Proazulenes, azulene and colchicine as fluorescent dyes for study of cellular interactions in allelopathy
Russian Academy of Sciences Institute of Cell Biophysics, Institutskaya Str., 3, Pushchino, Moscow region, 142290, Russia Email firstname.lastname@example.org
Fluorescence of allelochemicals could be a tool for the study of allelopathic mechanisms at the cellular level. Moreover, there are supposed to be valuable products in the chemical industry, such as natural pharmaceuticals and pesticides that are based on the allelopathy knowledge. This paper gives one possible example of the cellular models: vegetative microspores of Equisetum arvense and pollen of Hippeastrum hybridum. Allelochemicals and pharmaceuticals with 7-membered rings, such as sesquiterpene lactones proazulenes artemisinine, austricine, gaillardine, grosshemine and azulene as well as relative alkaloid colchicine, selectively fluoresce upon interaction with different cellular compartments after the histochemical staining of fixed or vital samples. This makes them suitable as potential fluorescent dyes. The substances have characteristics that may be used in enterprises producing pharmaceuticals, pesticides and fluorescent dyes.
Fluorescence of allelochemicals could be a tool for the study of allelopathic mechanisms at the cellular level.
Allelochemicals, fluorescent dyes, vegetative microspores, pollen, sesquiterpene lactones
Many plant allelochemicals are valuable as medicinal, pesticidal and biologically active compounds. Among the allelochemicals, containing 7-membered rings, proazulenes artemisinine from Artemisia annua L., austricine from Achillea millefolium L., gaillardine from Gaillardia pulchella Foug. and Inula oculus-christi L., grosshemine from Grosshemia genera and Centaurea ruthenica Lam. , azulenes from Achillea millefolium, Artemisia and Matricaria genera as well as relative alkaloid colchicine from genera Colchicium, Merendra, Ramond and Gloriosa and others, are known to depress germination of seeds, pollen and vegetative microspores (Vaughan and Vaughan, 1988; Chen et al., 1990; Konovalov, 1995; 1996; Roshchina et al., 1998; Roshchina, 2004). The compounds can also be pharmaceuticals and components of cosmetic preparations due to their bactericidal and antiinflammatory characteristics. These allelochemicals may fluoresce (Bhattacharyya et al., 1986; Roshchina, 2004; 2005), and their fluorescent features may be applied to the study of allelopathic mechanisms (Roshchina, 2004). In this publication the ability of the above-mentioned substances to be fluorescent markers and dyes is considered using some examples.
The objects of the study were microspores, which were used as cellular models of cell-acceptors (Roshchina, 2004): generative microspores (male gametophyte or pollen) Hippeastrum hybridum L.(Herb.) collected in the green-house and vegetative microspores of Equisetum arvense L. collected from meadow near the Oka river. As reagents, artemisinine, austricine, gaillardine and grosshemine provided by All Russian Institute of Medicinal Plants, azulene and colchicine given from Sigma were used. All these reagents were dissolved in ethanol (stock solutions were 10-3M) and then diluted with water.
The effects of the compounds on the fluorescence of microspores were examnied on object glasses (slides) as described earlier (Roshchina, 2004). All experiments were performed at room temperature 20-22 OC. The cells of pollen and developed pollen tubes, as well as germinated vegetative microspores were analysed with conventional and luminescence microscopes (Roshchina, 2004), as well as laser scannining confocal microscope (LSCM), according to Roshchina et al. (2004). Fluorescence from intact cells induced by ultra-violet light 360-380 nm, and fluorescence spectra of the intact cells were registered by microspectrofluorimeter as described earlier (Roshchina, 2004; Roshchina et al., 2004). In each treatment, ten microspores were used for measuring the maximal fluorescence.
Sesquiterpene lactones, and alkaloid having 7-membered rings (Fig.1) can fluoresce in solutions, mainly at blue wavelengths (at 420-460 nm). This fluorescence is related to the 7-membered ring(s) by the chromophore that is marked by a broken line-box in Fig.1 (Roshchina et al., 1998).
Figure 1. Formulae of compounds analysed.
These lipophilic compounds enter cells and interact with organelles. They retard microspore germination and the substances may bind with the nucleus of the cell (Roshchina, 2004; 2005). This is a possible mode of action. Interactions between the tested allelochemicals and the microspores were examined by observation of changes in cell fluorescence after treatment with the compounds.
