Plant CellPhysiol. 37(8): 149-153 (1996) JSPP 1996 Haustoria of Cuscuta japonica, a Holoparasitic Flowering Plant, Are Induced by the Cooperative Effects of Far-Red Light and Tactile Stimuli Yoshifumi Tada', Michizo Sugai 2 and Katsuhisa Furuhashi 1-3 1 Department of Biology, Faculty of Science, Niigata University, Niigata, 95-21 Japan 2 Department of Biology, Faculty of Science, Toyama University, Toyama, 93 Japan When seedlings of Cuscuta japonica were grown with Vigna radiata (the host plant) in a flower pot for 6 d under white light and then irradiated with far-red or blue light (ca. 6 [ano\ photons m~ 2 s" 1 ), the seedlings parasitized V. radiata. However, no parasitism of the seedlings was observed under red or white light or in darkness. The parasitic behavior of seedlings of C. japonica was observed even if an acrylic rod was used as a substitute for the host plant. Upon incubation under far-red light, the seedling twined tightly around the rod and developed haustoria towards it. Haustoria also developed when apical and subapical regions of seedlings were held between two glass plates that were about.7 mm apart and were irradiated with far-red light. However, no haustoria were induced by either the hold or irradiation alone. These results indicate that parasitism of Cuscuta japonica is controlled by the cooperative effects of two physical signals, far-red light and appropriate tactile pressure. Our findings suggest that parasitism by the genus Cuscuta involves a novel strategy. Key words: Cuscuta japonica Far-red light Haustoria Parasitism Tactile stimuli. The importance of chemical signals in host-parasite interactions has been demonstrated in studies of various parasitic animals and plants. The response of parasites to a chemical signal from the host can also be observed in some parasitic angiosperms (Stewart and Press 199). However, since such observations are limited to certain species of root parasites (Change and Lynn 1986, Steffens et al. 1982, 1986), it is unknown whether or not stem parasites require some chemical signal for parasitism (Stewart and Press 199). We showed previously that far-red light induces parasitism between individual dodder plants (Cuscuta) of the same species cultured in vitro (Furuhashi et al. 1995). It appeared that the dodder plant might not need any chemical signal for successful parasitism. However, it is important to ascertain whether this photocontrol of parasitism exists in interactions between a general host and its parasite. Therefore, we chose to study an experimental system in To whom correspondence should be addressed. vivo with seedlings of Cuscuta japonica as the parasite and Vigna radiata as the host plant. This system has many advantages. In particular the physiological state of the experimental materials is relatively uniform and we were able successfully to detect reversible effects of far-red light and red light on the parasitism and on the developmet of haustoria. Using seedlings of C. japonica, we examined the possibility that induction of haustoria might occur without any signal from a living organism by using artificial, non-biological materials. This report describes the induction of haustoria in C. japonica by two physical signals, far-red light and touch. Materials and Methods Plant materials Seeds of Cuscuta japonica were collected at Kashiwazaki City in Niigata Prefecture in the autum of 1992 and stored at 4 to 6 C for 1 or 2 years. Prior to use the seeds were soaked for 15 min in concentrated H 2 SO 4 for to increase the frequency of germination and washed with distilled water. Seeds of Vigna radiata were purchased from a seed wholesaler in Niigata Prefecture. The seeds were allowed to imbibe water on wet filter paper for 24 h before sowing. Seeds of both C. japonica and V. radiata were sown at a depth of 2.5 cm in vermiculite in a flower pot and incubated at 26 ± 1 C under continuous white light (ca. 5>mol photons m~ 2 s~') from fluorescent lamps (FL- 2SSW/18G; Hitachi, Tokyo, Japan). Seedlings of V. radiata and C. japonica appeared at the surface of vermiculite 4 and 5 d after sowing, respectively. On day 6, seedlings of C. japonica that showed excessive or retarded elongation were discarded. The frequency of parasitism was calculated as the ratio of the number of parasitizing seedlings to the total number of seedlings. More than twenty plants were usually used in each experiment. Criteria for parasitism Seedlings of C. japonica that had been irradiated with light at the chosen wavelength for 24 h were incubated under white light for 2d. A parasitizing seedling had a typical profile, with the seedling tightly twined around a stem of V. radiata, making a coil with a low pitch. Elongation of the apical region was very significantly retarded by the coiling and haustoria projected from the coiled region. The development of haustoria was examined under a light microscope. Light source Blue light was obtained by passing light from blue-type fluorescent lamps (FL-2S B; wavelength, 37-47 nm; Toshiba, Tokyo, Japan) through a blue acrylic filter (Acrylite 373; Mitsubishi Rayon, Tokyo, Japan). Red light was obtained by passing light from red-type fluorescent lamps (FL-2S R-F; wavelength, 6-7 nm; Matsushita, Osaka, Japan) through four layers of red cellophane (Nisseido, Tokyo, Japan). Far-red light was obtained by passing light from far-red-type fluorescent lamps 149
