Lipoxygenase in the Cells of Angiosperms

Ewa Szczuka¹, Aleksandra Seta¹, Marcin Domaciuk¹, Ewa Skórzyńska-Polit², Irena Giełwanowska³

¹Department of Plant Anatomy and Cytology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland

²Department of Plant Physiology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland

³Department of Plant Physiology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-719 Olsztyn, Poland

ABSTRACT

The enzyme lipoxygenase (LOX; EC 1.13.11.12) catalyzes dioxygenation of the long chain of fatty acids such as arachidic, linoleic, and α-linolenic acids, which contain a cis, cis-1,4-pentadiene structure. LOX has been found in all individual plant parts of angiosperms. Its isoforms occur in the cells of seeds, pods, seedlings, cotyledons, leaves, roots, fruits, young inflorescences, flowers (e.g., in the cells of petals, anthers, in the walls of the microsporangium at the stage of microspores and pollen grains in the loculus), young anthers, microspores, pollen grains, and potato tubers.

The use of the immunogold labelling technique allows precise evaluation of the localization of LOX on the cytological level in the cells of angiosperm plants. Investigations with this method have shown that LOX occurs in the cytosol (most often, the immunogold particles were distributed randomly in the cytoplasm) and vacuoles. The immunogold particles which revealed the presence of the enzyme were found to be associated with microsomal membranes and the plasma membrane and were also discovered in the close vicinity of mitochondria and near or within plastids (they were visible in the area of the prolammellar body) and chloroplasts. In the latter, LOX was found both in the envelope and in the stroma. Often, LOX was detected near (close to) the short endoplasmic reticulum elements – mainly RER (rough endoplasmic reticulum). Some single immunogold particles were observed at or in the area of the cell walls of all the investigated parts of angiosperm plants. LOX was also detected in the inner exine of the pollen grain and in places connecting exine layers of neighbouring pollen grains.

Immunolocalization of lipoxygenase in an electron microscope indicates a functioning „lipoxygenase pathway” in all cells of the investigated angiosperm plant parts. The intensity of the immunogold reaction may indirectly indicate differentiated activity of the enzyme in particular plant cells.

INTRODUCTION

Lipoxygenase (LOX; EC 1.13.11.12) is widely spread in the cells of living organisms belonging to different systematic groups. In plants, this enzyme catalyzes dioxygenation of the long chain of fatty acids such as arachidic, linoleic, and α-linolenic acids, which contain a cis, cis-1,4-pentadiene structure. Lipoxygenase strongly prefers free fatty acids as substrates, but it has also been found to have activity with polyunsaturated fatty acids (PUFAs) esterified to phospholipids and neutral lipids such as triglycerides (Feussner and Wasternack, 2002). Lipoxygenase plays a number of active roles in different processes during plant life. These numerous and extremely important roles decide, among others, even about the construction of the LOX pathway. A possible LOX pathway has been shown in Scheme 1.

Scheme 1. The LOX pathway. (The scheme was taken from Porta and Rocha-Sosa, 2002)

Lipoxygenase fulfils various functions in plants. In soybean, this enzyme plays a role in nitrogen storage and partitioning. LOX can serve as a vegetative storage protein, transiently storing nitrogen in the paraveinal mesophyll cell layer prior to its redistribution to vegetative or reproductive sink tissues (Feussner et al., 1997). The enzyme in dry soybean seeds has a direct impact on the level of protease inhibitors and their activity (Lima de Carvalho et al., 1999). LOX is believed to mediate the formation of superoxide anion in senescent plants (Lynch and Thompson, 1984). Other functions of LOX, including stress responses and pathogen defense, were presented earlier by Rosahal (1996) or Porta and Rocha-Sosa (2002). The numerous functions of LOX have been collected and arranged in Table 1.

