Planta
Planta (1988) 176:183 188
9 Springer-Verlag 1988
Desensitization by red and blue light of phototropism in maize coleoptiles Moritoshi Iino** Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, CA 94305, USA
Abstract. The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that these desensitization responses contribute to the inductiion of second positive curvature, a response induced by prolonged irradiation.
Key words: Adaptation (photosensory) - Blue light - Coleoptile (phototropism) - Phototropism Phytochrome and phototropism - Zea (phototropism). Introduction
A pretreatment with red light (RL) is known to shift the bell-shaped phototropic fluence-response *CIW-DPB Publication No. 1001 **Present address and address for correspondence: Tokyo Metropolitan University, Department of Biology, Fukazawa 2-1-1, Setagaya-ku, Tokyo 158, Japan Abbreviations." BL=blue light; RL=red light
curve for the first positive curvature of oat and maize coleoptiles to higher fluences, the maximal shift observed being about one order of magnitude (Zimmerman and Briggs 1963; Blaauw and Blaauw-Jansen 1964; Briggs and Chon 1966). This RL response is most likely mediated by phytochrome (Chon and Briggs 1966). Evidence that blue light (BL) can also induce a shift was first provided by Blaauw and Blaauw-Jansen (1970) in oat coleoptiles, and has recently been also shown in maize (Iino 1987). These RL- and BL-dependent responses follow distinct time courses : after a brief light pretreatment, the RL-dependent shift developed slowly over a period of a few hours (Blaauw and Blaauw-Jansen 1964; Chon and Briggs 1966), whereas the BL-dependent response was estimated to reach the maximum within 5 min, followed by a slow return to the initial level (Iino 1987). The shifts of the fluence-response curve to higher fluences indicate that the quantum effectiveness for the phototropic response is lowered (note that the shifts have been observed in the response curve plotted as a function of the logarithm of fluences). In general, this type of desensitization could be a refection of an underlying photosensory adaptation (see Iino 1987). In maize coleoptiles, however, it has been shown that BL pretreatment not only induces a simple displacement of the fluence-response curve to higher fluences but includes a reduction in the response maximum (Iino 1987). This was most clearly shown by a pretreatment with a highfluence bilateral BL, which led to the complete elimination of the bell-shaped fluence-response curve, followed by a gradual reappearance of the curve. The shift of the fluence-response curve described above accompanied the reappearance of the response. The findings indicated that the BLinduced desensitization occurs with respect to both the response level and the quantum effectiveness. Blaauw and Blaauw-Jansen (1970) showed a
184
BL-induced shift of the fluence-response curve using oat coleoptiles in which the phytochrome-mediated shift was fully developed by a 2-h pretreatment with RL. In the study with maize (Iino 1987), BL-sensitive responses were observed by using RLgrown plants and performing experiments under background irradiation with RL. Under this condition, the phytochrome-mediated desensitization remained saturated (Iino and Briggs 1984). The use of RL-treated plants, in which phytochrome-mediated desensitization is saturated, is advantageous since it allows the isolation of the BL-specific response. It has not yet been elucidated, however, whether or not BL-specific desensitization occurs in dark-adapted plants. The present study has been undertaken primarily to investigate BL-specific desensitization of the first positive curvature in coleoptiles of darkadapted maize seedlings. It is difficult to achieve this by using only BL to induce the desensitization because the phytochrome-mediated desensitization would also be induced by this BL. Another obvious problem is the complication arising from phytochrome-mediated phototropism, which is also induced by BL (Iino et al. 1984a). In the present study, therefore, a saturating RL pulse was applied immediately after the inductive BL pulse. The BLspecific responses may then be evaluated in comparison with the control obtained with the RL treatment alone. This protocol also allows us to test whether or not BL-specific and phytochromemediated desensitization responses are simultaneously induced in the same plant. Material and methods Plant material. Seedlings of maize (Zea mays L. cv. Trojan T 929; Pfizer Genetics, Doniphan, Neb., USA) were raised as described in Iino et al. (1984a). In brief, maize seeds (kernels) were rinsed in running tap water for 4 h, sown on moist Kimpak paper (Kimberly-Clark, Neenah, Wis., USA), and incubated at 25-26 ~ C under RL (0.15 g m o l - m - 2 . s 1). Two days after sowing, seedlings were selected for uniformity, and transplanted into trays (base, 21.5.6.5 cm2; height, 5 cm) filled with moist vermiculite. Ten to twelve (mostly twelve) seedlings were planted in a row in each tray, oriented in such a way that the flat side of each seed was perpendicular to the plane of the seed row. The seedlings were incubated thereafter at 25 ~ C in the dark for an additional day, and used for the experiments. Experimental treatments with light, and measurement of curvature. Phototropic stimulation with BL was done by applying unilateral light normal to the plane of the plant row. The direction of incidence of irradiation was approximately parallel to the longest transverse axis of the coleoptile. The duration of phototropic stimulation was always 30 s. Treatment with RL (14 g m o l . m - 2 .s-1) was accomplished by applying light from above. The seedlings were incubated for 100 min after photo-
M. Iino : Light-induced desensitization of coleoptile phototropism tropic stimulation, and the curvatures of the coleoptiles were measured from their photocopier images (Iino et al. 1984a). Plants were not exposed to any light, except the BL and RL for the experimental treatments, after the end of RL pre-incubation. Each datum point is the mean measurement obtained from seedlings in three trays. In each experiment, phototropic stimulation with a fluence of 0.60 gmol. m - 2, corresponding to the peak of the phototropic fluence-response curve, was included as a control treatment to check the comparability of the responses measured on different occasions (experimental conditions as described for the top panel of Fig. 1). Responses in the controls obtained on different occasions varied only slightly (mean curvatures ranged between 21 ~ and 24~ indicating high reproducibility of the responses.
