Storage Lipids in Developing and Germinating Pollen Grain of Flowering Plants

Plants utilize storage lipids as an energy source and to provide carbon during active metabolic periods. These lipids are typically stored as triacylglycerols (TAGs) in various sporophytic organs and tissues, and can be stored in a nearly anhydrous form due to their non-polar nature. The storage lipids are accumulated in specialized organelles called oil bodies (OBs), lipid bodies, lipid droplets or oil globules. Oleaginous plants' mature pollen grains contain abundant oil bodies in their vegetative cytoplasm. However, there is currently limited knowledge about the breakdown and role of these cellular structures in processes directly related to plant reproduction. Previous research on storage lipids in pollen has been limited and fragmented. Therefore, the purpose of this review is to summarize and evaluate current knowledge on OBs in pollen grains, and to highlight the importance of further investigation into the physiological and molecular nature of storage lipids in the reproductive biology of angiosperms.

Lipids are crucial components of cells and serve as important reserves in plants, providing carbon and energy during active metabolic periods. Triacylglycerols (TAGs) are the preferred lipid storage compounds for most eukaryotes due to their non-polar nature and ability to be stored in a nearly anhydrous form. Plants accumulate these storage lipids in specialized organelles called oil bodies (OBs), which have been found in various plant organs and tissues, including male and female gametophytes. However, little is known about the behavior, breakdown, and role of OBs in processes related to sexual plant reproduction, despite their presence in pollen grains of different species.

Previous studies on storage lipids biology in pollen have been limited and fragmented, but recent research has greatly improved our understanding of OB behavior and mobilization during sexual processes in higher plants. Therefore, the goal of this report is to summarize and evaluate current knowledge on pollen OBs, as well as to highlight the importance of further studies into the physiological and molecular nature of storage lipids in the reproductive biology of angiosperms.

The formation of pollen OBs is correlated with the kinetics of gene expression and protein synthesis of enzymatic markers of lipid biosynthesis. Developing pollen of Brassica napus demonstrated a more than five-fold increase in TAG levels from the first until the second pollen mitosis, which was temporally connected to OB accumulation in the cytoplasm and high expression of four lipid biosynthesis genes. In mature Brassica napus pollen, the fatty acid composition of the intracellular membrane and OB membrane was similar, suggesting shared fatty acid biosynthesis.

Seed oil bodies (OBs) and their components, such as triacylglycerols (TAGs), phospholipids (PLs), and oleosins, are formed within specialized microdomains of the endoplasmic reticulum (ER) via acyl-editing of fatty acyl chains in the nitrogenous phospholipids of the ER (Hsieh and Huang, 2004). Similarly, in pollen grains, the ER and vesicles proliferate extensively during cytoplasm maturation and expansion in the vegetative cell (Piffanelli et al., 1998). Previous studies have suggested a connection between the accumulation of compounds necessary for pollen development and pollen tube growth and organelles such as the ER, vacuoles, and Golgi (Rodríguez-García and Fernández, 1990; McCormick, 1993; Yamamoto et al., 2003). In Arabidopsis pollen, highly dilated rough ER (rER) surrounding OBs has been observed both before and after anthesis (Yamamoto et al., 2003), and in developing olive pollen, OBs were often found in direct contact with rER cisternae (Rodríguez-García and Fernández, 1990; Zienkiewicz et al., 2011), supporting the significant role of ER cisternae in the formation of pollen OBs and suggesting a common mechanism of OBs ontogeny in different plant tissues.

The current understanding of OBs' structure has been mainly derived from intensive studies of oilseeds of various species (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 suggests a rather universal structure of these organelles in higher plants. Both pollen and seed OBs are spherical organelles with sizes typically ranging from 0.1 to 2.5 μm, consisting of a TAG matrix surrounded by a single layer of PLs, with a few embedded unique proteins (Piffanelli et al., 1997; Jiang et al., 2007; Zienkiewicz et al., 2010) (Fig. 3).