Table 1. Active roles of LOX in several processes during plant life. (The table was taken from Porta and Rocha-Sosa, 2002)

Plant lipoxygenases are a frequent subject of study (for numerous references see Porta and Rocha-Sosa, 2002). Despite of this only a few investigators have focused on their localization. Meanwhile, the many functions of lipoxygenase itself and the many functions of the individual compounds of the LOX pathway seem to require for their explanation, examination of the localization of this enzyme on the cytological level, which can be done by electron microscopy and the immunogold labelling technique. For instance, to our knowlege, there exist only a few reports on LOX immunolocalization in different cells of plant organs (Wang et al., 1999; Leone et al., 2001: Szczuka et al., 2006; Skórzyńska-Polit et al., 2005; 2006). Therefore, in this paper we have focused our attention on this, important for knowledge of cell and organ processes occurring with the necessary presence of LOX.

MATERIAL AND METHODS

Plant material

Bulbs of Gagea lutea (L.) Ker.-Gaw. (Liliaceae) growing in a natural habitat in Stalowa Wola (south-western part of Poland) were used in the study. Both, flower buds and leaves of Gagea lutea (L.) Ker.-Gaw. were isolated from shoots growing from the bulbs under ground.

Light microscopy

Flower buds freshly excised from the bulbs and leaves cut into small segments were fixed in a mixture of ethanol and acetic acid (3:1). After that, they were washed in 70% alcohol, dehydrated, and embedded in paraffin wax. Then the samples were cut into 4 μm specimens and mounted on slides. Subsequently, they were stained with fast green and safranin (standard procedure) and examined in a light microscope.

Small segments (2-3 mm) from flower buds and leaves were fixed in 3.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.0 for 24 h at room temperature. The samples were postfixed in osmium tetroxide (OsO4), dehydrated in ethanol and aceton, and embedded in Spurr’s resin. Semithin sections of plant organs were stained with 0.1% toluidine blue in 0.5% sodium carbonate at about 60°C.

Immunolabelling

For immunogold labelling, small segments (2-3 mm) from plant organs were fixed in 2% formaldehyde (freshly prepared from paraformaldehyde) and 1% glutaraldehyde dissolved in PBS (0.1 M phosphate buffer, pH 7.4) for 24 h at 4^oC. The samples were rinsed several times in PBS and 0.5 M NH₄Cl in PBS, dehydrated in ethanol, embedded in LR White resin (Sigma), and polymerised at 60^oC overnight. Ultrathin sections were collected on nickel grids, treated with aqueous 0.56 M sodium periodate for 30 min, thoroughly washed with distilled water, and treated with 0.1 M HCl for 10 min followed by a 5 min water wash. Sections were incubated first in 1 % BSA in PBS for 30 min at room temperature, then with preimmune rabbit serum (Agrisera) diluted 1/1000 in PBS-BSA for 1 h at room temperature. After triplicate washing with PBS-BSA (each wash lasting 10 min), the sections were incubated with PBS-BSA containing rabbit anti-LOX antiserum diluted 1/1000 for 1 h and repeatedly washed with PBS-BSA. Goat anti-rabbit immunoglobulins conjugated to 10 nm gold particles (GAR – gold) (Sigma) were diluted 1/50 in PBS-BSA and then applied for 40 min at room temperature. Next, the sections were washed several times with PBS and redistilled water. As an additional control, samples were incubated with preserum and GAR-gold or GAR-gold only, omitting the primary antiserum. The sections were stained with 2 % uranyl acetate for 5 min and Reynolds reagent (lead nitrate and sodium citrate) for 1 min. All sections were examined using the transmission electron microscope.

RESULTS

Lipoxygenase was detected in different (organs) parts of the plants (Gymnosperms and Angiosperms), but due to the limited scope of this article, we only focused on parts of flower buds and leaves.

