Planta (1988)174:373-379

P l a n t a 9 Springer-Verlag ] 988

Transient reduction of responsiveness of blue-lightmediated hair-whorl morphogenesis in Acetabularia mediterranea induced by blue light Raincr Schmid*, Martin Tiinncrmann, and Evi-Marion Idziak Institut ffir Pflanzenphysiologie, Zellbiologie und Mikrobiologie, Freie Universit/it Berlin, K6nigin-Luise-Strasse 12-/6, D-1000 Berlin-33, Germany

Abstract. After a prolonged period of red light the formation of a new whorl of lateral hairs can be induced in Acetabularia mediterranea Lamouroux (=A. acetabulum (L.) Silva) by a pulse of blue light. It has previously been shown that the response to blue light obeys the law of reciprocity. In this paper we demonstrate that the responses to blue light are additive only within 10 rain after the onset of blue-light treatment, since the responsiveness of the cells is also affected by blue light. One hour after a short blue-light pulse the response to a second blue-light pulse has come to a minimum. After that, the responsiveness is restored in a refractory period of several hours. The fluenceresponse curves for hair-whorl formation at the time of minimum responsiveness are shifted parallel to the original fluence-response curves without preirradiation. Again, the law of reciprocity applies. This indicates an increased light requirement only for the same degree of hair-formation response. The sensitivity to blue light of the "reduction of responsiveness" response is higher by a factor of about 50 than the "induction of hairformation" response. Key words: Acetabularia - Blue light (pulse irradiation) - Hair formation - Acetabularia - Light responsiveness (change).

Introduction Many responses of organisms to blue light are transduced by mechanisms that show direct correlations to the number of incident quanta, indicating the validity of the Bunsen-Roscoe reciprocity * To whom correspondence should be addressed

law, i.e. the degree of response is identical whenever the product of photon-fluence rate and irradiation time is constant. In some of the blue-lightmediated phenomena the law of reciprocity holds only under restraints. Biphasic fluence-response curves have been reported for carotenogenesis of the fungi P h y c o m y c e s (Jayaram et al. 1979). Fusarium (Schrott 1980) and Neurospora (Schr0tt 1980, 1981). The two phases were interpreted as the consequence of the availability of material essential for the light-transduction chain. The amount of that material is changed by two processes of opposing nature: (i) the consumption of material in the first phase (reciprocity law applies) and (ii) its re-synthesis when its pool is empty (Schrott 1981). Limited validity of the reciprocity law has also been described for phototropism in coleoptiles of cereals (Briggs/960; Iino 1987) as well as in hypocotyls of dicotyledonous seedlings such as Lactuca and Arabidopsis (Steinitz et al. 1985; Steinitz and Poff 1986). In these systems only the first positive curvature follows the Bunsen-Roscoe law, while the second positive curvature is provoked only by long irradiation times. This fluence dependency resembles strongly that of the biphasic fluence-response curves of fungal carotenogenesis. Furthermore, evidence has recently been presented , derived from various pulse-irradiation experiments (Steinitz and Poff/986; Iino 1987), that the phototropic fluence-response relation may also be interpreted in a similar way. Disappearance of the photoreceptor or some limiting transducing intermediate during the early periods of photoreception and its regeneration after some time have been proposed. A recent interpretation suggests, conversely, the formation of an active photoproduct a n d its inactivation, combined with a process of transient desensitization (Iino 1987). Similar conclusions were derived also from pulsed blue-light effects on

