Lipotubuloids in ovary epidermis of Ornithogalum umbellatum act as metabolons: suggestion of the name ‘lipotubuloid metabolon’

Highlight

Lipotubuloids, cytoplasmic domains of Ornithogalum umbellatum ovary epidermis, have been called lipotubuloid metabolons, as within these regions the structures and enzymes associated with lipid metabolism have been documented.

Abstract

A metabolon is a temporary, structural–functional complex formed between sequential metabolic enzymes and cellular elements. Cytoplasmic domains called lipotubuloids are present in Ornithogalum umbellatum ovary epidermis. They consist of numerous lipid bodies entwined with microtubules, polysomes, rough endoplasmic reticulum (RER), and actin filaments connected to microtubules through myosin and kinesin. A few mitochondria, Golgi structures, and microbodies are also observed and also, at later development stages, autolytic vacuoles. Each lipotubuloid is surrounded by a tonoplast as it invaginates into a vacuole. These structures appear in young cells, which grow intensively reaching 30-fold enlargement but do not divide. They also become larger due to an increasing number of lipid bodies formed in the RER by the accumulation of lipids between leaflets of the phospholipid bilayer. When a cell ceases to grow, the lipotubuloids disintegrate into individual structures. Light and electron microscope studies using filming techniques, autoradiography with [³H]palmitic acid, immunogold labelling with antibodies against DGAT2, phospholipase D1 and lipase, and double immunogold labelling with antibodies against myosin and kinesin, as well as experiments with propyzamide, a microtubule activity inhibitor, have shown that lipotubuloids are functionally and structurally integrated metabolons [here termed lipotubuloid metabolons (LMs)] occurring temporarily in growing cells. They synthesize lipids in lipid bodies in cooperation with microtubules. Some of these lipids are metabolized and used by the cell as nutrients, and others are transformed into cuticle whose formation is mediated by cutinsomes. The latter were discovered in planta using specific anti-cutinsome antibodies visualized by gold labelling. Moreover, LMs are able to rotate autonomously due to the interaction of microtubules, actin filaments, and motor proteins, which influence microtubules by changing their diameter.

Introduction

A metabolon has been defined as a ‘temporary, structural–functional, supramolecular complex of sequential metabolic enzymes and cellular elements, in which metabolites are passed from one active site to another without complete equilibration with the bulk cellular fluids’ (Srere, 1985). This substrate channelling should decrease the transit time of intermediates, prevent their loss through diffusion, protect labile intermediates from solvent, and prevent their entrance into competing metabolic pathways (Deitmer and Becker, 2013). The proposed beneficial role of metabolons includes increasing the concentration of intermediates at the active sites of sequential enzymes and minimizing the reactive or potentially toxic intermediates (Srere, 1987; Winkel, 2004; Jørgensen et al., 2005; Moller, 2010; Sweetlove and Fernie, 2013; Singleton et al., 2014).

The term ‘metabolon’ has been used to describe various functional and structural complexes. In plants, the following have been called metabolons: escape of the synthase–cysteine complex, the Calvin cycle, cyanogenic glucoside synthesis, and the phenylpropanoid pathway (Jørgensen et al., 2005). In addition, the urea cycle of rat hepatocytes (Cheung et al., 1989), the citrate cycle of porcine liver (Shatalin et al., 1999), and the glycolytic pathway of rabbit skeletal muscle and mouse fibroblasts (Vértessy and Ovádi, 1987; Clegg and Jackson, 1990) have been termed metabolons. In Saccharomyces cerevisiae, a glycolytic metabolon stabilized by F-actin has been described (Araiza-Olivera et al., 2013). Examination of photosynthesis in Arabidopsis with radioactive CO₂ revealed that the photosynthetic complex functions as a metabolon (Szecowka et al., 2013). Similar complexes have been identified in spinach chloroplasts (Linden and Schilling 1984) and in sunflower (Ito and Mitsumori 1992), and tobacco (Hasunuma et al., 2010) leaves.