Gagea lutea (L.) Ker.-Gaw. is an early-spring monocotyledonous plant. In Polish climate conditions, it blooms in March. The morphological structure of the Gagea lutea plant and its flower at the anthesis stage is shown in Figures 1 and 2. The single Gagea lutea plant grows from a bulb (exactly from a shortened underground stem). The seedlings containing stems, leaves and flower buds develop inside the bulbs and grow out in early spring. Three different bulbs of Gagea lutea with shoots growing above the bulbs are visible in Fig. 3. Flowers of Gagea lutea have a structure typical of monocotyledons. The centrally positioned pistil is surrounded by six stamens and the same number of petals. In the mature flower, the stamen comprises an anther inclusive of microsporangia and the intervening connective (Fig. 4), and the filament. As shown in Fig. 4., the Gagea lutea anther consists of four microsporangia. Each microsporangium is surrounded by the anther wall, which is built of the epidermis, endothecium, middle layer, and tapetum. In the loculus, microspores are present. Stamens and anthers are enveloped by petals. Initially, a single stamen develops from the primordium, which is built of mertistematic tissue. A transverse section of the developing anther is shown in Fig. 5. The meristematic tissue of the future microsporangia is lightly stained with toluidine blue. Such a developing anther is surrounded by the petals (Fig. 6). Figures 3, 5, and 6 show the stages of the development of leaves (Fig. 3), anther (Fig. 5), and petals (Fig. 6) which were used to examine the localization of lipoxygenase with the electron transmission microscope (TEM), shown in this paper.

Immunogold LOX PAb localization in the cells of the anther (at the stage of the young stamen) of Gagea lutea shows the singular gold particles in the cytoplasm, in the vacuole (Fig. 7), and in the area of the cell wall (Fig. 8). The density of the gold particles that revealed the presence of lipoxygenase was very low. Similarly to the cells of the young, developing stamen, the single immunogold particles were found in the cytoplasm (Fig. 9), vacuoles, and the cell wall (Figs. 10 and 11) of petal cells.

In a young leaf, 1-3 cm long, numerous immunogold particles were observed in the dense cytoplasm with ribosomes of the parenchyma cell. The particles were distributed randomly in the cytoplasm or gathered near short ER elements and the outer plastid membrane (Figs. 12 and 13). Single immunogold particles were visible in the close vicinity of mitochondria or small vacuoles. Some immunogold particles were observed at the cell wall, organelle membranes, or even inside the plastids, and in the area of the prolammellar body.

In order to determine the degree of specificity of the immunogold reaction, a control reaction including all the procedures was carried out. The control reaction was conducted omitting incubation with the primary antibody. Only single gold particles (a few per one nickel grid) were found in the specimens. In most grid meshes (like in the figure shown in this paper) no gold particles were present (Fig. 14).

DISCUSSION

As it was mentioned in the introduction to this paper, plant lipoxygenases are a very frequent subject of study (for numerous references see Porta and Rocha-Sosa, 2002). The researchers have shown the occurrence of LOX in plants using various methods. For example, the simplest method of LOX localization is determination of the enzyme activity in individual plant parts. The enzyme activity in a plant extract can be measured using (i) methods based on oxygen uptake (manometric or polarographic techniques), (ii) methods based on formation of conjugated diens, or (iii) determination of hydroperoxides (Grossman and Zakut, 1979).

Another method used in the investigations concerned determination of lipoxygenase isoenzymes is carried out by electrophoresis (SDS-PAGE, native PAGE, IEF) (Grossman and Zakut, 1979; Heinisch et al., 1996; Smith et al., 1997). Additionally, LOX activity can be determined by distinguishing between lipoxygenase and heme proteins. Cyanide was suggested as a selective inhibitor for distinguishing between them in the oxidation of fatty acids, but according to Grossman and Zakut (1979) lipoxygenase activity is also sensitive to cyanide. These authors cited another method of distinguishing the activities of heme and non-heme proteins, which is based on the different effects of linoleate on the fluorescence of these catalysts.

As reported in the Results section of this paper, the localization of LOX was carried out by using the immunogold labelling technique. This method (i.e. the immunogold LOX PAb localization method) allows to evaluate the localization of LOX on the cytological level in the cells of all angiosperm plants. For example, the occurrence of lipoxygenase in different parts and types of anther cells had been reveales with this method (Szczuka et al., 2004, 2006).

