Cadiz Allelopathy Group (GAC), Dept. of Organic Chemistry, Faculty of Sciences University of Cadiz. Apdo. 40, 11510 – Puerto Real, Cádiz, Spain. www2.uca.es/dept/quimica_organica/ Email email@example.com
The last results of our ongoing research project on inductors of the germination in Orobanche species are presented. In continuation of our last results and findings, we have explored the molecular reasons for the O. cumana-sunflower recognition mechanism. A group of novel sesquiterpene lactones that mimics the molecular structure of strigolactones have been synthesized and their ability to induce germination in several Orobanche species has been tested. The lactones tested present a lactone-enol-γ-lactone moiety similar to that of strigolactones. Also, the influence of heteroatoms different from oxygen in this system has been explored. Factors such as the molecular volume are under study and the results of the corresponding germination bioassays have been analysed in terms of host-parasite specificity and host recognition.
Several sesquiterpene lactones having a lactone-enol-γ-lactone moiety similar to that of strigolactones have been synthesized. Their ability to induce germination of several Orobanche species is discussed and related with the changes induced in their chemical structure.
sesquiterpene lactones, SAR studies, Orobanche spp., parasitic weeds
Parasitic weeds of the genus Orobanche constitute a serious threat to many economically important crops. They induce high yield losses in crops such as sunflower, legumes, and greens. However, their importance does not come only from the economic point of view, but also because of their unique relationship with their hosts. Allelopathy is understood as a chemical interaction or communication between plants and the organisms living in their environment (Rice, 1984; IAS, 1996). In this sense, the parasitic weed-host recognition mechanism is a good example of allelopathy, according to the actual definition and hypothesis. Parasitic weeds use specific compounds exuded by their host to recognize their presence in the surroundings (Joel, 1995). These compounds induce the germination of dormant seeds and also help them to locate the host roots for attachment following an increasing concentration direction (chemotropism) (Fate, 1990; Chang, 1986). However, it is unlikely that these compounds were synthesized and exuded by the plant with this purpose; rather they should be considered as defence products. Co-evolution of parasitic plants and their host should led to a situation in which parasites take advantage of these compounds to recognize a viable host by which they could complete their life cycle.
To date, few compounds have been isolated and characterized as inductors from natural hosts, all of them belonging to plants being parasitized by members of the Orobanchaceae and Striga families. Many of these host plants have been investigated, several inductors characterized, and their structures reviewed recently (Galindo, 2004). Interestingly, all of these “natural inductors” belong to the same skeletal type, named strigolactones (Figure 1). Other natural products and synthetic derivatives have been also found to induce broomrape or witchweed germination. However, none of them have been isolated from their typical hosts.
As it can be seen in Figure 1, all of these compounds share a common backbone and present a lactone-enol-γ-lactone moiety (rings C and D). This special disposition has been identified as the bioactiphore of the molecule and a molecular mechanism for its attachment has been proposed (Mangnus, 1992).
Figure 1. Broomrape and witchweed germination inductors isolated from natural hosts.
In continuation of our research project on allelopathic sunflower (Macías, 2002) we are interested in all the aspects of its chemical ecology. Consequently, we have investigated its relationship with Orobanche cumana, as it is the specific host of sunflower and causes big yield losses in Spain and other sunflower-producing countries, especially in Europe (Pujadas-Salvá, 2000). However, no germination inductors have been isolated or characterized from sunflower so far. In continuation with our previous studies (de Luque, 2000; Galindo, 2001) we are exploring the possibility of sesquiterpene lactones as specific germination inductors of O. cumana in sunflower. Previously, we have demonstrated that sesquiterpene lactones specifically induce germination of O. cumana but not in other Orobanche species. However, it has been reported that sesquiterpene lactones also induce germination in some Striga species. We are currently exploring the molecular requirements of sesquiterpene lactones with different backbones for germination activity and found some new clues.
Herein we present the last findings of our ongoing research project on Orobanche germination inductors. Modifications in the guaianolide skeleton aiming to mimic the lactone-enol-γ-lactone moiety typical of strigolactones have been introduced and the results in the bioactivity and specificity on Orobanche species are shown.
Synthesis of strigolactone-like sesquiterpene lactones.
The synthetic methodology used is depicted in Scheme 1. Starting from dehydrocostuslactone we have performed a synthetic pathway where the key point is the obtention of a 1,3-dicarbonyl compounds (7-10 and 19,20).
Scheme 1. Synthetic pathway to get access to strigolactone-like guaianolides.
Seed germination bioassay.
