Planta 9 Springer-Verlag 1988
Planta (1988) 176:189-195
Phototropic fluence-response relations for Avena coleoptiles on a clinostat Benjamin Steinitz*, Th~r~se Best, and Kenneth L. Poff MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312, USA
Abstract. Phototropism o f A r e n a sativa L. has been characterized using a clinostat to negate the gravitropic response. The kinetics for development of curvature was measured following induction by a single pulse of blue light (BL), five pulses of BL at 20-min intervals, and this same pulsed-light regime following a 2-h red light (RL) pre-irradiation. A final curvature of about 14 ~ is expressed within 180 min following the single pulse; a final curvature of about 62 ~ in about 240 min following five pulses without pre-irradiation; and a curvature of over 125 ~ in 360 rain following five pulses after the R L pre-irradiation. For seedlings not pre-irradiated, the final curvature to five pulses of BL at a total fluence of 9.4 p m o l . c m --2 increases with time of darkness between pulses up to 15 min; with seedlings pre-irradiated with RL, curvature increased more slowly with time of darkness between pulses to a maximum at 35 min. The final curvature induced by a constant fluence of 9.4 pmol. c m - 2 increases linearly with time between the first pulse and last pulse of a five-pulse sequence. The curvature induced by a single BL pulse with a 5min R L co-irradiation increases with fluence to a maximum of about 60 ~ at about 10 p m o l . c m 2, and then decreases to 0 ~ at about 200 pmol. cm-2. Curvature induced by five BL pulses following a 2-h R L pre-irradiation increased with fluence from a threshold of about 0.05 p m o l . c m - 2 to a maximum of 90 ~ at about 10 p m o l . c m -2, and then gradually decreased with fluence to 50 ~ at 1 000 p m o l . c m -2. Based on these data, it is concluded that the initial photoproduct formed by a BL pulse has a limited lifetime, that there is a kinetic limitation " d o w n s t r e a m " of the photoreceptor * Permanent address: Department of Ornamental Horticulture, Agricultural Research Organization, Volcani Center, Bet-Dagan 50-250, Israel Abbreviations." BL = blue light; RL = red light.
pigment for phototropism, and that the additive effect of pulsed BL is distinct from the potentiating effect of R L on phototropism. Thus, any degree of curvature from 0 ~ to over 90 ~ may be induced by a fluence in the ascending arm of what is traditionally called the "first positive" phototropic response.
Key words: A r e n a (phototropism) - Blue light Coleoptile - Phototropism (fluence response) Red light
Introduction A great deal of the effort which has gone into the study of phototropism over the past century has centered on the measurement of the fluence-response relationship and on efforts to understand that relationship. The so-called "first positive" curvature, which is seen in response to short exposures of light at a relatively low fluence rate, consists of an ascending and a descending arm. The "second positive" curvature is seen in response to long exposures of light at a relatively high fluence rate (Dennison 1979). Since reciprocity is valid in the fluence range for "first positive" curvature, the response is limited by the absorption of quanta, and the quantum requirements can appropriately be assessed. However, curvature is limited in this fluence range, typically not exceeding 30 ~ (Steyer 1967; Blaauw and Blaauw-Jansen 1970; Baskin 1986; Baskin and Iino 1987; Noguchi and Hasegawa 1987), and thus technically limiting the ability to measure the quantum requirements for the induction of any substantial degree of bending (i.e., > 30~ In the higher fluence range in which the "second positive" curvature is observed, a much stronger bending response (up to 90 ~ is ob-
190
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
served. Unfortunately, in this range, the response is limited by the time of irradiation, not by the quanta absorbed (Everett and Thimann 1968; Pickard et al. 1969). Thus, reciprocity is not valid, and the quantum requirements cannot be assessed in this fluence range. Because of these apparent differences observed in different fluence ranges, "first positive" and "second positive" phototropism have come to be considered separate responses. In Arabidopsis thaliana, it has been possible to overcome these problems using pulsed blue light (BL) if the pulses are separated by a dark period of 15 20 min. With such a protocol, large angles of curvature may be obtained in response to a total fluence which falls within the fluence range associated with the "first positive" response. Moreover, with this protocol, reciprocity is valid for the entire fluence range, such that the quantum requirements for phototropism can indeed be evaluated. These results have been ascribed to a kinetic limitation in the transduction sequence, and a short lifetime of an initial physiologically active photoproduct (Steinitz and Poff 1986). Since the great majority of the work which has been reported on phototropism has been with the coleoptiles of a few grasses, we wished to extend the study of phototropism in response to pulsed light to the Arena sativa coleoptile, one of the more frequently used materials. One obvious reason for the previous frequent use of such material is that the gravitropic response is extremely sensitive in coleoptiles which have been exposed to red light (RL). Thus, these coleoptiles are very erect, permitting small angles of phototropic curvature to be measured with relative ease. However, it is evident that the gravitropic response of the shoot works against the phototropic response of that shoot to unilateral light. For this reason, and since clinostats have been used to negate this effect of gravity in few studies of phototropism (Shen-Miller and Gordon 1967; Heathcote and Bircher 1987; Nick and Sch~.fer 1988), we decided to measure phototropism with clinostats being used to change continuously the direction of the gravitational vector. Red light not only alters the response of the plant to gravity (Feldman and Briggs 1987), but has also been reported to alter the phototropic response (Kang and Burg 1974; Pohl and Russo 1984; Hofmann and Schfifer 1987). We therefore included exposure of the seedlings to RL as an additional variable. In this paper, we report the results of experiments designed to measure phototropism by Arena coleoptiles to pulsed BL under several different RL regimes, and with the gravitational response negated through the use of clinostats.
