ABSTRACT
In plants, storage lipids serve as energy source and provide carbon equivalents for periods of active metabolism. They can be stored in different sporophytic organs and tissues usually in the form of triacylglycerols (TAGs), which are non-polar and can be stored in a nearly anhydrous form. Plants accumulate the storage lipids in specialized organelles called oil bodies (OBs), lipid bodies, lipid droplets or oil globules. Mature pollen grain of oleaginous plants present copious oil bodies in the vegetative cytoplasm. However, little is known about the behavior, breakdown and role of these cellular structures in processes directly connected to sexual plant reproduction. Up to now, data on storage lipids biology in pollen were rather few and fragmentary. The aim of this review is to sum up and verify the current knowledge on pollen grain OBs as well as to expose the significance of further studies on physiological and molecular nature of storage lipids in reproductive biology of angiosperms.
Introduction
Lipids are major and vital cellular constituents. In plants, stored lipids reserves play an important role in the life cycle by providing carbon and energy equivalents for periods of active metabolism (Graham 2008; Murphy 2012). For most eukaryotes, the preferred storage compounds are lipids in the form of triacylglycerols (TAGs), which are non-polar and can be stored in a nearly anhydrous form. Plants accumulate the storage lipids in specialized organelles called oil bodies (OBs), lipid bodies, lipid droplets or oil globules (Murphy 2012). These organelles have been found in different sporophytic plant organs and tissues, like oil seeds and oleaginous fruits (Huang 1994) as well as in cells of both, male (Piffanelli et al. 1998; Rodríguez-García et al. 2003; Zienkiewicz et al. 2010) and female gametophyte (Wu et al. 1999; Jiang et al. 2009). However, despite the obvious presence of OBs in pollen grains of different species (Fig. 1) little is known about the behavior, breakdown and role of these cellular structures in processes directly connected to sexual plant reproduction.
So far, data on storage lipids biology in pollen were rather few and fragmentary. Recent studies of our group strongly improved our knowledge on behavior and mobilization of pollen OBs and proved the high importance of storage lipids during sexual processes in higher plants. Thus, the major goal of this report is to sum up and verify the current knowledge on pollen OBs as well as to expose the significance of further studies on physiological and molecular nature of storage lipids in reproductive biology of angiosperms.
The Biogenesis and Structure of Pollen Oil Bodies—Facts, Models and Theory
Formation of pollen OBs is correlated with the kinetics of gene expression and protein synthesis of enzymatic markers of lipid biosynthesis (Piffanelli et al. 1997). More than five-fold increase in the TAGs levels was demonstrated in developing pollen of Brassica napus from the first until the second pollen mitosis (Piffanelli et al. 1997). These events were temporally connected with OBs accumulation in pollen cytoplasm and with high expression of four lipid biosynthesis genes SAD, EAR, CYB5 and ACP, encoding stearoyl ACP desaturase, enoyl ACP desaturase, cytochrome b5, and acyl carrier protein, respectively (Piffanelli et al. 1997). In mature Brassica napus pollen fatty acid composition of the intracellular membrane and OBs membrane was found to be similar, which may reflect their shared fatty acid biosynthesis (Piffanelli et al. 1997, 1998).
The biogenesis of seed OBs and their constituents TAGs, PLs and oleosins occurs in specialized endoplasmatic reticulum (ER) microdomains and involves acyl-editing of fatty acyl chains within the nitrogenous phospholipids of the ER (Hsieh and Huang 2004). In the pollen grain the intracellular membrane systems is characterized by extensive proliferation of ER and vesicles during maturation and expansion of the cytoplasm in the vegetative cell (Piffanelli et al. 1998). Previously, it was suggested that ER as well as vacuoles and Golgi are linked to the accumulation of compounds necessary for pollen development and pollen tube growth (Rodríguez- García and Fernández 1990; McCormick 1993; Yamamoto et al. 2003). Indeed, highly dilated rough ER (rER) surrounding OBs was observed before and after anthesis in Arabidopsis pollen (Yamamoto et al. 2003). Similarly in the developing olive pollen OBs were often found in the direct contact with rER cisternae (Fig. 2) (Rodríguez-García and Fernández 1990; Zienkiewicz et al. 2011). These data support the significant role of ER cisternae in the formation of pollen OBs and suggest common mechanism of OBs ontogeny in different plants tissues.
