Journal of Experimental Botany, Vol. 52, No. 364, pp. 2227-2234,
November 1, 2001
© 2001 Oxford University Press
Original Papers |
Sunflower (Helianthus annuus L.) response to broomrape (Orobanche cernua Loefl.) parasitism: induced synthesis and excretion of 7-hydroxylated simple coumarins
1 Agricultural and Plant Biochemistry Research Group, Departamento Bioquímica y Biología Molecular, ETSIAM, University of Córdoba, Apdo 3048, 14080 Córdoba, Spain
2 Institute for Sustainable Agriculture, CSIC, Córdoba, Spain
Received 7 March 2001; Accepted 3 July 2001
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Abstract |
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The interaction of the parasitic plant Orobanche cernua with resistant and susceptible cultivars of Helianthus annuus L. was investigated. Using different bioassays to evaluate the early stages of the parasite life cycle (germination, attachment, penetration, and establishment), differences were observed between O. cernua-resistant and O. cernua-susceptible sunflower varieties. Germination of O. cernua seeds in the presence of resistant sunflower roots was approximately half that of germination in the presence of susceptible roots, and germinated seeds displayed enhanced browning symptoms. Parasite radicles or host-tissue around the contact point turned brown after O. cernua attachment to sunflower roots, especially in the resistant varieties. These observations suggested the possible accumulation of toxic compounds as a defence strategy in the resistant sunflower varieties. Sunflower 7-hydroxylated simple coumarins may play a defensive role against O. cernua parasitism by preventing successful germination, penetration and/or connection to the host vascular system. This hypothesis is supported by the following data: (i) coumarins inhibited the in vitro germination of O. cernua seeds induced by the strigol analogue GR24 and caused a browning reaction in germinated seeds and (ii) resistant sunflowers accumulated higher levels of coumarins in roots and excreted greater amounts than susceptible varieties in response to O. cernua infection.
Key words: Coumarins, Orobanche, plant defence, plant stress, sunflower.
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Introduction |
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Approximately 1% of flowering plants (nearly 4000 species) are parasitic on other plant species and some, such as Striga, Orobanche and Cuscuta, cause significant loss of yield (Molau, 1995
; Riches and Parker, 1995; Nickrent et al., 1998). However, studies on parasitic angiosperms and their interaction with host plants are limited compared with the intensive research carried out into other symbiotic associations involving plants. At the molecular level, research has focused on identifying and characterizing the host signals that induce germination and haustorium induction (Butler, 1995; Estabrook and Yoder, 1998; Yoder, 1999).
Plant resistance to parasitic plants, both non-host and host-specific, seems to be a complex multifactorial process. This interplay is dependent on the host (species, varieties and populations), the parasite (species and races or populations), and biotic and abiotic environmental factors (Cubero et al., 1994). Taking into account the complex life cycle of the parasite (Parker and Riches, 1993), a range of host plant strategies for parasite resistance may be proposed. A potential strategy might be directed at interrupting specific developmental stages such as germination, haustorial induction, attachment, penetration and connection to the vascular system, development, emergence, and flowering (Jorrín et al., 1999). The earlier stages may be the most efficient targets for resistance, and low production of germination stimulants as a resistance or avoidance mechanism is well documented (Butler, 1995). In addition to germination, alternative mechanisms operate in varieties resistant to Striga and Orobanche. It has been reported that once parasite seeds have germinated and become attached to the host root, either root tissue penetration and connection to the vascular tissue or tubercle development are prevented in resistant hosts (Lane et al., 1997; Goldwasser et al., 1997). Other typical plant defence responses against pathogenic microorganisms (Jackson and Taylor, 1996) are also induced in response to parasitic plant infection. These include host cell browning around the penetration site (Dörr et al., 1994; Lane et al., 1997; Goldwasser et al., 1997), phytoalexin accumulation (Wegmann et al., 1991; Lane et al., 1997; Westwood et al., 1998), lignification and cell wall phenolic deposition (Antonova, 1994; Ish-Shalom Gordon et al., 1994; Mayer et al., 1997; Goldwasser et al., 1999), and pathogenesis-related protein induction (Joel and Portnoy, 1998). These inducible mechanisms, as for phytopathogens (Jackson and Taylor, 1996), may result in the restriction of infective structures to small areas immediately surrounding the penetration site or prevention of their connection to the vascular system.