374

R. Schmid et al.: Change of blue-light responsiveness in Acetabularia

stomatal opening (Iino et al. 1985), although desensitization was not obvious in this case. These examples demonstrate that an apparent loss of reciprocity as a result of blue irradiation could probably be a common phenomenon. Indeed, there is a tendency to generalize these observations as being characteristic of the blue-lighttransducing system (Iino et al. 1985; Iino 1987). On the other hand, a generalization may be unjustiffed since other experimental systems are described that obey the reciprocity law even under extremely long periods of blue light of up to 96 h or even more (Liining and Dring 1975; Ltining and Neshul 1978; Liining 1980), while others appear to require continuous irradiation and do not respond to single blue-light pulses at all (for example: morphogenesis of fern sporelings, Mohr 1956; respiration enhancement in Chlorella, Kowallik 1967; greening of tobacco cell cultures, Richter and Wessel 1985). In a previous paper we introduced the siphonaceous green alga Acetabularia mediterranea as a suitable model system for studies of the blue-light dependency of cellular photomorphogenesis (Schmid et al. 1987). It has been demonstrated that, after a prolonged period of culture in red light, growth and morphogenesis of lateral hairs can be induced by a short blue-light stimulus. The action spectrum for this photomorphogenetic response agrees with those of other blue-light effects. Parallelism of fluence-response curves for different wavelengths indicates the involvement of only one photoreceptor pigment. It has been shown that the response of hair-whorl formation is a function of the incident quanta at a given wavelength. This obediance to the reciprocity law, however, appears to fail after extended irradiation programs (visible beyond about 10 min), according to orienting experiments. Since this behaviour appears to correspond to that of other organisms, where the sensitivity to blue light is altered by the effective radiation itself, we investigated the nature of the limitations to the law of reciprocity that occur in Acetabularia. These studies are the subject of the present publication. Material and methods All methods for the culture of Acetabularia mediterranea Lamouroux (= A. acetabulum (L.) Silva) as well as the irradiation sources and schemes were described by Schmid et al. (1987). The principle of the procedure was a preculture of the cells for two weeks in white light followed by one week of red light. After that, blue light was used for the induction of growth and hair development, and after a further day in red light, the number of cells with new hair whorls could be evaluated.

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Fig. 1. Fluence-response curves for hair-whorl induction in Acetabularia. After the standard pretreatment (white followed by red light) a blue irradiation was given as indicated and the percentage of cells with new hairs was determined 1 d later. Standard blue light (broad banded) was used. The duration of the irradiation was either 10 min or 1 h. Fluence rates were varied using neutral density filters to give the fluences indicated on the abscissa When intermittent irradiation programs were used, red light was used during the intervals. Unless stated otherwise, each datum point represents the mean of five independent measurements. In the present investigations we used, besides the standard blue-light source (1.6.10- 5 m o l - m - 2 . s - 1, standard p h o t o n fluence rate), mainly light from a xenon high-pressure lamp (XBO 450 W; Osram, Miinchen, F R G ) filtered through an interference filter with transmission maximum at 451 nm (DIL; Schott, Mainz, FRG). The p h o t o n fluence rate of this source was set to 1.13-10- 6 m o l - m - 2. s - 1. Whenever fluence rates other than this were used, it is indicated in the legends.

Results

Irradiation times in those former experiments that showed validity of the law of reciprocity (Schmid et al. 1987) were in the range of seconds to only a few minutes. When irradiation periods were extended to 1 h, using low fluence rates of blue light, the fluence-response curve was shifted towards reduced effectiveness of the blue irradiation by a factor of about 1.5 (Fig. 1). Obviously, the responsiveness to blue light had changed between 10 min and 1 h. When a non-saturating blue-light period was divided into two or more individual pulses which were intermittently distributed over a period of 2 h (red light during the intervals) we observed that the effects were not additive (Fig. 2). A comparison with the effects caused by the single partial pulses showed no effect at all of the second blue-light pulse when the interval between both partial irradiations was long. A higher responsiveness to the following pulses was found with shorter intervals

R. Schmid et al. : Change of blue-light responsiveness in Acetabularia

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Fig. 2. Effect of various intermittent stimuli of blue light on hair-whorl formation in Acetabularia. After the standard pretreatment the cells were induced to form a hair whorl by blue light (2- J 0 - v tool. m - 2. s - 1, standard blue-light source). After I d more in red light the n u m b e r of cells with new hairs was determined. Insert shows the irradiation scheme. Blue light was given either as a single pulse of 150 s or was equally distributed over a period of 2 h by two, five, or ten pulses, each of the duration of one half, one fifth or one tenth of that of the 150-s pulse, respectively. Red light was given during the intervals. Closed circles give the effect of the intermittent irradiations. The columns at the side of each value show the responses caused by only one of the individual partial stimuli. Bars= +SD

