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On the light requirement for replication of plastids in polytrichum
Plant Science Letters, 3 (1974) 81--85 © Elsevier Scientific Publishing Qompany, Amsterdam -- Printed in The Netherlands
ON THE LIGHT REQUIREMENT FOR REPLICATION OF PLASTIDS IN POLYTRICHUM
LEE B. KASS and DOMINICK J. PAOLILLO JR.
Section o f Genetics, Development and Physiology, Cornell University, Ithaca, N. Y. 14850
(U.S.A.) (Received November 26th, 1973) (Revision received January 24th, 1974)
SUMMARY
The chloroplasts of germinating spores of the moss Polytrichum replicate in darkness to a lesser extent than in light. A stable number of plastids is attained by 48 h in darkness, i.e. the p!astids become light-dependent with respect to replication. Light stimulates the plastids to replicate. When red light is used, the promotive effect of the light can be completely reversed by far red light. INTRODUCTION
Light has been shown to be important in chloroplast development with phytochrome mediation involved in some of the developmental events [3, 4}. Hahn and Miller [ 5] reported a light requirement for plastid replication m the germinating spores of the moss Polytrichum. Partial reversibility of the promotive effect ot red light by far red light indicated that phytochrome mediation may be involved. Our results with similar materials show that plastid replication in Polytrichum spores does occur in the dark, although only ~o a limited extent. Ironically, thi~ finding makes it possible to develop an experimental system in which the demonstration of red--far red reversibility of plastid replication is more cl,-~r cut than that given by Hahn and Miller [ 5]. MATERIALS ~ND METHODS
Field-ripened capsules of Polytrichum c o m m u n e Hedw. were collected from Texas Hollow, Schuyler County, N.Y. in July and August, 1972. Spores were harvested as previously described t6], after the capsules were sterilized for 5 min in 5% Clorox solution and dried under sterile conditions. Spores were dusted onto a medium consisting of Voth's solution No. 5 solidified with 1% Difco Bacto agar, with and without 2% sucrose. Cultures were sampled by scraping the spores or germlings from the agar with a metal spatula and 81
preparing a water m o u n t for microscopic observation. Counts of plastid number were made using fluorescence microscopy, with a Zeiss microscope and an Osram HBO 200/4 lamp, with excitation filters BG 3 and BG 12 (blue) and barrier filter 53 (yellow). Fluorescence microscopy allows accurate counts of chloroplast number in unswollen and ungerminated spores because the plastids fluoresce red, and are easily visible through the spore wail. Control cultures were grown at. 11 800 lux under continuous fluorescent illumination (Sylvania, 40 W, warm white) and in the dark (wrapped in aluminum foil, on the same growth rack). Experiments on red--far red reversibility were performed after a 48-h incubation of the spores in the dark. Cultures were then placed into the following light regimes (1) continuous darkness; (2) continuous white light at 11 800 lux; (3) red light for 15 min, every 6 h, followed by darkness, (4) red light for 15 rain followed immediately by far red light for 15 min, e w r y 6 h, followed by darkness. The experiment was replicated to verify the results. The growth temperature was 25 °, throughout. Light filters were obtained from Rosco Laboratories, Harrison, N.Y. The filter for red light consisted of 1 medium red (No. 823) Roscolene filter plus 1 layer of red cellophane. The red light source was 2 Sylvania 40 W cool white fluorescent tubes, one from each side of the chamber at an angle of 45 ° from the horizontal. The filter for far red light consisted of 1 medium red Roscolene filter sandwiched between 2 dark urban blue (No. 866) Roscolene filters. The far red light source consisted of a row of 100 W incadescent bulbs placed 25 cm above the cultures, with a 10-cm layer of water as a cooling iilter, in a clear plexiglas container. The intensities of red and far red light were 0.00211 