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Short-term Phytochrome Response in Seed Germination
Institut fijr Botanik und Pharmazeutische Biologie der Universitat Erlangen-Niirnberg
Short-term Phytochrome Response in Seed Germination WOLFGANG HAUPT
and
ROBERT SCHEUERLEIN
With 2 figures Received July 1,1977 . Accepted July 15, 1977
Summary The germination of the photoblastic seeds of Lactuca sativa can be induced by a red flash in the millisecond range already. The induction is given either by one single flash or by tWO flashes, separated by a dark interval of 20 s, the total energy being equal under both these conditions. Dose response curves for single and double flash experiments differ very little from each other, but the saturation level is significantly higher in the double flash experiments. However, the effect of continuous irradiation of 20 s and longer saturates at a still higher level. We suggest to consider dark reactions of phytochrome intermediates as being responsible for the observed results.
Key words: phytochrome, seed germination, short-term effect, intermediates.
Introduction
In the phytochrome induced chloroplast movement in the green alga Mougeotia, and BRETZ (1976) found an enhancement effect if two R1) flashes were applied consecutively instead of simultaneously. This enhancement effect requires the dark interval exceeding 100 ms and is saturated with a dark interval in the range of 1 to 10 s. Obviously, the photoconversion of phytochrome by the first flash starts a dark reaction as a result of which the second flash becomes more effective. HAUPT
One of the possible interpretations suggests that the second flash acts on intermediates of the phytochrome photoconversion Pr -+ Pf/) induced by the first flash. If this assumption is true, the same or a very similar effect should be found in any phytochrome response of other systems. Another interpretation of the Mougeotia effect, however, suggests a change in the proportion of free to bound 1) The following abbreviations will be used: R = red, 2max ca. 650 nm; FR = far-red, 2> 700 nm; P r , P fr = phytochrome in the red absorbing and in the far-red absorbing form, respectively.
z. PJlanzenphysiol. Bd. 85. S. 445-450. 1977.
446
WOLFGANG HAUPT and ROBERT SCHEUERLEIN
phytochrome as a result of the first flash. In this case, that effect might be restricted to the Mougeotia system by the following reason: The chloroplast orientation requires an intracellular absorption gradient, and this in turn depends on phytochrome being dichroic and bound to the membrane. There is nearly no other phytochrome response known until now where bound phytochrome is necessary already during light absorption. Thus, it could be helpful to know whether the enhancement effect of Mougeotia can be found in other phytochrome responses as well. In the present paper, therefore, the germination of lettuce seeds will be investigated; this system has been chosen because here it is possible to work with reasonably small light fields. This is necessary in order to obtain, from one single flash, sufficient energy per area for phototransformation of phytochrome.
Material and Methods The seeds (exactly speaking: achenes) of Lactuca sativa, cultivar «Perax» were obtained from Fa. Sperl, Niirnberg, harvested 1974 and used in the experiments 1975. During the whole time of the experiments, the stock of the seeds was kept in the refrigerator (5 ± 1 DC). The coat (combined seed and fruit coat) is black in this cultivar, single white coated seeds were discarded before starting the experiments. In green safe-light, 25 or 50 seeds each were placed in a regular pattern in small petri dishes (5 cm cj) with 3 layers of white filter paper (SCHLEICHER and SCHULL, Selecta 595) and 1.5 ml quartz distilled water. The seeds covered an area with 2 cm cj), the micropylar pole always pointed outward. This close arrangement of the seeds had a slight inhibitory effect on the rate of germination, but this effect was constant within an experiment. Moreover, experiments with 25 or 50 seeds in each petri dish did not differ in the basic results, although showing slightly different absolute values. After this preparation, the seeds were kept in 24.5 ± 0.5 DC in complete darkness except for the experimental light treatments after 4 hours. 36 hours later, the germination rate was counted, a seed being considered as germinated if the coat has been disrupted and the radicula is visible. Under these conditions, the dark germination rate is about 30 % whereas after saturating red light about 80 % of the seeds germinate. This suggests that the unirradiated seeds already have part of their phytochrome in the P fr form, and this is confirmed by the fact that saturating FR can reduce the germination of the dark controls to less than 10 0/0. Hence, all experiments were started with 10 min FR, obtained by a projector «Prado» with a glass filter «RG 9» (3 mm) and a heat filter «KG b (2 mm), both by Schott (Mainz); the intensity at the seed level was 12.5 W· m-2 • Seeds with only this far-red irradiation are taken as .dark controls» in the experiments to be described. This FR preirradiation was followed by the flash inductions. We used two different types of flash lamps alternatively (but only one type within one experiment): Agfatronic 220 A and Uno mat 5000. The flash light was made monochromatic either by an interference filter AL 649 (Schott, band width 21 nm) or by a broad band interference filter K-65 (Balzers, band width 50 nm). Neutral glass filters (Schott) reduced the intensity as desired. If the intensity were to reduce by 5 to 10 Ofo only, microscopic slides were used successfully. The distance between the front face of the flash lamp and the seeds was kept constant to 4 cm. The resulting irradiance could be measured by a flashmeter «Sixtron 2, Electronic»
z. Pjlanzenphysiol. Bd. 85. S. 445-450.1977.