Light sources. Red light for pre-incubation : light from Sylvania (Danvers, Mass., USA) red fluorescent tubes was filtered through one layer each of No. I and No. 14 Cinemoid (Rank Strand Electrics, London, UK). Blue light for phototropic stimulation: light from a projector (600H with an ELH 300W lamp; Eastman-Kodak, Rochester, N.Y., USA) was passed through a blue glass filter (No. 5-60, 5 mm thick; Corning Glass Works, Corning, N.Y.). Red light for experimental treatment: light from Sylvania red fluorescent tubes was filtered through a layer of red Plexiglas (No. 2423; Rohm and Haas, Philadelphia, Pa., USA) and a layer of Cinemoid No. 1. Fluence rates of the BL for phototropic stimulation were controlled with calibrated neutral-density glass filters (Balzers, Marlborough, Mass., USA). Photon fluence rates were measured using a quantum photometer (LI-185A; Lambda Instruments Corp., Lincoln, Neb., USA).
Results
Figure I (top panel) shows the typical bell-shaped fluence-response curve for first positive curvature. In this experiment, dark-adapted plants were irradiated with 30 s unilateral BL, followed immediately by 30 s irradiation with RL (14 gmol.m -2. s-1) from above. The phototropic fluence-response curve obtained without RL treatment showed a negative curvature at high fluences, a consequence of gravitropic compensation for the phytochrome-mediated positive phototropism induced in the mesocotyl (Iino et al. 1984a, b). This apparent negative curvature could be eliminated here by the RL treatment. The fluence-response curve in Fig. 1 (top panel) is essentially identical to the one obtained previously with an RL treatment given immediately before phototropic stimulation (Iino etal. 1984a). The effect of a high-fluence BL pulse (105 gmol.m -2, given unilaterally) on the subsequent phototropic fluence-response relationships was investigated with varied dark periods between the high-fluence pulse and the inductive pulse of unilateral BL. An RL pulse (30 s, 14 gmol.m -2. s -1) was applied immediately after the highfluence BL pulse. To evaluate the BL-specific re-
M. Iino: Light-induced desensitization of coleoptile phototropism 30
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sponse, controls with only the RL pulse were made. This R L treatment, the same as that used above to eliminate phytochrome-mediated curvature responses, is also sufficient to saturate the phytochrome-mediated desensitization response (Sch/ifer et al. 1984). The high-fluence BL pulse, although given unilaterally, did not induce any apparent response: i.e., this fluence is at the bottom of the descending arm of the fluence-response curve (Fig. 1, top panel). Figure 1 shows results obtained with intervals of 4, 8, 16, 32 and 60 min. The fluence-response curves, including those in the R L controls (obtained for 16, 32 and 60 min), showed complex changes with increasing time intervals. The most conspicuous is the effect of BL pretreatment showing the elimination and subsequent reappearance of the phototropic response. When phototropic stimulation was given 4 min after the high-fluence BL pulse, only a very slight response, if any, could be found. With a further delay before phototropic stimulation, the bell-shaped fluence-response curve began to appear and the peak height increased progressively (Fig. 1, 8-60 min). It is also clear that, in the course of the reappearance of the fluenceresponse curve, the responses at higher fluences (around 10 g m o l . m -2) were greater than those in the RL controls (Fig. 1, 16-60 min). In most cases, the unilateral BL irradiation for pretreatment and that for phototropic stimulation were made from the same side of the coleoptile (solid circles in Fig. 1, 16-60 min). When phototropic stimulation was made at 16 min from the side opposite to that of pretreatment, the resulting fluence-response curve (data shown with solid squares) was similar, though perhaps not identical, to that found when the irradiation was given from
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Fig. 1. Effects of pretreatment with a high-fluence BL pulse on the phototropic fluence-response curve for first positive curvature of dark-adapted maize coleoi~tiles. Curvature was measured 100 min after phototropic stimulation (30 s) with unilateral blue light (BL). Top panel: fluence-response curve obtained without BL pretreatment. Red light (30 s, 420 l-Lmolm -2) was given from above immediately after phototropic stimulation. Different symbols indicate separate experiments. Lower panels: fluence-response curves with BL pretreatment. Following unilateral irradiation with a high-fluence BL pulse (30 s, 105 gmol.m-2), phototropic stimulation was given at the time indicated in each panel and measured from the onset of high-fluence BL to that of phototropic stimulation (solid circles). Red light (30 s, 420 gmol.m -2) was given from above immediately after the high-fluence BL pulse. Controls without the BL pretreatment were obtained by providing the RL at comparable times (open circles). The normalized curve in the top panel is shown for comparison (dotted lines). Each datum point is the mean obtained from 30-36 seedlings. Vertical bars = + S E
186