Oleosins, which are classified into two distinct classes, H- and L-(high and low molecular weight) oleosins, are the major proteins associated with seed OBs (Tzen and Huang, 1992). The H-form differs from the L-form by having an insertion of 18 residues in their C-terminal domain. Most oleosins are relatively small proteins, with molecular weights 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 a "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 has been 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).

The presence of oleosin in tapetum and pollen from various species has been reported in previous studies (Ross and Murphy 1996; Alché et al. 1999; Kim et al. 2002; Jiang et al. 2008). Putative oleosin isoforms were identified in rapeseed and a novel group of oleosins was found in Arabidopsis pollen. A unique oleosin was also discovered as the major protein in lily pollen oil bodies (OBs). Sequence alignment revealed that the C-terminal domain of seed H-oleosins contains an insertion of 18 residues that is absent in lily and Arabidopsis pollen oleosins. Phylogenetic tree analysis further showed that lily pollen oleosin of gametophytic origin is distinct from oleosins found in seed oil bodies and tapetum, which are sporophytic in origin, and may represent a pollen-specific oleosin.

Oleo-pollenins, a family of proteins highly expressed in tapetal cells of anthers, have an N-terminal domain (oleosin-like domain) that is initially similar to the central hydrophobic domain of seed oleosin. These proteins are associated with tapetal lipid droplets via their oleosin-like domain until the tapetal cells undergo apoptosis. The oleosin-like domain is then removed by a specific peptidase to form the mature protein, pollenin, which is transferred to the outer wall of the pollen grains. Pollenins are the most abundant proteins in the pollen coat and are required for rapid hydration of Arabidopsis pollen grain.

Apart from oleosin, two minor proteins, caleosin and steroleosin, have been identified in the seed OBs fraction. Caleosins belong to a large gene family found ubiquitously in higher plants and in several lipid-accumulating fungi. All caleosins contain a calcium-binding site known as the helix-loop-helix EF hand motif, a central hydrophobic region with a potential lipid-binding domain, and a C-terminal region including several conserved protein phosphorylation sites. Caleosin is located on the OBs surface or associated with the ER-subdomain and potentially contributes to OBs stability. It may also be involved in signal transduction via calcium binding or phosphorylation/dephosphorylation in processes such as membrane expansion, lipid trafficking, OBs biogenesis, and degradation. Caleosin possesses peroxigenase activity, suggesting its involvement in phytooxylipin biosynthesis and biotic and abiotic stress response. Monocot seed OBs contain an additional N-terminal appendix of approximately 40-70 residues, making them larger than those in dicotyledonous seed OBs. A unique caleosin isoform distinct from that in seed OBs has been identified in OBs from pollen of lily and olive. However, olive pollen caleosin, similar to seed caleosins, co-localizes with ER structures, is able to bind calcium in vitro, and shows similar structural conformation in OBs membrane like its seed counterpart. Thus, despite their different molecular structures, seed and pollen caleosins seem to have rather conserved functions in OBs formation and stabilization.

Steroleosin, a minor protein associated with oil bodies (OBs), contains 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. Sterol-binding dehydrogenases are implicated in signal transduction in different plant tissues, and it is suggested that in seeds, they specifically facilitate the mobilization of OBs during germination. However, so far, no steroleosin has been found in OBs from generative tissues of higher plants.

In flowers, the anthers are the major lipid-accumulating organs, where pollen development occurs. The anther consists of meiotic cells (microspores or pollen grains) 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. The anther tapetum plays a secretory role in sporogenesis and is involved in pollen wall and pollen coat formation.

Pollen development consists of two major phases: microsporogenesis and microgametogenesis. 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. The mature pollen grain comprises a generative cell or two sperm cells, completely enclosed within the 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. Entomophilous pollen grains accumulate relatively more lipids than anemophilous pollen grains, which accumulate starch as their main reserve. OBs are present in pollen grains of various species and are synthesized mainly in the vegetative cell of the pollen grain. However, OBs have been observed also in both pollen cells of lily and only in the generative cytoplasm in Polystachia pubescens. The accumulation of lipid reserves takes place following the rapid lipid biosynthesis soon after the vacuolation stage of the microspore.