Short-term phytochrome response
447
(Gossen, Erlangen) in relative units and transformed into absolute units using the manufacturers information about the spectral sensitivity of the sensor and using the measured spectral transmission of the interference and glass filters. The light field was homogenously illuminated with a deviation ;;;;; ± 10010 as demonstrated by exposing photographic material with a series of white light flashes filtered through different neutral density filters. If the flash lamps were started repeatedly in short intervals (double flash irradiations), the flash energies deviated significantly from the energy of a single flash up to about 10010. Under identical conditions, these deviations were reproducible. They, therefore, could be predicted and compensated for by inserting the proper neutral density filters. Still remaining differences were considered in the graphs by shifting the data points slightly to each other. Since we aimed at proving or disproving the enhancement effect of double flash irradiation in our system, as compared to the Mougeotia system, we had to ensure saturation of the dark reaction between the two flashes. We therefore used a constant dark interval of 20 s. This is far beyond the critical point in Mougeotia, and hence the dark reaction in question obviously is saturated. Both flashes were given with the same flash lamp from the same direction. For comparison, only one flash with double the energy is given; this corresponds to the two simultaneously given flashes in Mougeotia. In contrast to Mougeotia, it was not possible, of course, to irradiate the nearly intransparent seeds from two opposite sides either simultaneously or consecutively and still to reach the same phytochrome molecules.
Results By establishing dose respone curves, the effect of single and double flash irradiations can be compared. This is shown in fig. 1 for 25 seeds per petri dish and the Agfatronic flash lamp with the Schott interference filter, using a logarithmic energy scale. The data points of both single and double flash irradiation are close together, only in the upper part of the curve the double flash seems to be more effective than the single flash. A similar experiment is shown in fig. 2, but with 50 seeds per petri dish and the Unomat flash lamp with the Balzers broad-band interference filter as the light source. In this experiment, obviously saturation has been reached. The curves clearly indicate that the saturation level is higher after the double flash than after the single flash irradiation, the difference being highly significant and amounting to about 13 0/0. It should be stressed, however, that these saturation levels are valid for the flash experiments only; after continuous R irradiations with 5 Wm- 2 during 20 to 300 seconds, still higher response levels have been reached. Fundamentally, the same results have been obtained in two further experiments not given in detail here. In a preliminary experiment, the total energy has been distributed among the two flashes not equally (50 : 50 %), but in varying relations from 10 : 90 to 90 : 10 %. In no case could the maximal effect of the 50 : 50 distribution be reached. The small differences, however, are not statistically significant; hence, any conclusion cannot be drawn from this experiment.
z. PJlanzenphysiol. Ed. 85. S. 445-450.1977.
448
WOLFGANG HAUPT
and
ROBERT SCHEUERLEIN
SATURATING
80
e- - -
- - - -
RED
- - - _
%
DOUBLE
-",'" '"
60
/
'"
",,,,Q
SINGLE
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z
2 ~
z
i
a:
-
40
w
CJ
20
100
500
W.s.m- 2
Fig. 1
80
SATURATING
RED
_.•
e _._. _. _. _. _.-._._.-'-' -' _. _. _. _. A A ."",..'-.