the same side. It was nevertheless evident that with the phototropic stimulation from the opposite side, a response exceeding that in the control also occurred at high fluences. The following conclusions are reached. First, a high-fluence BL pulse eliminates the phototropic responses to subsequent unilateral stimulation; afterwards, the phototropic response reappears gradually. Second, a shift of the fluence-response curve to higher fluences accompanies the disappearance and reappearance of the response. The phototropic fluence-response curves in the RL controls indicated that the RL treatment given 16 min before the inductive pulse of unilateral BL did not measurably affect the fluence-response curve, but the treatment given 30 and 60 min before induced progressively a shift of the curve to higher fluences and an increase in the peak height (Fig. 1). Comparisons of the responses in BL-treated plants and those in RL controls make it clear that the shift induced by BL occurs far earlier than that induced by RL. This was most evident in the results obtained at 16 min (Fig. 1). At this time, no significant RL-induced shift was observed, but the BL-induced shift was already evident. A small peak found at 8 min in the high-fluence range indicates that the shift is induced very rapidly. The results demonstrate that in dark-adapted maize coleoptiles, phytochrome-mediated and BL-specific shifts of the fluence-response curve can be induced simultaneously, and that the two responses develop following different time courses. Discussion
Desensitization and regeneration responses following a BL pulse. The previous study with RLadapted maize coleoptiles (Iino 1987) has shown (1) that pretreatment with high-fluence BL abolishes phototropic responses to an immediately following unilateral BL pulse, but the responsiveness to the unilateral pulse is gradually restored in such a way that the height of the bell-shaped fluenceresponse curve increases gradually, (2) that during the course of this reappearance of the response, the fluence-response curve is positioned at higher fluences compared to that induced without BL pretreatment, and (3) that when a sufficient time (12 h) is inserted between the high-fluence pulse and the inductive unilateral pulse, both height and position of the fluence-response curve are close to those found without the BL pretreatment. The present study demonstrates that these properties of the first positive curvature are indeed also pres-
M. Iino: Light-induced desensitization of coleoptile phototropism
ent in dark-adapted coleoptiles, a material most commonly used in phototropism research. The regeneration of responsiveness following a preceding pulse appears to be a common property of BL responses of plants, as demonstrated in the BL-induced stomatal response in Commelina (Iino et al. 1985) and BL-induced cell division in Adiantum protonemata (Iino et al. 1988) in which, after stimulation with a saturating pulse, responsiveness to another pulse was gradually restored. Steinitz and Poff (1986) arrived at a similar conclusion from studies of the phototropism of Arabidopsis hypocotyls showing that responses to multiple pulses were optimized when certain time intervals were provided between the pulses. Iino (1987) and Iino et al. (1985, 1988) interpreted the regeneration response in terms of a photoproduct which is produced by pulse stimulation and is converted back to the initial reactant(s) by a dark reaction. In these studies, mathematical functions were formulated based on the interpretations and were fitted to the data from two-pulse experiments to yield the predicted values of the rate constant of the dark reaction. Steinitz and Poff (1986) interpreted their results with multiple pulses by assuming that an active photoproduct decays in the dark and that a reactant (either the photoreceptor or an intermediate) for the photoproduct is re-synthesized in the dark. Although these authors did not assume that the reactant is regenerated from the photoproduct, the interpretation is in principle consonant with that described above. Another feature of the first positive curvature - the reduction of the sensitivity to BL by BL pretreatment - has so far been demonstrated in oat and maize coleoptiles (Blaauw and Blaauw-Jansen 1970; Iino 1987; present study) although not in other BL responses (Iino et al. 1985, 1988). However, a recent study of BL-induced hair-whorl formation in Acetabularia has shown that sensitivity to BL is reduced by a preceding stimulation with a low-fluence BL pulse (Schmid et al. 1988). The relationship between first positive curvature and second positive curvature (phototropism induced by prolonged stimulation) has long been obscure (see Iino 1987). It has recently been argued that second positive curvature can be explained with the two features of first positive curvature described above, i.e., the regeneration of responsiveness and the reduction of the BL sensitivity (Steinitz and Poff 1986; Iino 1987). Computation of the predicted response curves for long irradiation based on the results from pulse experiments are straightforward when BL-induced sensitivity changes are not apparent (Iino et al. 1985, 1988).
M. Iino: Light-induced desensitization of coleoptile phototropism
In the case of phototropism where sensitivity changes are thought to play an important role, such an approach is not easy. The changes in the light-sensitivity parameter (such as m in Iino 1987) during long irradiation must be determined before the predicted "second positive" responses can be computed for a test of the hypotlhesis.