The current model of OBs structure have risen from intensive studies in seeds of several species, mainly those in oilseeds (Tzen and Huang 1992; Tzen et al. 1993; Murphy 2001; Purktova et al. 2008; Tzen 2012). The presence of OBs across a wide range of organisms and their highly conserved molecular composition, suggest rather universal structure of these organelles in higher plants. Pollen OBs as well as seed OBs are spherical organelles with a size usually ranging from 0.1 to 2.5 μm and consist of a TAG matrix, surrounded by a single layer of phospholipids (PLs) with a few embedded unique proteins (Piffanelli et al. 1997; Jiang et al. 2007; Zienkiewicz et al. 2010) (Fig. 3).
Oleosins are the major proteins associated with seed OBs and are classified in two distinct classes, H- and L-(high and low molecular weight) oleosins (Tzen and Huang 1992). The H-form differs from the L-form by having insertion of 18 residues in their C-terminal domain. Most oleosins are relatively small proteins with molecular weight ranging from 15 to 26 kDa depending on the isoforms and plant species (Huang 1996; Murphy 1993). All oleosins have three domains: a hydrophilic N-terminal domain, a hydrophobic central domain containing ‘proline knot’ motif in its center, and a hydrophilic C-terminal domain (Tzen et al. 1993; Abell et al. 2004). Oleosins play an essential role in the stability of seed OBs, preventing coalescence of OBs during seed desiccation (Leprince et al. 1998). It was demonstrated that the size of OBs is correlated with oleosin content in seeds and might be regulated by oleosins (Ting et al. 1996; Siloto et al. 2006; Shimada et al. 2008).
In addition to seeds, oleosin has been found in tapetum and pollen of different species (Ross and Murphy 1996; Alché et al. 1999; Kim et al. 2002; Jiang et al. 2008). Putative oleosin isoforms were found in the pollen of rapeseed (Roberts et al. 1995) and a novel group of oleosins was identified in Arabidopsis pollen (Kim et al. 2002). A unique oleosin was found as the major protein of lily pollen OBs (Jiang et al. 2007). Sequence alignment showed that insertion of 18 residues in the C-terminal domain of seed H-oleosins is absent in the lily and Arabidopsis pollen oleosins. Moreover, phylogenetic tree analysis indicated that lily pollen oleosin of gametophytic origin is different from oleosins found in seed oil bodies and tapetum (sporophytic) and might represent a pollen-specific oleosin (Jiang et al. 2007).
Oleosin-like proteins or oleo-pollenins are a family of proteins whose members are highly expressed in tapetal cells of anthers (Robert et al. 1994; Alché et al. 1999; Ross and Murphy 1996; Murphy 2001). Oleo-pollenins initially contain the N-terminal domain (oleosin-like domain) that is similar to the central hydrophobic domain of seed oleosin (Murphy 2001). These proteins are associated with tapetal lipid droplets via their oleosin-like domain until the tapetal cells undergo apoptosis. At this point, the oleosinlike domain is removed by a specific peptidase to form the mature protein, pollenin, which is transferred to the outer wall of the pollen grains (Ting et al. 1998). Pollenins are the most abundant proteins in the pollen coat and are required for rapid hydration of Arabidopsis pollen grain (Mayfield and Preuss 2000).