Here, we suggest the term ‘lipotubuloid metabolon’ (LM) as a complex of enzymes similar to those mentioned above but also combined with specific cellular structures enclosed together in one cytoplasmic domain, the lipotubuloid.

Structure and function of lipotubuloids as LMs

Specific cytoplasmic regions called lipotubuloids (Kwiatkowska, 1971a , b) can also act as metabolons and have been termed LMs. They were first described in Ornithogalum umbellatum ovary epidermis cells (Fig. 1). They are rich in ribosomes forming numerous polysomes and rough endoplasmic reticulum (RER). Moreover, they contain numerous lipid bodies entwined with microtubules, which in turn are connected by myosin and kinesin to actin filaments (Kwiatkowska et al., 2013). In LMs, there are also very few mitochondria, Golgi structures, or microbodies (peroxisomes and glyoxysomes). On the basis of the literature concerning lipotubuloids, it can be concluded that these are very intricate complexes, structurally and functionally integrated by the cytoskeleton, that form lipid and cuticle elements.

Fig. 1.

O. umbellatum lipotubuloid metabolon (LM). (A) In living cells. (B) After OsO₄ fixation. l, LM; n, nucleus; no, nucleolus. Bar, 10 µm. (Reproduced from Kwiatkowska et al., 2012b . Originally published in: Montanaro G, Lichio B, eds. Advances in selected plant physiology aspects. InTech, Rijeka, 365–388, under CC BY 3.0 licence. Available from: http://dx.doi.org/10.5772/34442)

Lipotubuloids appear in very young cells of ovary epidermis as a result of aggregation of single lipid bodies (Fig. 2). Subsequently, along with the growing cell, they increase in size because endoplasmic reticulum (ER)-mediated formation of new lipid bodies takes place in them as a result of lipid accumulation between the leaflets of the phospholipids bilayer (Fig. 3) (Kwiatkowska et al., 2012a ). In ovaries changing into fruit, when epidermal cells stop growing, lipotubuloids disintegrate into separate lipid bodies (Fig. 2), following the disappearance of the surrounding microtubules (Kwiatkowska, 1971a ). Thus, the microtubules create a system that holds the whole domain together (Kwiatkowska et al., 2007).

Fig. 2.

O. umbellatum ovary epidermis and LM development. (A) Epidermis from 1mm long ovary with a few gathering lipid bodies. (B–D) LMs increasing in size. (E) Cells with spherical LMs. (F, G) Cells that have ceased growing, with (F) LMs at initial stages of disintegration (F), followed by LM disintegration (G). L, lipotubuloid; Lb, lipid bodies; N, nucleus. Reproduced from Kwiatkowska et al., 2007. Role of DNA endoreduplication, lipotubuloids, and gibberellic acid in epidermal cell growth during fruit development of Ornithogalum umbellatum. Journal of Experimental Botany 58, 2023–2031, by permission of the Society of Experimental Biology.

Fig. 3.

Scheme showing an LM fragment adhering to the tonoplast, showing ER-mediated formation of new lipid bodies, autolytic vacuoles (AV), cytoplasmic filament (cf), cutinsome (Cs), endoplasmic reticulum (ER), Golgi structures (GA), lipid bodies in statu nascendi (iLb); lipid bodies (Lb) surrounded by a half unit membrane, entwined by microtubules (mt), a mitochondrion (M), polysomes (Pr), ribosomes (r), tonoplast (t), and vacuoles (V).

Before lipotubuloid breakdown, typical autolytic vacuoles appear inside them (Fig. 4). They are surrounded by microtubules and contain lipase and acid phosphatase, which digest the lipotubuloid elements (Kwiatkowska et al., 2011a , 2012b ).

Fig. 4.

O. umbellatum ovary epidermis electron microscopy image of a LM, showing autolytic vacuoles (AV), endoplasmic reticulum (ER), lipid bodies (Lb) surrounded by a half unit membrane, entwined by microtubules (mt), microbody (Mb), mitochondrion (M), vacuoles (V), and tonoplast (t). Bar, 300nm.