Influences of red light. In the present study it has been demonstrated that RL-dependent and BL-dependent shifts of the photototropic fluence-respouse curve to higher fluences can be induced simultaneously, and confirmed that the shift induced by RL develops slowly (Blaauw and Blaauw-Jansen 1964; Chon and Briggs 1966) whereas that induced by BL develops rapidly and is relatively transient (Iino 1987). The results also indicate that the shift by BL can exceed that induced by a saturating fluence of RL. This in turn indicates that RL-induced and BL-induced responses are additive. The model presented in the preceding paper (Iino 1987) can be extended to incorporate the phenomenon of RL-induced shift of the fluence-respouse curve, by assuming that the phytochrome system, as postulated for the shift induced by BL, reduces the quantum effectiveness for the photoproduct formation. It is not required that the earliest reaction points affected in the phototropic sensory transduction chain by phytochrome and the BL system be the same. This extension of the model provides, as discussed below, an explanation for an unresolved problem concerning the relationship between first and second positive curvatures. It is known that the second positive curvature of oat coleoptiles is stimulated by a 2-h pretreatment with RL, despite the desensitization of the first positive curvature by the same treatment (Zimmerman and Briggs 1963). This phenomenon may be explained in the following way. In RLpretreated plants, the BL sensitivity of the photoproduct formation is lowered by both phytochrome and the BL system whereas in the nontreated plants, only the BL-dependent desensitization occurs. The phytochrome-mediated desensitization would be induced by the BL used for phototropic stimulation; however, it would not develop to a substantial extent, because of the slow development of the phytochrome-mediated response during the stimulation times commonly used for the induction of the second positive curvature. If any significant concentration difference in the photoproduct between the irradiated and shaded sides were to be reached under the steady-state condition, it would be achieved at lower fluence rates
187
of BL in the RL-treated plants, leading to a greater second positive response. Conflicting results have been reported concerning the effect of RL on the peak height of the fluence-response curve. Previous investigations with maize (same cultivar) under similar experimental conditions showed a curve displacement in response to RL that was accompanied with some increase (Sch/ifer et al. 1984) or no increase (Hofmann and Sch/ifer 1987) of the peak height. The results obtained here appear to show a still larger peak increase than that described by Sch/ifer et al. (1984). The reason for these disagreements is not clear. The RL effect to increase the peak height and that to shift the fluence-response curve may follow different time courses; the response in peak height measured at I h (Fig. 1) decreases by 2 h (Sch/ifer et al. 1984; Hofmann and Schiller 1987) whereas the shift of the curve is sustained over 2 h. Unlike the effect of RL in shifting the fluenceresponse curve, the change in the peak height may be an indirect effect of RL, arising for example from changes in the growth-rate distribution along the coleoptile (Curry et al. 1956; Iino 1982). The author is indebted to Dr. Winslow R. Briggs for generous encouragement during this research.
References Blaauw, O.H., Blaauw-Jansen, G. (1964) The influence of red light on the phototropism of Arena coleoptiles. Acta Bot. Need. 13, 541-552 Blaauw, O.H., Blaauw-Jansen, G. (1970) Third positive (c-type) phototropism in the Arena coleoptiles. Acta Bot. Neerl. 19, 764-776 Briggs, W.R., Chon, H.P. (1966) The physiological versus the spectrophotometric status of phytochrome in corn coleoptiies. Plant Physiol. 41, 1159-1166 Chon, H.P., Briggs, W.R. (1966) Effect of red light on the phototropic sensitivity of corn coleoptiles. Plant Physiol. 41, 1715-1724 Curry, G.M., Thimann, K.V., Ray, P.M. (1956) The base curvature response of Arena seedlings to the UV. Physiol. Plant 9, 429-440 Hofmann, E., Sch/ifer, E. (1987) Red light-induced shift of the fluence-response curve for first positive curvature of maize coleoptiles. Plant Cell Physiol. 28, 37~45 Iino, M. (1982) Action of red light on indole-3-acetic acid status and growth in coleoptiles of etiolated maize seedlings. Planta 156, 21-32 Iino, M. (1987) Kinetic modelling of phototropism in maize coleoptiles. Planta 171, 110 126 Iino, M., Briggs, W.R. (1984) Growth distribution during first positive phototropic curvature of maize coleoptiles. Plant Cell Environ. 7, 97-104 Iino, M., Briggs, W.R., Sch~ifer, E. (1984a) Phytochrome-mediated phototropism in maize seedling shoots. Planta 160, 41-51 Iino, M., Nakagawa, Y., Wada, M. 0988) Blue light-regulation
188 of cell division in Adiantum protonemata: kinetic properties of the photosystem. Plant Cell Environ. 11 (in press) Iino, M., Ogawa, T., Zeiger, E. (1985) Kinetic properties of the blue-light response of stomata. Proc. Natl. Acad. Sci. USA 82, 8019-8023 Iino, M., Sch/ifer, E., Briggs, W.R. (1984b) Photoperception sites for phytochrome-mediated phototropism of maize mesocotyls. Planta 162, 477M79 Sch/ifer, E., Iino, M., Briggs, W.R. (1984) Red-light-induced shift of the fluence-response curve for first positive phototropic curvature of maize coleoptiles. In: Blue light effects on biological systems, pp. 476M79, Senger, H., ed. Springer, Berlin Heidelberg
M. Iino: Light-induced desensitization of coleoptiIe phototropism Schmid, R., Tiinnermann, M., Idziak, E.-M. (1988) Transient reduction of responsiveness of blue-light mediated hair whorl morphogenesis in Acetabularia mediterranea induced by blue light. Planta 174, 373-379 Steinitz, B., Poff, K.L. (1986) A single positive phototropic response induced with pulsed light in hypocotyls of Arab# dopsis thaliana seedlings. Planta 168, 305-315 Zimmerman, B.R., Briggs, W.R. (1963) Phototropic dosageresponse curves for oat coleoptiles. Plant Physiol. 38, 248 253 Received 9 October 1987; accepted 31 May 1988
Planta (1988) 176:183 188
9 Springer-Verlag 1988
Desensitization by red and blue light of phototropism in maize coleoptiles Moritoshi Iino** Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, CA 94305, USA
Abstract. The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that these desensitization responses contribute to the inductiion of second positive curvature, a response induced by prolonged irradiation.