In the cells of the developing Gagea lutea anther and petal, the immunogold LOX PAb localization shows the presence of singular gold particles in the cytoplasm, in the vacuole and in the area of the cell wall. Similarly, in both investigated developing parts of flower buds the gold particles revealing lipoxygenase were not numerous. This event indicates a low intensity of immunoreaction and indirectly, a low activity of lipoxygenase.

In contrast, to the developing anther and petal in the flower bud, the immunogold LOX Pab reaction in a young leaf was very intense. Numerous immunogold particles were found in the dense cytoplasm of the parenchyma cell (mesophyll). Sometimes they were distributed randomly in the cytoplasm, but very often they gathered near short ER elements and the outer plastid membrane. In the mesophyll cells of young leaves, single immunogold particles were visible in the close vicinity of mitochondria or small vacuoles. Some single immunogold particles were observed at the cell wall, organelle membranes, or even inside the plastids, and in the area of the prolammellar body.

As it was mentioned earlier, the enzyme LOX is widely spread in the cells of organisms belonging to different systematic groups of plants, animals and fungi. In plant parts of angiosperms, lipoxygenase or its isoforms occur in young, developing organs, in mature organs, and also in degenerating parts of plants. The presence of lipoxygenase was observed in the cells of seeds, pods, seedlings, cotyledons, leaves, roots, fruits, young inflorescences, flowers (e.g., in the cells of petals, anthers, in the walls of the microsporangium at the stage of microspores, and pollen grains in the loculus), young anthers, microspores, pollen grains, and potato tubers. Additionally, our observations are partially supported also by results obtained by other authors. For instance, Feussner et al. (1995) localized the enzyme within the chloroplast using immunocytochemical analysis. Lipoxygenase associated with the thylakoid membrane was found in tomato fruits (Bowsher et al., 1992). LOXs were localized in the stroma, and substantial LOX activity was detected in the chloroplast envelope fraction. In cotyledons, besides soluble LOXs, particulate LOXs were also found in microsome membranes, plasma membranes and lipid bodies (Feussner and Wasternack, 2002). Importantly, the above results on LOX localization were only of marginal interest to the mentioned authors. It should also be emphasized that LOX localization in cells is very problematic, largely because soluble lipoxygenases tend to adhere to membranes nonspecifically (Siedow and Girvin, 1980).

As shown in this paper, immunolocalization of lipoxygenase in an electron microscope indicates a functioning „lipoxygenase pathway” in all cells of the investigated angiosperm plant parts. The intensity of the immunogold reaction indirectly indicates differentiated activity of the enzyme in particular plant cells (Szczuka and Skórzyńska, 2008). In the plant cells investigated in this paper, the intensity of the immunogold reaction was comparatively low. Therefore, we may assume that the activity of lipoxygenase in the tissues of the investigated young plant organs in comparison to the activity of lipoxygenase in mature plant organs is relatively low. Nevertheless, we would like to underline, that at the moment knowledge concerning LOX localization on the cytological level is still insufficient and further investigation is necessary.

ACKNOWLEDGEMENTS

We would like to thank producer for developing antibody used in the experimental part of this paper. Polyclonal antibody against antigen LOX was produced by Agrisera, SE-911 21 Vännäs, Sweden, www.agrisera.se

LITERATURE

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Feussner I. and Wasternack C. 2002. The lipoxygenase pathway. Annu. Rev. Plant Biol., 53: 275-297.

Feussner I., Hause B., Vörös K., Parthier B. and Wasternack C. 1995. Jasmonate-induced lipoxygenase forms are localized in chloroplasts of barley leaves (Hordeum vulgare cv Salome). Plant J., 7: 949-957.

Feussner I., Balkenhohl T.J., Porzel A., Kühn H. and Wasternack C. 1997. Structural elucidation of oxygenated storage lipids in cucumber cotyledons - implication of lipid body lipoxygenase in lipid mobilization during germination. J. Biol. Chem., 272: 21635-21641.