Investigations of [³H]palmitic acid incorporation at the cellular level have shown that lipotubuloids as LMs are the site of intensive lipid synthesis, which was shown by selective labelling of these domains and visualized with silver grains during the classic process of photographic development. After 6h of incubation in medium without palmitic acid, silver grains corresponding to metabolized lipids became scattered all over the cell (Figs 5 and 6A, B) (Kwiatkowska et al., 2012b ), while in a cross-section of the epidermis, the remaining silver grains could be seen in a cuticular layer on the cell wall as cuticle components, cutin, and waxes (Kwiatkowska et al., 2011a, 2012a ). The grains indicated that lipids disappeared following organic solvent extraction of fats (Kwiatkowska, 1972a, 2004). This gave rise to the suggestion that lipotubuloids as the sites of lipid synthesis should be termed metabolons.

Fig. 5.

Fragment of O. umbellatum ovary epidermis LM with palmitic acid (PA) and cutinsomes (Cs), narrow microtubules (Nmt), phospholipase D (PLD), endoplasmic reticulum (ER), polysomes (Pr), wide microtubules (Wmt), actin filament (Af), diacylglycerol acyltransferase 2 (DGAT2), lipase (Lp), kinesin (k), and myosin (m).

Fig. 6.

Lipotubuloids as metabolons: cytochemical identification of palmitic acid and enzymes participating in lipid biosynthesis in epidermal cells of O. umbellatum ovary. (A, B) Autoradiography with [³H]palmitic acid incorporation (according to Kwiatkowska, 1972b ): lipotubuloids labelled during incubation (A) and scattering labelling throughout a cell during the post-incubation period (B). Bars, 10 µm. (C–I) Immunogold electron microscopy: (C) DGAT2 (Reproduced from Kwiatkowska et al., 2011a . Lipid bodies in lipotubuloids of Ornithogalum umbellatum ovary epidermis contain diacylglicerol acyltransferase 2 (DGAT2) and lipase, incorporate ³H-palmitic acid and are connected with cuticle synthesis. Trends in Cell and Molecular Biology 6, 97–108); (D) phospholipase D1 localized in lipotubuloid at microtubules and at the periphery of lipid bodies (according to Kwiatkowska et al., 2012a ); bar, 100nm; (E) lipase identified in autolytic vacuoles and at the lipid body periphery of lipotubuloids; bar, 500nm (Reproduced from Kwiatkowska et al., 2011a. Lipid bodies in lipotubuloids of Ornithogalum umbellatum ovary epidermie contain diacylglicerol acyltransferase 2 (DGAT2) and lipase, incorporate ³H-palmitic acid and are connected with cuticle synthesis. Trends in Cell and Molecular Biology 6, 97–108); (F) cutinsomes labelling the lipotubuloids, cytoplasm and cell wall; bar, 500nm (according to Kwiatkowska et al., 2014); (G) labelled cutinsome in the territory of a lipotubuloid; bar, 100nm (according to Kwiatkowska et al., 2014); (H) colocalization of myosin (large gold grains) and kinesin (small gold grains in white rings) in lipotubuloids; bar, 100nm (Reproduced from Kwiatkowska et al., 2013. Immunogold method evidences that kinesin and myosin bind to and coupe microtubules and actin filaments in lipotubuloids of Ornithogalum umbellatum ovary epidermis. Acta Physiologiae Plantarum 35, 1967–1977. With kind permission of Springer Science+Business Media). Specificities of antibodies were verified with Western blotting, with results of analyses included for the appropriate images. In (C) and (E): lane 1, SDS-PAGE of the epidermis cell extract; lane 2, Western blotting of the epidermis cell extract probed with anti-DGAT2 antibody diluted 1:100 (C) or anti-lipase antibody diluted 1:200 (E); lane 3, molecular mass standards and their weights in kDa. In (H) in left and right inserts: lanes 1, SDS-PAGE of the epidermis cell extract; lanes 2, pre-immune serum; lanes 3, anti-myosin antibody diluted 1:50 (right) and anti-kinesin antibody diluted 1:50 (left); lanes 4, molecular mass standards and their weights in kDa. Arrows indicate the detected proteins; the exposure time for each antibody was 20min. AV, autolytic vacuole; c, cytoplasm; cl, cuticular layer; cp, cuticle proper; Cs, cutincome; cw, cell wall; l, lipotubuloid; Lb, lipid body; mt, microtubules; n, nucleus; V, vacuole.