Key words: Adaptation (photosensory) - Blue light - Coleoptile (phototropism) - Phototropism Phytochrome and phototropism - Zea (phototropism). Introduction
A pretreatment with red light (RL) is known to shift the bell-shaped phototropic fluence-response *CIW-DPB Publication No. 1001 **Present address and address for correspondence: Tokyo Metropolitan University, Department of Biology, Fukazawa 2-1-1, Setagaya-ku, Tokyo 158, Japan Abbreviations." BL=blue light; RL=red light
curve for the first positive curvature of oat and maize coleoptiles to higher fluences, the maximal shift observed being about one order of magnitude (Zimmerman and Briggs 1963; Blaauw and Blaauw-Jansen 1964; Briggs and Chon 1966). This RL response is most likely mediated by phytochrome (Chon and Briggs 1966). Evidence that blue light (BL) can also induce a shift was first provided by Blaauw and Blaauw-Jansen (1970) in oat coleoptiles, and has recently been also shown in maize (Iino 1987). These RL- and BL-dependent responses follow distinct time courses : after a brief light pretreatment, the RL-dependent shift developed slowly over a period of a few hours (Blaauw and Blaauw-Jansen 1964; Chon and Briggs 1966), whereas the BL-dependent response was estimated to reach the maximum within 5 min, followed by a slow return to the initial level (Iino 1987). The shifts of the fluence-response curve to higher fluences indicate that the quantum effectiveness for the phototropic response is lowered (note that the shifts have been observed in the response curve plotted as a function of the logarithm of fluences). In general, this type of desensitization could be a refection of an underlying photosensory adaptation (see Iino 1987). In maize coleoptiles, however, it has been shown that BL pretreatment not only induces a simple displacement of the fluence-response curve to higher fluences but includes a reduction in the response maximum (Iino 1987). This was most clearly shown by a pretreatment with a highfluence bilateral BL, which led to the complete elimination of the bell-shaped fluence-response curve, followed by a gradual reappearance of the curve. The shift of the fluence-response curve described above accompanied the reappearance of the response. The findings indicated that the BLinduced desensitization occurs with respect to both the response level and the quantum effectiveness. Blaauw and Blaauw-Jansen (1970) showed a
184
BL-induced shift of the fluence-response curve using oat coleoptiles in which the phytochrome-mediated shift was fully developed by a 2-h pretreatment with RL. In the study with maize (Iino 1987), BL-sensitive responses were observed by using RLgrown plants and performing experiments under background irradiation with RL. Under this condition, the phytochrome-mediated desensitization remained saturated (Iino and Briggs 1984). The use of RL-treated plants, in which phytochrome-mediated desensitization is saturated, is advantageous since it allows the isolation of the BL-specific response. It has not yet been elucidated, however, whether or not BL-specific desensitization occurs in dark-adapted plants. The present study has been undertaken primarily to investigate BL-specific desensitization of the first positive curvature in coleoptiles of darkadapted maize seedlings. It is difficult to achieve this by using only BL to induce the desensitization because the phytochrome-mediated desensitization would also be induced by this BL. Another obvious problem is the complication arising from phytochrome-mediated phototropism, which is also induced by BL (Iino et al. 1984a). In the present study, therefore, a saturating RL pulse was applied immediately after the inductive BL pulse. The BLspecific responses may then be evaluated in comparison with the control obtained with the RL treatment alone. This protocol also allows us to test whether or not BL-specific and phytochromemediated desensitization responses are simultaneously induced in the same plant. Material and methods Plant material. Seedlings of maize (Zea mays L. cv. Trojan T 929; Pfizer Genetics, Doniphan, Neb., USA) were raised as described in Iino et al. (1984a). In brief, maize seeds (kernels) were rinsed in running tap water for 4 h, sown on moist Kimpak paper (Kimberly-Clark, Neenah, Wis., USA), and incubated at 25-26 ~ C under RL (0.15 g m o l - m - 2 . s 1). Two days after sowing, seedlings were selected for uniformity, and transplanted into trays (base, 21.5.6.5 cm2; height, 5 cm) filled with moist vermiculite. Ten to twelve (mostly twelve) seedlings were planted in a row in each tray, oriented in such a way that the flat side of each seed was perpendicular to the plane of the seed row. The seedlings were incubated thereafter at 25 ~ C in the dark for an additional day, and used for the experiments. Experimental treatments with light, and measurement of curvature. Phototropic stimulation with BL was done by applying unilateral light normal to the plane of the plant row. The direction of incidence of irradiation was approximately parallel to the longest transverse axis of the coleoptile. The duration of phototropic stimulation was always 30 s. Treatment with RL (14 g m o l . m - 2 .s-1) was accomplished by applying light from above. The seedlings were incubated for 100 min after photo-
M. Iino : Light-induced desensitization of coleoptile phototropism tropic stimulation, and the curvatures of the coleoptiles were measured from their photocopier images (Iino et al. 1984a). Plants were not exposed to any light, except the BL and RL for the experimental treatments, after the end of RL pre-incubation. Each datum point is the mean measurement obtained from seedlings in three trays. In each experiment, phototropic stimulation with a fluence of 0.60 gmol. m - 2, corresponding to the peak of the phototropic fluence-response curve, was included as a control treatment to check the comparability of the responses measured on different occasions (experimental conditions as described for the top panel of Fig. 1). Responses in the controls obtained on different occasions varied only slightly (mean curvatures ranged between 21 ~ and 24~ indicating high reproducibility of the responses.