Grossman S. and Zakut R. 1979. Determination of the activity of lipoxygenase (lipoxidase). Methods Biochem. Anal., 25: 303-329.

Heinisch O., Kowalski E., Ludwig H. and Tauscher B. 1996. Staining for soybean lipoxygenase activity in electrophoretic gels. Fat/Lipids, 5: 183-184.

Lima de Carvalho W., Goreti de Almeida Oliveira M., Goncalves de Barros E. and Moreira, M.A. 1999. Lipoxygenases affect protease inhibitor levels in soybean seeds. Plant Physiol. Biochem., 37: 497-501.

Lynch D.V. and Thompson J.E. 1984. Lipoxygenase-mediated production of superoxide anion in senescing plant tissue. FEBS Lett., 173: 251-254.

Porta H. and Rocha-Sosa M. 2002. Plant lipoxygenases. Physiological and molecular features. Plant Physiol., 130: 15–21.

Rosahal S. 1996. Lipoxygenase in plants – their role in development and stress response. Z. Naturforsch., 51c: 123-138.

Schmitt N.F. and van Mechelen J.R. 1997. Expression of lipoxygenase isoenzymes in developing barley grains. Plant Sci., 128: 141-150.

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Szczuka E., Skórzyńska-Polit E., Pawlikowska-Pawlęga B., Sobieska J. and Gawron A. 2004. Localization of lipoxygenase in the anther of Gagea lutea. Materials of 18^th International Congress on Sexual Plant Reproduction. Beijing (China) 2004: 53.

Szczuka E., Skórzyńska-Polit E., Pawlikowska-Pawlęga B., Sobieska J. and Gawron A. 2006. Localization of lipoxygenase in the anther of Gagea lutea. Acta Biol. Cracov., 48: 19-26.

Skórzyńska-Polit E., Pawlikowska-Pawlęga B., Szczuka E., Drążkiewicz M. and Krupa Z. 2006. Localization and activity of lipoxygenase in Arabidopsis thaliana plants under heavy metal stress. Plant Growth Reg. 48: 29-39.

Skórzyńska-Polit E., Pawlikowska-Pawlęga B., Szczuka E, Plak A. and Melke J. 2005. Localization and activity of lipoxygenase in Cd-treated seedlings of Phaseolus coccineus. Acta Soc. Bot. Pol. 3: 199-207.

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LEGENDS

Fig. 1. General habit of the Gagea lutea (L.) Ker.-Gaw. plant.

Fig. 2. Gagea lutea. Bud and flower at anthesis stage.

Fig. 3. Three different bulbs of Gagea lutea with shoots growing above the bulbs.

Fig. 4. A transverse section of a Gagea lutea flower bud. Note the four microsporangia (M) of the anther enveloped with thin petals (PL). Fast green and safranin staining. 270x.

Fig. 5. A transverse section of the developing anther. Note the meristematic tissue of the future microsporangia. Stained with toluidine blue. 380x.

Fig. 6. Fragments of petals (PL) of a Gagea lutea flower – transverse section. Stained with toluidine blue. 380x.

Figs. 7 – 14. Immunolabelling to lipoxygenase in Gagea lutea.

Figs. 7 and 8. Immunogold LOX PAb localization in the cells of the anther (at the stage of young stamen) of Gagea lutea. Note singular gold particles (arrows) in the cytoplasm (Fig. 7) and in the area of the cell wall (Fig. 8). M – mitochondrion, V – vacuole, CW – cell wall.

Figs. 9 – 11. A portion of a petal cell of Gagea lutea. Single immunogold particles (arrows) in the cytoplasm (C) (Fig. 9), vacuoles, and the cell wall (Figs. 10 and 11).

Figs. 12 and 13. Portions of a mesophyll cell. Note singular gold particles (arrows) near and in the plastid (P) in the cytoplasm (C) (arrows), near RER (arrowheads) and in the area of the cell wall (CW). PB – prolammellar body.

Fig. 14. A mespophyll cell. A control micrograph of the cytoplasm (C) with a fragment of the plastid (P), and the nucleus (N).