15 Induction of haustoria in Cuscuta japonica (FL-2S FR-74; wavelength, 67-86 nm; Toshiba) through an acrylic filter (Acrylite 12, Mitsubishi Rayon). Measurements of the fluence rate of light were made with a silicon photocell, as described in a previous paper (Furuhashi et al. 1995). Fluence rates were adjusted by passage of light through black net to approximately 6//mol photons m~ 2 s~'. The glass-plate method for induction of haustoria For artificial induction of haustoria, apical and subapical regions of five seedlings were held between two glass plates (2cmx 1 cm, 1.7 mm in thickness) under white light and the lower parts of the stems that protruded from the plate were treated carefully to protect them from injury. The distance between the two plates was kept constant by putting pieces of filter paper (.7 mm in thickness) between them. The plates were placed horizontally in an incubator. The upper side of the plates was irradiated with far-red light for 2h at 26±1 C. After irradiation, the plates and plants were kept for 2 d in darkness. The number of haustoria that developed in the swirl that formed at the apical and subapical regions of the seedlings was counted under a light microscope. Results Parasitism of seedlings of Cuscuta japonica on Vigna radiata When seedlings of C. japonica and V. radiata (the host plant) were grown together in a flower pot in sunlight, almost all of the seedlings of C. japonica parasitized V. radiata. However, no parasitism at all was observed if the same experiment was carried out under white light from fluorescent lamps. The white light did not induce parasitism even if it was supplied as various alternating periods of light and darkness. These results suggest that the light environment is important for the parasitism by seedlings of C. japonica, as was also found in the case of parasitism of Cuscuta plants cultured in vitro (Furuhashi et al. 1995). We examined the effects of light quality on the induction of parasitism. When seedlings of C. japonica that had been grown for 6 days under white light from fluorescent lamps were incubated under continuous far-red or blue light (ca. 6^molm~ 2 s~') for 2d, the seedlings parasitized V. radiata (Fig. 1). Far-red light was more effective than blue light in inducing parasitism. Parasitism was also observed when irradiation with far-red or blue light for 1 d was followed by 2 d under white light. By contrast, no parasitism at all was observed when these seedlings were irradiated with white or red light or incubated in darkness (Table 1). To examine whether the effect of far-red light was canceled by red light, we irradiated seedlings of C. japonica alternately with one hour of far-red light and one hour of red light. The effect of far-red light on the parasitism was canceled by red light supplied immediately after the far-red light. This effect could be reversed when far-red light followed the red light (Table 2). Thus, far-red/red reversibility was clearly observed in the photo-induction of parasitism. The development of haustoria on the seedlings began Table 1 Effects of light quality on the parasitism of seedlings of Cuscuta japonica Light Blue Red Far-red White Darkness Rate of parasitism % Seedlings of C. japonica were grown with Vigna radiata for 6 d under white light. Then they were irradiated with light of various colors (ca. 6/miol photons m" 2 s"') for 24h and subsequently incubated at 26±1 C for 2d under white light (ca. 5^mol m~ 2 s~') fromfluorescentlamps. The rate of parasitism (%) was calculated as the ratio of the number of parasitizing seedlings to the total number of seedlings, multiplied by 1. within 24 h after C. japonica had entwined the host plant and continued for 2 or 3 d. If the entwining seedlings artificially detached from the host plant after 24 h, a second attachment by the newly elongated stem began upon exposure to far-red light. Moreover, some haustoria developed at the initial entwined region that had formerly been attached to the host plant (Fig. 2). This observation suggests that the development of haustoria progresses sequentially once it has been triggered. Parasitic behavior of seedlings of C. japonica in the presence of acrylic rods To determine whether some chemical signal from the host plant is needed for parasitism of C. japonica, we examined the parasitic behavior of the seedlings upon exposure to non-living materials. When seedlings were grown beside acrylic rods in a flower pot under white light from fluorescent lamps, the Table 2 Reversible effects of far-red light and red light on the parasitism of seedlings of Cuscuta japonica Light treatment - Rl 1 1 1 - FR 1 1 - FR 1 56 72 Rate of parasitism (%) 14 33 3 - Rl Seedlings of C. japonica and V. radiata were irradiated alternately with far-red and red light (ca. 6/umol m~ 2 s~"'). Afterwards, seedlings were incubated at 26± 1 C in darkness. Parasitism was examined at 48 h after the start of light treatments. and Rl indicate irradiation with far-red and red light, respectively, for 1 h. The rate of parasitism was calculated as described in the footnote to Table 1.