Material and methods Plants. Seeds (caryopses) of Arena sativa L., cv. Victory
CI 2020, were obtained from the U.S. Department of Agriculture Branch Experiment Station in Aberdeen, Id. After de-husking, the seeds were soaked 2 h in distilled water and placed in Petri dishes, embryo up, on four layers of Whatman No. 41 filter paper (Whatman International, Maidstone, Kent, UK) moistened with distilled water. The seeds were incubated for another 22 h in a Sherer Gro Lab (Sherer-Gillett, Marshall, Mich., USA) under continuous white light at 25 ~ C. Following the 24 h soaking and incubation period, single seedlings at the same developmental stage were planted into 5-ml glass vials filled with 1% (w/v) solidified Difco Bacto Agar (Difco Laboratories, Detroit, Mich., USA) in distilled water. The vials with seedlings were placed in transparent plastic boxes (19.9. 7.5 cm 3) containing a small amount of water, covered, and incubated in darkness at 25 ~ C. Phototropic stimulation began 50 h after sowing. Photostimulation and development of bending took place at 25 ~ C in darkness with relative humidity controlled at 90-100%. The vials with seedlings were transferred onto holders of the clinostat and rotated about a horizontal axis (perpendicular to the gravitational vector) at 55 s. rotation- ~ (1.1 rpm). The long axis of the seedlings was parallel to the axis of rotation. Thus, the seedlings were horizontal, but rotated such that any side of the seedling was directed downward only transiently. The clinostat was equipped with a switch which was triggered at each rotation. An electronic counter registered the revolutions and was connected to a shutter permitting an actinic irradiation at predetermined times. Seedlings were mounted on the clinostat such that the actinic irradiation was in the transverse plane passing perpendicular to the vascular bundles. Any RL preirradiation was administered from above prior to placement on the clinostat. The phototropic stimulation, and the co-irradiation with RL when applicable, were administered while the seedlings were rotating on the clinostat and rotation of the plants continued until completion of the development of curvature. Light sources. White light in the growth chamber, at 125 mmol-
m z s ~, was provided by General Electric (Cleveland, Oh., USA) Deluxe Cool-White F48TI2/CWX/HO fluorescent tubes. Red light for the pre-irradiation (at 240 pmol. c m - 2. s- ~) was from a Sylvania Gold F15T12-GO tube (GTE Products Corp., Danvers, Mass., USA), filtered through a red cellophane transmitting light in the 560-720 nm range with peak transmission at 630 nm. Red-light co-irradiation (at 37 pmol.cm -z. s- 1) was from a source consisting of a projector equipped with a General Electric 300-W ELH multimirror quartzline lamp used in combination with a 3-cm layer of aqueous cupric-sulfate solution (5%, w/v) and a 628nm interference (half-bandwidth of 10 nm). Phototropism was induced by a unilateral light stimulus from a Leitz projector (Leitz, Wetzlar, FRG) equipped with a 250-W BLV Halogen lamp and a 449-nm interference filter (half-bandwidth of 10nm; PTR Optics, Waltham, Mass., USA). Blue-light fluence rates were varied by changing the distance between the plants and the light source and by using neutral density filters. Blue-light exposure times (10 ms to 2.5 s) were varied with a UniBlitz 262 shutter connected to a UniBlitz model 310B shutter timer control (A.W. Vincent Associates, Rochester, N.Y., USA). For co-irradiation, the BL and RL were started at the same time, but were not necessarily of the same duration. Fluence rates of RL and BL were measured with either a model 68 Kettering radiometer (Laboratory Data Control, Riviera Beach, Fla., USA) or an IL 700A radiometer (International Light, Newburyport, Mass., USA), while white
B. Steinitz et al. : Fluence-response relations for phototropism in Arena light was measured with a Li-Cor LI-185A radiometer (Li-Cor, Lincoln, Neb., USA). Measurement o f curvature. The time course of phototropic bending (Fig. 1) was followed by time-lapse infrared photography. Photographs were taken with a Pentax camera (Pentax Corp., Englewood, Colo., USA) using Kodak (Eastman-Kodak, Rochester, N.Y., USA) BW IR film. A flash-lamp covered with a Kodak 87C wranen infrared filter was used as an infrared light source. Thus the infrared " l i g h t " was at wavelengths longer than 800 nm. The camera was situated such that its focal plane was parallel to the bending direction of the coleoptiles. Curvatures were measured on the enlarged negative images. In some experiments, 360 min after onset of the phototropic stimulus, seedlings were gently attached to a sticky transparent tape, with the direction of bending parallel to the tape surface. The tape was inserted into a photographic enlarger and the curvature traced and measured from the enlarged shadowgraph of the seedlings. Mean curvature was cahzulated from measurements made on :10-:14 seedlings at each exposure. Experiments were repeated four times such that, for each exposure, four mean values were obtained. The data in the figures represent the final mean value of these m e a n s + o n e standard error of the mean (SE).
Results
The kinetics o f p h o t o t r o p i c curvature by the Arena coleoptile depends on the experimental conditions employed. In most cases in the literature, curvature has been measured 100 min after onset of the phototropic stimulation (e.g. Zimmerman and Briggs 1963; Blaauw and Blaauw-Jansen 1970). However, it takes 5-6 h for the complete phototropic curvature of Arena coleoptiles to develop on a clinostat (Shen-Miller and Gordon 1967). We therefore measured the kinetics of bending in seedlings rotated on a clinostat. Although all of the seedlings received the same total fluence (94 p m o l . c m -2) of unilateral BL, this fluence was delivered using three different types of phototropic stimulations: (i) a single 2.5-s BL pulse at a fluence rate of 3 7 . 6 p m o l . c m - 2 - s -~, administered to seedlings which had previously been kept in darkness; (ii) five 0.5-s pulses at 2 0 - 9 intervals and a fluence rate of 37.6 p m o l - c m - ~- s - 1, administered to seedlings which had previously been kept in darkness; and (iii) the same BL regime as in (ii), but administered immediately following a 2-h R L pre-irradiation from above. The results (Fig. 1) show a different time-course of development of curvature in each case. Seedlings exposed to a single BL pulse achieved a maximum curvature of only 14~ about 180 min following the stimulus. When the same total fluence was applied in a sequence of five pulses, development of curvature required approx. 250 9 following the first pulse, yet coleoptiles curved up to 62 ~ Finally, coleoptiles pre-irradiated with R L responded to the same pulsed-BL regime
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Fig. 1. Time course for phototropic bending in oat coleoptiles. Bending was induced in all cases by a total fluence of 9.4 pmol. cm -2 of BL. 9 Seedlings stimulated by a single phototropic BL pulse, with no exposure to RL; 9 Seedlings stimulated with five pulses of BL (1.88 pmol-cm -2 per pulse) at 20-min intervals, with no exposure to RL; A Seedlings stimulated with five BL pulses (:l.88 pmol.cm -2 per pulse) at 20-min intervals, with RL pre-treatment for 2 h prior to phototropic stimulation
with a curvature of over 120 ~ but required 360 min following the first flash to attain that curvature. Consequently, in the subsequent experiments, curvature was always measured 6 h after the beginning of the unilateral BL stimulation. The results in Fig. 1 indicate that the response to a given fluence is larger when applied as a sequence of pulses than when applied as a single pulse. In order to obtain the maximum bending response to a given fluence administered as pulsed light, it is necessary to measure the optimum time interval between successive pulses in a sequence. To this end, seedlings were exposed to a unilateral BL stimulus at a constant total fluence of 9.4 pmol.cm -2. This stimulus was administered either as a single 0.5-s pulse at a fluence rate of 18.8 pmot.cm 2.s-~, or as a sequence of five 0A-s pulses at a fluence rate of 18.8 p m o l - c m - 2 . s -~. in this experiment, seedlings were included which had not received any R L treatment and seedlings pre-irradiated for 2 h with R L from above prior to the BL phototropic stimulation. The curvature induced by a single pulse was nearly 40 ~ in nonRL-treated seedlings compared to 73 ~ in R L preirradiated seedlings (Fig. 2). In non-RL-treated seedlings, stimulated with pulsed BL, a strong increase in response to the same fluence was observed with increasing time of darkness separating
192
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
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(o, n), or to a sequence of five pulses of 1.88 pmol.cm -2 per pulse (o; 9 of BL. The phototropic stimulation was applied to either seedlings kept in darkness up to the time of stimulation (o, 9 or seedlings which received 2 h RL pre-irradiation before the stimulation ([], m). Curvature was measured 6 b after onset of the phototropic stimulation
successive BL pulses. This increase leveled o u t w h e n time between pulses exceeded 15 min. Coleoptiles which received a R L p r e - i r r a d i a t i o n also s h o w e d an increased c u r v a t u r e u n d e r pulsed BL conditions, b u t required a significantly longer duration o f d a r k n e s s between successive pulses to reflect t h a t i n c r e m e n t in bending. We f o u n d 35 m i n to be the o p t i m u m interval o f d a r k n e s s between successive BL pulses in these seedlings (Fig. 2). T h e c u r v a t u r e induced b y a c o n s t a n t fluence o f 9.4 p m o l . c m - 2 , a d m i n i s t e r e d either as a single pulse or with five pulses, is p l o t t e d in Fig. 3 as a function o f the d u r a t i o n o f the period between the first a n d last pulse. These d a t a were t a k e n f r o m the results presented for n o n - R L - t r e a t e d seedlings in Fig. 2. It is evident t h a t the degree o f c u r v a t u r e
Fig. 3. The dependence of phototropic curvature on the duration of the phototropic stimulation in oat coleoptiles. Curvature was induced by a constant fluence (9.4 pmol.cm-2), and the duration of stimulation measured as the time elapsed between the first and the last pulse of a five-pulse sequence. o Fluence administered in a single pulse (0.5 s at 18.8 pmol. cm-2.s-1).
