Cynthia Suárez

Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination

In plant organs and tissues, the neutral storage lipids are confined to discrete spherical organelles called oil bodies. Oil bodies from plant seeds contain 0.6–3% proteins, including oleosins, steroleosins, and caleosins. In this study, a caleosin isoform of ∼30 kDa was identified in the olive pollen grain. The protein was mainly located at the boundaries of the oil bodies in the cytoplasm of the pollen grain and the pollen tube. In addition, caleosins were also visualized in the cytoplasm at the subapical zone, as well as in the tonoplast of vacuoles present in the pollen tube cytoplasm. The cellular behaviour of lipid bodies in the olive pollen was also monitored during in vitro germination. The number of oil bodies decreased 20-fold in the pollen grain during germination, whereas the opposite tendency occurred in the pollen tube, suggesting that oil bodies moved from one to the other. The data suggest that this pollen caleosin might have a role in the mobilization of oil bodies as well as in the reorganization of membrane compartments during pollen in vitro germination.

Structural organization and cytochemical features of the pistil in Olive (Olea europaea L.) cv. Picual at anthesis

Pistil structure and composition are critical in recognizing and permitting the germination of suitable pollen grains. We have studied the structure of the different component tissues of the pistil, their organization and cytochemical features of olive flowers, Olea europaea L., at anthesis, an essential first step for understanding the processes of pollen-pistil interaction and fertilization. The pistil from olive cv. Picual trees is characterized by a wet bilobed stigma, a solid style and a bilocular ovary containing four ovules. The stigma is composed of external multicellular papillae and a non-papillate inner region of secretory cells. An exudate is observed on the surface of the papillae at anthesis, the moment when the flowers (first) open, but the anthers are not yet dehiscent. The inner secretory cells of the stigma and those of the stylar transmitting tissue are continuous, constituting a funnel-shaped zone which extends from within the stigma to the style base. The outer surface of the ovary and style epidermis is surrounded by a cuticle layer, while internally, the locule wall, formed by the innermost cells of the endocarp, consists of two layers of periclinally oriented cells with thicker cell walls. Starch granules are distributed differentially, concentrated most densely in the style (adjacent to the vascular bundles), in the distil region of the ovary, and in the micropylar ends of the ovules. Well-developed vascular bundles are present in the lower part of the stigma, the style and in the pericarp of the ovary. The histochemical identification of sugars and lipid substances within and around the vascular bundles suggests that they are involved in the transport of these materials. Ultrastructural observations confirm the presence of exudates on the papillar surface and confirm the secretory characteristics of the inner stigmatic cells. They also demonstrate marked differences in size, form, and vacuolar and cytoplasmic contents among the cells of the various style and upper ovary tissues. We provide the first detailed cytological description at anthesis of all the olive pistil tissues, indicating the structural and cytochemical basis for the pistil behavior which will transpire during the progamic phase.

Cellular localization and levels of pectins and arabinogalactan proteins in olive ( Olea europaea L.) pistil tissues during development: implications for pollen–pistil interaction

Cell wall components in the pistil are involved in cell–cell recognition, nutrition and regulation of pollen tube growth. The aim of this work was to study the level, whole-organ distribution, and subcellular localization of pectins and arabinogalactan proteins (AGPs) in the olive developing pistil. Western blot analyses and immunolocalization with fluorescence and electron microscopy were carried out using a battery of antibodies recognizing different types of pectin epitopes (JIM7, JIM5, LM5, and LM6) and one anti-AGPs antibody (JIM13). In the olive pistil, highest levels of acid esterified and de-esterified pectins were observed at pollination. Moreover, pollination was accompanied by a slight decrease of the galactose-rich pectins pool, whereas arabinose-rich pectins were more abundant at that time. An increased expression of AGPs was also observed during pollination, in comparison to the pistil at the pre-anthesis stage. After pollination, the levels of pectins and AGPs declined significantly. Inmunofluorescence localization of pectins showed their different localization in the olive pistil. Pectins with galactose residues were located mainly in the cortical zones of the pistil, similar to the neutral pectins, which were found in the parenchyma and epidermis. In turn, the neutral pectins, which contain arabinose residues and AGPs, were localized predominantly in the stigmatic exudate, in the cell wall of secretory cells of the stigma, as well as in the transmitting tissue of the pistil during the pollination period. The differences in localization of pectins and AGPs are discussed in relation to their roles during olive pistil developmental course.

