieties, we successfully validated 16 genetic markers. Additionally, machine learning tools demonstrated that 9 out of the 16 validated markers have a high predictive ability for fruit weight. As a result, our work provides, for the first time, a set of 9 well-validated genetic markers suitable for use in marker-assisted selection during the early stages of olive breeding programs.

Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialise some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as those corresponding to RNA polymerase II in olive trees. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olives. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development through the synthesis of this type of RNAs.

Nitro‐fatty acids (NO ₂ ‐FAs) are potent electrophiles that react with nucleophilic residues in proteins (cysteines, histidines and lysines) via a process known as nitroalkylation. This reversible post‐translational modification (PTM) functions as a selective signalling mechanism by modulating the structure, transport, and catalysis of target proteins. In the present work, in vivo nitroalkylated proteins targets by nitro‐oleic acid (NO ₂ ‐OA), nitro‐linoleic acid (NO ₂ ‐LA) and NO ₂ ‐Ln have been identified in different Arabidopsis development, including the seed, vegetative, generative and senescence stages. The results reveal that nitroalkylation is a PTM that occurs throughout the development of Arabidopsis, primarily during the vegetative and generative stages. This modification predominantly affects proteins involved in energy metabolism, redox coenzymes and stress responses. Although all three NO ₂ ‐FAs interact with the three amino acids mentioned, cysteine is the primary target. However, its adducts are significantly more labile and redox‐sensitive than those formed with histidine or lysine. Overall, these results suggest that nitroalkylation constitutes a reversible post‐translational modification linked to the redox state. This regulation could participate in modulating the plasticity of energy flow, contributing to maintaining homoeostasis throughout the life cycle and resilience to environmental stress. Data are available via ProteomeXchange with identifier PXD060471.

Transcription is carried out in most eukaryotes by three multimeric complexes (RNA polymerases I, II and III). However, plants contain two additional RNA polymerases (IV and V), which have evolved from RNA polymerase II. RNA polymerases II, IV and V contain both common and specific subunits that may specialize some of their functions. In this study, we conducted a search for the genes that putatively code for the specific subunits of RNA polymerases IV and V, as well as the corresponding of RNA polymerase II in olive. Based on the homology with the genes of Arabidopsis thaliana, we identified 13 genes that putatively code for the specific subunits of polymerases IV and V, and 16 genes that code for the corresponding specific subunits of polymerase II in olive. The transcriptomic analysis by RNA-Seq revealed that the expression of the RNA polymerases IV and V genes was induced during the initial stages of fruit development. Given that RNA polymerases IV and V are involved in the transcription of long non-coding RNAs, we investigated their expression and observed relevant changes in the expression of this type of RNAs. Particularly, the expression of the intergenic and intronic long non-coding RNAs tended to increase in the early steps of fruit development, suggesting their potential role in this process. The positive correlation between the expression of RNA polymerases IV and V subunits and the expression of non-coding RNAs supports the hypothesis that RNA polymerases IV and V may play a role in fruit development throughout the synthesis of this type of RNAs.