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(Fig. 2). Whether two pulses were separated by a period of about 2 h or ten pulses separated by 13-rain intervals each, none of the intermittent irradiations produced the response induced by the single continuous irradiation, though the total photon fluence was identical. In a further set of experiments, the interval between a first pulse o f low effectiveness and a second one of medium effectiveness was varied (Fig. 3A, B). Between the two blue-light pulses, standard red light was given, although, when using darkness, the results were principally the same (data not shown). We found that the responsiveness to the second blue-light pulse exhibited a typical behaviour. After a period of about 10 min, where strict additivity of intermittent irradiations could be shown (Table 1), the ability to respond decreased rapidly and came to a minimum l h after the first blue-light treatment (Fig. 3). This was followed by a refractory period of more than 3.5 h, characterized by a slow increase in the responsiveness to blue light. The time course of all these changes appeared to be largely independent of the fluence of the first irradiation (compare Figs. 3A and B with 3C). When different fluences were used in the first pulse and the two irradiations together totalled the same amount of blue-light fluence, the shapes of the curves were virtually identical (shown for two examples in Fig. 3 A and B). Only the low-

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interval befweenlSfand 2ndpu!.se Fig. 3 A - C . Time course of the change in the responsiveness of blue-light-mediated hair-whorl formation in Acetabularia after its induction by blue light. Cells were irradiated according to the standard procedure. Blue light (451 nm) of a total duration of 30 s was split into two pulses ( 5 + 2 5 s in A, 1 0 + 2 0 s in B) and the interval between the pulses was varied. In C, a combination of pulses of 30 s and 60 s was used. During the intervals, red light was given. The n u m b e r of cells with new hairs was determined after 1 d more in red light. Bars= +SD

est level, detectable after an interval of i h, differed due to the number of hair whorls induced by the first pulse. The similar shapes of the curves (Fig. 3) indicated that the reduction of responsiveness was already saturated by blue light. Indeed, a fluenceresponse curve showed that very low amounts of blue light were saturating and that the hair-formation response could not be suppressed completely (Fig. 4). The saturation level was found to be between 10 and 20% whorl formation (see also Fig. 3 A). This remaining response was not the inductive effect of the first pulse, since even with

R. Schmid et al. : Change of blue-light responsiveness in Acetabularia

376

1. Effect of intermittent blue irradiation, distributed equidistantly over a period of 10 min, o n the additivity of the response of hair-whorl formation in Acetabularia

Table

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A fluence-response curve that was determined in the late refractory period, i.e. 5 h after the first light pulse, was not distinguishable from that obtained without any pre-irradiation (not shown). This indicated a restoration of the original properties of the system. Whether it was complete could not be deduced from these data for statistical reasons. A restoration has also been shown by a repetition of the experiment with various interval durations. When, 5 h after a short blue-light pulse, a second short blue-light pulse was given and, after that, the responsiveness tested upon various intervals, a decrease could again be observed that exhibited the same time course as in the experiments with only one single pre-irradiation (Fig. 6, compare also with Fig. 3). Discussion

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Fig. 4. Dependence of the reduction of responsiveness of bluelight-mediated hair-whorl formation in Acetabularia on the a m o u n t of blue light in the first pulse. Cells were preirradiated according to the standard procedure. After that short blue-light pulses of different durations were given (0.5-10 s, as indicated by the scale on top of the figure). The response of hair formation to blue light 1 h after the first blue-light pulse (point of minimum of responsiveness, see Figs. 2, 3) was tested by a sec~ ond stimulus of 25 s duration. The dashed line gives the response to a single pulse of 25 s of blue light

a photon fluence of 3.10 -6 m o l - m -2 only 5% whorl formation was obtained (Fig. 1). Reducing the responsiveness could be half-saturated by a fluence (at 451 nm) as small as 3.10 -7 m o l ' m -2 which is about one-fiftieth (2%) of the fluence required to induce 50% of the cells for hair-whorl formation (see Fig. 6 for comparison). Fluences above 10 - 6 mol. m - 2 were saturating. An irradiation that saturated the decrease of responsiveness caused a parallel shift in the fluence-response curve for hair-whorl formation towards lower-fluence effectiveness (Fig. 5). The hair-formation response 1 h after a first blue-light pulse again followed the law of reciprocity (shown for a response of about 50%, Table 2).

