Oleosins in Tapetum Cells and the Novel Organelle Tapetosome

The presence of oleosins in tapetum cells of anthers in Arabidopsis and Brassica was discovered a decade ago from unintended gene cloning results

(deOliverira et al. 1993; Roberts et al. 1994). The finding was unexpected because tapetum cells were not known to contain OBs similar to those in seeds. Subsequently, these oleosins were found to be present in a novel, neutral lipid-containing organelle, which has been termed the tapetosome because of its unique presence in the tapetum of plants (Wu et al. 1997). To date, the presence of tapetal oleosins is limited to species of the insect/self-pollinating Brassicaceae family, especially Brassica and Arabidopsis.

In Arabidopsis, nine genes encode the tapetal oleosins, eight of which are in tandem on chromosome 5 (Fiebig et al. 2004; Kim et al. 2002; Schein et al. 2004). One of these genes is highly expressed to produce an oleosin of 53 kDa, which represents about 70% of all tapetal oleosins. Most of the other Arabidopsis tapetal oleosins are smaller (10-23 kDa), but one has 115 kDa. As expected, Brassica has a similar oleosin gene system (Roberts et al. 1994; Ross and Murphy 1996; Ruiter et al. 1997), and the most active gene (ortholog of the Arabidopsis gene encoding the 53-kDa oleosin) produces a major oleosin of 45 or 48 kDa (from the B. rapa AA genome or B. oleracea CC genome, respectively). Genes encoding the tapetal oleosins have undergone rapid evolution that altered the N- and C-terminal regions but not the hairpin regions, as the genes encoding seed oleosins do. Findings of these evolutionary changes reiterate that the N- and C-terminal regions of oleosins have minimal constraints for protein structures, and thus functions.

The tapetum is a one-cell layer enclosing the anther locule, in which microspores mature to become pollen. Tapetum cells are the only anther sporophytic cells that are metabolically very active and control maturation of microspores. At an early stage of anther development, the tapetum cells are specialized for active secretion and contain abundant RER and secretory vesicles. At a late stage of anther development, at least in Brassicaceae species, the cells become a temporary storehouse of ingredients to be deposited onto maturing pollen as pollen coat. The tapetum cells at this late stage of development are packed with two predominant storage organelles, the elaioplasts and tapetosomes (Owen and Makaroff 1995; Platt et al. 1998; Polowick and Sawhney 1990). The elaioplasts, of 3-4 ^m in diameter, are specialized plas-tids largely devoid of thylakoids but filled with small spherical lipid droplets of steryl esters enclosed by the structural protein PAP. Although elaioplasts of similar morphology can be found in nontapetum cells, such as fruit and petal cells, tapetosomes are unique to the tapetum cells. Each spherical tapeto-some, of 2-3 ^m in diameter, has oleosin-coated TAG droplets associated with vesicles derived from ER. These oleosin-coated TAG droplets are similar in structure and constituents to seed OBs.

The contents of tapetosomes and elaioplasts are selectively retained and discharged to the anther locule after death of the tapetum cells during the final stage of anther development. Oleosins, but not TAGs, of tapetosomes and steryl esters, and not the structural protein PAP of elaioplasts are selectively retained and transferred to the pollen surface, forming the bulk of pollen coat

(Wu et al. 1997, 1999). The rationale and mechanism for the selectivity are unclear. It is intriguing that in seed OBs, TAGs are the prime ingredient for physiological function and oleosins are the accessories, whereas in tapeto-somes, oleosins may be the main element for physiological function (to be described) and TAGs are the accessories. The tapetum TAGs disappear after death of the cells, and their function and metabolic fate are unknown. They may be used as an energy source for active metabolism of the tapetum cells. Their fatty acids could also be used to produce jasmonic acid as a floral maturation hormone, or alkanes as one of the two major lipid constituents (the other being the elaioplast steryl esters) for deposition onto maturing pollen. These possibilities are testable with Arabidopsis mutants defective in tapetum TAG synthesis or degradation.

Although the steryl esters and other lipids on pollen form a useful waterproofing layer, the function of the abundant oleosins there is less clear. In Brassica, the predominant 45/48-kDa oleosin on pollen has been cleaved selectively into two fragments, one containing the N-terminal portion and the central hydrophobic stretch, and the other the long hydrophilic C-terminal portion (Ross and Murphy 1996; Ting et al. 1998). Whether other smaller oleosins on pollen are cleaved is not known. The cleavage may be fortuitous in mutation and have no physiological relevance. The most abundant oleosin on pollen has a large size (53 kDa in Arabidopsis and 45/48 kDa in Brassica) owing to its possession of numerous repeats of short peptides at its C terminus. Each of these repeats possesses several glycine residues, which again makes the protein glycine-rich.

Because of its glycine-rich nature, it has been speculated that this pollen oleosin (and extrapolating to other oleosins) might interact the cell walls of the stigma. Such a speculation should be taken with caution. Oleosins have undergone rapid evolutionary changes, and both tapetal and seed oleosins have repeats of short peptides at their C termini; some of these repeats have high glycine contents, whereas others do not. The rapidity and extensiveness of changes at the C termini may reflect the minimal structural constraints on this part of the protein to perform functions. The high glycine contents at the C termini of oleosins may be fortuitous, and certainly the glycine-rich C termini in some seed oleosins do not have an apparent function for interaction with cell walls. In fact, the short repeats at C termini of the most abundant tapetum oleosins have not only a high glycine content but also high serine and lysine contents, making the oleosin also serine-rich and lysine-rich (the Arabidopsis 53-kDa oleosin has 26, 16, and 14 mol %, and the Brassica 48-kDa oleosin 21, 16, and 11 mol % of glycine, serine, and lysine, respectively).

