1 University of Tsukuba, Email firstname.lastname@example.org
2 National Institute for Agro-Environmental Sciences
3Institute of Sakura Sciences,
4Institute of Space and Astronautical Science /JAXA
Interactions between plants and other organisms are well known, and in particular those involving species producing allelochemicals. The effect of gravity on allelopathy has not been examined. We employed a 3D-clinorotation device to generate pseudo-microgravity and to study this question. If the biosynthesis, emission, transport and sensing mechanism associated with allelochemicals are affected by gravity, many organisms and ecological systems might exhibit different behaviour under microgravity experienced in space. Velvet bean (Mucuna pruriens L.) and sunflower (Helianthus annuus L.) have strong allelopathic properties. The growth of lettuce seedlings, as an index of the allelopathic potency, was compared between the 3D-clinostated (pseudo-microgravity) group and a control group. The allelopathic potential of velvet bean and sunflower against lettuce seedling was reduced in microgravity. The synthesis and release of the allelochemicals were also reduced in both species grown under pseudo-microgravity.
This is the first report on suppression of synthesis and release of allelochemicals under pseudo-microgravity, which suggests interactions among organisms may differ in outer space from the terrestrial ones.
Allelochemicals, 3D-clinostat, pseudo-microgravity, sunflower, velvet bean plant
During the evolutionary process, many interactions between organisms have evolved. There are interactions which can either depress or enhance the growth of micro-organisms and other plants. Allelopathy is an interaction between plants and other living organisms that is mediated by specific chemicals released from that plant species (Rice 1984). Allelopathy can be a difficult subject of study, because it is regulated by many parameters, which are difficult to control in experiment.
The possibility of space exploration, accompanied by the establishment of human bases on extraterrestrial bodies may become a reality in the near future. As life support systems in a spacecraft or outpost on the surface of extraterrestrial planets are being designed, the ecology of selected plant species that will be grown will require a better understanding of allelopathic and other biological interactions among those species in microgravity environments. The stability of such ‘artificial’ ecological systems must be sufficient to sustain human life. Gravity is one of terrestrial environmental factors that affect biological systems. We examined the effect of microgravity on created by the 3D-clinorotation allelopathy (Yamashita et al. 1997). Velvet bean (Mucuna pruriens L.) and sunflower (Helianthus annuus L.) have strong allelopathic properties (Rice 1984; Fujii 1999). Their allelopathic potential on lettuce seedlings under pseudo-gravity was studied. The growth of lettuce seedlings was compared between the 3D-clinostat adapted group and a control group.
Mucuna pruriens (L.) DC. var. utilis (Wall ex Wight) Burk (Velvet bean) - Lactuca sativea L. (lettuce), and Helianthus annuus L. (sunflower) were used as plant materials. The growth of lettuce plants grown in the presence of either velvet bean or sunflower under either normal gravity or pseudo-gravity conditions was examined.
Velvet bean plant was pre-incubated for two weeks before being transplanted to Agripot (Kilin, Tokyo) for the intercropped incubation described previously (Tomita-Yokotani et al. 2003). The intercropped plants were incubated for four days under normal gravity or pseudo-microgravity, respectively. The growth of lettuce seedling under these two conditions was compared to the growth of lettuce without velvet bean plant. In the case of sunflower achene, twenty achenes were pre-incubated on the agar medium under two conditions. The achenes were removed and twenty lettuce seeds were incubated on the agar medium where sunflower achenes were removed. After the four days of incubation, growths of root and shoot of lettuce seedlings were measured. Procedure of this process is shown in Figures 1A and B.
Extraction and purification of candidate substances from velvet bean plant and sunflower achene
Allelopathic substances, L-DOPA and sundiversifolide, from velvet bean and sunflower, respectively, were analysed with a protocol described in Figures 2-A and B, respectively (Fujii et al. 1991; Ohono et al. 2001). The extraction and purification of allelochemicals from velvet bean plant and sunflower achene exudates are shown in Figures 1-A and B. In the case of velvet bean plant, the 5 mm tip of its root was first homogenized with ethanol including 0.1 % TFA (trifluoroacetic acid). After the filtration, the filtrate was concentrated to dryness. After the extract solved in distilled water, it was filtered through Mol-Cut (Millipore, LGC, Yonezawa) in order to remove chemicals with higher molecular weight. Low molecular weight fraction was further purified on a ODS mini-column, C18 Sep-Pak cartridge (Waters), with 100 % water. In the case of sunflower achene, the agar medium was extracted with ethyl acetate. The extracts were concentrated to dryness, and further purified on a ODS mini-column, C18 Sep-Pak cartridge, with 40-100 % methanol/water.
Analysis of their candidate substances, L-DOPA and sundiversifolide, in velvet bean and sunflower
L-DOPA and sundiversifolide were analysed by HPLC and LC-ESI+/MS. The condition for analysis of L-DOPA and sunndiversifolide is described in Figures 2A and B.
Figure 3-A shows the results of allelopathic interaction between velvet bean and lettuce. The allelopathic action was reduced under the pseudo-microgravity condition created by the 3D-clinolotion. In the case of the sunflower – lettuce system, allelopathy was also suppressed under the pseudo-microgravity compared to the control (Figure 3-B). L-DOPA was reported as a allelopathic substance in velvet bean plant (Fujii 1999). The amount of this substance decreased in plants grown under the pseudo-microgravity conditions (Figure 4A) compared to the control plants. Similarly, the amount of sundiversifolide, one allelochemical produced by sunflower, decreased under the pseudo-microgravity condition (Figure 4B).
Figure 1. Protocol of bioassay in velvet bean (A) and sunflower (B).
Figure 2. The procedures for the extraction of candidate allelopathic substances, L-DOPA from velvet bean (A) and sundiversifolide from sunflower (B).
Figure 3. Effects of pseudo – microgravity on the lettuce root and shoot growth. A-Together with velvet bean plant. B-After planted on the agar medium of twenty numbers of sunflower achene incubated for one day. Bars indicate average +-SE.
Figure 4. Chromatograms of L-DOPA (A-1 and A-2) and sundiversifolide (B-1 and B-2), in velvet bean and sunflower achene, respectively. A-1 and B-1 are HPLC and LC-ESI/MS chromatograms ([M+Na]+) of the control plants, and A-2 and B-2 are that of plants grown under pseudo-microgravity.
The allelopathic interaction between the velvet bean – lettuce and sunflower – lettuce systems was compared under two gravitational conditions. Vervet bean plants and sunflower achene had reduced allelopathic potentials under pseudo-microgravity. The amount of allelochemicals released from either velvet bean or sunflower was reduced in the experiment groups grown under microgravity. The reduction of allelopathic substances released from root tip of velvet bean plant and sunflower achene under microgravitational environment created by the 3D-clinorotation suggests that allelopathic interactions may be affected in space.
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