- Top of page
- Materials and Methods
The root parasitic plants Orobanche and Striga spp. are devastating pests in agricultural production throughout the world (Joel et al., 2007). These root parasites depend on host plants for nutrients and water and cannot survive without parasitizing hosts. Their tiny seeds contain limited resources so that the parasites must connect to hosts within a week of germination. The seeds of these parasites germinate only when they perceive host-derived chemicals, termed ‘germination stimulants’, released from plant roots. The first described germination stimulant for Striga, named strigol, was isolated from the root exudates of a false host, cotton (Gossypium hirsutum) (Cook et al., 1966, 1972), and later identified from genuine hosts, sorghum (Sorghum bicolor), maize (Zea mays), and proso millet (Pennisetum glaucum) (Siame et al., 1993). Subsequently, sorgolactone was isolated from S. bicolor root exudates (Hauck et al., 1992). Alectrol was purified from cowpea (Vigna unguiculata) root exudates (Müller et al., 1992) and recently identified as orobanchyl acetate (Xie et al., 2008a). The first described Orobanche germination stimulant, orobanchol, was isolated by Yokota et al. (1998) from red clover (Trifolium pratense) root exudates. Recently, 2′-epiorobanchol and solanacol were characterized from root exudates of tobacco (Nicotiana tabacum), a host of Phelipanche ramosa (formally called Orobanche ramosa) (Xie et al., 2007). In addition, two novel stimulants, sorgomol (Awad et al., 2006; Xie et al., 2008b) and a putative didehydro-orobanchol (strigol) isomer (Sato et al., 2003; Xie et al., 2007), were identified in the root exudates of several Poaceae species and the Solanaceae species N. tabacum and tomato (Solanum lycopersicum). The structure of this didehydro-orobanchol isomer has not yet been clarified. These strigol-related germination stimulants are collectively called strigolactones (Fig. 1).
Among the strigolactones, 5-deoxystrigol was originally isolated as a branching factor of arbuscular mycorrhizal (AM) fungi from root exudates of Lotus japonicus (Akiyama et al., 2005). We also identified 5-deoxystrigol as one of major germination stimulants of S. bicolor, Z. mays, and pearl millet (Pennisetum typhoideum) (Awad et al., 2006).
The AM fungi, which are obligate symbionts, are incapable of completing their life cycle without residing in their host roots. After spore germination and hyphal growth, the hyphal branching of AM fungi occurs in the vicinity of host roots. Because the phenomenon does not occur in the vicinity of roots of nonhosts, including rapeseed (Brassica napus) and white lupin (Lupinus albus), hyphal branching is considered to be the host recognition process (Giovannetti et al., 1993). In addition, strigolactones were found to induce a rapid increase in mitochondrial density and changes in the shape and movement of the organelles in AM fungi (Besserer et al., 2006). Such activation of the mitochondria might lead to the oxidation of lipids, which are the main form of carbon storage in AM fungal spores. Therefore, strigolactones may be crucial components of root exudates that switch on lipid catabolism at the presymbiotic stage of the fungus (Besserer et al., 2006; Akiyama, 2007). Furthermore, strigolactones induce gene expression of Gigaspora margarita CuZn superoxide dismutase (GmarCuZnSOD) (Lanfranco et al., 2005) and chemotrophic growth of Glomus mosseae hyphae (Sbrana & Giovannetti, 2005). The AM association is by far the most widespread association between microorganisms and higher plants. Within the angiosperms, at least 80% of the species are able to form AM symbioses (Harrison, 2005). Therefore, if strigolactones are indispensable for host recognition of AM fungi, they may be widely distributed in the plant kingdom (Akiyama, 2007). However, characterizations of strigolactones have been conducted for only a few plant species as plants exude trace amounts of unstable strigolactones. Accordingly, it is necessary to clarify the distribution of strigolactones in the plant kingdom to understand the chemical communications in the rhizosphere between plants and AM symbionts, and plants and root parasites.