An oleosin molecule may serve dual functions on pollen and subsequently on the stigma because of its amphipathic property. Its N- and C-terminal portions are hydrophilic/amphipathic, and its central portion is hydrophobic. The overall amphipathic oleosin can act as an emulsifying agent to uniformly coat the pollen with steryl esters, alkanes, flavonoids, and other ingredients. It may also aid in water uptake for germination after the pollen grain has landed on the stigma. Brassicaceae species have dry stigmas, and water must be drawn from the stigma interior to the pollen for germination and tube growth. Steryl esters and other neutral lipids are not amphipathic enough to be able to act as a wick. However, the abundant and amphipathic oleosins (and/or flavonoids) could act in this manner. On the basis of these two proposed functions, the mutational addition of repeats of short peptides, which are all fairly hydrophilic, to the C termini and fragmentation of the Brassica 45/48-kDa oleosins into two halves do not affect the function of the oleosins. The proposed functions are also in agreement with the observation that the pollen of an Arabidopsis null mutant in the major pollen-coat oleosin does not hydrate efficiently on the stigma (Mayfield and Preuss 2000). This partial loss of function could have been due to the lack of sufficient oleosins on the pollen to serve as a wick and/or the pollen coat not having been properly emulsified. Overall, the major structural constraints on oleosins to perform the proposed functions are a long hydrophobic stretch to interact with the TAG droplets in tapetosomes (not a function per se but for storage in the organelles) and an amphipathic molecule to emulsify the pollen coat materials and take up water from stigma. All the observed mutational changes on tapetal oleosins have not affected these constraints and are thus extensive because of the lack of selective pressure.

Tapetosomes have a unique morphology (Platt et al. 1998; Wu et al. 1997). Transmission electron microscopy has revealed that in situ each tapetosome has a nonhomogeneous interior whose internal structures cannot be recognized. However, these structures can be observed clearly after the tapeto-somes has been isolated and subjected to osmotic swelling. A tapetosome consists of oleosin-coated TAG droplets associated via ionic linkage with ER-derived vesicles (Fig. 2). Isolated tapetosomes, after a high- or low-pH treatment, can be subfractionated into TAG droplets (which contain oleosins and TAGs), and membranous vesicles (which possess ER-derived calreticulin and luminal binding protein).

Tapetosomes are synthesized via a special mechanism, as revealed in a recent study with immunofluorescence microscopy and transmission electron microscopy (Hsieh and Huang 2005). During early development of a tape-tum cell, the ER luminal protein calreticulin exists as a network, and contains no oleosins. Subsequently, oleosins appear together with calreticulin in the ER network, which possesses centers with a high ratio of oleosin to cal-reticulin. Transmission electron microscopy shows that at this stage massive ER cisternae interconnect the numerous maturing tapetosomes in a cell. Finally, the ER network largely disappears, and solitary tapetosomes containing oleosins and calreticulin prevail. These and other (Platt et al. 1998) microscopical studies, along with findings from subcellular fractionation, allow for a model depicting the biogenesis of tapetosomes from RER (Fig. 2). Initially,

Tapetum Mechanism

Fig. 2 Model for the synthesis of a tapetosome in Brassica tapetum cells. a formation of an oleosin-coated oil droplet from RER by a mechanism similar to that in Fig. 1c. Each oil droplet consists of an oil matrix (light grey) enclosed by a layer of PL (dark) and oleosins (medium grey). b Association of several budding oil droplets and ER cisternae. c A maturing tapetosome containing detached ER vesicles. d A mature tapetosome (modified from Hsieh and Huang 2004)

Fig. 2 Model for the synthesis of a tapetosome in Brassica tapetum cells. a formation of an oleosin-coated oil droplet from RER by a mechanism similar to that in Fig. 1c. Each oil droplet consists of an oil matrix (light grey) enclosed by a layer of PL (dark) and oleosins (medium grey). b Association of several budding oil droplets and ER cisternae. c A maturing tapetosome containing detached ER vesicles. d A mature tapetosome (modified from Hsieh and Huang 2004)

TAG droplets are produced via an ER-budding mechanism identical to that in maturing seeds. These TAG droplets are covered by oleosins and PLs. As many are produced they converge. More ER cisternae are connected to the droplet clusters and eventually break off as vesicles. As a consequence, a tapetosome is formed. During the peak period of tapetosome formation, all the maturing tapetosomes in the cell are interconnected via ER cisternae.

The function of the abundant ER-derived vesicles in tapetosomes remains to be elucidated. These vesicles possess the same basic constituents of cal-reticulin and luminal binding protein as the ER cisternae do. The vesicles in tapetosomes may aid in the transfer of oleosins from lysed tapetum cells to the pollen surface. They may possess proteins that would subsequently exert action on the stigma, such as incompatibility factors and other signaling proteins. They may contain ions such as calcium and boron for the pollen surface; these ions would subsequently modulate the cell wall structures of the stigma. Or, they may contain flavonoids and other secondary metabolites for the pollen surface. Such pollen-surface metabolites are well known, but of uncertain function. Subcellular fractionation and modern microscopy should be used to test the presence of these ingredients in the tapetosome vesicles.

Future studies on the tapetal oleosins and tapetosomes should aim at expanding the existing findings to non-Brassicaceae species, pinpointing the roles of oleosins on pollen, and examining the contents of the ER-derived vesicles in tapetosomes. Working hypotheses exist and are testable. In addition, use of Arabidopsis mutants defective in individual constituents will aid these tests.

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