Light sources. Red light for pre-incubation : light from Sylvania (Danvers, Mass., USA) red fluorescent tubes was filtered through one layer each of No. I and No. 14 Cinemoid (Rank Strand Electrics, London, UK). Blue light for phototropic stimulation: light from a projector (600H with an ELH 300W lamp; Eastman-Kodak, Rochester, N.Y., USA) was passed through a blue glass filter (No. 5-60, 5 mm thick; Corning Glass Works, Corning, N.Y.). Red light for experimental treatment: light from Sylvania red fluorescent tubes was filtered through a layer of red Plexiglas (No. 2423; Rohm and Haas, Philadelphia, Pa., USA) and a layer of Cinemoid No. 1. Fluence rates of the BL for phototropic stimulation were controlled with calibrated neutral-density glass filters (Balzers, Marlborough, Mass., USA). Photon fluence rates were measured using a quantum photometer (LI-185A; Lambda Instruments Corp., Lincoln, Neb., USA).
Results
Figure I (top panel) shows the typical bell-shaped fluence-response curve for first positive curvature. In this experiment, dark-adapted plants were irradiated with 30 s unilateral BL, followed immediately by 30 s irradiation with RL (14 gmol.m -2. s-1) from above. The phototropic fluence-response curve obtained without RL treatment showed a negative curvature at high fluences, a consequence of gravitropic compensation for the phytochrome-mediated positive phototropism induced in the mesocotyl (Iino et al. 1984a, b). This apparent negative curvature could be eliminated here by the RL treatment. The fluence-response curve in Fig. 1 (top panel) is essentially identical to the one obtained previously with an RL treatment given immediately before phototropic stimulation (Iino etal. 1984a). The effect of a high-fluence BL pulse (105 gmol.m -2, given unilaterally) on the subsequent phototropic fluence-response relationships was investigated with varied dark periods between the high-fluence pulse and the inductive pulse of unilateral BL. An RL pulse (30 s, 14 gmol.m -2. s -1) was applied immediately after the highfluence BL pulse. To evaluate the BL-specific re-
M. Iino: Light-induced desensitization of coleoptile phototropism 30
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sponse, controls with only the RL pulse were made. This R L treatment, the same as that used above to eliminate phytochrome-mediated curvature responses, is also sufficient to saturate the phytochrome-mediated desensitization response (Sch/ifer et al. 1984). The high-fluence BL pulse, although given unilaterally, did not induce any apparent response: i.e., this fluence is at the bottom of the descending arm of the fluence-response curve (Fig. 1, top panel). Figure 1 shows results obtained with intervals of 4, 8, 16, 32 and 60 min. The fluence-response curves, including those in the R L controls (obtained for 16, 32 and 60 min), showed complex changes with increasing time intervals. The most conspicuous is the effect of BL pretreatment showing the elimination and subsequent reappearance of the phototropic response. When phototropic stimulation was given 4 min after the high-fluence BL pulse, only a very slight response, if any, could be found. With a further delay before phototropic stimulation, the bell-shaped fluence-response curve began to appear and the peak height increased progressively (Fig. 1, 8-60 min). It is also clear that, in the course of the reappearance of the fluenceresponse curve, the responses at higher fluences (around 10 g m o l . m -2) were greater than those in the RL controls (Fig. 1, 16-60 min). In most cases, the unilateral BL irradiation for pretreatment and that for phototropic stimulation were made from the same side of the coleoptile (solid circles in Fig. 1, 16-60 min). When phototropic stimulation was made at 16 min from the side opposite to that of pretreatment, the resulting fluence-response curve (data shown with solid squares) was similar, though perhaps not identical, to that found when the irradiation was given from
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Fig. 1. Effects of pretreatment with a high-fluence BL pulse on the phototropic fluence-response curve for first positive curvature of dark-adapted maize coleoi~tiles. Curvature was measured 100 min after phototropic stimulation (30 s) with unilateral blue light (BL). Top panel: fluence-response curve obtained without BL pretreatment. Red light (30 s, 420 l-Lmolm -2) was given from above immediately after phototropic stimulation. Different symbols indicate separate experiments. Lower panels: fluence-response curves with BL pretreatment. Following unilateral irradiation with a high-fluence BL pulse (30 s, 105 gmol.m-2), phototropic stimulation was given at the time indicated in each panel and measured from the onset of high-fluence BL to that of phototropic stimulation (solid circles). Red light (30 s, 420 gmol.m -2) was given from above immediately after the high-fluence BL pulse. Controls without the BL pretreatment were obtained by providing the RL at comparable times (open circles). The normalized curve in the top panel is shown for comparison (dotted lines). Each datum point is the mean obtained from 30-36 seedlings. Vertical bars = + S E
186