9 Fluence administered in five pulses (5 pulses of 0.1 s at 18.8pmol.cm-2.s-1). Seedlings were not exposed to RL. Curvature was measured 6 h after onset of phototropic stimulation is a linear function o f the time between the first and last pulse in the sequence u p to a stimulus period o f 60 m i n (15 m i n between pulses). T h e same relationship exists for R L - p r e - i r r a d i a t e d seedlings up to a stimulus period o f 1 4 0 9 (35 m i n between pulses). T h e fluences o f BL used in the a b o v e experim e n t s (Figs. 1, 2) are all in the r a n g e o f fluences ascribed to the traditional " f i r s t p o s i t i v e " response. W h e n the s a m e fluence was applied as pulsed light, a m u c h higher c u r v a t u r e was induced t h a n the c u r v a t u r e s r e p o r t e d in the literature a n d f o u n d b y us (Fig. 1) to a single pulse o f the s a m e fluence. Since the results indicated the b e n d i n g response to be strongest in R L - t r e a t e d seedlings, we u n d e r t o o k to redefine the p h o t o t r o p i c fluence response relations in R L - i r r a d i a t e d coleoptiles. T w o fluence-response curves were o b t a i n e d (Fig. 4). F o r one fluence-response curve, c u r v a t u r e was induced by a sequence o f five equal unilateral BL pulses, with 35 m i n d a r k n e s s between successive pulses in
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
193
Discussion
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Fig. 4. Fluence-response curves for the induction of phototropic curvature in oat coleoptiles. Curvature was induced either by a single exposure to unilateral BL (o), or by a sequence of 5 unilateral pulses (e). The response of seedlings stimulated with pulsed-light is presented as a function of the total fluence adminstered in the sequence. Each sequence consisted of five pulses, all of which were of one combination of pulse length and fluence rate. Successive pulses in a series were separated by 35 min of darkness. Seedlings received either 2 h of RL preirradiation preceding the phototropic stimulation (o), or a 5min RL co-irradiation, starting with the beginning of the phototropic stimulation (o). Curvature was measured 6 h after onset of the BL irradiation
the sequence. For a second fluence-response curve, curvature was induced by a single unilateral BL pulse. In the first case, the pulsed BL was applied following a 2-h RL pre-irradiation; in the second case the single BL pulse was given together with a 5-rain RL co-irradiation. In both instances the fluence-response curve consists of an ascending and a descending arm. The threshold fluence value for the induction of curvature under pulsed BL is about 0.05 pmol-cm -2. The peak response, to both single-pulse and pulsed-light stimulation, is approx. 10 p m o l . c m - 2. The peak curvature was higher when induced by pulsed-light than when induced by non-pulsed light, reaching a value of 90 ~ in response to pulsed light. In the descending arm, a second 0 ~ curvature value was observed at 200 pmol.cm -2. However, this occurred only in coleoptiles stimulated with a single BL pulse; the descending arm in coleoptiles exposed to pulsed BL was found to be shifted to higher fluences. Within the limitations of fluence rates and exposure times employed, we could not reach under pulsed BL a sufficiently high fluence to obtain an indifferent curvature response with the seedlings rotating on the clinostat.
It is difficult to design an experiment which will permit one to assess cleanly the transduction pathway for phototropism in plants in the absence of other complicating systems. For example, in Arena, there is a dramatic effect of RL both on phototropism and on gravitropism. The usual protocol has been to pre-irradiate seedlings with RL, thereby increasing the gravitropic sensitivity in order to have erect coleoptiles at the beginning of the phototropism experiment. Many RL effects on phototropism have been reported. Phytochrome mediates a RL-stimulated phototropic response in the mesocotyl (Iino et al. 1984); a preirradiation with RL has been reported to increase the amplitude of the phototropic response to BL (Blaauw-Jansen 1958; Blaauw and Blaauw-Jansen 1964); and a pre-irradiation with RL has been reported to raise the threshold for the phototropic response to BL (Zimmerman and Briggs 1963; Chon and Briggs 1966; Curry 1969). Since phytochrome absorbs BL, one must conservatively assume that the BL irradiation which induces phototropism is absorbed also by phytochrome. Given this, it was necessary to demonstrate that a sequence of BL pulses induces a curvature greater than that of a single pulse, and that this induction of greater curvature is different from that known to be mediated via phytochrome. These conditions have indeed been met. Five pulses of BL induce a curvature considerably greater than that induced by a single blue pulse (Figs. 1, 2). However, pre-irradiation with RL has an effect which is clearly in addition to that of the multiple pulses. Curvature of seedlings pre-irradiated with RL and then exposed to a sequence of BL pulses is consistently greater than that of seedlings exposed only to the BL pulses (Figs. 1, 2). The kinetics for the development of curvature in response to five pulses of BL alone is different from that obtained when the seedlings are pre-irradiated with RL (Fig. 1). In addition, the dependence of curvature on the period of time in darkness separating the pulses is clearly different for seedlings receiving the pre-irradiation with RL than for those receiving only the BL pulses (Fig. 2). We therefore conclude that subdividing a given BL pulse into a sequence of pulses of the same total fluence results in a phototropic curvature greater than that induced by the single pulse, and that this effect is separate from and in addition to that induced by RL. Based on the results presented here, Arena coleoptiles show the same response to pulsed BL which
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B. Steinitzet al. : Fluence-responserelations for phototropism in Arena