The authors' current research involves the sunflower–Orobanche cernua Loefl. (Orobanche cumana Wallr.; Pujadas and Thalouarn, 1998) interaction. O. cernua parasitism is the most serious threat to cultivated sunflower in Mediterranean and Eastern European countries. It has been exacerbated by the appearance of new and more virulent races, which overcome the resistance of most commercial varieties (Alonso et al., 1996). As available control methods achieve only limited success, there is a need to find resistant cultivars, and hence, to identify the mechanisms involved in generating resistance.
The data presented in this paper suggest that sunflower 7-hydroxylated simple coumarins (scopoletin, its glucosyl conjugate scopolin, and ayapin) may play a defensive role against O. cernua by preventing germination and connection of the parasite. These coumarins are stress-induced, multidefensive secondary metabolites (Jorrín and Prats, 1999), reported as being phytoalexins (Tal and Robeson, 1986), insect-feeding deterrents (Olson and Roseland, 1991) and allelochemicals (Tang et al., 1995).
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Materials and methods |
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Chemicals
Scopoletin was obtained from Sigma. Ayapin was synthesized from esculetin as described previously (Tal and Robeson, 1986
). Scopolin was provided by Dr MA Fliniaux (Université de Jules Verne Picardie, France) and GR24 by Dr B Zwanenburg (University of Nijmegan, Netherlands).
Plant material and growth conditions
The sunflower varieties Agrosur (confectionery, white seed coat, susceptible to O. cernua) and Cortés (oily, black seed coat, resistant to the O. cernua populations tested) were provided by Eurosemillas S.A. (Córdoba, Spain). Sunflower seeds were germinated and grown as previously described (Tena et al., 1984). O. cernua populations were collected from infected sunflower (cv. Florasol 92 and J6 Arburg) fields between 1994 and 1996. Once cleaned, the seeds were stored in darkness at room temperature until use. The susceptible or resistant character of the sunflower varieties was confirmed by artificial infection in pot assays (Domínguez et al., 1996). The susceptible variety supported O. cernua growth throughout maturation and flowering, whereas only small tubercles were occasionally observed on the roots of the resistant sunflower variety.
Orobanche cernua seed germination bioassays
O. cernua seeds were germinated on wet paper (Whatman GF/C) in Petri dishes with GR24 (Mangnus et al., 1992) or on agar in the presence of isolated sunflower roots.
The paper method is a modification of that described for O. crenata (Van Hezewijk et al., 1994). Fifty O. cernua seeds were evenly spread on glass fibre paper and placed in small Petri dishes (5.5 cm diameter). For conditioning the seeds, 0.2 ml of 0.3 mM MES buffer, pH 6.0, was added, the dishes sealed with parafilm, wrapped with aluminium foil and kept in darkness at 25 °C for 11 d. After this conditioning period, 0.2 ml of either 30 µM of the germination stimulant GR24 (optimal concentration for germination), either alone or in combination with coumarins and other phenolic compounds at different concentrations, were added to each Petri dish and then incubated as indicated above. Germination was evaluated 4 d later using a binocular microscope. Germination was expressed as a percentage of the total seeds. Seeds were considered to be germinated when the germ tube was at least 0.1 mm long.