Induction of haustoria in Cuscuta japonica 151 Fig. 1 Parasitism of seedlings of C. japonica on Vigna radiata. Seedlings were precultured for 6 d under continuous white light from fluorescent lamps and then they were incubated for 2 d under continuous far-red light (6j/mol photons m~ 2 s~'). Arrows show coils where parasitism has occurred. Fig. 2 Development of a haustorium of C. japonica after artificial detachment from the host plant. A haustorium is visible on the stem of a seedlings of C. japonica. This kind of haustorium was observed in large numbers in subapical regions of seedlings that had entwined V. radiata and had been artificially removed after 24 h. Bar=l mm. seedlings elongated and parasitic behavior (entwining and the development of haustoria) was not observed. However, if the flower pot was irradiated by continuous far-red or blue light, each seedling wrapped itself tightly around an acrylic rod and projected haustoria towards it (Figs. 3, 4). This far-red light or blue light-induced parasitic behavior was observed with almost all of the experimental plants. By contrast, entwining and the development of haustoria did not occur under red or white light or in darkness. The ability of far-red light to induce parasitic behavior was observed even if the seedlings were exposed to far-red light for only 2 h and then incubated in darkness. These results indicate that parasitism of C. japonica is triggered by far-red or blue light and does not require any chemical signal from the host plant. Induction of haustoria using glass plates Parasitic entwining and development of haustoria by seedlings of Fig. 3 Parasitic behavior of C. japonica with an acrylic rod. When seedlings were grown for 6 d under white light from fluorescent lamps and then transferred to continuous far-red light, each seedling tightly entwined an acrylic rod (arrows), simulating the reaction to the host plant.
152 Induction of haustoria in Cuscuta japonica Fig. 6 Development of haustoria in the swirl at the apex of a seedling of C. japonica. Many haustoria (arrows) that project towards the center of the swirl are visible. Some haustoria have invaded the apical tissues of the adjoining stem. Bar= 1 mm. Fig. 4 Projection of haustoria of C. japonica towards an acrylic rod. Many haustoria (arrows) projected towards the acrylic rod. Bar=l mm. C. japonica upon contact with acrylic rods were induced by far-red light. However, far-red light failed to induce haustoria when individual seedlings were incubated in test tubes. These results suggest that induction of haustoria requires some other signal in addition to far-red light. We developed an appropriate method for investigation of such a signal. Apical and subapical regions of seedlings of C. japonica, grown for 7 d under white light from fluorescent lamps, were held between two glass plates that were approximately.7 mm apart and placed horizontally for vertical irradiation by far-red light. After irradiation for 2 h, the seedlings were incubated for 2 d in darkness. Figure 5 shows the result. The apical regions of seedlings coiled anti-clockwise and many haustoria projected to the center of each swirl (Fig. 6). The direction of the swirl was determined by gravity and did not depend on the direction of irradiation. These responses were observed in more than 8% of the experimental plants. By contrast, when seedlings between two glass plates were incubated under red or white light or in darkness, the apical regions of the seedlings elongated randomly and no development of haustoria was observed. Furthermore, far-red/red reversibility was also demonstrated for the formation of swirls and the development of haustoria (data not shown). Discussion Fig. 5 Swirls formed at the apical and subapical regions of seedlings of C. japonica. When apical and subapical regions of seedlings were held between two glass plates and irradiated with farred light, the apical region coiled anti-clockwise and formed a swirl. Details of the experimental procedure are given in the Materials and Methods. Bar=l cm. It has been generally accepted that parasitism is achieved by a complicated process that involves host-parasite interactions. In some parasitic angiosperms, parasitism is usually initiated by interactions between a chemical signal from the host and the receptor of the parasite. In the root parasite Striga asiatica, 2,6-dimethoxy-/?-benzoquinone