was described previously in Arabidopsis thaliana. A kinetic limitation or relaxation process is evident in both organisms, somewhere in the transduction process. It is this process that imposes the requirement for a 15-20-rain period of darkness between pulses. It is also likely that this process is the basis for many of the characteristics of the "second positive" curvature. Curvature to a fluence in the "second positive" region is linearly dependent upon the time of irradiation for Arena (Pickard et al. 1969) and for Arabidopsis (Steinitz and Poff 1986). An analogous time-dependence of curvature on the duration of the stimulus period for pulsed light has been demonstrated for Arabidopsis (Steinitz and Poff 1986), and in this present work for Arena (Fig. 3). In this experimental procedure using pulsed light, the stimulus period consists mainly of darkness. Had this period of darkness been filled by an irradiation, the curvature would not have been increased; rather, the apparent quantum requirements for curvature would have been increased and reciprocity invalidated. The data in Fig. 2 show that a given sub-saturating fluence of light induces a maximal curvature when divided into pulses at about 15-min intervals, and that the minimal curvature is induced by a given fluence when applied as a single pulse. We interpret Fig. 2 to mean that the physiologically active photoproduct is unstable, decaying with time in about 15 rain. Were the photoproduct stable, the maximum curvature would have been induced by a single pulse exerting its influence over the entire time of the experiment. This is the same conclusion that was also reached for Arabidopsis by Steinitz and Poff (1986), based on similar data. If our conclusion be correct that the photoproduct is unstable, then we can further conclude that one specific effect of RL is to decrease the rate of decay of the photoproduct. This is based on the difference in dependence of curvature on time in darkness between pulses for RL-pre-irradiated seedlings and those which received no pre-irradiation (Fig. 2). The curvature was less with a 45-rain time of darkness between pulses than when that time was 35 min for the RL-pre-irradiated seedlings. We attribute this decrease to the experimental design. Curvature was measured 6 h after onset of the phototropic stimulation. After the fifth pulse at 45-rain intervals, the plants had 3 h left until the end of the experiment. This 3 h was likely insufficient to permit complete development of curvature to the last pulse. This implies that RL preirradiation also slows the rate of curvature development to a given BL pulse, and this also would be concluded from the data in Fig. 1. Thus, a RL
pre-irradiation slows the development of curvature, and also slows the rate of decay of the physiologically active photoproduct. As a result of RL decreasing the rate of decay of the physiologically active photoproduct, this photoproduct would be expected to function longer following RL pre-irradiation or with RL coirradiation. One would then anticipate that a single BL pulse alone would result in a lower curvature than would the same pulse following RL pre-irradiation or with RL co-irradiation. It is not surprising, therefore, that the response to a single BL pulse of 9.4 pmol.cm -2 is a curvature of about 10~ (Fig. 1), while the response to a single BL pulse of the same fluence with RL co-irradiation is about 50 ~ (Fig. 4). We have previously noted the similarities between phototropism and stomata opening induced by pulsed BL (Steinitz and Poff 1986). The overlapping area of similarities is extended, by the results of the present study, to the influence of RL on the response induced by BL. Co-irradiation with RL increases BL-mediated malate formation in Vicia faba guard cells (Ogawa et al. 1978) and enhances BL-mediated stomata opening (conductance level) in wheat (Karlsson 1986) and in Commelina communis (Assmann 1988). In the latter case, RL co-irradiation significantly extends the time required to reach maximum conductance following the inductive BL pulse, and extends the time required for the conductance level to return to the level found in darkness. These observations indicate that the life-time of the physiologically active photoproduct formed in BL in stomates is increased by RL. The fluence-response curve for curvature to five pulses of BL by RL pre-irradiated seedlings (Fig. 4) shows a threshold fluence (minimum curvature required for curvature) and fluence for peak curvature of approx. 0.05 p m o l . c m - 2 and 1 0 p m o l . c m -z, respectively. These are the same values as those which we have obtained (data not shown), and those previously reported (e.g., Blaauw and Blaauw-Jansen 1970) for Arena in response to single pulses of BL by non pre-irradiated seedlings. Thus, we see no shift in threshold for the phototropic response to BL induced by RL. We see a clear increase in response induced by the RL pre-irradiation (Figs. 1, 2), and by a RL coirradiation (Fig. 4). The descending arm of the fluence-response curve is elevated, showing higher curvature for the seedlings receiving five pulses than that obtained with those receiving a single pulse (Fig. 4). At present, we have inadequate data to formulate an hypothesis to account fully for
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
this change in the descending arm. However, it seems inescapable that multiple systems or muItiple effects of BL are involved in this descending arm. The data presented here in no sense reduce the apparent complexity of the interrelated systems of gravitropism, phototropism to BL, and the RL effects which we assume to be phytochrome-mediated. To study these interrelated phenomena will require the systematic variation of each parameter or the study of single system or parameter in the absence of the others. Through the use of a clinostat, the gravitropic response has been eliminated in this study. To eliminate the other systems will require the use of mutants lacking specific responses to light. This work was supported by the U.S. Department of Energy under Contract No. DE-AC02-76ER0-1338.