The agar germination bioassay is a modification of the procedure published earlier for Striga (Hess et al., 1992). Petri dishes (9 cm diameter) containing 25 ml of autoclaved 0.7% agar were used. Isolated sunflower roots (5–6 cm long) from 5–8-d-old seedlings were placed on the surface of the agar in the centre of the plate. 200 non-conditioned O. cernua seeds were spread evenly over the surface of the agar. Petri dishes were sealed with parafilm, covered with aluminium foil and kept in darkness at 25 °C for up to 15 d. O. cernua seed germination was evaluated using a binocular microscope. The number of germinated seeds and the maximum germination distance (i.e. the distance between the host root and the most distant germinated O. cernua seed) was recorded. Germination was expressed as a percentage of the total seeds. A similar bioassay was performed to test the germination of O. ramosa in the presence of sunflower roots and that of O. cernua in the presence of tobacco, potato and Helianthus tuberosus. The number of O. cernua germinated seeds showing a browning reaction in the radicle, was recorded from day 7, and expressed as a percentage of total germinated seeds.
Orobanche cernua attachment bioassay
Seven–ten-day-old sunflower plants were root-infected with GR24-germinated O. cernua seeds using a plastic-tray bioassay, which is a modification of that reported for Striga (Lane et al., 1991). Two layers of filter paper underneath one circular layer of glass paper (Whatman GF/C, 15 cm diameter) were placed in plastic trays (210x150x40 mm) filled with perlite. Sunflower seedlings were placed on the tray with the roots resting on the glass fibre paper and the hypocotyl on one edge of the tray. The upper surface of the root was covered with a circular layer of glass paper containing O. cernua seeds (5 mg, either GR24-germinated or non-germinated) making sure there was sufficient contact between the sunflower root and the O. cernua seeds. The trays were wrapped with aluminium foil and kept under the same conditions for plant growth. During the course of the experiment, nutrient solution (De la Guardia and Benlloch, 1980) was added to the perlite to maintain high humidity conditions. The course of the infection process was followed visually using a binocular microscope. At various times post-inoculation, the root and the aerial part of the sunflower plants were separated, washed with tap water to eliminate non-attached O. cernua seeds, dried with filter paper, frozen in liquid nitrogen, and stored at -70 °C until use. The glass papers in contact with the roots were submerged in methanol for 24 h in order to dissolve organic compounds excreted by the root.
Coumarin extraction and analysis
Coumarins were extracted from the roots and the aerial part of the sunflower plants (Gutierrez et al., 1995; Gutierrez-Mellado et al., 1996). Three consecutive extractions (acetone, acetone and 1:1 acetone-methanol) were performed. Shoot samples were depigmented by partitioning with hexane:ether (6:4, v:v). Samples were vacuum-dried, redissolved in methanol and applied to a Lichrosphere 100 RP-18 HPLC column. Elution was performed with an acetonitrile:water gradient and coumarins were detected by fluorescence (excitation and emission wavelength of 340 and 430 nm, respectively) (Gutierrez et al., 1995; Gutierrez-Mellado et al., 1996).
Statistics
The experimental design was completely randomized. Data were tested for homogeneity and analysis of variance and differences of means (Tukey, P<0.05) were performed (Statistics for Windows 1.0, 1996).
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Results |
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O. cernua seed germination on agar in the presence of isolated sunflower roots
O. cernua seeds germinated in Petri dishes containing agar in the presence of isolated sunflower roots. Germination became visible after 4 d, and maximum values were reached on day 8 (Fig. 2A
). The radicle elongated for 4 d, reaching a maximum length of 2.0–3.0 mm during the course of the experiment (Fig. 1A) with O. cernua seeds collected from cultivar J6 Arburg. The percentage germination and maximum distance of germinated seeds from the roots were higher in the presence of the susceptible cultivar (45% germination and 3.8±0.6 cm distance) than from the resistant cultivar (25% germination and 2.6±0.4 cm distance). Similar percentages of germination and distance for resistant and susceptible sunflower varieties were observed in the other O. cernua populations tested (maximum germination percentages 22% and 38% in the presence of resistant and susceptible hosts, respectively; data not shown).