The fluorescence of all the compounds upon interaction with microspores was analysed. On the object glass under luminescent microscope, sesquiterpene lactones had a weak fluorescence in blue (400-430 nm), while untreated vegetative microspores fluoresced in blue-green and in red (450-500 nm and 680 nm), and pollen grains in blue-green (at 490-510 nm). Most significant emission was for artemisinine, gaillardine, grosshemine, azulene and colchicine. Sesquiterpene lactones were bound on the cell surface, and in some cases, after staining with artemisinine, gaillardine and azulene, light emission by nuclei and chloroplasts (in vegetative microspores) was seen. Figs 2 and 3 shows examples of the interaction of artemisinine, azulene and colchicine with cells of microspores that were estimated from the changes in cellular fluorescence seen from their fluorescence spectra. Artemisinine and azulene were bound on the cell surface that emitted blue light, but fluorescence of nuclei was also seen (Fig.2). Artemisinine also stained elaters of vegetative microspores (the surface structures for the anchoring microspores to a substrate such as soil). The fluorescence of cells treated with the compounds increased significantly in comparison with untreated microspores and pure dyes lying on the object glass. When blue-fluorescing (maximum at 420-460 nm) allelochemicals bound with pollen grains, a new intensive maximum arose at 550 nm. Similar results were obtained for gaillardine and grosshemine. In contrast, austricine changed colour from blue to yellow-orange with a maximum at 550 nm, the fluorescence intensity in this spectral region was not increased (Roshchina, 2004).
Possible interactions of the compounds with cellular organelles should be considered because artemisinine, azulene and gaillardine also marked the nucleus. Azulene is known to react with the cell wall, leading to the appearance of orange-red emission (Roshchina et al., 1995) whereas DNA-containing structures such as nuclei and chloroplasts fluoresced in blue or blue-green (Roshchina, 2004). Thus, similar sesquiterpene lactones related to proazulenes and azulenes can penetrate into the cell and, binding with some organelles, induce the enhanced fluorescence that makes clear the cellular mechanisms of actions for the allelochemicals, based on the site of the fluorescence location.
Figure 2. Left- The fluorescence spectra of vegetative microspores of Equisetum arvense stained with artemisinine (10-5M) and generative microspores (pollen) of Hippeastrum hybridum stained with azulene (10-5M). 1 - Pure compound in solution; 2 - Untreated microspore; 3, 4, 5. Relatively surface , nucleus , elaters in microspore after the treatment with allelochemical. Right – the LSCM image ( excitement with 458 nm) of fluorescing pollen grain treated with azulene (bar – 20 μm), the surface of the spore (orange-red emission) and large nucleus in a centre (blue-green emission) are seen.
Figure 3. LSCM images of Hippeastrum hybridum pollen tube (a,b,c) and vegetative microspore of Equisetum arvense (d) stained with colchicine 10-7 M. The laser excitation wavelength 458 nm. a) Fluorescing parts of pollen grain and pollen tube. b) The pollen tube with the fluorescing spermium, which is seen on the tip of the tube (shown by arrow); c) One of the spermium in the pollen tube (tubulin-related bundles are seen); d) Microspore with elaters (the cell surface and some parts of elaters fluoresce in blue-green). Right - fluorescent spectra of the microspore where 1 and 2 –relatively untreated spore and solution of colchicine on the object glass; 3. after the treatment of microspore with colchicine. White line bars for a, b, c and d are 50, 100, 10 and 20 μm, relatively.
Colchicine induced blue-green fluorescence of elaters in vegetative micospores and some bundles of cytoplasm in microspores and pollen tubes, especially near cell wall and spermia (Fig. 3). The fluorescence spectra show 5-6 times increased fluorescence in blue-green (460-490 nm) after treatment. Perhaps, fluorescing parts of the vegetative microspore, pollen grain and developing pollen tube are related to monomers of tubulin, which is marked with colchicine (Roshchina, 2005). In living cells, these monomers are present constantly around microtubules, where tubulin is in polymerized form.
They are best known as toxins - mitotic agents that induce polyploidy, but in many cases they act as herbicides, and can retard a germination of seeds, vegetative microspores and pollens (Vaughan and Vaughan, 1988; Roshchina, 2005).
Since proazulenes ,azulene and colchicine and its derivatives are able to pass through the plasmic membrane , interaction with cellular components DNA- containing organelles or tubulin of cytoplasm and organelles is supposed. Major mechanisms behind their action consist of interaction with nucleic acid directly or indirectly (through structural or contractile proteins), and in their influence on the protein synthesis. In order to understand the possible mechanisms of their action, it is essential to determine which intercellular components could interact with allelochemicals.
Moreover, these compounds may be applied as fluorescent dyes for histochemical staining of all types of cells (animal, microbial and plant). Luminescent microscopy is applied in many fields of biology, and may be used in allelopathy. In the pharmaceutical industry, azulenes and some proazulenes are produced by dehydration of sesquiterpenes from plant essential oils in the presence of oxidants whereas colchicine is produced by isolation from plants or is synthesised from sesquiterpene lactones and phenylalanine or tyrosine. Similar enterprises could profit from the additional production of the fluorescent dyes.
Histochemical staining of cells with some allelochemicals as fluorescent dyes is a useful tool in the study of allelopathic mechanisms. Production of the compounds, not only as medical drugs, but also as histochemical dyes could have value in pharmaceutical enterprises.
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