gcal/cm 2/rain and 0.0524 gcal/cm 2/min respectively at the level of the cultures, as measured with a Kipp and Zonen (Delft, Holland) thermopile solarimeter. The far red light used in this study was tested for its ability to suppress germination of Black Beauty squash (zucchini) obtained from Agway, Inc., Syracuse, N.Y. This variety of squash fails to germinate in far red light [9]. Germination in the light and dark was 85% for our seeds, but only 11% of the seeds germinated in far red light. This difference is highly significant, statistically, using the significance test based on a normal distribution [2]. RESULTS
General observations on the morphology o f germination Spores of Polytrichum commune cultured on agar show germ tube emergence by 48 h. The distal end of the growing filament becomes a rhizoid in at least some germlings by approx. 96 h in light with sucrose, and by 120 h in light without sucrose. Rhizoid differentiation is also evident at 120 h in darkness, in the presence of sucrose. No germ tubes emerge in the dark on unsupplemented mineral medium. The spores contain 2--4 chloroplasts at the time they are harvested from 82
their capsules. The plastids 8f the spore and the young germ tube are subsphezical and dark green. As the rhizoid begins to differentiate one observes in the participating cells numerous light green, small chloroplasts which are more filiform than spherical. The formation of these pale green plastids seems to involve the subdivision of the large, dark green plastids. At the opposite end of the germ tube, the plastids remain dark green, and here a protonema forms, at right angles to or opposite the rhizoid. The chloroplasts in a p r o t o n e m a are like those in the spore. It is necessary to keep in mind the distinction between replication of large, dark green plastids vs. the formation of small, pale green plastids as are found in the rhizoids. The conclusions drawn in this study are intended to apply to the replication of the large chloroplasts in the spore and y o u n g germ tube. Counts of plastids made at 120 h are sometimes influenced by the early stages of rhizoid formation, b u t these effects are taken into account in the interpretation of the data. Time course o f chloroplast replication By 8 h culture in white light, a significant change in plastid mtmber can be detected using the two-tailed Z test, of Alder and Roessler [ 1 ]. By 48 h the plastids are too numerous to count accurately. This is not the case in the dark. Although the results lea~'e no question t h a t replication has occurred, a plateau is reached at about 48 h (Fig. 1). This stable number is maintained until 120 h, when the germ tubes express themselves as rhizoids and the small plastids t h a t are produced in the cells at the tip of the filamcnt contribute to an increase in plastid number, ~.' they are counted.
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Fig. 1. Changes in mean number of plastids per spore (or germling) in light with sucrose ( - o - ) and in the dark with sucrose (- ~ - ) and without sucrose ( - • -). Each point represents a c o u n t o f 60 spores or germlings, except for the initial number, which is based on a count of 500 spores. The variability in mean number of chloroplasts between 48 and 96 h in dark is not statistically significant. using the two-tailed Z test of Aflder and Roessler
[z]. Fig. 2. The effect o f continuous light (- '~ -), intermittent red light (- ~ -), red followed by far red light (-/~; -), and continuous darkness (- • -) on plastid number in spores incubated 48 h in darkness. Each point is based on a count of 500 spores or germlings. The differences in red light v s . other treatments at 96 and 120 h are highly significant according to the twotailed Z test of Alder and Roessler [1 ].
83
Sucrose did not affect plastid number, but was required for germ tube emergence,in the dark. It seems, then, that growth of a germ tube is not causally related to plastid replication, because the same amount of replication occurs when the germ tube does not emerge.