We have developed a specific and rapid analytical method for known strigolactones using high-performance liquid chromatography (HPLC) connected to tandem mass spectrometry (LC/MS/MS) (Sato et al., 2003). LC/MS/MS analyses revealed the presence of 5-deoxystrigol in the root exudates of S. bicolor, Z. mays, and P. typhoideum (Awad et al., 2006). Besserer et al. (2006) identified sorgolactone as a branching factor in S. bicolor root exudates using LC/MS/MS. Furthermore, using LC/MS/MS, we demonstrated that nutrient deficiencies affect strigolactone exudation: in T. pratense, phosphorus (P) deficiency significantly promoted orobanchol exudation (Yoneyama et al., 2007a), while in S. bicolor, nitrogen (N) deficiency as well as P deficiency enhanced 5-deoxystrigol exudation (Yoneyama et al., 2007b).
To date, we have examined a wide range of plant species including crops, weeds, and even trees for the production of strigolactones and have found that plants produce diverse mixtures of known and unknown strigolactones. Although some of these unknown strigolactones have been purified and subjected to structural elucidation (Yokota et al., 1998; Xie et al., 2007, 2008a,b), at least several novel strigolactones remain to be characterized (K. Yoneyama, unpublished). Therefore, to compare strigolactone production among different plant species, the major strigolactones in each plant species should first be identified. As in the case of the Poaceae species (Awad et al., 2006), the major strigolactones of plant species within the same family are expected to be similar. We thus focused on the Fabaceae for the comparison of strigolactone production within the family as we had already identified major strigolactones produced by T. pratense (Yokota et al., 1998).
In this paper we extended the characterization of strigolactones in root exudates of 12 Fabaceae plant species, including L. albus, a nonhost of AM fungi, by comparing retention times of germination stimulants on reverse-phase (RP)-HPLC with those of standards and by using LC/MS/MS. In addition, the effects of N deficiency and P deficiency on strigolactone exudation by L. albus were examined to clarify whether the regulation of strigolactone exudation is related to the nutrient acquisition strategy of plants.
- Top of page
- Materials and Methods
In the present study, strigolactones exuded from 12 Fabaceae plants were characterized by an RP-HPLC separation–germination assay and by LC/MS/MS. Orobanchyl acetate (alectrol) was detected in root exudates from all of these plants, and most of them exuded orobanchol and 5-deoxystrigol. Therefore, orobanchol, orobanchyl acetate, and 5-deoxystrigol appear to be major germination stimulants in the Fabaceae. In addition to these strigolactones, a didehydro-orobanchol isomer and solanacol were identified in the root exudates from four and one species, respectively. Furthermore, two novel stimulants, one from P. sativum and the other from L. albus root exudates, were detected. The stimulant found in the root exudate of P. sativum seems to be present in the root exudates of A. sinicus, A. hypogaea, C. arietinum, M. sativa, and V. faba. After several steps of purification, this stimulant appeared to be a novel strigolactone with a molecular weight of 404 Da, while its structure has not yet been fully elucidated as the amounts purified from P. sativum root exudates were not sufficient for comprehensive spectroscopic analyses.
In the case of P. sativum, although distinct peaks of the didehydro-orobanchol, orobanchol, orobanchyl acetate, and 5-deoxystrigol were observed in the MRM chromatogram (Fig. 3a), germination stimulation activities were not detected in the RP-HPLC fractions corresponding to the retention times of orobanchol and 5-deoxystrigol. Only a weak germination stimulation activity was associated with fraction 16, corresponding to the retention time of orobanchyl acetate (Fig. 3b). This implies that these strigolactones contributed to the overall germination stimulation activity in the root exudate only to a small extent as compared with the novel strigolactone eluted in fractions 12 and 13 and orobanchyl acetate in fraction 16.