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) or susceptible Agrosur (
) sunflower roots. (A) Germinated seeds of Orobanche cernua population J6 Arburg; values expressed as a percentage of the total seeds. (B) Germinated seeds with browning symptoms, values expressed as percentage of the germinated seeds. Data are means of five independent replicates ±SE.
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Changes in some of the germinated O. cernua seeds started to be observed on day 7. Radicles turned from colourless and translucid to brownish-yellow, elongation ceased, and Orobanche development stopped (Fig. 1B
). At 14 d, the number of seeds showing these changes was higher in the presence of roots from the resistant variety (90% of germinated seeds) than the susceptible variety (75% of germinated seeds) (Fig. 2B). Browning was visible earlier in the presence of the resistant variety (over 75% of germinated seeds at day 11) than its susceptible counterpart (50% of germinated seeds at day 11). The germination process was also quantified using an identical germination bioassay with other plant and Orobanche species. The percentage of O. cernua seed germination in the presence of isolated roots from non-host plants (Solanum tuberosum, Nicotiana tabacum and Helianthus tuberosus), and that of O. ramosa (sunflower non-pathogen) in the presence of sunflower roots was, in all cases, lower than 2% (data not shown).
Orobanche cernua seed germination on filter paper in the presence of GR24 and coumarins
O. cernua seeds germinated on filter paper in the presence of the strigol analogue GR24. The percentage of germination (around 90%) was much higher than that obtained in the presence of isolated sunflower roots, although no visible differences were observed between germinated seeds in these two bioassays. Unlike the findings reported for O. crenata (Van Hezewijk et al., 1994), O. cernua germination did not require a previous conditioning period, although a more homogeneous and a higher percentage of germination was obtained with conditioned seeds. A low percentage of germination was obtained in the absence of GR24 (negative control, from 2–6%) and in the presence of low concentrations (1–30 µM) of scopoletin, ayapin, chlorogenic acid, isoliquiritigenin, and naringenin (from 10–18%; data not shown). The effect of scopoletin, ayapin, scopolin, chlorogenic acid, isoliquiritigenin, and naringenin on O. cernua seed germination induced by GR24was also evaluated. Both scopoletin and ayapin at concentrations of 0.01–1 mM inhibited GR24-induced germination (Fig. 3). The percentage of germination in the presence of these coumarins depended on the concentration used. Ayapin was a more potent inhibitor than scopoletin at all the concentrations tested. A combination of the two coumarins, at the same final concentration, led to a higher germination inhibition, thus showing an additive effect on the inhibition of the GR24-induced seed germination. In the presence of the glucosyl scopoletin derivative, scopolin, at 0.5 and 0.1 mM, the percentage of germination was quite close to that obtained in the presence of GR24 alone (79% and 84%, respectively). Neither chlorogenic acid, nor isoliquiritigenin and naringenin inhibited GR24-induced germination, with germination percentages higher than 75% at the 1 mM concentration tested (data not shown). Scopoletin and ayapin, when added to GR24-germinated O. cernua seeds, stopped radicle elongation and caused browning of the radicle in a similar way to that observed in the presence of isolated sunflower roots (data not shown).
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) or scopoletin+ayapin (
) at different concentrations. Data correspond to the J6 Arburg population and are expressed as a percentage of germination of the total seeds. Germination in MES buffer without GR24 was lower than 5%, and with GR24 reached 93%. Values are means of four independent replicates ±SE.
Orobanche cernua attachment bioassay
The early stages of the infection process (from attachment to tubercle formation) can be monitored with this bioassay using a binocular microscope (Fig. 1C–E). O. cernua successfully invaded the susceptible variety and small tubercles, 17–23 per plant, were established 18–25 d after inoculation (Fig. 1D). In the resistant variety, the O. cernua radicle elongated and adhered to the surface of the root as in the susceptible interaction, but within 3–5 d after inoculation, the O. cernua radicle became brown (Fig. 1E). No tubercle formation was observed in the roots of the resistant variety.