60
z
o
..... et Z
i
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/
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Fig. 2
z. PJlanzenphysiol. Bd. 85. S. 445-450. 1977.
500
1000
Short-term phytochrome response
449
Discussion On a first, uncritical view, the results on seeds seem to be in accordance with the results on Mougeotia (HAUPT and BRETZ): Two flashes, separated by a saturating dark interval, are more effective than a single flash with double the intensity (or than two flashes given simultaneously). If, however, we look more closely to the dose response curves, fundamental differences will become obvious. First, quantitatively: In Mougeotia, double flashes which nearly saturate the response if given separately, can be nearly ineffective if given simultaneously (HAUPT and BRETZ, fig. 1 and 2). With our seeds, in contrast, only small quantitative differences are observed. Second, qualitatively: In Mougeotia, the dose-response curves for consecutively and for simultaneously applied flashes are shifted to each other along the abscissa (BRETZ, in preparation). In seeds, however, these curves are shifted along the ordinate. In the lower part, these curves differ only slightly, but they saturate at clearly different levels. This is important by methodical reasons also: Even if we should have failed to fully compensate for the different sources of error in administering equal energies, this could mean only unimportant shifts of the curves along the abscissa, but the saturation levels would remain uninfluenced. To explain the effect In seeds, we need not discuss any hypothesis involving phytochrome dichroism. In the multicellular tissue of the seed and its strongly absorbing coat, so much light scattering will occur that the phytochrome molecules in all orientations will have the same probability of absorbing the light, even if polarized light were given. In consequence, we need not consider free and bound phytochrome as an explanation of our effect insofar as different orientation of these two phytochrome populations is concerned (random versus dichroic orientation). As the most likely explanation, we make use of the intermediates in the photoconversion Pr--->-P fr . According to KENDRICK and SPRUIT (1974) and SPRUIT et al. (1975), P r is transformed by light into the intermediate P 09S • This first product undergoes dark reactions which partly transform it to the next intermediate and finally to PIr, but which partly also bring it back to Pro These reactions, originally investigated in low temperatures, are very fast in room temperature, at least as long as free (dissolved) phytochrome is concerned. If, therefore, red light is given during seconds or minutes, P 69S may undergo transformation to P lr nearly quantitatively. Fig. 1 and 2: Seed germination as induced by a single red flash or by two equal red flashes separated by a 20 s dark interval «
z. PJlanzenphysiol. Bd. 85. S. 445-450.1977.
450
WOLFGANG HAUPT and ROBERT SCHEUERLEIN
After a very short saturating flash, however, a measurable amount of P 69S can revert to P" and hence the next flash can increase the P lr level via P 69S • Since the finally resulting Plr level is responsible for the physiological dark reactions which finally result in control of germination, the germination rate should reflect these differences. This is in accordance with our results: A single flash saturates at a germination rate of about 57 % and a second flash after 20 s increases the saturation level to 70 0/0. It could be predicted that a third flash would again increase this level to some degree. This experiment is still lacking. But instead - and again in accordance with the theory - with continuously given red light (20 to 300 s), a germination rate of about 80 Ofo is reached. In conclusion, phytochrome intermediates which until now are known from physical measurements only, have to be taken into consideration in physiological experiments also, at least if the irradiation times are comparable with the rate constants of the dark reactions. It should be important, therefore, to know these rate constants for room temperature. On the other hand, physiological experiments are in progress to further confirm our explanation in lettuce seeds. References HAUPT, W. and N. BRETZ: Short-term Reactions of Phytochrome in Mougeotia? Planta (Berl.) 128, 1-3 (1976). KENDRICK, R. E. and C. ]. P. SPRUIT: Inverse Dark Reversion of Phytochrome: An Explanation. Planta (Berl.) 120,265-272 (1974). SPRUIT, C. ]. P., R. E. KENDRICK, and R. ]. COOKE: Phytochrome Intermediates in Freezedried Tissue. Planta (Berl.) 127, 121-132 (1975).
Prof. Dr. W. HAUPT, SchloBgarten 4, D-8520 Erlangen.
z. P/lanzenphysiol. Bd. 85. S. 445-450.1977.
