Inflorescence Emergence and Flowering Response of Olive Cultivars Grown in Olive Reference Collection of Portugal (ORCP).

In olive trees, fluctuations in the onset of phenological stages have been reported due to weather conditions. The present study analyses the reproductive phenology of 17 olive cultivars grown in Elvas (Portugal) in 3 consecutive years (2012-2014). Through 2017-2022, the phenological observations continued with four cultivars. The phenological observations followed the BBCH scale. Over the course of the observations, the bud burst (stage 51) occurred gradually later; a few cultivars did not follow this trend in 2013. The flower cluster totally expanded phase (stage 55) was achieved gradually earlier, and the period between stages 51-55 was shortened, especially in 2014. Date of bud burst showed a negative correlation with minimum temperature (Tmin) of November-December, and, in 'Arbequina' and 'Cobrançosa', the interval stage 51-55 showed a negative correlation with both the Tmin of February and the Tmax of April, whereas in 'Galega Vulgar' and 'Picual' there was instead a positive correlation with the Tmin of March. These two seemed to be more responsive to early warm weather, whereas 'Arbequina' and 'Cobrançosa' were less sensitive. This investigation revealed that olive cultivars behaved differently under the same environmental conditions and, in some genotypes, the ecodormancy release may be linked to endogenous factors in a stronger way.

Regulation of sterol content and biosynthetic gene expression during flower opening and early fruit development in olive.

Phytosterols are lipophilic membrane components essential not only for diverse cellular functions but also are biosynthetic precursors of the plant hormone, brassinosteroid (BR). However, the interaction between phytosterol and BR during early fleshy-fruit growth remains largely uncharacterized. In olive, phytosterols are important lipids because they affect oil quality, but phytosterol composition during flowering and early fruit development has not been explored. Here, we first investigated the temporal changes in phytosterol composition, and biosynthetic gene expression that occurred during olive flower opening and early fruit growth. Next, we analyzed the interrelationship between phytosterol and BR, whose levels we manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz). In this report, the profiling of phytosterol measurement revealed that ß-sitosterol is the most abundant in olive reproductive organs. Our data demonstrate that both OeCYP51 and OeSMT2 genes are upregulated during floral anthesis in good agreement with the rise in cholesterol and ß-sitosterol contents in olive flower. By contrast, the OeCYP51 and OeSMT2 genes displayed different expression patterns during early olive-fruit development. Furthermore, our data show that exogenous EBR enhanced the early olive-fruit growth, as well as the OeSMT2 transcript and ß-sitosterol levels, but decreased the OeCYP51 transcript, squalene, campesterol and cholesterol levels, whereas the Brz treatment exerted the opposite effect. Overall, our findings indicate an up-regulation of ß-sitosterol biosynthesis by BR at the transcriptional level during early olive-fruit growth, providing a valuable tool to unravel the physiological function of SMT2 in future studies.

Modulation of sphingolipid long-chain base composition and gene expression during early olive-fruit development, and putative role of brassinosteroid.

Sphingolipids are abundant membrane components and signalling molecules in various aspects of plant development. However, the role of sphingolipids in early fleshy-fruit growth has rarely been investigated. In this study, we first investigated the temporal changes in sphingolipid long-chain base (LCB) content, composition, and gene expression that occurred during flower opening and early fruit development in olive (Olea europaea L. cv Picual). Moreover, the interaction between sphingolipid and the plant hormone, brassinosteroid (BR), during the early fruit development was also explored. For this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, sphingolipid LCB content, and gene expression were examined in olive fruit at 14 days post-anthesis (DPA). We here show that sphingolipid with C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation are quantitatively the most important sphingolipids in olive reproductive organs. In this work, the total LCB amount significantly decreased at the anthesis stage, but olive sphingosine-1-phosphate lyase (OeSPL) gene was expressed exclusively in flower and upregulated during the anthesis, revealing an association with the d18:1(8E) accumulation. However, the LCB content increased in parallel with the upregulation of the expression of genes for key sphingolipid biosynthetic and LCB modification enzymes during early fruit development in olive. Likewise, we found that EBR exogenously applied to olive trees significantly stimulated the fruit growth rate whereas Brz inhibited fruit growth rate after 7 and 14 days of treatment. In addition, this inhibitory effect could be counteracted by the application of EBR. The promotion of early fruit growth was accompanied by the down-regulation of sphingolipid LCB content and gene expression in olive fruit, whereas Brz application raised levels of sphingolipid LCB content and gene expression in olive fruit after 7 and 14 days of treatment. Thus, our data indicate that endogenous sphingolipid LCB and gene-expression levels are intricately controlled during early fruit development and also suggest a possible link between BR, the sphingolipid content/gene expression, and early fruit development in olive.

Sphingolipid Distribution, Content and Gene Expression during Olive-Fruit Development and Ripening.

Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB [t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.