References Assmann, S.N. (1988) Enhancement of the stomatal response to blue light by red light, reduced intercellular concentration of COz, and low vapor pressure differences. Plant Physiol. 87, 226-231 Baskin, T.R. (1986) Redistribution of growth during nutation and phototropism in the pea epicotyL Planta 169, 406-414 Baskin, T.R., Iino, M. (1987) An action spectrum in the blue and ultraviolet for phototropism in alfalfa. Photochem. Photobiol. 46, 127-136 Blaauw-Jansen, G. (1958) The influence of red and far-red light on growth and phototropism of the Arena seedling. Acta Bot. Neerl. 8, 1-39 Blaauw, O.H., Blaauw-Jansen, G. (1964) The influence of red light on the phototropism of Arena coleoptiles. Acta Bot. Neert. 13, 541-552 Blaauw, O.H., Blaauw-Jansen, G. (t970) The phototropic response of Arena coteoptiles. Acta Bot. Neerl. 19, 755 763 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. (1969) Phototropism. In: The physiology of plant growth and development, pp. 243-273, Wilkins, M.B., ed. McGraw-Hill, New York Dennison, D_S. (1979)Phototropism. In: Encyclopedia of plant physiology, N.S., vol. 7: Physiology of movements, pp. 506566, Haupt, W., Feinleib, M.E., eds. Springer, Berlin Heidelberg New York
195
Everett, M., Thimann, K.V. (1968) Second-positive phototropism in Arena coleoptiles. Plant Physiol. 43, 1786-1792 Feldman, L.J., Briggs, W.R. (1987) Light-regulated gravitropism in seedling roots of maize. Plant Physiol. 83, 241-243 Heathcote, D.G., Bircher, B.W. (1987) Enhancement of phototropic response to a range of light doses in Triticum aestivum coleoptiles in clinostat-simulated microgravity. Planta 170, 249-256 Hofmann, E., Schfifer, 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., Briggs, W.R. and Sch/ifer, E. (1984) Phytochromemediated phototropism in maize seedling shoots. Planta 160, 41-51 Kang, B.G., Burg, S.P. (1974) Red light enhancement of the phototropie respone of etiolated pea stems_ Plant Physiol. 53, 445-448 Karlsson, P.E. (1986) Blue light regulation of stomata in wheat. I. Influence of red background illumination and initial conductance level. Physiol. Plant. 66, 202-206 Nick, P., SchS.fer, E. (1988) Interaction of gravi- and phototropic stimuIation in the response of maize (Zea mays L.) coleoptiles. Planta 173, 213-220 Nogucbi, H., Hasegawa, K. (1987) Phototropism in hypocotyls of radish. IiI. Influence of unilateral or bilateral illumination of various light intensities on phototropism and distribution of cis- and trans-raphanusanins and raphanusamide. Plant Physiol. 83, 672-675 Ogawa, T., Ishikawa, H., Shimada, K., Shibata, K. (1978) Synergistic action of red and blue light and action spectra for malate formation in guard cells of Viciafaba L. Planta 142, 61-65 Pickard, B.G., Ditson, K., Harrison, V., Donegan, E. (1969) Second positive phototropic response patterns of the oat coleoptile. Planta 88, 1-33 Pohl, U., Russo, V.E.A. (1984) Phototropism. In: Membranes and sensory transduction, pp. 231-330, Colombetti, G., Lend, F., eds. Plenum Press, New York Shen-Miller, J., Gordon, S.A. (1967) GravitationM compensation and the phototropic response of oat coleoptiles. Plant Physiol. 42, 352-360 Steinitz, B., Poff, K.L. (]986) A single positive phototropic response induced with pulsed light in hypocotyls of Arabidopsis thaliana seedlings. Planta 168, 305-315 Steyer, B. (1967) Die Dosis-Wirkungsrelation bei geotroper und phototroper Reizung: Vergleich von Mono- mit Dicotyledonen. Planta 77, 277-286 Zimmerman, B.K., Briggs, W.R. (1963) Phototropic dosageresponse curves for oat coleoptiles. Plant Physiol. 38, 237247 Received 7 April; accepted 15 June 1988
Planta (1988) 176:189-195
Phototropic fluence-response relations for Avena coleoptiles on a clinostat Benjamin Steinitz*, Th~r~se Best, and Kenneth L. Poff MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312, USA
Abstract. Phototropism o f A r e n a sativa L. has been characterized using a clinostat to negate the gravitropic response. The kinetics for development of curvature was measured following induction by a single pulse of blue light (BL), five pulses of BL at 20-min intervals, and this same pulsed-light regime following a 2-h red light (RL) pre-irradiation. A final curvature of about 14 ~ is expressed within 180 min following the single pulse; a final curvature of about 62 ~ in about 240 min following five pulses without pre-irradiation; and a curvature of over 125 ~ in 360 rain following five pulses after the R L pre-irradiation. For seedlings not pre-irradiated, the final curvature to five pulses of BL at a total fluence of 9.4 p m o l . c m --2 increases with time of darkness between pulses up to 15 min; with seedlings pre-irradiated with RL, curvature increased more slowly with time of darkness between pulses to a maximum at 35 min. The final curvature induced by a constant fluence of 9.4 pmol. c m - 2 increases linearly with time between the first pulse and last pulse of a five-pulse sequence. The curvature induced by a single BL pulse with a 5min R L co-irradiation increases with fluence to a maximum of about 60 ~ at about 10 p m o l . c m 2, and then decreases to 0 ~ at about 200 pmol. cm-2. Curvature induced by five BL pulses following a 2-h R L pre-irradiation increased with fluence from a threshold of about 0.05 p m o l . c m - 2 to a maximum of 90 ~ at about 10 p m o l . c m -2, and then gradually decreased with fluence to 50 ~ at 1 000 p m o l . c m -2. Based on these data, it is concluded that the initial photoproduct formed by a BL pulse has a limited lifetime, that there is a kinetic limitation " d o w n s t r e a m " of the photoreceptor * Permanent address: Department of Ornamental Horticulture, Agricultural Research Organization, Volcani Center, Bet-Dagan 50-250, Israel Abbreviations." BL = blue light; RL = red light.
pigment for phototropism, and that the additive effect of pulsed BL is distinct from the potentiating effect of R L on phototropism. Thus, any degree of curvature from 0 ~ to over 90 ~ may be induced by a fluence in the ascending arm of what is traditionally called the "first positive" phototropic response.
Key words: A r e n a (phototropism) - Blue light Coleoptile - Phototropism (fluence response) Red light
Introduction A great deal of the effort which has gone into the study of phototropism over the past century has centered on the measurement of the fluence-response relationship and on efforts to understand that relationship. The so-called "first positive" curvature, which is seen in response to short exposures of light at a relatively low fluence rate, consists of an ascending and a descending arm. The "second positive" curvature is seen in response to long exposures of light at a relatively high fluence rate (Dennison 1979). Since reciprocity is valid in the fluence range for "first positive" curvature, the response is limited by the absorption of quanta, and the quantum requirements can appropriately be assessed. However, curvature is limited in this fluence range, typically not exceeding 30 ~ (Steyer 1967; Blaauw and Blaauw-Jansen 1970; Baskin 1986; Baskin and Iino 1987; Noguchi and Hasegawa 1987), and thus technically limiting the ability to measure the quantum requirements for the induction of any substantial degree of bending (i.e., > 30~ In the higher fluence range in which the "second positive" curvature is observed, a much stronger bending response (up to 90 ~ is ob-
190
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
served. Unfortunately, in this range, the response is limited by the time of irradiation, not by the quanta absorbed (Everett and Thimann 1968; Pickard et al. 1969). Thus, reciprocity is not valid, and the quantum requirements cannot be assessed in this fluence range. Because of these apparent differences observed in different fluence ranges, "first positive" and "second positive" phototropism have come to be considered separate responses. In Arabidopsis thaliana, it has been possible to overcome these problems using pulsed blue light (BL) if the pulses are separated by a dark period of 15 20 min. With such a protocol, large angles of curvature may be obtained in response to a total fluence which falls within the fluence range associated with the "first positive" response. Moreover, with this protocol, reciprocity is valid for the entire fluence range, such that the quantum requirements for phototropism can indeed be evaluated. These results have been ascribed to a kinetic limitation in the transduction sequence, and a short lifetime of an initial physiologically active photoproduct (Steinitz and Poff 1986). Since the great majority of the work which has been reported on phototropism has been with the coleoptiles of a few grasses, we wished to extend the study of phototropism in response to pulsed light to the Arena sativa coleoptile, one of the more frequently used materials. One obvious reason for the previous frequent use of such material is that the gravitropic response is extremely sensitive in coleoptiles which have been exposed to red light (RL). Thus, these coleoptiles are very erect, permitting small angles of phototropic curvature to be measured with relative ease. However, it is evident that the gravitropic response of the shoot works against the phototropic response of that shoot to unilateral light. For this reason, and since clinostats have been used to negate this effect of gravity in few studies of phototropism (Shen-Miller and Gordon 1967; Heathcote and Bircher 1987; Nick and Sch~.fer 1988), we decided to measure phototropism with clinostats being used to change continuously the direction of the gravitational vector. Red light not only alters the response of the plant to gravity (Feldman and Briggs 1987), but has also been reported to alter the phototropic response (Kang and Burg 1974; Pohl and Russo 1984; Hofmann and Schfifer 1987). We therefore included exposure of the seedlings to RL as an additional variable. In this paper, we report the results of experiments designed to measure phototropism by Arena coleoptiles to pulsed BL under several different RL regimes, and with the gravitational response negated through the use of clinostats.