In the LC/MS/MS analyses, each strigolactone was monitored with the MRM channel specific to it at an independent sensitivity. For example, in Fig. 3a, the ion intensity in the channel for orobanchyl acetate (1.95 × 107) was 100-fold larger than those for orobanchol (1.15 × 105) and 5-deoxystrigol (7.14 × 104). Given the fact that, in electrospray ionization (ESI) mass spectrometry, ion intensities are not always proportional to the concentrations of analytes, and the ionization efficiency of orobanchol is c. 10-fold lower than those of orobanchyl acetate and 5-deoxystrigol, the amounts of orobanchyl acetate would be at least 10-fold larger than those of orobanchol and 5-deoxystrigol. We could not increase amounts of root exudate samples for RP-HPLC separation, because the activity was distributed broadly in many fractions when an excessive amount of sample was loaded for HPLC.
Similar results were obtained with the root exudates of G. max, P. vulgaris, and V. faba, where 5-deoxystrigol was detected by LC/MS/MS but germination stimulation activity was not observed in the fraction(s) corresponding to the retention time of 5-deoxystrigol. In addition to the differences in the contents of strigolactones as discussed above, the differences in sensitivity of root parasite seeds to various strigolactones and the presence of inhibitors also affected the results of germination assays. In fact, O. minor used in the germination assays was c. 100-fold more sensitive to orobanchol than to 5-deoxystrigol; 0.1 nM orobanchol elicited c. 60% germination and a similar level of germination was observed at 10 nM 5-deoxystrigol.
Although many authors have suggested that strigolactones are distributed widely in the plant kingdom, the isolation and identification of strigolactones have been hampered by the extremely low concentrations produced and exuded by host roots as well as their relative instability. The recent development of an analytical method using LC/MS/MS enables identification and quantification of known strigolactones (Sato et al., 2003, 2005; Awad et al., 2006; Besserer et al., 2006; Yoneyama et al., 2007a,b; Xie et al., 2007) and, in addition, the search for novel strigolactones (Awad et al., 2006; Xie et al., 2007). Here we demonstrated that the 12 Fabaceae plants, including the nonmycotrophic L. albus, exude at least three strigolactones, and that, among the known strigolactones, orobanchol, orobanchyl acetate, and 5-deoxystrigol are the major ones produced by these plants grown hydroponically.
It is intriguing that all the plants examined so far exude mixtures of at least two strigolactones. Although the question of why plants produce and exude mixtures of strigolactones has not yet been answered, quantitative and/or qualitative differences in the strigolactone compositions may be one of the key factors determining the host specificity of AM fungi and root parasites. Therefore, the effects of various combinations of strigolactones on both hyphal branching of AM fungi and seed germination of root parasites need to be examined.
Orobanchol and orobanchyl acetate (alectrol) were first isolated from root exudates of T. pratense and V. unguiculata as germination stimulants, respectively. Orobanchol was also identified in root exudates from Solanaceae plants including N. tabacum (Xie et al., 2007) and S. lycopersicum (Sato et al., 2003), and from the Compositae marigold (Targetes patula) (K. Yoneyama, unpublished). Orobanchyl acetate was detected in several Compositae plants (K. Yoneyama, unpublished). However, these strigolactones have not been found in the root exudates from any members of the Poaceae examined to date (Awad et al., 2006; K. Yoneyama, unpublished). These results suggest that orobanchol and orobanchyl acetate seem to be distributed in dicotyledons but not in monocotyledons.