In addition to oleosin, two minor proteins namely caleosin and steroleosin have been identified in seed OBs fraction (Frandsen et al. 1996; Lin et al. 2002; Lin and Tzen 2004). Caleosins belong to a large gene family found ubiquitously in higher plants and in several lipid-accumulating fungi, such as Neurospora crassa and Aspergillus nidulans (Murphy 2001). All caleosins contain, a calcium-binding site known as helix-loop-helix EF hand motif capable of binding a single calcium atom, a central hydrophobic region with a potential lipid-binding domain, and a C-terminal region including several conserved protein phosphorylation sites (Frandsen et al. 1996; Naested et al. 2000). Caleosin is located on the OBs surface or associated with ER-subdomain (Naested et al. 2000). This protein potentially contributes to OBs stability and might be involved in signal transduction via calcium binding or phosphorylation/dephosphorylation in processes such a membrane expansion, lipid trafficking or OBs biogenesis and degradation (Chen et al. 1999; Poxleitner et al. 2006). Moreover, it was demonstrated that caleosin possess peroxigenase activity, suggesting its involvement in phytooxylipin biosynthesis and biotic and abiotic stress response (Hanano et al. 2006; Kim et al. 2011). Caleosins in monocot seed OBs contain an additional N-terminal appendix of approximately 40–70 residues, therefore are larger than those in dicotyledonous seed OBs (Liu et al. 2005; Chen et al. 2012). Recently, a unique caleosin isoform distinct from that in seed OBs has been identified in OBs from pollen of lily and olive (Olea europaea L.) (Jiang et al. 2008; Zienkiewicz et al. 2010, 2011). However, olive pollen caleosin, similar to seed caleosins co-localize with ER structures, is able to bind calcium in vitro and shows similar structural conformation in OBs membrane like its seed counterpart (Zienkiewicz et al. 2011). Thus, despite different molecular structure, seed and pollen caleosins seem to have rather conserved functions in OBs formation and stabilization.
A second minor OBs-associated protein is steroleosin. These proteins contain a small N-terminal OBs anchoring domain and a large soluble sterol binding dehydrogenase domain that belongs to a super-family of pre-signal proteins (Lin et al. 2002; Tzen et al. 2003). Sterol-binding dehydrogenases are implicated in signal transduction in different plant tissues and in the seed it is suggested that they specifically facilitate mobilization of OBs during germination (Lin et al. 2002). So far no steroleosin have been found in OBs from generative tissues of higher plants.
Storage Lipids Behavior during Pollen Development
The major lipid-accumulating organs of flowers are the anthers, where pollen development occurs. The anther consists of the meiotic cells (microspores or pollen grain) at the center, surrounded by the tapetum and by the anther wall somatic layers (sporophytic tissues) namely, from outside to inside, epidermis, endothecium and middle layers (Goldberg et al. 1993). The anther tapetum plays a secretory role in sporogenesis and is involved in pollen wall and pollen coat formation (Scott et al. 2004; Zhu et al. 2008).
Pollen development consists of two major phases: microsporogenesis and microgametogenesis (McCormick 1993). This process begins when pollen mother cells (PMC) produce a tetrad of haploid microspores after meiosis, which are encased in a callose (β-1, 3-glucan) wall. After callose degradation microspores are released into the anther loculus and after a period of microspore maturation, they undergo mitosis to finally produce pollen grains. Mature pollen grain comprises a generative cell or two sperm cells, completely enclosed within cytoplasm of the vegetative cell. During the long period of pollen maturation, the vegetative cell accumulates storage compounds like carbohydrates and lipids, which will be used for pollen germination and early pollen tube elongation (Bednarska 1988; McCormick 1993; Rodríguez-García et al. 2003; Zienkiewicz et al. 2010, 2011, 2013). The entomophilous pollen grains accumulate relatively more lipids than anemophilous pollen grains, which accumulate starch as their main reserve (Baker and Baker 1979; Piffanelli et al. 1998). The presence of OBs in pollen grains has been reported in several species such as Brassica napus, Tradescantia bracteata, Gossypium hirsutum, Lilium longiflorum, Arabidopis thaliana or Olea europaea (Mepham and Lane 1970; Charzyńska et al. 1989; Wetzel and Jensen 1992; Van Aelst et al. 1993; Rodríguez-García et al. 2003; Jiang et al. 2007; Zienkiewicz et al. 2011). Pollen OBs are synthesized mainly in the vegetative cell of the pollen grain as it has been reported in Olea europaea (Rodríguez-García et al. 2003), Brassica napus (Charzyńska et al. 1989) or Arabidopsis thaliana (Owen and Makaroff 1995). However, OBs have been observed also in both pollen cells of lily (Nakamura and Miki- Hirosige 1985) and only in the generative cytoplasm in Polystachia pubescens (Schlag and Hesse 1992). The accumulation of lipid reserves takes place following the rapid lipid biosynthesis, soon after the vacuolation stage of the microspore (Fig. 4) (Evans et al. 1992; Piffanelli et al. 1997; Rodríguez- García et al. 2003; Zienkiewicz et al. 2011).