Coumarin induction in response to O. cernua infection
Root tissue from sunflower plants cvs Agrosur and Cortés grown in perlite mainly accumulated scopolin (around 25–30 nmol g-1 FW), with scopoletin not detected and ayapin present in a very small amount (below 3 nmol g-1 FW). The transfer of plants to the trays and inoculation with Orobanche seeds increased the amount of scopolin detected in the roots, with a peak after 24 h followed by a decrease. At all times, levels were higher in infected than in uninfected plants, and reached maximum levels in the resistant rather than in the susceptible varieties (Fig. 4). Ayapin in roots only increased in the resistant, infected variety, with values close to 9 nmol g-1 FW after 4 d, and decreasing to near zero on day 7. Scopoletin in roots was below the detection limit of the methodology used in all cases.
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), and resistant (
), sunflower varieties. Data are presented as nmol of the corresponding coumarin g-1 FW on different days after inoculation with Orobanche. Open symbols correspond to non-infected plants and closed symbols to infected plants. Values are mean of four independent replicates ±SE.Excreted coumarins were quantified after elution from the filter paper in contact with the sunflower root. The amount of scopolin excreted in non-infected susceptible and resistant control plants was much lower than that accumulated in root tissue (less than 35 nmol g-1 FW; Fig. 4
). Inoculation caused an increase in the amount of scopolin excreted, this effect being more notable in the resistant (134 nmol g-1 FW) than in the susceptible (55 nmol g-1 FW) varieties. The amount of ayapin excreted was much higher than that accumulated in roots (values between 10 and 50 nmol g-1 FW for the susceptible and resistant varieties, respectively). Inoculation stimulated a marked increase in the amount of excreted ayapin in the inoculated, resistant sunflower roots only, with values close to 150 nmol g-1 FW at 7 d post-inoculation. The amount of scopoletin excreted was much lower than scopolin or ayapin; in all cases, the highest concentration was recorded at 1 d post-inoculation, with no differences between sunflower varieties or between treatments (18–30 nmol g-1 FW); thereafter, values decreased to near control values (Fig. 4). The coumarin content was also analysed in the aerial part of the plant between 4 and 7 d post-inoculation. In plants grown in pots, as indicated above for the roots, scopolin was the major coumarin (around 20 nmol g-1 FW, with no differences between varieties), with ayapin and scopoletin below the sensitivity limit of the detection method used. Inoculation only caused significant increases in coumarin content in the resistant variety, with scopolin values of 240–260 nmol g-1 FW, and ayapin 14–23 nmol g-1 FW.
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Discussion |
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The possible mechanisms for preventing parasitic plant infection were studied in the host-specific interaction between sunflower and Orobanche cernua using a susceptible and a resistant variety. The susceptible variety supported Orobanche growth to mature flowering plants whereas the formation of the tubercle was only rarely observed in the resistant variety. This suggests that resistance mechanisms appear to be established during the early developmental stages of the parasite, from germination to connection and tubercle formation. Similar observations have been reported in other Orobanche and Striga interactions (Dörr et al., 1994
; Lane et al., 1997; Goldwasser et al., 1997).
Isolated sunflower roots from both the resistant and the susceptible variety stimulated O. cernua seed germination (Fig. 2A), but not that of O. ramosa (data not shown). By contrast, O. cernua seed germination was not observed in the presence of other plant species tested (Solanum tuberosum, Nicotiana tabacum and Helianthus tuberosus) (data not shown). These data indicate that the H. annuu–O. cernua interaction is species-specific at the germination stage. The percentage and distance of O. cernua seed germination was much lower in the presence of the resistant variety than the susceptible one (Fig. 2A). These differences may be interpreted as evidence of decreased stimulant production between both varieties. Other studies involving different sunflower varieties and Orobanche populations report no differences in germination (Dörr et al., 1994); some studies using other Orobanche spp. even report that resistant varieties induced germination at higher rates than susceptible ones (Goldwasser et al., 1997; unpublished results from this laboratory). Although it seems clear that in the sunflower–O. cernua interaction, unlike that reported for Striga (Butler, 1995), resistance is not established at the germination stage, a low production of germination stimulant could be relevant, since it might entail a reduction in the number of parasite individuals attacking the host plant.