Short-term Phytochrome Response in Seed Germination WOLFGANG HAUPT
and
ROBERT SCHEUERLEIN
With 2 figures Received July 1,1977 . Accepted July 15, 1977
Summary The germination of the photoblastic seeds of Lactuca sativa can be induced by a red flash in the millisecond range already. The induction is given either by one single flash or by tWO flashes, separated by a dark interval of 20 s, the total energy being equal under both these conditions. Dose response curves for single and double flash experiments differ very little from each other, but the saturation level is significantly higher in the double flash experiments. However, the effect of continuous irradiation of 20 s and longer saturates at a still higher level. We suggest to consider dark reactions of phytochrome intermediates as being responsible for the observed results.
Key words: phytochrome, seed germination, short-term effect, intermediates.
Introduction
In the phytochrome induced chloroplast movement in the green alga Mougeotia, and BRETZ (1976) found an enhancement effect if two R1) flashes were applied consecutively instead of simultaneously. This enhancement effect requires the dark interval exceeding 100 ms and is saturated with a dark interval in the range of 1 to 10 s. Obviously, the photoconversion of phytochrome by the first flash starts a dark reaction as a result of which the second flash becomes more effective. HAUPT
One of the possible interpretations suggests that the second flash acts on intermediates of the phytochrome photoconversion Pr -+ Pf/) induced by the first flash. If this assumption is true, the same or a very similar effect should be found in any phytochrome response of other systems. Another interpretation of the Mougeotia effect, however, suggests a change in the proportion of free to bound 1) The following abbreviations will be used: R = red, 2max ca. 650 nm; FR = far-red, 2> 700 nm; P r , P fr = phytochrome in the red absorbing and in the far-red absorbing form, respectively.
z. PJlanzenphysiol. Bd. 85. S. 445-450. 1977.
446
WOLFGANG HAUPT and ROBERT SCHEUERLEIN
phytochrome as a result of the first flash. In this case, that effect might be restricted to the Mougeotia system by the following reason: The chloroplast orientation requires an intracellular absorption gradient, and this in turn depends on phytochrome being dichroic and bound to the membrane. There is nearly no other phytochrome response known until now where bound phytochrome is necessary already during light absorption. Thus, it could be helpful to know whether the enhancement effect of Mougeotia can be found in other phytochrome responses as well. In the present paper, therefore, the germination of lettuce seeds will be investigated; this system has been chosen because here it is possible to work with reasonably small light fields. This is necessary in order to obtain, from one single flash, sufficient energy per area for phototransformation of phytochrome.
Material and Methods The seeds (exactly speaking: achenes) of Lactuca sativa, cultivar «Perax» were obtained from Fa. Sperl, Niirnberg, harvested 1974 and used in the experiments 1975. During the whole time of the experiments, the stock of the seeds was kept in the refrigerator (5 ± 1 DC). The coat (combined seed and fruit coat) is black in this cultivar, single white coated seeds were discarded before starting the experiments. In green safe-light, 25 or 50 seeds each were placed in a regular pattern in small petri dishes (5 cm cj) with 3 layers of white filter paper (SCHLEICHER and SCHULL, Selecta 595) and 1.5 ml quartz distilled water. The seeds covered an area with 2 cm cj), the micropylar pole always pointed outward. This close arrangement of the seeds had a slight inhibitory effect on the rate of germination, but this effect was constant within an experiment. Moreover, experiments with 25 or 50 seeds in each petri dish did not differ in the basic results, although showing slightly different absolute values. After this preparation, the seeds were kept in 24.5 ± 0.5 DC in complete darkness except for the experimental light treatments after 4 hours. 36 hours later, the germination rate was counted, a seed being considered as germinated if the coat has been disrupted and the radicula is visible. Under these conditions, the dark germination rate is about 30 % whereas after saturating red light about 80 % of the seeds germinate. This suggests that the unirradiated seeds already have part of their phytochrome in the P fr form, and this is confirmed by the fact that saturating FR can reduce the germination of the dark controls to less than 10 0/0. Hence, all experiments were started with 10 min FR, obtained by a projector «Prado» with a glass filter «RG 9» (3 mm) and a heat filter «KG b (2 mm), both by Schott (Mainz); the intensity at the seed level was 12.5 W· m-2 • Seeds with only this far-red irradiation are taken as .dark controls» in the experiments to be described. This FR preirradiation was followed by the flash inductions. We used two different types of flash lamps alternatively (but only one type within one experiment): Agfatronic 220 A and Uno mat 5000. The flash light was made monochromatic either by an interference filter AL 649 (Schott, band width 21 nm) or by a broad band interference filter K-65 (Balzers, band width 50 nm). Neutral glass filters (Schott) reduced the intensity as desired. If the intensity were to reduce by 5 to 10 Ofo only, microscopic slides were used successfully. The distance between the front face of the flash lamp and the seeds was kept constant to 4 cm. The resulting irradiance could be measured by a flashmeter «Sixtron 2, Electronic»
z. Pjlanzenphysiol. Bd. 85. S. 445-450.1977.