The latest research has supported our previous hypothesis, namely that LMs are involved in cutin synthesis (Kwiatkowska et al., 2014). They are the site where cutinsomes, cutin-building blocks, are formed (Fig. 6G) as a result of self-assembly and self-esterification of hydroxy fatty acids (Heredia-Guerrero et al., 2008). In O. umbellatum, they were identified with the use of anti-cutinsome-specific antibodies (Domínguez et al., 2010) and visualized using gold particles in electron microscopy. LMs, due to their progressive-rotary movement, transport cutinsomes to the vicinity of a cell wall (Fig. 6F, G). The cutinsomes from the cytoplasm (Figs 4–6 and 9 in Kwiatkowska et al., 2014) then move through the plasmolemma by non-vesicular contact and through the cell wall to the cuticle, probably with the use of transporters.

Lipid metabolism

Ultrastructural autoradiography has shown that radioactive palmitic acid in LMs becomes incorporated in the lipid body periphery (Fig. 5) (Kwiatkowska et al., 2011a, 2012a ). This area of lipid bodies contains an enzyme, diacylglycerol acyltransferase 2 (DGAT2) (Figs 5 and 6C), mediating the last stage of lipid synthesis, which was revealed in lipid bodies and RER by the immunogold method with antibodies against this enzyme. DGAT2 is synthesized on ER ribosomes (Fig. 5) (Kwiatkowska et al., 2011a , 2012a ). In Arabidopsis thaliana, in meristematic cells, the immunogold technique revealed the presence of DGAT2 in the ER and in lipid bodies (Kwiatkowska et al., 2012c). Similar results for DGAT2 were obtained by Kuerschner et al. (2008) in lipid bodies of oleate-loaded COS-7 cells, and by McFie et al. (2011) in murine cells, in Saccharomyces cerevisiae (Jacquier et al., 2011), and in Caenorabditis elegans (Xu et al., 2012), as well as in yeast (Aymé et al., 2014). In lipotubuloids of O. umbellatum, in the same area where DGAT2 acts, i.e. in the lipid body periphery, lipase was also revealed with a specific antibody as a part of the metabolon (Figs 5 and 6E) (Kwiatkowska et al., 2011a , 2012b ). Thus, it is presumed that synthesized lipids are temporarily stored in lipid bodies and then used as nutrients and cuticle-building blocks by dynamically growing but not dividing epidermis cells, which enlarge 30-fold during ovary development (Kwiatkowska, 1971a ). Simultaneously, the volume of LMs grows proportionally and nuclear DNA becomes endoreduplicated to the 8C level (Kwiatkowska et al., 2007). Development of an ovary epidermis and of the forming fruit is closely related to the growth dynamic of the whole organ.

Role of microtubules in lipid synthesis

Microtubules seem to cooperate with lipid bodies in lipid synthesis (Fig. 5), transporting precursors and enzymes synthesized in the RER to the lipid bodies, which was shown by the following: (i) silver grains first appeared over microtubules after a short [³H]palmitic acid incubation and later were observed over lipid bodies; (ii) blockade of [³H]palmitic acid incorporation into LMs by propyzamide, an inhibitor of microtubule function, resulting in inhibition of lipid synthesis precursor delivery; and (iii) the presence of gold grains near the microtubules after DGAT2 (Fig. 6C) and phospholipase D1 (Fig. 6D) specific immunogold labelling reactions (Fig. 5) (Kwiatkowska et al., 2012a ). Microtubule involvement was also observed in macrophages showing the formation of lipid droplets as an immunological response (Pacheco et al., 2007).