Induction of haustoria in Cuscuta japonica 153 acts as a chemical signal for recognition of the host plant (Chang and Lynn 1986). Xenognosins A and B and soyasapogenol B also serve as chemical signals in Agalinis purpurea (Steffens et al. 1982, 1986). However, details of the mechanisms of signal recognition have not been elucidated, and our present understanding of parasitism in angiosperms is limited. In the case of stem parasites, such as dodder plants, it is unknown whether or not some chemical signal is required for the induction of parasitism (Stewart and Press 199). In this study, we showed that the parasitic behavior of seedlings of Cuscuta japonica, a stem parasite, can be controlled by far-red or blue light. Moreover, we observed the reversible effects of far-red and red light on the parasitism. No parasitism was observed when seedlings were incubated under white light from fluorescent lamps, although the parasitism is frequently observed when plants are grown in sunlight. This discrepancy seems to result from the quality of light from fluorescent lamps which contains relatively little far-red light as compared with red light. The ratio of far-red to red light might be important for the induction of parasitism. Our experimental system, using seedlings of C. japonica, provides a new approach as well as confirming the results of a previous study (Furuhashi et al. 1995). We found that the development of haustoria is induced by the cooperative effects of two physical signals, far-red light and appropriate contact pressure. Our results indicate that C. japonica does not require any chemical or physical signal from the host plant, at least at the early stages of the parasitism, and they suggest that the development of haustoria can be interpreted as a type of photomorphogenesis. This study yielded the first evidence that a physical signal can induce haustoria in stem parasites. Furthermore, our findings suggest that the genus Cuscuta has a novel strategy for parasitism that differs from those of other parasite plants. Lane and Kasperbauer (1965) found that twining of seedlings of Cuscuta indicora is induced under blue or farred light and they proposed that phytochome participates in the twining. We showed here that the development of haustoria on seedlings of Cuscuta japonica is induced by far-red or blue light. The reversible effects of far-red light and red light suggests the participation of phytochrome in this phenomenon, although it is unclear whether or not the effect of blue light is mediated by phytochrome. Further studies of action spectra using a spectrograph are underway to characterize the putative participation of phytochrome. This study provides new approaches to investigations of parasitism. For example, the glass-plate method allows the synchronized formation of haustoria on seedlings of C. japonica. This synchronized formation of haustoria provides a potentially useful system for studies of the molecular biology of the development of haustoria in the genus Cuscuta. The authors are grateful to Prof. L.G. Hickok of the University of Tennessee for his careful checking of the manuscript. This work was supported in part by a General Research Grant (no. 68449) to K.F. from the Ministry of Education, Science and Culture, Japan. References Chang, M. and Lynn, D.G. (1986) Haustoria and the chemistry of host recognition in parasitic angiosperms. /. Chem. Ecol. 12: 561-579. Furuhashi, K., Kanno, M. and Morita, T. (1995) Photocontrol of parasitism in a parasitic flowering plant, Cuscuta japonica Chois, cultured in vitro. Plant Cell Physiol. 36: 533-536. Lane, H.C. and Kasperbauer, M.J. (1965) Photomorphogenic responses of dodder seedlings. Plant Physiol. 4: 19-116. Steffens, J.C., Lynn, D.G., Kamat, V. and Riopel, J.L. (1982) Molecular specificity of haustorial induction in Agalinis purpurea (L) Raf (Scrophulariaceae). Ann. Bot. 5: 1-7. Steffens, J.C., Lynn, D.G. and Riopel, J.L. (1986) A haustorial induction for the root parasite Agalinis purpurea. Phytochemistry 25: 2291-2298. Stewart, G.R. and Press, M.C. (199) The physiology and biochemistry of parasitic angiosperms. Annu. Rev. Plant Physiol. Plant Mot. Biol. 41: 127-151. (Received April 1, 1996; Accepted August 21, 1996)