Short-term phytochrome response
447
(Gossen, Erlangen) in relative units and transformed into absolute units using the manufacturers information about the spectral sensitivity of the sensor and using the measured spectral transmission of the interference and glass filters. The light field was homogenously illuminated with a deviation ;;;;; ± 10010 as demonstrated by exposing photographic material with a series of white light flashes filtered through different neutral density filters. If the flash lamps were started repeatedly in short intervals (double flash irradiations), the flash energies deviated significantly from the energy of a single flash up to about 10010. Under identical conditions, these deviations were reproducible. They, therefore, could be predicted and compensated for by inserting the proper neutral density filters. Still remaining differences were considered in the graphs by shifting the data points slightly to each other. Since we aimed at proving or disproving the enhancement effect of double flash irradiation in our system, as compared to the Mougeotia system, we had to ensure saturation of the dark reaction between the two flashes. We therefore used a constant dark interval of 20 s. This is far beyond the critical point in Mougeotia, and hence the dark reaction in question obviously is saturated. Both flashes were given with the same flash lamp from the same direction. For comparison, only one flash with double the energy is given; this corresponds to the two simultaneously given flashes in Mougeotia. In contrast to Mougeotia, it was not possible, of course, to irradiate the nearly intransparent seeds from two opposite sides either simultaneously or consecutively and still to reach the same phytochrome molecules.
Results By establishing dose respone curves, the effect of single and double flash irradiations can be compared. This is shown in fig. 1 for 25 seeds per petri dish and the Agfatronic flash lamp with the Schott interference filter, using a logarithmic energy scale. The data points of both single and double flash irradiation are close together, only in the upper part of the curve the double flash seems to be more effective than the single flash. A similar experiment is shown in fig. 2, but with 50 seeds per petri dish and the Unomat flash lamp with the Balzers broad-band interference filter as the light source. In this experiment, obviously saturation has been reached. The curves clearly indicate that the saturation level is higher after the double flash than after the single flash irradiation, the difference being highly significant and amounting to about 13 0/0. It should be stressed, however, that these saturation levels are valid for the flash experiments only; after continuous R irradiations with 5 Wm- 2 during 20 to 300 seconds, still higher response levels have been reached. Fundamentally, the same results have been obtained in two further experiments not given in detail here. In a preliminary experiment, the total energy has been distributed among the two flashes not equally (50 : 50 %), but in varying relations from 10 : 90 to 90 : 10 %. In no case could the maximal effect of the 50 : 50 distribution be reached. The small differences, however, are not statistically significant; hence, any conclusion cannot be drawn from this experiment.
z. PJlanzenphysiol. Ed. 85. S. 445-450.1977.
448
WOLFGANG HAUPT
and
ROBERT SCHEUERLEIN
SATURATING
80
e- - -
- - - -
RED
- - - _
%
DOUBLE
-",'" '"
60
/
'"
",,,,Q
SINGLE
","C
z
2 ~
z
i
a:
-
40
w
CJ
20
100
500
W.s.m- 2
Fig. 1
80
SATURATING
RED
_.•
e _._. _. _. _. _.-._._.-'-' -' _. _. _. _. A A ."",..'-.
60
z
o
..... et Z
i
a:
40
/
w
.