The data presented here support the hypothesis of a host defence mechanism capable of preventing germination or development of the germinated parasitic seed. Thus, in the presence of isolated sunflower roots, some germinated O. cernua seeds stopped growing, turned brown and died (Fig. 1B), the percentage of seeds turning brown being higher in the presence of isolated roots from the resistant sunflower variety (Fig. 2B). This effect may be accounted for by host excretion of toxic compounds, a phenomenon that has previously been reported (Whitney, 1978). This allelopathic strategy has been extensively documented in the case of crop–weed competitiveness studies (Einhellig, 1995).
The absence of tubercle formation on the roots of the resistant variety, but not in the presence of the susceptible one, was observed using the tray attachment bioassay (Fig. 1D). In the resistant sunflower variety, browning of the attached Orobanche radicle and of the sunflower root around the attachment point was clearly visible (Fig. 1E). A similar reaction has been reported for sunflower and other resistant host varieties (Dörr et al., 1994; Goldwasser et al., 1997; Lane et al., 1997), and has been associated with the induced synthesis of phytoalexins or other toxic compounds whose accumulation may block radicle penetration through the host root, preventing connection to the host vascular tissue and causing parasite death.
The data presented here clearly indicate that, in sunflower, the induced synthesis, accumulation and excretion of toxic compounds seems to be an important defensive mechanism for preventing parasitic plant infection. The existence of defensive compounds effective against microorganisms, insects and competitive neighbouring plants has been demostrated in studies of the 7-hydroxylated simple coumarins (Rice, 1974; Murray et al., 1982; Tang et al., 1995; Wilson and Rice, 1968; Tal and Robeson, 1986; Urdangarín et al., 1999). The data obtained here suggest that the sunflower coumarins scopoletin, its glucosyl derivative, scopolin, and ayapin may also be part of the sunflower defence strategy against parasitic Orobanche, by acting as allelochemicals (preventing O. cernua seed germination or killing germinated seeds) or phytoalexins (preventing root penetration and connection to the vascular system). The hypothesis that phytoalexins may act as defensive compounds against parasitic weeds was first proposed by Wegmann (Wegmann et al., 1991) in experiments with sunflower and chickpea. In contrast to the results reported here, accumulation of scopoletin was observed with no indication of the presence of ayapin. No data concerning excretion was presented. More recently, phytoalexin induction in response to Striga has been reported (Lane et al., 1995). Whether scopoletin and ayapin production in sunflower plants is a clear factor of resistance or simply an unrelated defence response needs to be confirmed. So far, other typical plant defence responses have been reported to be induced as a consequence of parasitic plant infection (Mayer et al., 1997; Joel and Portnoy, 1998; Goldwasser et al., 1999). How effective the phytoalexin response is in preventing parasitism depends on the parasite. For instance, this does not seem to be the case of tobacco resistance to Orobanche (Westwood et al., 1998).
O. cernua also prompted an increase in scopolin and ayapin in the aerial portion of the plant. Previous results on coumarin synthesis in isolated organs (roots, stems and leaves) and whole plants have shown that aerial signals, metabolic precursors or the coumarins themselves are translocated from the aerial part of the plant (Gutiérrez-Mellado et al., 1997).
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Acknowledgments |
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This work was supported by the Spanish CICYT (AGF95-0103) and the Junta de Andalucía (AGR 0164). The authors thank Dr MA Fliniaux for kindly providing scopolin, Dr D Joel for kindly providing Orobanche seeds, and Dr P Bolwell for his critical reading of the manuscript.
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Notes |
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3 To whom correspondence should be addressed. Fax: +34 957 218563. E-mail: bf1jonoj{at}uco.es
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