Autonomic motion of LMs

There is one more function of microtubules, namely that they are the driving force of autonomic rotation of LMs, which also exhibit progressive movement depending on cytoplasm streaming (Kwiatkowska, 1972a ; Kwiatkowska et al., 2009). LM rotation is not driven by cyclosis but by driving forces inside LMs, which was shown by the following phenomena: (i) 2,4-dinitrophenol, which stops ATP synthesis, blocked cyclosis after 15–16.5min and the rotary motion of LMs after 25min; (ii) measurements made by means of a filming technique revealed that the peripheral speed of LM rotation in some instances was 6.2 times higher than the maximum speed of cytoplasmic motion in the same cell (Kwiatkowska, 1972a ).

It seems that the autonomous LM rotary movement, which is multidirectional and goes around varying axes, might be mediated by the motor proteins kinesin and myosin, as well as actin filaments, which influence microtubules, increasing or decreasing their diameter as a result of the microtubule varying monomer sizes and the distance between them (Kwiatkowska et al., 2009). In LMs in which rotary movement was blocked by 2,4-dinitrophenol, only microtubules of medium size were present (Kwiatkowska et al., 2009). Ali et al. (2008) showed that when both motor proteins were connected to the same cargo, as in the case of LMs (Fig. 5) in which kinesin and myosin were simultaneously revealed with the double immunogold labelling method (Fig. 6H), the electrostatic interaction of myosin with microtubules increased the motor activity of kinesin. It was supposed that the collaboration of motor proteins with actin filaments and microtubules made autonomic high-peripheral-speed rotary motion of LMs in epidermal cells possible. It is obvious that the progressive-rotary movement of LMs plays a biologically significant role that greatly facilitates both the entry of components necessary for lipid and cuticle synthesis and the release of nutritional and building substances to a cell at the proper time and place (Kwiatkowska et al., 2013).

Conclusions

To the best of our knowledge, in the literature concerning metabolons there is no description of structures similar to LMs. However, it seems that they can be justifiably recognized as metabolons due to the fact that LMs exist temporarily, only during cell growth, and their function and structure correlate exactly with the definition of a metabolon proposed by Srere (1985) and the characteristics described by others (see Introduction).

The facts observed using filming techniques and immuno- and cytochemical methods (with Western blot controls) using specific antibodies against lipid-metabolized enzymes, as well as antibodies recognizing motor proteins and cutinsomes (Fig. 6), indicate that LMs are closely integrated structures with very complex functions including: (i) lipid synthesis and lipolysis in lipid bodies; (ii) temporary lipid storage in lipid bodies; (iii) the formation of new lipid bodies, microtubules, and ER; (iv) facilitation of lipid transport and metabolism as nutrients and building blocks; (v) the synthesis and transport of cuticle components; and (vi) the generation of autonomous rotary motion. All these processes take place within one cytoplasmic domain, the lipotubuloid, constituting the metabolon. Functions of LMs are adjusted to cell growth and increase in their number of intracellular components, and in the area of a wall and cuticle.

Perspectives

To date, the abovementioned processes have been observed only in O. umbellatum due to the exceptional stability of their microtubules, which in LMs of this species are coated with a polysaccharide layer (Kwiatkowska et al., 2011c). Further research is planned to examine lipotubuloids (described previously as ‘elaioplasts’) containing the same components of LMs as in O. umbellatum but with less stable microtubules in Haemanthus albiflos (Kwiatkowska et al., 2010), Funkia sieboldiana, Vanilla planifolia, and Althaea rosea (Kwiatkowska et al., 2011b ). This research will allow us to determine whether lipotubuloids in these species have similar functions to those in LMs in O. umbellatum.

It is worth noting that, in the 19th and 20th centuries, ‘elaioplasts’, which were usually not plastids, were described in around 120 plant species, and new examples of LMs are sure to be identified in future research, and their involvement in the formation of cuticle elements is likely to turn out to be a more common phenomenon.

Glossary

Abbreviations:

ER

endoplasmic reticulum

LM

lipotubuloid metabolon

RER

rough endoplasmic reticulum.