Red--far red reversibility of light-induced plastid multiplication The results of the experiment are presented in Fig. 2. Significant plastid replication occurred in both inte_rmittent red light and continuous white light. Red followed by far red light totally prevented any increase in plastid number. There was a slight rise in plastid replication at 120 h in continuous darkness possibly due to the beginnings of rhizoid differentiation. In spores of Onoclea, red light promotes growth of the rhizoid, and far red light negates the~effect of red light [10]. In our experiments far red light may be inhibiting rhizoid formation and therefore the lack of increase in plastid number as compared with the number in total darkness at 120 h. DISCUSSION
Hahn and Miller [5] reported that the plastids in spores of Polytrichum do not multiply in darkness, but we find that this is not exactly the case. Initial counts of plastid number are not reported Lq their paper. Rather remarkably, all the treatments they used seem to have yielded the same plastid number after 48 h of incubation. This included both continuous darkness and continuous light. Our own cultures in the light gave much higher numbers of plastids per spore or germling compared with those in the dark. We cannot account for this evident difference between their results and ours, unless it involves differences in handling of the spores. We have tried to copy their techniques using static liquid culture, and cannot use them to obtain reproducible results. But static liquid culture is not optimal for rapid, vigorous growth of Polytrichum sporelings. On the other hand our data are in basic agreement with those of Hahn and Miller [5] if one concentrates on the period between 48 and 96 h of incubation. Our results support the contention that the promotion of plastid replication by red light is far red reversible. It has been shown that a Polytrichum spore contains only one piastid directly after meiosis [ 11 ]. Because the mature spore contains 2--4 plastids, one or more replications can occur during ripening. If the mature spores are cultured in the light, the plastids replicate in essentially all spores [8]. If the mature spores are incubated in the dark the mean number of plastids increases but does not double. Therefore, some fraction of the initial population of plastids cannot multiply without light. By 48 h, all the plastids are in this category because replication is halted by that time in darkness. It is this circum. stance that allows for a successful demonstration of red--far red reversibility. because all the plastids nave become light-sensitive, and no further alteration in plastid number occurs in the dark, until rhizoids differentiate. This gives additional support to the concept derived from the results with inhibitors of 84
plastid replication in light and dark [ 8] that the chloroplasts of Polytrichum exist in two phases with respect to their ability to replicate: (1) a lightsensitive phase and (2) a light-insensitive phase. We do not k n o w the nature of the light response (presumably mediated by p h y t o c h r o m e ) t h a t occurs in the plastid. Because the plastids of the spore are chloroplasts [ 11 ] we are n o t dealing with the structural changes that require light in the greening of etioplasts [3]. But some parallel may exist with the p h y t o c h r o m e mediation of plastid development in the lag phase of greening in etioplasts [ 3], and with the p h y t o c h r o m e mediation of the stimulation of RNA and protein synthesis in developing plastids of higher plants [ 4]. Recently evidence derived from isolated plastids has been presented to argue t h a t "a p h y t o c h r o m e mechanism exists, if not inside the plastid, then on the plastid envelopes" [ 12]. Finally, we wish to point out that as cytodifferentiation occurs in the germling, the plastids that are in cells that participate in rhizoid formation divide themselves into many small plastids that have a light green color. Because this occurs in the darkness ~s well as in light, it is evident that the restraint on plastid division imposed by darkness depends on the state of diff:rentiation o f the cells that contain the plastids. As in Onoclea [ 10], p h y t o c h r o m e controls differ in the rhizoid as compared to other cells of the germling. ACKNOWLEDGMENT
This research was supported by a National Science Foundation grant, GB-25097. REFERENCES
1 H.L. Alder and E.B. Roessler, Intro6rction to Probability and Statistics. Freeman, San Francisco, 1960, p. 117. 2 N.T.J. Bailey, Statistical Methods in Biology, Wiley, New York, 1959, p. 38. 3 L. Bogorad, in T.W. Goodwm(Ed.), Biochemistry of Chloroplasts, Vol. II, Academic Press, New York, i967., p. 615. 4 N. Scott, R. Munns, D. Graham and R.M. Smillie, in N.K. Boar~man, A.W. Linnano and R.M. Smillie (Eds.), Autonomy and Biogenesis of Mitoehondria and Chloroplasts, North Holland, Amsterdam, 1971, p. 383. 5 L.W. Hahn and J.H. Miller, Physiol. Plant., 19 (1966) 134. 6 D.J. Paolillo Jr. and R.H. Jagels, Bryologist, 72 (1969) 444. 7 P.D. Voth, Bot. Gaz., 104 (1943) 591. 8 L.B. Kass and D.J. Paolillo Jr., Z. Pflanzenphysiol., (1974) (in press). 9 J. Boisard and R. Malcoste, Ph37siol. V~g., 8 (1970) 565. 10 J.H. Miller and P.M. Miller, Plant and Cell Physiol., 4 (1963) 65. 11 D.J. Paolillo Jr., Cytologia, 34 (1969) 133. 12 F.A.M. Wellburn and A.R. Wellburn, New Phytol., 72 (1973) 55.