Material and methods Plants. Seeds (caryopses) of Arena sativa L., cv. Victory
CI 2020, were obtained from the U.S. Department of Agriculture Branch Experiment Station in Aberdeen, Id. After de-husking, the seeds were soaked 2 h in distilled water and placed in Petri dishes, embryo up, on four layers of Whatman No. 41 filter paper (Whatman International, Maidstone, Kent, UK) moistened with distilled water. The seeds were incubated for another 22 h in a Sherer Gro Lab (Sherer-Gillett, Marshall, Mich., USA) under continuous white light at 25 ~ C. Following the 24 h soaking and incubation period, single seedlings at the same developmental stage were planted into 5-ml glass vials filled with 1% (w/v) solidified Difco Bacto Agar (Difco Laboratories, Detroit, Mich., USA) in distilled water. The vials with seedlings were placed in transparent plastic boxes (19.9. 7.5 cm 3) containing a small amount of water, covered, and incubated in darkness at 25 ~ C. Phototropic stimulation began 50 h after sowing. Photostimulation and development of bending took place at 25 ~ C in darkness with relative humidity controlled at 90-100%. The vials with seedlings were transferred onto holders of the clinostat and rotated about a horizontal axis (perpendicular to the gravitational vector) at 55 s. rotation- ~ (1.1 rpm). The long axis of the seedlings was parallel to the axis of rotation. Thus, the seedlings were horizontal, but rotated such that any side of the seedling was directed downward only transiently. The clinostat was equipped with a switch which was triggered at each rotation. An electronic counter registered the revolutions and was connected to a shutter permitting an actinic irradiation at predetermined times. Seedlings were mounted on the clinostat such that the actinic irradiation was in the transverse plane passing perpendicular to the vascular bundles. Any RL preirradiation was administered from above prior to placement on the clinostat. The phototropic stimulation, and the co-irradiation with RL when applicable, were administered while the seedlings were rotating on the clinostat and rotation of the plants continued until completion of the development of curvature. Light sources. White light in the growth chamber, at 125 mmol-
m z s ~, was provided by General Electric (Cleveland, Oh., USA) Deluxe Cool-White F48TI2/CWX/HO fluorescent tubes. Red light for the pre-irradiation (at 240 pmol. c m - 2. s- ~) was from a Sylvania Gold F15T12-GO tube (GTE Products Corp., Danvers, Mass., USA), filtered through a red cellophane transmitting light in the 560-720 nm range with peak transmission at 630 nm. Red-light co-irradiation (at 37 pmol.cm -z. s- 1) was from a source consisting of a projector equipped with a General Electric 300-W ELH multimirror quartzline lamp used in combination with a 3-cm layer of aqueous cupric-sulfate solution (5%, w/v) and a 628nm interference (half-bandwidth of 10 nm). Phototropism was induced by a unilateral light stimulus from a Leitz projector (Leitz, Wetzlar, FRG) equipped with a 250-W BLV Halogen lamp and a 449-nm interference filter (half-bandwidth of 10nm; PTR Optics, Waltham, Mass., USA). Blue-light fluence rates were varied by changing the distance between the plants and the light source and by using neutral density filters. Blue-light exposure times (10 ms to 2.5 s) were varied with a UniBlitz 262 shutter connected to a UniBlitz model 310B shutter timer control (A.W. Vincent Associates, Rochester, N.Y., USA). For co-irradiation, the BL and RL were started at the same time, but were not necessarily of the same duration. Fluence rates of RL and BL were measured with either a model 68 Kettering radiometer (Laboratory Data Control, Riviera Beach, Fla., USA) or an IL 700A radiometer (International Light, Newburyport, Mass., USA), while white
B. Steinitz et al. : Fluence-response relations for phototropism in Arena light was measured with a Li-Cor LI-185A radiometer (Li-Cor, Lincoln, Neb., USA). Measurement o f curvature. The time course of phototropic bending (Fig. 1) was followed by time-lapse infrared photography. Photographs were taken with a Pentax camera (Pentax Corp., Englewood, Colo., USA) using Kodak (Eastman-Kodak, Rochester, N.Y., USA) BW IR film. A flash-lamp covered with a Kodak 87C wranen infrared filter was used as an infrared light source. Thus the infrared " l i g h t " was at wavelengths longer than 800 nm. The camera was situated such that its focal plane was parallel to the bending direction of the coleoptiles. Curvatures were measured on the enlarged negative images. In some experiments, 360 min after onset of the phototropic stimulus, seedlings were gently attached to a sticky transparent tape, with the direction of bending parallel to the tape surface. The tape was inserted into a photographic enlarger and the curvature traced and measured from the enlarged shadowgraph of the seedlings. Mean curvature was cahzulated from measurements made on :10-:14 seedlings at each exposure. Experiments were repeated four times such that, for each exposure, four mean values were obtained. The data in the figures represent the final mean value of these m e a n s + o n e standard error of the mean (SE).
