Here, we used fluorescence microscopy for the in situ localization of Ca2+ ions in intact olive pistils after Fluo- 3 AM injection into inflorescences. Fluo-3 AM, similar to other calcium indicators (like those from the Fura family or Indo-1) must be introduced into the examined cells, and this step is a prerequisite to measure intracellular Ca2+ ions by using microscopy imaging techniques. To introduce this dye into intact pistils, we injected the Fluo-3 solution directly into olive inflorescences. To date, this is the first report on using a Ca2+-sensitive dye in the form of an acetoxymethyl ester to follow Ca2+ behaviour in plant reproductive organs. The presence of the dye inside the cells of the olive pistil indicates the following: (1) The amount of dye solution used was sufficient to penetrate the tissues of the inflorescence peduncle, whole flowers, and floral organs. (2) The concentration of Fluo-3 esters introduced into the inflorescence tissues was enough to eliminate the previously reported potential problem of Fluo-3 ester hydrolysis by cell wall hydrolases [27, 28].
As far as we know, there are no data in the literature reporting the Ca2+ content in whole pistils during their development in angiosperms. Most of the studies on Ca2+ in pistil tissues focused on the period of full maturity and are frequently restricted to defined parts of the pistil, particularly the stigma and ovary [4, 16, 21, 32].
It is well known that Ca2+ is involved in multiple intracellular and intercellular signalling pathways [2, 33]. At the earliest analyzed stage of olive flower development (stage 1), the levels of Ca2+ were quite low. This is probably because buds at this stage are tightly closed and practically isolated from any external biotic and abiotic factors. Furthermore, at this stage, the main task of the flower bud is to complete the growth and maturation of anthers and the pistil. Consequently, the intensity of the signalling events in the stigma of the flower bud is low. As progress in flower development occurred, resulting in gradual petal whitening and flower opening (stage 2), an increase in Ca2+ levels, in parallel with its appearance in the stigma, was observed. At this time of olive flower development, we observed the following: (1) the beginning of exudate production and secretion by papillae cells and (2) accumulation of lipids, pectins, arabinogalactan proteins, and other components in the stigmatic tissues [29, 30]. Such increase in the metabolic activity of stigmatic tissues requires intensification of signalling events, in which Ca2+ is thought to be a key player. At this stage of flower development, we showed the accumulation of Ca/Sb precipitates in the vacuoles of the stigma cells as well as in the intracellular spaces between them. The stigmatic surface is the main place for signal exchange between pollen and stigma. Ca2+ ions are more abundant in the receptive stigmas than in the non-receptive surfaces [16, 34–36]. The highest levels of Ca2+ accumulation were observed in olive stigmatic tissues at the time of pollination. Because in the olive the stigmatic receptivity is closely related with the pollination time, our results support a positive correlation between the Ca2+ levels in the stigmatic exudates and the receptivity state of the stigma in the olive . Thus, we propose that the grade of fluorescence intensity of the incorporated Fluo-3 AM could be used as a potential marker of the degree of stigma receptivity.
The strong decrease of the Ca2+ pool in the pistil at the last stages of pistil development coincides with the degradation of the stigma tissues. The decay of the stigma is the first step in the flower senescence process, which involves structural, biochemical, and molecular changes that lead to programmed cell death (PCD) [37–39]. Flower senescence is also known to be regulated by several signalling pathways involving Ca2+. The presence of Ca2+ in the stigmatic exudate at the end of the anthesis period might suggest that this cation is necessary for the onset of the senescence process . Indeed, Serrano et al.  reported that at the latest stage of olive flower development, once the stigma was completely brown, papillae cells exhibit PCD symptoms as a result of the incompatibility reaction between pollen and papillae stigma cells. In our opinion and according to our results, the papillae cells death is rather a consequence of their developmental program and the Ca2+ accumulation observed in these cells might be one of the PCD hallmarks during stigma senescence.
Significant changes in the stylar Ca2+ pool were also observed at the time of anther dehiscence (stage 4). The Ca2+ labelling in the style was temporally correlated with the receptive phase of the stigma and pollination, since the stigmatic surface was covered with many pollen grains. It supports the involvement of the transmitting tissue in Ca2+ delivery for pollen tube growth. It is well known that pollen tube growth requires Ca2+ ions from the extracellular environment under both in vitro and in vivo conditions [22, 41]. Indeed, the presence of Ca2+ in the style has been reported in Petunia hybrida  and in tobacco . The implication of Ca2+ in pollen tube growth and its guidance during the progamic phase has also been reported in other species [7, 22, 19, 42, 43]. In already pollinated flowers (stage 5), the stigmatic and stylar pool of Ca2+ decreased significantly in comparison to that in stage 4. The low levels of detectable Ca2+ along the style in the olive at this time of the reproduction course indicate that pollen tube growth through the stylar tissues is already complete.
The most striking features of Ca2+ distribution in the olive pistil were observed in the ovary at the time of pollination (stage 4) and fertilization (stage 5). Ca2+ was observed to specifically accumulate in one of the four ovules present in the ovary, whereas the remaining ovules showed no labelling. This localization pattern was observed in more than 80% of the ovaries at stage 4 and in more than 95% of the ovaries at stage 5. It has been established that the micropyle contains high levels of Ca2+, which closely correlate with fertility and serve probably as an attractant for the growing pollen tube . In Nicotiana and Plumbago, the Ca2+ concentration in the micropylar regions reached the peak when the pollen tube arrives [32, 44]. Chudzik and Snieżko  proposed that such an accumulation of Ca2+ may serve as a marker of ovule receptivity. Indeed, at stage 4, in situ accumulation of ovular Ca2+ was observed to start at the micropylar region. However, the presence of this specific "single-ovular" Ca2+ labelling was still observed at the post-anthesis stage of flower development (stage 5) when most of the flowers were successfully fertilized. According to the previous observations that in olive only 1 or 2 (in exceptional cases) ovules are fertilized , we suggest that the observed Ca2+ localization pattern might indicate which ovule will be fertilized or has been already fertilized.
It is well known that post-fertilization events leading to fruit formation include changes in the tissue developmental programs, which implicate a continuous exchange of signals between different types of cells . Ca2+ has been shown to play a crucial role in processes such as egg cell activation [20, 47], gamete fusion [20, 48], or embryo sac degeneration [44, 49]. Given that, we propose that Ca2+ fluorescence can be used as a specific marker of fertilized ovules in multiovular ovaries. However, calcium level could remain high after fertilization of this ovule, so further experiments will be necessary to elucidate which explanation is the correct one.