85
ON THE LIGHT REQUIREMENT FOR REPLICATION OF PLASTIDS IN POLYTRICHUM
LEE B. KASS and DOMINICK J. PAOLILLO JR.
Section o f Genetics, Development and Physiology, Cornell University, Ithaca, N. Y. 14850
(U.S.A.) (Received November 26th, 1973) (Revision received January 24th, 1974)
SUMMARY
The chloroplasts of germinating spores of the moss Polytrichum replicate in darkness to a lesser extent than in light. A stable number of plastids is attained by 48 h in darkness, i.e. the p!astids become light-dependent with respect to replication. Light stimulates the plastids to replicate. When red light is used, the promotive effect of the light can be completely reversed by far red light. INTRODUCTION
Light has been shown to be important in chloroplast development with phytochrome mediation involved in some of the developmental events [3, 4}. Hahn and Miller [ 5] reported a light requirement for plastid replication m the germinating spores of the moss Polytrichum. Partial reversibility of the promotive effect ot red light by far red light indicated that phytochrome mediation may be involved. Our results with similar materials show that plastid replication in Polytrichum spores does occur in the dark, although only ~o a limited extent. Ironically, thi~ finding makes it possible to develop an experimental system in which the demonstration of red--far red reversibility of plastid replication is more cl,-~r cut than that given by Hahn and Miller [ 5]. MATERIALS ~ND METHODS
Field-ripened capsules of Polytrichum c o m m u n e Hedw. were collected from Texas Hollow, Schuyler County, N.Y. in July and August, 1972. Spores were harvested as previously described t6], after the capsules were sterilized for 5 min in 5% Clorox solution and dried under sterile conditions. Spores were dusted onto a medium consisting of Voth's solution No. 5 solidified with 1% Difco Bacto agar, with and without 2% sucrose. Cultures were sampled by scraping the spores or germlings from the agar with a metal spatula and 81
preparing a water m o u n t for microscopic observation. Counts of plastid number were made using fluorescence microscopy, with a Zeiss microscope and an Osram HBO 200/4 lamp, with excitation filters BG 3 and BG 12 (blue) and barrier filter 53 (yellow). Fluorescence microscopy allows accurate counts of chloroplast number in unswollen and ungerminated spores because the plastids fluoresce red, and are easily visible through the spore wail. Control cultures were grown at. 11 800 lux under continuous fluorescent illumination (Sylvania, 40 W, warm white) and in the dark (wrapped in aluminum foil, on the same growth rack). Experiments on red--far red reversibility were performed after a 48-h incubation of the spores in the dark. Cultures were then placed into the following light regimes (1) continuous darkness; (2) continuous white light at 11 800 lux; (3) red light for 15 min, every 6 h, followed by darkness, (4) red light for 15 rain followed immediately by far red light for 15 min, e w r y 6 h, followed by darkness. The experiment was replicated to verify the results. The growth temperature was 25 °, throughout. Light filters were obtained from Rosco Laboratories, Harrison, N.Y. The filter for red light consisted of 1 medium red (No. 823) Roscolene filter plus 1 layer of red cellophane. The red light source was 2 Sylvania 40 W cool white fluorescent tubes, one from each side of the chamber at an angle of 45 ° from the horizontal. The filter for far red light consisted of 1 medium red Roscolene filter sandwiched between 2 dark urban blue (No. 866) Roscolene filters. The far red light source consisted of a row of 100 W incadescent bulbs placed 25 cm above the cultures, with a 10-cm layer of water as a cooling iilter, in a clear plexiglas container. The intensities of red and far red light were 0.00211 gcal/cm 2/rain and 0.0524 gcal/cm 2/min respectively at the level of the cultures, as measured with a Kipp and Zonen (Delft, Holland) thermopile solarimeter. The far red light used in this study was tested for its ability to suppress germination of Black Beauty squash (zucchini) obtained from Agway, Inc., Syracuse, N.Y. This variety of squash fails to germinate in far red light [9]. Germination in the light and dark was 85% for our seeds, but only 11% of the seeds germinated in far red light. This difference is highly significant, statistically, using the significance test based on a normal distribution [2]. RESULTS