The increase of OB numbers during pollen development is positively correlated with high levels of OBs-associated proteins (Zienkiewicz et al. 2011). The expression of three genes encoding oleosins was found in the microspores of Arabidopsis thaliana (Kim et al. 2002). Oleosin mRNAs were detectable also in the olive developing microspore and pollen grain (Alché et al. 1999). In contrast, lily pollen oleosin is accumulated at further steps of pollen maturation but not at the pre-meiosis and microspore stages (Jiang et al. 2007). Possible function of these oleosins could be stabilization of OBs during pollen development and maturity. The level of olive pollen caleosin continuously increase after the asymmetric mitosis of microspore and during the subsequent steps of pollen maturation, and is positively correlated with increasing number of OBs inside developing pollen (Zienkiewicz et al. 2011).
OBs Mobilization during Pollen Germination
Fertilization in flowering plants relies on the growth and elongation of the pollen tube, which delivers the sperm cells to the female gametophyte in the ovule (Russell 1991). Rapid elongation of the pollen tube demands an energy production and biosynthetic capacity (Taylor and Hepler 1997). The mobilization of storage OBs resumes at a more rapid rate following germination and growth of the pollen tube (Piffanelli et al. 1998). It has been proposed that OBs serves as energy supply for the pollen tubes growth and as source for the rapid synthesis of membrane lipids after germination (Dorne et al. 1988; Zienkiewicz et al. 2013). In hydrated olive pollen, OBs polarize towards the exine and aperture and move to the emerging pollen tube, which is most likely caused by cytoplasmic streaming (Rodríguez- García et al. 2003). In the olive, OBs mobilization starts after pollen hydration and progress during the pollen tube growth (Zienkiewicz et al. 2010, 2013). During this period, the number of OBs decrease almost 20-fold in the pollen grain, whereas the opposite tendency is observed in the pollen tube, suggesting that oil bodies moved from the pollen grain towards the growing pollen tube as soon as the pollen grain begins to germinate (Fig. 5). After 12 h of in vitro germination the OBs were almost completely metabolized (Zienkiewicz et al. 2010). Moreover, sugar removal from the germinating medium did not influence pollen tube growth rate, suggesting that OBs are sufficient as carbon supply for proper, early pollen tube growth (Zienkiewicz et al. 2013).
In mature pollen grain, OBs are frequently in close contact with the ER cisternae (Rodríguez-García and Fernández 1990), which persist during pollen germination and may facilitate mobilization of the OBs into membrane components. After lily pollen germination the OBs were individually surrounded by tubular membrane structures, encapsulated in the vacuoles (Jiang et al. 2007). These results suggested that degradation of OBs during pollen tube elongation might be carried out by vacuolar digestion. The apparent fusion of OBs with vacuoles has also been reported during seed germination in Arabidopsis (Poxleitner et al. 2006) and proposed as part of the TAG mobilization. Moreover, it was found that caleosin participates in OBs-vacuole interactions (Poxleitner et al. 2006). In the pollen tube of the olive, caleosin was localized in the intracellular membranes and in the tonoplast (Zienkiewicz et al. 2010). Therefore, it might be possible that caleosin mediate OB-vacuole membrane fusion in pollen tube (Zienkiewicz et al. 2010). Caleosin was detected in olive pollen during the whole germination process and its level decreased coincidentally with the reduction in the number of OBs present in the pollen tube (Zienkiewicz et al. 2010).
