Mecanismos de rechazo del polen incompatible en olivo (olea europaea l.)

Resumen

Olive trees (Olea europaea L.) have been cultivated for thousands of years in the Mediterranean area and are of paramount importance to both the culture and economy of the region. Spain is the world's main olive oil producer. Among the different Spanish cultivars, Picual is not only the most widely grown variety but also the most important in terms of production. The economical viability of olive-oil production depends directly upon the success of the fruit harvest, which is determined, among other factors, by the proportion of male to female flowers, climatic conditions during fruit setting and self-incompatibility. Self-incompatibility is an intraspecific reproductive barrier for preventing self-fertilization in angiosperms. There are basically two distinct systems of SI: gametophytic and sporophytic, according to their different molecular and genetic control mechanisms. Due to the fact that self-incompatibility systems are not homologous, it is essential to complement the studies carried out with model plants, such as Nicotiana alata or Papaver rhoeas, with data obtained from species of economic importance such us the olive, for which we have very limited molecular and cellular self-incompatibility data. Within this context, a better understanding of the reproductive biology of this species, in particular of the mechanisms controlling self-incompatibility, are important to our being able to resort to the most suitable pollinizers in orchards hosting practically only one olive cultivar. Thus the main aim of this thesis has been to arrive at a better understanding of the reproductive biology of Olea europaea L., Picual cv., paying special attention to the mechanisms involved in hindering incompatible pollen-tube growth. To this end we used different cellular, biochemical and molecular methods to achieve the following objectives: 1) to examine the cellular and molecular changes induced by pollination; 2) to determine enzymatic activities controlling pollen-pistil interaction; 3) to study the molecules and mechanisms involved in the rejection of incompatible pollen. Our structural and histochemical observations showed that the olive pistil presents all the typical features of species displaying stylar gametophytic self-incompatibility response: its pollen is bicellular, the style is solid, the surface of the stigma is covered by an exudate, composed primarily of lipids and proteins, and although a large number of pollen grains germinate and grow in the stigma, only one or two are able to pass through the style. A detailed cytological and cytochemical study showed that the exudate appeared at anthesis, after the disruption of the cuticle covering the stigmal cells, and also the pollen tubes induced a loss of reserve material and vacuolization of stigmal and stylar cells. Detection in situ of enzymatic activity showed that peroxidase, esterase and acid phosphatase were active at the stigma surface before anthesis, demonstrating that the olive presents early receptivity. The highest activity of these enzymes was found when the anthers of the flower were still closed, which avoids self pollination at this early stage, thus encouraging cross-pollination. In addition, ribonuclease activity was detected in the extracellular matrix of styles, as well as inside pollen tubes growing in the upper part of the style. The tests applied to discover whether the rejection of incompatible pollen was associated to programmed cell death (TUNEL assay, DNA degradation analysis, caspase-3-like detection) revealed that the growth of tubes of incompatible pollen is halted in the stylar area in a way that suggests the involvement of this process. Furthermore, any pollen, even when sterile, seemed to accelerate programmed cell death in papillar cells. The detection of reactive oxygen species and nitric oxide by fluorochromes highlighted the fact that these reactive species play a part in pollen-pistil interaction through their control of programmed cell death. We also found that these molecules closely control each other's production during pollen-stigma interaction and that a link exists between peroxynitrite-dependent tyrosine nitration and programmed cell death in self-incompatibility in the olive tree.