the same side. It was nevertheless evident that with the phototropic stimulation from the opposite side, a response exceeding that in the control also occurred at high fluences. The following conclusions are reached. First, a high-fluence BL pulse eliminates the phototropic responses to subsequent unilateral stimulation; afterwards, the phototropic response reappears gradually. Second, a shift of the fluence-response curve to higher fluences accompanies the disappearance and reappearance of the response. The phototropic fluence-response curves in the RL controls indicated that the RL treatment given 16 min before the inductive pulse of unilateral BL did not measurably affect the fluence-response curve, but the treatment given 30 and 60 min before induced progressively a shift of the curve to higher fluences and an increase in the peak height (Fig. 1). Comparisons of the responses in BL-treated plants and those in RL controls make it clear that the shift induced by BL occurs far earlier than that induced by RL. This was most evident in the results obtained at 16 min (Fig. 1). At this time, no significant RL-induced shift was observed, but the BL-induced shift was already evident. A small peak found at 8 min in the high-fluence range indicates that the shift is induced very rapidly. The results demonstrate that in dark-adapted maize coleoptiles, phytochrome-mediated and BL-specific shifts of the fluence-response curve can be induced simultaneously, and that the two responses develop following different time courses. Discussion
Desensitization and regeneration responses following a BL pulse. The previous study with RLadapted maize coleoptiles (Iino 1987) has shown (1) that pretreatment with high-fluence BL abolishes phototropic responses to an immediately following unilateral BL pulse, but the responsiveness to the unilateral pulse is gradually restored in such a way that the height of the bell-shaped fluenceresponse curve increases gradually, (2) that during the course of this reappearance of the response, the fluence-response curve is positioned at higher fluences compared to that induced without BL pretreatment, and (3) that when a sufficient time (12 h) is inserted between the high-fluence pulse and the inductive unilateral pulse, both height and position of the fluence-response curve are close to those found without the BL pretreatment. The present study demonstrates that these properties of the first positive curvature are indeed also pres-
M. Iino: Light-induced desensitization of coleoptile phototropism
ent in dark-adapted coleoptiles, a material most commonly used in phototropism research. The regeneration of responsiveness following a preceding pulse appears to be a common property of BL responses of plants, as demonstrated in the BL-induced stomatal response in Commelina (Iino et al. 1985) and BL-induced cell division in Adiantum protonemata (Iino et al. 1988) in which, after stimulation with a saturating pulse, responsiveness to another pulse was gradually restored. Steinitz and Poff (1986) arrived at a similar conclusion from studies of the phototropism of Arabidopsis hypocotyls showing that responses to multiple pulses were optimized when certain time intervals were provided between the pulses. Iino (1987) and Iino et al. (1985, 1988) interpreted the regeneration response in terms of a photoproduct which is produced by pulse stimulation and is converted back to the initial reactant(s) by a dark reaction. In these studies, mathematical functions were formulated based on the interpretations and were fitted to the data from two-pulse experiments to yield the predicted values of the rate constant of the dark reaction. Steinitz and Poff (1986) interpreted their results with multiple pulses by assuming that an active photoproduct decays in the dark and that a reactant (either the photoreceptor or an intermediate) for the photoproduct is re-synthesized in the dark. Although these authors did not assume that the reactant is regenerated from the photoproduct, the interpretation is in principle consonant with that described above. Another feature of the first positive curvature - the reduction of the sensitivity to BL by BL pretreatment - has so far been demonstrated in oat and maize coleoptiles (Blaauw and Blaauw-Jansen 1970; Iino 1987; present study) although not in other BL responses (Iino et al. 1985, 1988). However, a recent study of BL-induced hair-whorl formation in Acetabularia has shown that sensitivity to BL is reduced by a preceding stimulation with a low-fluence BL pulse (Schmid et al. 1988). The relationship between first positive curvature and second positive curvature (phototropism induced by prolonged stimulation) has long been obscure (see Iino 1987). It has recently been argued that second positive curvature can be explained with the two features of first positive curvature described above, i.e., the regeneration of responsiveness and the reduction of the BL sensitivity (Steinitz and Poff 1986; Iino 1987). Computation of the predicted response curves for long irradiation based on the results from pulse experiments are straightforward when BL-induced sensitivity changes are not apparent (Iino et al. 1985, 1988).
M. Iino: Light-induced desensitization of coleoptile phototropism
In the case of phototropism where sensitivity changes are thought to play an important role, such an approach is not easy. The changes in the light-sensitivity parameter (such as m in Iino 1987) during long irradiation must be determined before the predicted "second positive" responses can be computed for a test of the hypotlhesis.