Results
The kinetics o f p h o t o t r o p i c curvature by the Arena coleoptile depends on the experimental conditions employed. In most cases in the literature, curvature has been measured 100 min after onset of the phototropic stimulation (e.g. Zimmerman and Briggs 1963; Blaauw and Blaauw-Jansen 1970). However, it takes 5-6 h for the complete phototropic curvature of Arena coleoptiles to develop on a clinostat (Shen-Miller and Gordon 1967). We therefore measured the kinetics of bending in seedlings rotated on a clinostat. Although all of the seedlings received the same total fluence (94 p m o l . c m -2) of unilateral BL, this fluence was delivered using three different types of phototropic stimulations: (i) a single 2.5-s BL pulse at a fluence rate of 3 7 . 6 p m o l . c m - 2 - s -~, administered to seedlings which had previously been kept in darkness; (ii) five 0.5-s pulses at 2 0 - 9 intervals and a fluence rate of 37.6 p m o l - c m - ~- s - 1, administered to seedlings which had previously been kept in darkness; and (iii) the same BL regime as in (ii), but administered immediately following a 2-h R L pre-irradiation from above. The results (Fig. 1) show a different time-course of development of curvature in each case. Seedlings exposed to a single BL pulse achieved a maximum curvature of only 14~ about 180 min following the stimulus. When the same total fluence was applied in a sequence of five pulses, development of curvature required approx. 250 9 following the first pulse, yet coleoptiles curved up to 62 ~ Finally, coleoptiles pre-irradiated with R L responded to the same pulsed-BL regime
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with a curvature of over 120 ~ but required 360 min following the first flash to attain that curvature. Consequently, in the subsequent experiments, curvature was always measured 6 h after the beginning of the unilateral BL stimulation. The results in Fig. 1 indicate that the response to a given fluence is larger when applied as a sequence of pulses than when applied as a single pulse. In order to obtain the maximum bending response to a given fluence administered as pulsed light, it is necessary to measure the optimum time interval between successive pulses in a sequence. To this end, seedlings were exposed to a unilateral BL stimulus at a constant total fluence of 9.4 pmol.cm -2. This stimulus was administered either as a single 0.5-s pulse at a fluence rate of 18.8 pmot.cm 2.s-~, or as a sequence of five 0A-s pulses at a fluence rate of 18.8 p m o l - c m - 2 . s -~. in this experiment, seedlings were included which had not received any R L treatment and seedlings pre-irradiated for 2 h with R L from above prior to the BL phototropic stimulation. The curvature induced by a single pulse was nearly 40 ~ in nonRL-treated seedlings compared to 73 ~ in R L preirradiated seedlings (Fig. 2). In non-RL-treated seedlings, stimulated with pulsed BL, a strong increase in response to the same fluence was observed with increasing time of darkness separating
192
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
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Fig. Z. Phototropic curvature in oat coleoptiles as a function of the time of darkness between successive light pulses. Seedlings were exposed to either a single pulse of 9.4 pmol-cm -2
(o, n), or to a sequence of five pulses of 1.88 pmol.cm -2 per pulse (o; 9 of BL. The phototropic stimulation was applied to either seedlings kept in darkness up to the time of stimulation (o, 9 or seedlings which received 2 h RL pre-irradiation before the stimulation ([], m). Curvature was measured 6 b after onset of the phototropic stimulation
successive BL pulses. This increase leveled o u t w h e n time between pulses exceeded 15 min. Coleoptiles which received a R L p r e - i r r a d i a t i o n also s h o w e d an increased c u r v a t u r e u n d e r pulsed BL conditions, b u t required a significantly longer duration o f d a r k n e s s between successive pulses to reflect t h a t i n c r e m e n t in bending. We f o u n d 35 m i n to be the o p t i m u m interval o f d a r k n e s s between successive BL pulses in these seedlings (Fig. 2). T h e c u r v a t u r e induced b y a c o n s t a n t fluence o f 9.4 p m o l . c m - 2 , a d m i n i s t e r e d either as a single pulse or with five pulses, is p l o t t e d in Fig. 3 as a function o f the d u r a t i o n o f the period between the first a n d last pulse. These d a t a were t a k e n f r o m the results presented for n o n - R L - t r e a t e d seedlings in Fig. 2. It is evident t h a t the degree o f c u r v a t u r e
Fig. 3. The dependence of phototropic curvature on the duration of the phototropic stimulation in oat coleoptiles. Curvature was induced by a constant fluence (9.4 pmol.cm-2), and the duration of stimulation measured as the time elapsed between the first and the last pulse of a five-pulse sequence. o Fluence administered in a single pulse (0.5 s at 18.8 pmol. cm-2.s-1).
9 Fluence administered in five pulses (5 pulses of 0.1 s at 18.8pmol.cm-2.s-1). Seedlings were not exposed to RL. Curvature was measured 6 h after onset of phototropic stimulation is a linear function o f the time between the first and last pulse in the sequence u p to a stimulus period o f 60 m i n (15 m i n between pulses). T h e same relationship exists for R L - p r e - i r r a d i a t e d seedlings up to a stimulus period o f 1 4 0 9 (35 m i n between pulses). T h e fluences o f BL used in the a b o v e experim e n t s (Figs. 1, 2) are all in the r a n g e o f fluences ascribed to the traditional " f i r s t p o s i t i v e " response. W h e n the s a m e fluence was applied as pulsed light, a m u c h higher c u r v a t u r e was induced t h a n the c u r v a t u r e s r e p o r t e d in the literature a n d f o u n d b y us (Fig. 1) to a single pulse o f the s a m e fluence. Since the results indicated the b e n d i n g response to be strongest in R L - t r e a t e d seedlings, we u n d e r t o o k to redefine the p h o t o t r o p i c fluence response relations in R L - i r r a d i a t e d coleoptiles. T w o fluence-response curves were o b t a i n e d (Fig. 4). F o r one fluence-response curve, c u r v a t u r e was induced by a sequence o f five equal unilateral BL pulses, with 35 m i n d a r k n e s s between successive pulses in
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
193
Discussion
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Fig. 4. Fluence-response curves for the induction of phototropic curvature in oat coleoptiles. Curvature was induced either by a single exposure to unilateral BL (o), or by a sequence of 5 unilateral pulses (e). The response of seedlings stimulated with pulsed-light is presented as a function of the total fluence adminstered in the sequence. Each sequence consisted of five pulses, all of which were of one combination of pulse length and fluence rate. Successive pulses in a series were separated by 35 min of darkness. Seedlings received either 2 h of RL preirradiation preceding the phototropic stimulation (o), or a 5min RL co-irradiation, starting with the beginning of the phototropic stimulation (o). Curvature was measured 6 h after onset of the BL irradiation
the sequence. For a second fluence-response curve, curvature was induced by a single unilateral BL pulse. In the first case, the pulsed BL was applied following a 2-h RL pre-irradiation; in the second case the single BL pulse was given together with a 5-rain RL co-irradiation. In both instances the fluence-response curve consists of an ascending and a descending arm. The threshold fluence value for the induction of curvature under pulsed BL is about 0.05 pmol-cm -2. The peak response, to both single-pulse and pulsed-light stimulation, is approx. 10 p m o l . c m - 2. The peak curvature was higher when induced by pulsed-light than when induced by non-pulsed light, reaching a value of 90 ~ in response to pulsed light. In the descending arm, a second 0 ~ curvature value was observed at 200 pmol.cm -2. However, this occurred only in coleoptiles stimulated with a single BL pulse; the descending arm in coleoptiles exposed to pulsed BL was found to be shifted to higher fluences. Within the limitations of fluence rates and exposure times employed, we could not reach under pulsed BL a sufficiently high fluence to obtain an indifferent curvature response with the seedlings rotating on the clinostat.