Young cells of Acetabularia mediterranea which are preirradiated for several days with red light respond to a short blue irradiation by an increase in growth rate and, later on, by the development of a new whorl of hairs (Schmid et al. 1987). Although we have previously shown that the response obeyed the reciprocity law, the results of the pulse-irradiation experiments demonstrated that blue light was transduced additively into the morphogenetic response only within a short period of about 10 min (Table 1). After that, the responsiveness for additional blue light decreased drastically and was at a minimum about 1 h after the onset of blue light (Fig. 3). This transient reduction of responsiveness has to be regarded as the reason for the different fluence relationships when irradiation periods were extended beyond 10 min (Fig. 1). The properties of the time course of the change in the responsiveness indicate the presence of two processes with opposing effects. Both processes follow a blue irradiation: (i) a rapid process that is effective 10 rain after the onset of blue light, showing first-order decay kinetics, and being connected with a decrease of the inducibility of growth and morphogenesis; and (ii) a second slow process that restores the responsiveness to blue light. Whether the second process is mediated by blue light itself cannot be deduced from the data. The initial lagphase in the specific time course (Fig. 3) can be interpreted in two ways: either the process that causes the reduction of responsiveness starts at about 10 min after the beginning of blue light or it starts earlier and only becomes limiting to the responsiveness after that period. At the point of minimum responsiveness, i.e. 1 h after a first short blue-light pulse, the fluence-

R. Schmid etal. : Change of blue-light responsiveness in Acetabularia

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response curve for the induction of hair formation was shifted parallel towards lower effectiveness of light (Fig. 5). Since the response of hair-whorl development is qualitative rather than quantitative (response in the individuals is either positive or completely lacking), the steepness of the fluenceresponse curves depends on the degree of diversity of the threshold for blue light in the individual cells and, thus, is a property of the cell population used. As a consequence of the parallel shift of the fluence-response curve, the change in responsiveness occurred, statistically, to the same degree in all cells and was not a response of only part of the cell population. The parallel shift of the fluence-response curve allows no conclusions as to whether or not the photoreceptive system itself was affected by the blue-light treatment. If the photoreceptive system is affected, the inactivation cannot be an immediate light effect on the photoreceptor since it occurred during a constant time course, after induction by blue light, in red light (Fig. 3) and even

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lime affer 1sf purse [h] Fig. 6. Induction of a change of the responsiveness for hairwhorl formation close to the end of the refractory period, by a second, short blue-light pulse. Cells were pretreated according to the standard procedure and then irradiated by a short bluelight pulse (451 rim) of 5 s duration. After 5 h in red light, a second blue-light pulse of 5 s was given and the response for hair-whorl formation was induced, corresponding to the experiments shown in Figs. 3 and 4, by a blue-irradiation period of 15 s. After 24 h in red light the cells with new hair whorls were determined. Columns give the responses to the various pre-irradiations and controls. Scales on top of the figure indicate the respective durations of the intervals between the iighlL pulses (5 s) that reduced the responsiveness and those that were used to test the response (15 s)

in complete darkness (not shown). If, though, the photoreceptive system is affected, the deactivation is not at all complete. On the other hand, we cannot exclude that the desensitizing effect of blue light acts on properties of the light-transduction chain via a partial "functional" uncoupling from the photoreceptive process. So far, we have no notion for the molecular basis of such an uncoupling. As discussed below, the consumption of an intermediate and its re-synthesis cannot be acceptable interpretation. Because we could not decide from the results whether the receptive system or the reactive system was affected, we used the term

378

R. Schmid et al. : Change of blue-light responsiveness in Acetabularia

"responsiveness", which includes sensitivity as well as reactivity. Five hours after a first blue-light stimulus the responsiveness for hair-whorl development could again be lowered by a second stimulus during the same time course in which it was observed after only one blue-light pulse (Fig. 6; compare Fig. 3). Furthermore, the fluence-response curve for hairwhorl formation had shifted back to its original position (not shown). Thus, the system restores its original properties during the refractory period. As mentioned above, the time course for the change of responsiveness in Acetabularia allows the interpretation that it is composed of two opposite processes, i.e. loss and restoration of responsiveness. This might superficially be in agreement with the interpretations of other time-dependent changes of blue-light responses like carotenoid production in Neurospora (Schrott 1981) and phototropism of Arabidopsis (Steinitz and Poff 1986) and maize (Iino 1987). There are, however, striking differences with respect to the response in Acetabularia and the underlying mechanism, for various reasons, appears to be completely different. In the other organisms the amount of blue light required for a complete inactivation of the responsive system was in the range of that saturating the response itself under conditions where the reciprocity law holds. The desensitization appeared to be directly the result of blue-light irradiation. Thus, the conclusion that it is a component (photoreceptor or intermediate) of the same transduction chain which is affected is plausible. In Acetabularia, however, one fiftieth, i.e. only 2%, of the fluence for half-saturating hair formation was needed to half-saturate the reduction of responsiveness for hair-whorl formation. Obviously, the "reduction of responsiveness" had a light requirement that was quite different from the hair-formation response itself (the differences of the fluences that accomplish saturation are even larger). In the other responses, indeed, a transient insensitivity was demonstrated and even higher light dosages could not increase the responses. Since, in Acetabularia, the fluence relationship for hairwhorl formation was merely shifted (Fig. 5), it was not the suppression of the ability of the individual cells to respond that was provoked by the first blue irradiation, but rather their responsiveness was lowered. Since a full hair-formation response could still be obtained, the complete inactivation of the photoreceptor or the depletion of the transduction chain from an intermediate, as proposed at least for some of the other systems, could not be the reason for these changes. The much lower