In germinating seeds OBs breakdown occurs by the action of hydrolytic enzymes such as phospholipase A, lipoxygenase and lipase (Eastmond 2006; Rudolph et al. 2011). Lipid mobilization is initiated by the activation of TAG lipases, which hydrolyze TAGs and release glycerol and fatty acids (Graham 2008). The free fatty acids are then transported to the glyoxysome where their β-oxidation occurs. In an alternative pathway of OBs breakdown, lipoxygenase activity leads to oxygenation of storage TAGs to their hydroperoxy derivatives, which are subsequently cleaved by lipases (Feussner et al. 2001). However, before lipase and LOX get access to the TAGs it requires the proteolytic degradation of structural proteins of OBs and partial degradation of phospholipid monolayer of this organelle presumably by a patatin-like phospholipase (Matsui et al. 1999; Rudolph et al. 2011). Recently, we have demonstrated that phospholipase A, lipoxygenase and lipase are also likely involved in OBs mobilization during pollen tube growth (Zienkiewicz et al. 2013). The presence of phospholipase A, lipoxygenase and lipase activities associated to the surface of OBs was demonstrated during olive pollen germination. Interestingly, ‘phospholipase A’ activity on the OBs surface was detected in mature and germinated pollen, meanwhile lipase activity and lipoxygenase protein were associated with OBs boundaries after germination (Zienkiewicz et al. 2013). These results suggest that the mobilization of storage lipids by lipase and lipoxygenase during pollen germination is promoted by a phospholipase A (Zienkiewicz et al. 2013). Moreover, the capacity of olive pollen to germinate was strongly hampered by lipoxygenase and lipase inhibitors (Rejón et al. 2012; Zienkiewicz et al. 2013). This effect was usually accompanied by an accumulation of OBs at the germinative aperture. Taken together, all these data support the essential role of storage lipids in pollen tube growth and a strong functional relationship between OBs mobilization pathway and pollen performance.
In oilseeds, after germination free fatty acids derived from TAGs produces acetyl-CoA, which is converted to sucrose through the glyoxylate cycle and gluconeogenesis (Eastmond et al. 2000). Two enzymes unique to the glyoxylate cycle, malate synthase (MS) and isocitrate lyase (ICL) have been localized in seed gloxysomes. The presence of glyoxysomelike microbodies (Charzyńska et al. 1989) and expression of MS and ICL genes was also indicated in developing Brassica napus pollen (Zhang et al. 1994). However, expression of both genes is not activated during pollen germination when OBs are mobilized (Zhang et al. 1994). The presence of a functional glyoxylate cycle during pollen germination has yet to be confirmed.
Conclusions
Lipids provide the structural basis for cell membranes and fuels for metabolism in all living organisms. Recently, it has been found that lipids function also as mediators in many plant processes including signal transduction, cytoskeletal rearrangements and membrane trafficking. These processes are crucial for cell survival, growth and differentiation and for plant response to water, temperature, salinity and pathogens. It should be clearly stated, that understanding of physiological and molecular nature of pollen performance is extremely important because of its direct connection with food production, industrial biotechnology and medicine. However, it can be seen that our knowledge of plant lipid biology during sexual plant reproduction is insufficient. Despite highly developed methods of molecular biology we still don’t know which, how many and how proteins associated to lipid metabolism are involved in cellular processes regulated by lipids. In this context pollen grain still remains unexplored. Thus, further investigations should be extended in order to explore the pollen machinery directly connected to conversion of storage lipids into substrates used in lipid signaling, energy production and cellular membranes synthesis during pollen performance, with a special emphasis on pollen-pistil interaction.