Seeds contained in the tip of a spatula were homogeneously dispersed in a Petri dish ( 55mm ∅) on Whatman GF/A paper. For preconditioning, 1 mL of a solution of 0.3 mM of 2-[N-Morpholino]ethanesulfonic acid was added to the filter, and the Petri dishes sealed to prevent drying and incubated in darkness at 20ºC for 11 days. Stock solutions were prepared with acetone and diluted with MES 0.3 mM to obtain a 10 µM (0.1 % acetone) solution; 0.1 and 1 µM solutions were prepared by diluting with MES 0.3 mM (0.1 % acetone aqueous solutions).
After the conditioning period, 500 µL of an aqueous solution of GR-24 (used as internal standard) or the test compounds were added to every Petri dish, sealed with parafilm and incubated for another 4 days in darkness at 20ºC. Germination was observed under a microscope (30x) and the germinated seeds expressed as a percentage of the total seeds. Germination was considered when the radicle was at least 0.2 mm long. Seeds used belong to O. cumana, O. ramosa, O. aegyptiaca, O. crenata, O. minor, O. densiflora, O. foetida, O. gracilis, and O. hederae.
Our findings confirm that sesquiterpene lactones without the second lactone ring commonly present in strigolactones are specific inductors of the germination of O. cumana. The introduction of this new moiety (compounds 11-14, 21 and 22) causes the lost of specificity. It is also important to remark that compounds without the α,β-unsaturated carbonyl system are also active, being this fact opposite to the general hypothesis of a Michael addition reaction as molecular mechanism of recognition for all Orobanche species. The high specificity of the sunflower-O. cumana interaction could rely on the ability of O. cumana to recognize sesquiterpene lactones with or without an α,β-unsaturated carbonyl system. In the last case, the presence of an oxygen atom in the same disposition as in strigolactones appears to be another key factor in the ability to induce seed germination.
It seems there are two mechanisms of host-recognition in Orobanche species: a general one based on Zwanenburg’s hypothesis and fitted by sesquiterpene-like compounds presenting a lactone-enol-γ-lactone moiety (strigolactones and modified sesquiterpene lactones), and the another one specific for sesquiterpene lactones and O. cumana. In this case, the presence of the lactone ring (with or without the conjugated double bond in the lactone ring) seems to be the bioactiphore.
IAS: International Allelopathy Society. (1996). Constitution and Bylaws. First World Congress on Allelopathy. A Science for the Future. Cadiz. Spain.
Chang, M; Netzly, DH; Butler, LG; Lynn, DG (1986). Chemical regulation of distance: characterization of the first natural host germination stimulant for Striga asiatica. Journal of the American Chemical Society. 108, 7858-7860.
Fate, G., Chang, M., Lynn, D. G. (1990). Control of germination in Striga asiatica: chemistry of spatial definition. Plant Physiology. 93, 207-207.
De Luqe, AP; Galindo, JCG; Macías, FA, and Jorrín, J (2000). Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination. Phytochemistry, 53, 45-50.
Galindo, JCG; de Luque, AP, Jorrín, J; Macías, FA (2001). SAR studies of sesquiterpene lactones as Orobanche cumana seed germination stimulants. Journal of Agricultural and Food Chemistry, 50, 1991-1917.
Galindo, JCG; Macías, FA; García-Díaz, MD; Jorrín, J (2004). Chemistry of Host-Parasite Interactions. In: Allelopathy: Chemistry and Mode of Action of Allelochemicals. Macías, FA; Galindo, JCG, Molinillo, JMG, Cutler, HG (Eds.). CRC Press, BocaRatón, FL. 125-148.
Macías, FA; Varela, RM; Torres, AM; Galindo, JLG; Molinillo, JMG (2002). Allelochemicals from sunflower. Chemistry, Bioactivity, and Applications. In: Recent Advances in Allelochemical Research. Mallik, A and Inderjit (Eds.). Birkhauser Verlog AG, Switzerland, 73-83.
Mangnus, EM, and Zwanenburg, B (1992). Tentative molecular mechanism for germination stimulation of Striga and Orobanche seeds by strigol and its synthetic analogues. Journal of Agricultural and Food Chemistry. 40, 1066-1070.
Joel, D. M., Steffens, J. C. Matthews, D. E. (1995). Germination of weedy root parasites. In: Seed Development and Germination. Kiegel, J. and Galili, G. (Eds.) Marcel Dekker, Inc., New York, pp. 567-598.
Pujadas-Salvá, AJ and Velasco, L (2000). Comparative studies on Orobanche cernua L. and Orobanche cumana Wallr. (Orobanchaceae) in the Iberian Peninsula. Botanical Journal of the Linnean Society. 134, 513-527
Rice, E. L. (1984). In: Allelopathy. 2nd. Ed. Oklahoma University Press