light requirement of the response of "reduction of responsiveness" is a further argument against such an interpretation. The changes of sensitivity in fungal carotenogenesis, and higher-plant phototropism are fluence-dependent, while the reduction of responsiveness rather was time-dependent in Acetabularia. Furthermore, the former changes include periods where the sensitivity to blue light is dependent bn the irradiation time and does not obey the reciprocity law. We could show for Acetabularia that for the response of hair-whorl formation the law of reciprocity also applied at times when the responsiveness (1 h after a first short blue light pulse) was at its minimum. This observation indicates that, at any point of time, the evaluation of the effective light by the organism was a function of the number of absorbed photons, though the responsiveness to the same amount of light changed by a factor of about two. The most recent model proposed for blue-lightdependent changes of the sensitivity for maize phototropism (Iino 1987) differs from other explanations of the phenomena by the assumption of an active photoproduct that reverts to the inactive form in a light-independent process accompanied by a process of blue-light-induced desensitization. Whenever this hypothesis also assigns for other blue-light-mediated responses the desensitization process is applicable to Acetabularia. Our conditions, however, did not allow conclusions about the formation or stability of a photoproduct. References Briggs, W.R. (1960) Light dosage and phototropic responses of corn and oat coleoptiles. Plant Physiol. 35, 951-962 Iino, M. (1987) Kinetic modelling of phototropism in maize coleoptiles. Planta 171, 110-126 Iino, M., Ogawa, T., Zeiger, E. (1985) Kinetic properties of the blue-light response of stomata. Proc. Natl. Acad. Sci. USA 82, 8019-8023 Jayaram, M., Presti, D., Delbrfick, M. (1979) Light induced carotene synthesis in Phycomyces. Exp. Mycol. 3, 42-52 Kowallik, W. 0967) Action spectrum for an enhancement of endogenous respiration by light in Chorella. Plant Physiol. 42, 672-676 Lfining, K (1980) Critical levels of light and temperature regulating the gametogenesis of three Laminaria ssp. (Phaeophyceae). J. Phycol. 16, 1-15 Liining, K., Dring, M.E. (1975) Reproduction, growth, and photosynthesis of gametophytes of Laminaria saceharina grown in blue and red light. Mar. Biol. 29, 195-200 Lfining, K., NeshuI, M. (1978) Light and temperature demands for growth and reproduction of laminarian gametophytes in Southern and Central California. Mar. Biol. 45, 297-305 Mohr, H. (1956) Die Abhfingigkeit des Protonemawachstums und der ProtonemapolaritS.t bei Farnen vom Licht. Planta 47, 127-158

R. Schmid et al. : Change of blue-light responsiveness in Acetabularia Richter, G., Wessel, K. (1985) Red light inhibits blue lightinduced chloroplast development in cultured plant cells at the mRNA level. Plant Mol. Biol. 5, 175-182 Schmid, R., Idziak, E.-M., T/innermann, M. (1987) Action spectrum for the blue-light-dependent morphogenesis of hair whorls in Acetabularia mediterranea. Planta 171, 96-103 Schrott, E.L. (1980) Dose response and related aspects of carotenogenesis in Neurospora crassa. In: The blue light syndrome, pp. 309-318, Senger, H., ed. Springer, Berlin Heidelberg New York

379

Schrott, E.L. (1981) The biphasic fluence response of carotenogenesis in Neurospora crassa: Temporary insensitivity of the photoreceptor system. Planta 151, 371-374 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 Steinitz, B., Ren, Z., Poff, K.L. (1985) Blue and green lightinduced phototropism in Arabidopsis thaliana and Lactuca sativa L. seedlings. Plant Physiol. 77, 248-251 Received 23 September, accepted 8 December 1987