5-Deoxystrigol, originally isolated as a hyphal branching inducer for AM fungi from the root exudate of Lotus japonicus (Akiyama et al., 2005), has been shown to be one of major strigolactones in monocotyledonous plants including S. bicolor, Z. mays and P. typhoideum (Awad et al., 2006). As all the Fabaceae plants examined in this study, with the exception of T. incarnatum, were found to exude 5-deoxystrigol, 5-deoxystrigol appears to be widely distributed in both monocotyledons and dicotyledons. This is in good agreement with the proposed biosynthetic pathway for strigolactones, originating from carotenoids, where 5-deoxystrigol serves as the precursor of all the other known strigolactones (Matusova et al., 2005; Bouwmeester et al., 2007). The oxidation of 5-deoxystrigol produces mono-hydroxy-strigolactones such as orobanchol, strigol, and the recently identified sorgomol (Xie et al., 2008b). It is likely that sorgomol is then converted to sorgolactone by subsequent oxidation and decarboxylation (Xie et al., 2008b; Rani et al., in press). Among the plants examined to date, T. incarnatum, N. tabacum (Xie et al., 2007) and S. lycopersicum (unpublished data) did not produce detectable amounts of 5-deoxystrigol. It is interesting that these three plant species exuded solanacol, a strigolactone containing a benzene ring.
AM fungi supply mineral nutrients, especially P, to host plants by extending beyond the depletion zone for P around the root, the external mycelia improving P absorption. However, AM fungi are absent under all environmental conditions in the Brassicaceae and Chenopodiaceae, and are also quite rare or absent in many members of the Proteaceae and other typical root cluster-forming plant species including L. albus (Marschner, 1993). The proximity of roots of nonhosts including L. albus did not elicit hyphal branching of AM fungi (Giovannetti et al., 1993). An early hypothesis that nonhost plants do not induce the morphogenetical event suggested that roots of nonhosts secrete compounds into the rhizosphere that inhibit AM colonization (Fontenla et al., 1999). Schreiner & Koide (1993) showed that members of Brassicaceae have the potential to produce significant quantities of antifungal compounds in roots, probably isothiocyanates. An alternative explanation for the lack of AM colonization of nonhosts is that nonhosts fail to produce branching factors required by AM fungi for host recognition. In this study, it was demonstrated that the nonmycotrophic L. albus grown hydroponically also produces and exudes strigolactones, and therefore the hypothesis that nonhosts of AM fungi fail to exude strigolactones may be excluded.
The amounts of strigolactones released from L. albus, however, were quite low compared with mycotrophic plants. Indeed, the amounts of orobanchol and orobanchyl acetate, per unit root fresh weight, released from L. albus grown under P starvation were only 1/24 000 and 1/4080 that from T. pratense, respectively. Although sizes and growth stages of plants and the composition of strigolactones produced were different and thus direct comparisons were not possible, the total amount (or activity) of all strigolactones exuded by L. albus would be c. 1/1000 that exuded by T. pratense. For example, in the case of T. pratense, approx. 80% germination of O. minor was induced at 500-µl equivalent of the control culture (1/2 Tadano & Tanaka) medium (Yoneyama et al., 2007a) and a similar level of germination was achieved at 450-ml equivalent of the control medium of L. albus (Fig. 4b).
Another clear difference between mycotrophic and nonmycotrophic plants was observed in the response of strigolactone exudation to nutrient deficiency. In the cases of T. pratense and S. bicolor, which are host plants of AM fungi, P deficiency (and also N deficiency in S. bicolor) significantly promoted strigolactone exudation (Yoneyama et al., 2007a,b). In the case of L. albus, however, P and N deficiency slightly reduced strigolactone exudation (Fig. 4). Such a decrease in strigolactone production may be attributable to a reduction in metabolic functions in L. albus under N and P deficiencies. In another nonmycotrophic plant, Spinacia oleracia, neither P nor N deficiency enhanced strigolactone production (K. Yoneyama, unpublished).
Recently, the strigolactone orobanchol was identified from the root exudates of Arabidopsis thaliana, a host of Orobanche spp. but not of AM fungi (Goldwasser et al., 2008). These results suggest that strigolactones have other unknown functions indispensable for the normal growth and development of plants themselves.
Legumes are thought to be very versatile in their symbioses (Sprent & James, 2007). Therefore, further work on the characterization of strigolactones from nodulating and nonnodulating legumes and legumes that produce ectomycorrhiza, and the effects of mineral nutrients on their strigolactone production, can be expected to unveil the functions of strigolactones in the rhizosphere community and in plants.