Characterization of a caleosin expressed during olive (Olea europaea L.) pollen ontogeny

Background: The olive tree is an oil-storing species, with pollen being the second most active site in storage lipid biosynthesis. Caleosins are proteins involved in storage lipid mobilization during seed germination. Despite the existence of different lipidic structures in the anther, there are no data regarding the presence of caleosins in this organ to date. The purpose of the present work was to characterize a caleosin expressed in the olive anther over different key stages of pollen ontogeny, as a first approach to unravel its biological function in reproduction. Results: A 30 kDa caleosin was identified in the anther tissues by Western blot analysis. Using fluorescence and transmission electron microscopic immunolocalization methods, the protein was first localized in the tapetal cells at the free microspore stage. Caleosins were released to the anther locule and further deposited onto the sculptures of the pollen exine. As anthers developed, tapetal cells showed the presence of structures constituted by caleosin-containing lipid droplets closely packed and enclosed by ER-derived cisternae and vesicles. After tapetal cells lost their integrity, the caleosin-containing remnants of the tapetum filled the cavities of the mature pollen exine, forming the pollen coat. In developing microspores, this caleosin was initially detected on the exine sculptures. During pollen maturation, caleosin levels progressively increased in the vegetative cell, concurrently with the number of oil bodies. The olive pollen caleosin was able to bind calcium in vitro. Moreover, PEGylation experiments supported the structural conformation model suggested for caleosins from seed oil bodies. Conclusions: In the olive anther, a caleosin is expressed in both the tapetal and germ line cells, with its synthesis independently regulated. The pollen oil body-associated caleosin is synthesized by the vegetative cell, whereas the protein located on the pollen exine and its coating has a sporophytic origin. The biological significance of the caleosin in the reproductive process in species possessing lipid-storing pollen might depend on its subcellular emplacement. The pollen inner caleosin may be involved in OB biogenesis during pollen maturation. The protein located on the outside might rather play a function in pollen-stigma interaction during pollen hydration and germination.