It is difficult to design an experiment which will permit one to assess cleanly the transduction pathway for phototropism in plants in the absence of other complicating systems. For example, in Arena, there is a dramatic effect of RL both on phototropism and on gravitropism. The usual protocol has been to pre-irradiate seedlings with RL, thereby increasing the gravitropic sensitivity in order to have erect coleoptiles at the beginning of the phototropism experiment. Many RL effects on phototropism have been reported. Phytochrome mediates a RL-stimulated phototropic response in the mesocotyl (Iino et al. 1984); a preirradiation with RL has been reported to increase the amplitude of the phototropic response to BL (Blaauw-Jansen 1958; Blaauw and Blaauw-Jansen 1964); and a pre-irradiation with RL has been reported to raise the threshold for the phototropic response to BL (Zimmerman and Briggs 1963; Chon and Briggs 1966; Curry 1969). Since phytochrome absorbs BL, one must conservatively assume that the BL irradiation which induces phototropism is absorbed also by phytochrome. Given this, it was necessary to demonstrate that a sequence of BL pulses induces a curvature greater than that of a single pulse, and that this induction of greater curvature is different from that known to be mediated via phytochrome. These conditions have indeed been met. Five pulses of BL induce a curvature considerably greater than that induced by a single blue pulse (Figs. 1, 2). However, pre-irradiation with RL has an effect which is clearly in addition to that of the multiple pulses. Curvature of seedlings pre-irradiated with RL and then exposed to a sequence of BL pulses is consistently greater than that of seedlings exposed only to the BL pulses (Figs. 1, 2). The kinetics for the development of curvature in response to five pulses of BL alone is different from that obtained when the seedlings are pre-irradiated with RL (Fig. 1). In addition, the dependence of curvature on the period of time in darkness separating the pulses is clearly different for seedlings receiving the pre-irradiation with RL than for those receiving only the BL pulses (Fig. 2). We therefore conclude that subdividing a given BL pulse into a sequence of pulses of the same total fluence results in a phototropic curvature greater than that induced by the single pulse, and that this effect is separate from and in addition to that induced by RL. Based on the results presented here, Arena coleoptiles show the same response to pulsed BL which
194
B. Steinitzet al. : Fluence-responserelations for phototropism in Arena
was described previously in Arabidopsis thaliana. A kinetic limitation or relaxation process is evident in both organisms, somewhere in the transduction process. It is this process that imposes the requirement for a 15-20-rain period of darkness between pulses. It is also likely that this process is the basis for many of the characteristics of the "second positive" curvature. Curvature to a fluence in the "second positive" region is linearly dependent upon the time of irradiation for Arena (Pickard et al. 1969) and for Arabidopsis (Steinitz and Poff 1986). An analogous time-dependence of curvature on the duration of the stimulus period for pulsed light has been demonstrated for Arabidopsis (Steinitz and Poff 1986), and in this present work for Arena (Fig. 3). In this experimental procedure using pulsed light, the stimulus period consists mainly of darkness. Had this period of darkness been filled by an irradiation, the curvature would not have been increased; rather, the apparent quantum requirements for curvature would have been increased and reciprocity invalidated. The data in Fig. 2 show that a given sub-saturating fluence of light induces a maximal curvature when divided into pulses at about 15-min intervals, and that the minimal curvature is induced by a given fluence when applied as a single pulse. We interpret Fig. 2 to mean that the physiologically active photoproduct is unstable, decaying with time in about 15 rain. Were the photoproduct stable, the maximum curvature would have been induced by a single pulse exerting its influence over the entire time of the experiment. This is the same conclusion that was also reached for Arabidopsis by Steinitz and Poff (1986), based on similar data. If our conclusion be correct that the photoproduct is unstable, then we can further conclude that one specific effect of RL is to decrease the rate of decay of the photoproduct. This is based on the difference in dependence of curvature on time in darkness between pulses for RL-pre-irradiated seedlings and those which received no pre-irradiation (Fig. 2). The curvature was less with a 45-rain time of darkness between pulses than when that time was 35 min for the RL-pre-irradiated seedlings. We attribute this decrease to the experimental design. Curvature was measured 6 h after onset of the phototropic stimulation. After the fifth pulse at 45-rain intervals, the plants had 3 h left until the end of the experiment. This 3 h was likely insufficient to permit complete development of curvature to the last pulse. This implies that RL preirradiation also slows the rate of curvature development to a given BL pulse, and this also would be concluded from the data in Fig. 1. Thus, a RL
pre-irradiation slows the development of curvature, and also slows the rate of decay of the physiologically active photoproduct. As a result of RL decreasing the rate of decay of the physiologically active photoproduct, this photoproduct would be expected to function longer following RL pre-irradiation or with RL coirradiation. One would then anticipate that a single BL pulse alone would result in a lower curvature than would the same pulse following RL pre-irradiation or with RL co-irradiation. It is not surprising, therefore, that the response to a single BL pulse of 9.4 pmol.cm -2 is a curvature of about 10~ (Fig. 1), while the response to a single BL pulse of the same fluence with RL co-irradiation is about 50 ~ (Fig. 4). We have previously noted the similarities between phototropism and stomata opening induced by pulsed BL (Steinitz and Poff 1986). The overlapping area of similarities is extended, by the results of the present study, to the influence of RL on the response induced by BL. Co-irradiation with RL increases BL-mediated malate formation in Vicia faba guard cells (Ogawa et al. 1978) and enhances BL-mediated stomata opening (conductance level) in wheat (Karlsson 1986) and in Commelina communis (Assmann 1988). In the latter case, RL co-irradiation significantly extends the time required to reach maximum conductance following the inductive BL pulse, and extends the time required for the conductance level to return to the level found in darkness. These observations indicate that the life-time of the physiologically active photoproduct formed in BL in stomates is increased by RL. The fluence-response curve for curvature to five pulses of BL by RL pre-irradiated seedlings (Fig. 4) shows a threshold fluence (minimum curvature required for curvature) and fluence for peak curvature of approx. 0.05 p m o l . c m - 2 and 1 0 p m o l . c m -z, respectively. These are the same values as those which we have obtained (data not shown), and those previously reported (e.g., Blaauw and Blaauw-Jansen 1970) for Arena in response to single pulses of BL by non pre-irradiated seedlings. Thus, we see no shift in threshold for the phototropic response to BL induced by RL. We see a clear increase in response induced by the RL pre-irradiation (Figs. 1, 2), and by a RL coirradiation (Fig. 4). The descending arm of the fluence-response curve is elevated, showing higher curvature for the seedlings receiving five pulses than that obtained with those receiving a single pulse (Fig. 4). At present, we have inadequate data to formulate an hypothesis to account fully for
B. Steinitz et al. : Fluence-response relations for phototropism in Arena
this change in the descending arm. However, it seems inescapable that multiple systems or muItiple effects of BL are involved in this descending arm. The data presented here in no sense reduce the apparent complexity of the interrelated systems of gravitropism, phototropism to BL, and the RL effects which we assume to be phytochrome-mediated. To study these interrelated phenomena will require the systematic variation of each parameter or the study of single system or parameter in the absence of the others. Through the use of a clinostat, the gravitropic response has been eliminated in this study. To eliminate the other systems will require the use of mutants lacking specific responses to light. This work was supported by the U.S. Department of Energy under Contract No. DE-AC02-76ER0-1338.
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