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

Abstract

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.

Figure 1

Sudan black B staining of neutral lipids in sections from olive anthers. Light microscopy sections (A-F) -and enlarged views (A'-F')- of olive anthers at the PMC (A and A'), Te (B and B'), Mi (C and C'), YP (D and D'), MBP (E and E') and MP (F and F') stages. Oil bodies are indicated by arrowheads, while circles denote lipidic masses. Ap: pollen aperture; Cy: cytoplasm; En: endothecium; Ep: epidermis; GC: generative cell; L: anther locule; MBP: mid bicellular pollen grain; Mi: microspore; MP: mature pollen grain; N: microspore nucleus; PMC: pollen mother cell; T: tapetum; Te: tetrad; VN: vegetative nucleus; YP: young pollen grain.

Figure 2

Caleosin expression pattern during olive anther development. (A) Coomassie-stained gel of total proteins from olive anthers at the pollen mother cell (PMC), tetrad (Te), microspore (Mi), young pollen grain (YP), mid bicellular pollen grain (MBP) and mature pollen grain (MP) stages. (B) Western blot as in figure 2A probed with a FL anti-Clo3 Ab, followed by an anti-rabbit IgG Alexa 633-conjugated secondary Ab. A band of about 30 kDa (arrow) was recognized by the FL Ab. (C) Densitometric data corresponding to the 30 kDa band from figure 2B.

Figure 3

Fluorescence microscopy localization of caleosin in the olive anther. Sections from olive anthers at the PMC (A), Te (B), Mi (C), YP (D), MBP (E) and MP (F) stages were incubated with a FL anti-clo3 Ab, followed by an anti-rabbit IgG-Alexa Fluor 488-conjugated secondary Ab. Differential interference contrast (DIC) images of serial sections were also obtained to better visualize the different tissues of the anther. En: endothecium; Ep: epidermis; L: anther locule; MBP: mid bicellular pollen grain; Mi: microspore; MP: mature pollen grain; PMC: pollen mother cell; T: tapetum; Te: tetrad; YP: young pollen grain.

Figure 4

Transmission electron microscopy localization of caleosin in the olive tapetum. Sections from olive anthers at the young microspore (A), vacuolated microspore (B), young pollen grain (C) and mature pollen grain (D) stages were incubated with a FL anti-Clo3 Ab, followed by an anti-rabbit IgG-15 nm gold conjugated secondary Ab. Oil body-associated caleosin is indicated by arrowheads, while circles denote ER-associated caleosin. Note that: i) the tapetal cell vesicles fused with the plasma membrane (star), releasing their content to the loculus, and ii) oil bodies merged (arrows) to produce larger OBs and lipoid masses (asterisks). CW: cell wall; En: endothecium; L: anther locule; TC: tapetal cell; T: tapetum.

Figure 5

Transmission electron microscopy localization of caleosin in the olive pollen and locular fluid. Sections from olive anthers at the mid microspore (A), young pollen grain (B), mid bicellular pollen grain (C) and mature pollen grain (D) stages were treated as in figure 4. Gold labelling is indicated by arrowheads. (E) Control reaction by omitting the FL anti-Clo3 Ab showing the absence of gold labelling. CW: cell wall; Ex: exine; In: intine; L: anther locule; OB: oil body; PC: pollen coat; S: starch; TC: tapetal cell.

Figure 6

Subcellular distribution of caleosin in the olive pollen grain. Immunoblots were probed with a FL anti-Clo3 Ab, followed by an anti-rabbit IgG Alexa 633-conjugated secondary Ab. A single band of about 30 kDa was detected in all pollen fractions (arrow). MF: microsomal fraction; OB: oil body fraction; PC: pollen coat proteins.

Figure 7

Effects of calcium ions on electrophoretic mobility of olive pollen caleosin. Effects of calcium ions on electrophoretic mobility of caleosin isolated from pollen OBs (A) and pollen coat (B). Proteins were extracted from pollen OBs and pollen coat and pre-treated with EGTA. Ten μg of proteins were incubated with CaCl₂ (left lane), CaCl₂ followed by EGTA (middle lane), or both CaCl₂ and EGTA simultaneously (right lane). Immunoblots were probed with a FL anti-Clo3 Ab, followed by an anti-rabbit IgG Alexa 633-conjugated secondary Ab. Arrows show caleosin bands. Protein markers (kDa) are indicated on the left.

Figure 8

PEGylation of olive pollen OBs and immunodetection of caleosin by Western blotting. Oil bodies were isolated from olive pollen, incubated with PEG-MAL (5,000 Da), run on a 7.5% polyacrylamide Bis-Tris gel and transferred onto a PVDF membrane. Oil body-associated caleosin was immunodetected using an αN anti-Clo3 Ab, followed by an anti-rabbit IgG-DyLight 549 conjugated secondary Ab. One prominent higher molecular weight band (arrow), which corresponds to one modified Cys residue, was visible in PEGylated OBs but not in the control (i.e. non-PEGylated OBs).

Figure 9

PEGylation of olive pollen OBs and localization of caleosin by fluorescence microscopy. (A) Localization of caleosin on PEGylated (+) and non-PEGylated (-) OBs from pollen using a FL anti-Clo3 caleosin Ab, followed by a secondary Ab conjugated with DyLight 549. After PEGylation, the accessibility of the FL anti-Clo3 Ab was hampered and no fluorescence was observed. (B) Localization of caleosin as above but using an N-terminal (αN) anti-Clo3 caleosin Ab. After PEGylation, the primary Ab was able to bind to caleosin.