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

BACKGROUND AND AIMS Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination. METHODS Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies. KEY RESULTS Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes. CONCLUSIONS Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube-pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.

Morphological, histological and ultrastructural changes in the olive pistil during flowering

Sexual reproduction is essential for the propagation of higher plants. From an agronomical point of view, this is a particularly key process because fertilization guarantees fruit formation in most cultivated fruit species. In the olive, however, in spite of its agricultural importance, little attention has been paid to the study of sexual reproduction. In order to investigate the cellular and molecular mechanisms that regulate pollen–pistil interactions in the olive during the progamic phase, it is essential to first have a good knowledge of the reproductive structures involved in such interactions. This study characterizes the anatomical and ultrastructural changes in the olive pistil, beginning from the young pistil developing within the bud until the time of petal loss and visible stigma senescence. We have correlated changes in the pistil with a series of defined floral developmental stages and determined that olive pistil structures cannot be considered completely mature and ready to be pollinated and fertilized until the onset of anthesis. Our results clearly show histological and ultrastructural variation during the diverse flowering events. We discuss whether the changes observed might influence or result from pollen–pistil interactions during the progamic phase.

Whole-Organ analysis of calcium behaviour in the developing pistil of olive (Olea europaea L.) as a tool for the determination of key events in sexual plant reproduction

BackgroundThe pistil is a place where multiple interactions between cells of different types, origin, and function occur. Ca2+ is one of the key signal molecules in plants and animals. Despite the numerous studies on Ca2+ signalling during pollen-pistil interactions, which constitute one of the main topics of plant physiology, studies on Ca2+ dynamics in the pistil during flower formation are scarce. The purpose of this study was to analyze the contents and in situ localization of Ca2+ at the whole-organ level in the pistil of olive during the whole course of flower development.ResultsThe obtained results showed significant changes in Ca2+ levels and distribution during olive pistil development. In the flower buds, the lowest levels of detectable Ca2+ were observed. As flower development proceeded, the Ca2+ amount in the pistil successively increased and reached the highest levels just after anther dehiscence. When the anthers and petals fell down a dramatic but not complete drop in calcium contents occurred in all pistil parts. In situ Ca2+ localization showed a gradual accumulation on the stigma, and further expansion toward the style and the ovary after anther dehiscence. At the post-anthesis phase, the Ca2+ signal on the stigmatic surface decreased, but in the ovary a specific accumulation of calcium was observed only in one of the four ovules. Ultrastructural localization confirmed the presence of Ca2+ in the intracellular matrix and in the exudate secreted by stigmatic papillae.ConclusionsThis is the first report to analyze calcium in the olive pistil during its development. According to our results in situ calcium localization by Fluo-3 AM injection is an effective tool to follow the pistil maturity degree and the spatial organization of calcium-dependent events of sexual reproduction occurring in developing pistil of angiosperms. The progressive increase of the Ca2+ pool during olive pistil development shown by us reflects the degree of pistil maturity. Ca2+ distribution at flower anthesis reflects the spatio-functional relationship of calcium with pollen-stigma interaction, progamic phase, fertilization and stigma senescence.

Pollen and pistil features involved in the reproductive biology of the olive (Olea europaea L.)

Resumen de la comunicacion presentada al 28o International Horticultural Congress celebrado del 22 al 27 de 2010 en Lisboa.-- Sm09.028.