CJ
/
A /
/
/
/
/
/b.
20
100
W·s·m- 2
Fig. 2
z. PJlanzenphysiol. Bd. 85. S. 445-450. 1977.
500
1000
Short-term phytochrome response
449
Discussion On a first, uncritical view, the results on seeds seem to be in accordance with the results on Mougeotia (HAUPT and BRETZ): Two flashes, separated by a saturating dark interval, are more effective than a single flash with double the intensity (or than two flashes given simultaneously). If, however, we look more closely to the dose response curves, fundamental differences will become obvious. First, quantitatively: In Mougeotia, double flashes which nearly saturate the response if given separately, can be nearly ineffective if given simultaneously (HAUPT and BRETZ, fig. 1 and 2). With our seeds, in contrast, only small quantitative differences are observed. Second, qualitatively: In Mougeotia, the dose-response curves for consecutively and for simultaneously applied flashes are shifted to each other along the abscissa (BRETZ, in preparation). In seeds, however, these curves are shifted along the ordinate. In the lower part, these curves differ only slightly, but they saturate at clearly different levels. This is important by methodical reasons also: Even if we should have failed to fully compensate for the different sources of error in administering equal energies, this could mean only unimportant shifts of the curves along the abscissa, but the saturation levels would remain uninfluenced. To explain the effect In seeds, we need not discuss any hypothesis involving phytochrome dichroism. In the multicellular tissue of the seed and its strongly absorbing coat, so much light scattering will occur that the phytochrome molecules in all orientations will have the same probability of absorbing the light, even if polarized light were given. In consequence, we need not consider free and bound phytochrome as an explanation of our effect insofar as different orientation of these two phytochrome populations is concerned (random versus dichroic orientation). As the most likely explanation, we make use of the intermediates in the photoconversion Pr--->-P fr . According to KENDRICK and SPRUIT (1974) and SPRUIT et al. (1975), P r is transformed by light into the intermediate P 09S • This first product undergoes dark reactions which partly transform it to the next intermediate and finally to PIr, but which partly also bring it back to Pro These reactions, originally investigated in low temperatures, are very fast in room temperature, at least as long as free (dissolved) phytochrome is concerned. If, therefore, red light is given during seconds or minutes, P 69S may undergo transformation to P lr nearly quantitatively. Fig. 1 and 2: Seed germination as induced by a single red flash or by two equal red flashes separated by a 20 s dark interval «
z. PJlanzenphysiol. Bd. 85. S. 445-450.1977.
450
WOLFGANG HAUPT and ROBERT SCHEUERLEIN
After a very short saturating flash, however, a measurable amount of P 69S can revert to P" and hence the next flash can increase the P lr level via P 69S • Since the finally resulting Plr level is responsible for the physiological dark reactions which finally result in control of germination, the germination rate should reflect these differences. This is in accordance with our results: A single flash saturates at a germination rate of about 57 % and a second flash after 20 s increases the saturation level to 70 0/0. It could be predicted that a third flash would again increase this level to some degree. This experiment is still lacking. But instead - and again in accordance with the theory - with continuously given red light (20 to 300 s), a germination rate of about 80 Ofo is reached. In conclusion, phytochrome intermediates which until now are known from physical measurements only, have to be taken into consideration in physiological experiments also, at least if the irradiation times are comparable with the rate constants of the dark reactions. It should be important, therefore, to know these rate constants for room temperature. On the other hand, physiological experiments are in progress to further confirm our explanation in lettuce seeds. References HAUPT, W. and N. BRETZ: Short-term Reactions of Phytochrome in Mougeotia? Planta (Berl.) 128, 1-3 (1976). KENDRICK, R. E. and C. ]. P. SPRUIT: Inverse Dark Reversion of Phytochrome: An Explanation. Planta (Berl.) 120,265-272 (1974). SPRUIT, C. ]. P., R. E. KENDRICK, and R. ]. COOKE: Phytochrome Intermediates in Freezedried Tissue. Planta (Berl.) 127, 121-132 (1975).
Prof. Dr. W. HAUPT, SchloBgarten 4, D-8520 Erlangen.
z. P/lanzenphysiol. Bd. 85. S. 445-450.1977.