The presence and function of oil bodies (OBs) during pollen development and germination have been extensively studied. According to Zienkiewicz et al. (2011), high levels of OBs-associated proteins are positively correlated with an increase in OB numbers during pollen development. In Arabidopsis thaliana microspores, Kim et al. (2002) found that three genes encoding oleosins were expressed, while Alché et al. (1999) detected oleosin mRNAs in olive developing microspores and pollen grains. Jiang et al. (2007) reported that lily pollen oleosin is accumulated during later stages of pollen maturation, and not at pre-meiosis or microspore stages. The possible function of these oleosins is to stabilize OBs during pollen development and maturity. In contrast, the level of olive pollen caleosin continuously increases after asymmetric mitosis of microspores and during subsequent steps of pollen maturation, and is positively correlated with an increasing number of OBs in developing pollen (Zienkiewicz et al. 2011).

During pollen germination, OBs are rapidly mobilized to serve as an energy supply for pollen tube growth, and as a source for the rapid synthesis of membrane lipids (Dorne et al. 1988; Zienkiewicz et al. 2013). Hydrated olive pollen shows OBs polarizing towards the exine and aperture, and moving towards the emerging pollen tube, most likely due to cytoplasmic streaming (Rodríguez-García et al. 2003). OB mobilization starts after pollen hydration in the olive, and progresses during pollen tube growth (Zienkiewicz et al. 2010, 2013). During this period, the number of OBs decreases almost 20-fold in the pollen grain, whereas the opposite tendency is observed in the pollen tube, suggesting that OBs move from the pollen grain towards the growing pollen tube as soon as the pollen grain begins to germinate. After 12 hours of in vitro germination, the OBs are almost completely metabolized (Zienkiewicz et al. 2010). Sugar removal from the germinating medium does not influence pollen tube growth rate, suggesting that OBs are sufficient as a carbon supply for proper, early pollen tube growth (Zienkiewicz et al. 2013).

In mature pollen grain, OBs are frequently in close contact with the endoplasmic reticulum (ER) cisternae (Rodríguez-García and Fernández 1990), which persists during pollen germination and may facilitate mobilization of the OBs into membrane components. After lily pollen germination, OBs were individually surrounded by tubular membrane structures and encapsulated in vacuoles, suggesting that degradation of OBs during pollen tube elongation might be carried out by vacuolar digestion (Jiang et al. 2007). OB-vacuole membrane fusion in the pollen tube could be mediated by caleosin (Zienkiewicz et al. 2010). Caleosin was detected in olive pollen during the entire germination process, and its level decreased coincidentally with the reduction in the number of OBs present in the pollen tube (Zienkiewicz et al. 2010).

Hydrolytic enzymes such as phospholipase A, lipoxygenase, and lipase, have been shown to be involved in OBs breakdown during germination of seeds (Eastmond 2006; Rudolph et al. 2011). The activation of TAG lipases initiates lipid mobilization, which leads to the release of hydroperoxy derivatives of storage TAGs, which are subsequently cleaved by lipases to release fatty acids.

In addition to the enzymatic breakdown of OBs, studies have also shown the involvement of autophagy in OBs mobilization during pollen germination. Autophagy is a conserved catabolic process that mediates the degradation and recycling of cellular components, including organelles, under stress conditions or during nutrient deprivation (Yoshimoto et al. 2004). It has been suggested that autophagy may play a role in lipid mobilization from OBs in germinating Arabidopsis seeds (Huang et al. 2009). Similarly, autophagic vesicles have been observed surrounding and degrading OBs in germinating maize embryos (Thompson et al. 1995). Recent studies have also indicated that autophagy may play a role in OBs mobilization during pollen germination in Arabidopsis (Li et al. 2020) and tomato (Liu et al. 2021).

Overall, the mobilization of OBs during pollen germination is a complex process that involves the enzymatic breakdown of TAGs by lipases and LOX, as well as autophagic degradation. These processes are likely to be regulated by a variety of factors, including the availability of nutrients, hormonal signaling, and stress responses. Further studies are needed to fully understand the molecular mechanisms underlying OBs mobilization and its regulation during pollen germination.