Influences of red light. In the present study it has been demonstrated that RL-dependent and BL-dependent shifts of the photototropic fluence-respouse curve to higher fluences can be induced simultaneously, and confirmed that the shift induced by RL develops slowly (Blaauw and Blaauw-Jansen 1964; Chon and Briggs 1966) whereas that induced by BL develops rapidly and is relatively transient (Iino 1987). The results also indicate that the shift by BL can exceed that induced by a saturating fluence of RL. This in turn indicates that RL-induced and BL-induced responses are additive. The model presented in the preceding paper (Iino 1987) can be extended to incorporate the phenomenon of RL-induced shift of the fluence-respouse curve, by assuming that the phytochrome system, as postulated for the shift induced by BL, reduces the quantum effectiveness for the photoproduct formation. It is not required that the earliest reaction points affected in the phototropic sensory transduction chain by phytochrome and the BL system be the same. This extension of the model provides, as discussed below, an explanation for an unresolved problem concerning the relationship between first and second positive curvatures. It is known that the second positive curvature of oat coleoptiles is stimulated by a 2-h pretreatment with RL, despite the desensitization of the first positive curvature by the same treatment (Zimmerman and Briggs 1963). This phenomenon may be explained in the following way. In RLpretreated plants, the BL sensitivity of the photoproduct formation is lowered by both phytochrome and the BL system whereas in the nontreated plants, only the BL-dependent desensitization occurs. The phytochrome-mediated desensitization would be induced by the BL used for phototropic stimulation; however, it would not develop to a substantial extent, because of the slow development of the phytochrome-mediated response during the stimulation times commonly used for the induction of the second positive curvature. If any significant concentration difference in the photoproduct between the irradiated and shaded sides were to be reached under the steady-state condition, it would be achieved at lower fluence rates
187
of BL in the RL-treated plants, leading to a greater second positive response. Conflicting results have been reported concerning the effect of RL on the peak height of the fluence-response curve. Previous investigations with maize (same cultivar) under similar experimental conditions showed a curve displacement in response to RL that was accompanied with some increase (Sch/ifer et al. 1984) or no increase (Hofmann and Sch/ifer 1987) of the peak height. The results obtained here appear to show a still larger peak increase than that described by Sch/ifer et al. (1984). The reason for these disagreements is not clear. The RL effect to increase the peak height and that to shift the fluence-response curve may follow different time courses; the response in peak height measured at I h (Fig. 1) decreases by 2 h (Sch/ifer et al. 1984; Hofmann and Schiller 1987) whereas the shift of the curve is sustained over 2 h. Unlike the effect of RL in shifting the fluenceresponse curve, the change in the peak height may be an indirect effect of RL, arising for example from changes in the growth-rate distribution along the coleoptile (Curry et al. 1956; Iino 1982). The author is indebted to Dr. Winslow R. Briggs for generous encouragement during this research.
References Blaauw, O.H., Blaauw-Jansen, G. (1964) The influence of red light on the phototropism of Arena coleoptiles. Acta Bot. Need. 13, 541-552 Blaauw, O.H., Blaauw-Jansen, G. (1970) Third positive (c-type) phototropism in the Arena coleoptiles. Acta Bot. Neerl. 19, 764-776 Briggs, W.R., Chon, H.P. (1966) The physiological versus the spectrophotometric status of phytochrome in corn coleoptiies. Plant Physiol. 41, 1159-1166 Chon, H.P., Briggs, W.R. (1966) Effect of red light on the phototropic sensitivity of corn coleoptiles. Plant Physiol. 41, 1715-1724 Curry, G.M., Thimann, K.V., Ray, P.M. (1956) The base curvature response of Arena seedlings to the UV. Physiol. Plant 9, 429-440 Hofmann, E., Sch/ifer, E. (1987) Red light-induced shift of the fluence-response curve for first positive curvature of maize coleoptiles. Plant Cell Physiol. 28, 37~45 Iino, M. (1982) Action of red light on indole-3-acetic acid status and growth in coleoptiles of etiolated maize seedlings. Planta 156, 21-32 Iino, M. (1987) Kinetic modelling of phototropism in maize coleoptiles. Planta 171, 110 126 Iino, M., Briggs, W.R. (1984) Growth distribution during first positive phototropic curvature of maize coleoptiles. Plant Cell Environ. 7, 97-104 Iino, M., Briggs, W.R., Sch~ifer, E. (1984a) Phytochrome-mediated phototropism in maize seedling shoots. Planta 160, 41-51 Iino, M., Nakagawa, Y., Wada, M. 0988) Blue light-regulation
188 of cell division in Adiantum protonemata: kinetic properties of the photosystem. Plant Cell Environ. 11 (in press) Iino, M., Ogawa, T., Zeiger, E. (1985) Kinetic properties of the blue-light response of stomata. Proc. Natl. Acad. Sci. USA 82, 8019-8023 Iino, M., Sch/ifer, E., Briggs, W.R. (1984b) Photoperception sites for phytochrome-mediated phototropism of maize mesocotyls. Planta 162, 477M79 Sch/ifer, E., Iino, M., Briggs, W.R. (1984) Red-light-induced shift of the fluence-response curve for first positive phototropic curvature of maize coleoptiles. In: Blue light effects on biological systems, pp. 476M79, Senger, H., ed. Springer, Berlin Heidelberg
M. Iino: Light-induced desensitization of coleoptiIe phototropism Schmid, R., Tiinnermann, M., Idziak, E.-M. (1988) Transient reduction of responsiveness of blue-light mediated hair whorl morphogenesis in Acetabularia mediterranea induced by blue light. Planta 174, 373-379 Steinitz, B., Poff, K.L. (1986) A single positive phototropic response induced with pulsed light in hypocotyls of Arab# dopsis thaliana seedlings. Planta 168, 305-315 Zimmerman, B.R., Briggs, W.R. (1963) Phototropic dosageresponse curves for oat coleoptiles. Plant Physiol. 38, 248 253 Received 9 October 1987; accepted 31 May 1988