General observations on the morphology o f germination Spores of Polytrichum commune cultured on agar show germ tube emergence by 48 h. The distal end of the growing filament becomes a rhizoid in at least some germlings by approx. 96 h in light with sucrose, and by 120 h in light without sucrose. Rhizoid differentiation is also evident at 120 h in darkness, in the presence of sucrose. No germ tubes emerge in the dark on unsupplemented mineral medium. The spores contain 2--4 chloroplasts at the time they are harvested from 82
their capsules. The plastids 8f the spore and the young germ tube are subsphezical and dark green. As the rhizoid begins to differentiate one observes in the participating cells numerous light green, small chloroplasts which are more filiform than spherical. The formation of these pale green plastids seems to involve the subdivision of the large, dark green plastids. At the opposite end of the germ tube, the plastids remain dark green, and here a protonema forms, at right angles to or opposite the rhizoid. The chloroplasts in a p r o t o n e m a are like those in the spore. It is necessary to keep in mind the distinction between replication of large, dark green plastids vs. the formation of small, pale green plastids as are found in the rhizoids. The conclusions drawn in this study are intended to apply to the replication of the large chloroplasts in the spore and y o u n g germ tube. Counts of plastids made at 120 h are sometimes influenced by the early stages of rhizoid formation, b u t these effects are taken into account in the interpretation of the data. Time course o f chloroplast replication By 8 h culture in white light, a significant change in plastid mtmber can be detected using the two-tailed Z test, of Alder and Roessler [ 1 ]. By 48 h the plastids are too numerous to count accurately. This is not the case in the dark. Although the results lea~'e no question t h a t replication has occurred, a plateau is reached at about 48 h (Fig. 1). This stable number is maintained until 120 h, when the germ tubes express themselves as rhizoids and the small plastids t h a t are produced in the cells at the tip of the filamcnt contribute to an increase in plastid number, ~.' they are counted.
I .......
|
/ /
°a4~:
j"
jJ''"
,
1J f~
__-L .
24 b
48
72
96
1
.
24n
.
.
l . . . . .
48
1 .......
72
1. . . . .
9G
1. . . .
120
2
Fig. 1. Changes in mean number of plastids per spore (or germling) in light with sucrose ( - o - ) and in the dark with sucrose (- ~ - ) and without sucrose ( - • -). Each point represents a c o u n t o f 60 spores or germlings, except for the initial number, which is based on a count of 500 spores. The variability in mean number of chloroplasts between 48 and 96 h in dark is not statistically significant. using the two-tailed Z test of Aflder and Roessler
[z]. Fig. 2. The effect o f continuous light (- '~ -), intermittent red light (- ~ -), red followed by far red light (-/~; -), and continuous darkness (- • -) on plastid number in spores incubated 48 h in darkness. Each point is based on a count of 500 spores or germlings. The differences in red light v s . other treatments at 96 and 120 h are highly significant according to the twotailed Z test of Alder and Roessler [1 ].
83
Sucrose did not affect plastid number, but was required for germ tube emergence,in the dark. It seems, then, that growth of a germ tube is not causally related to plastid replication, because the same amount of replication occurs when the germ tube does not emerge.
Red--far red reversibility of light-induced plastid multiplication The results of the experiment are presented in Fig. 2. Significant plastid replication occurred in both inte_rmittent red light and continuous white light. Red followed by far red light totally prevented any increase in plastid number. There was a slight rise in plastid replication at 120 h in continuous darkness possibly due to the beginnings of rhizoid differentiation. In spores of Onoclea, red light promotes growth of the rhizoid, and far red light negates the~effect of red light [10]. In our experiments far red light may be inhibiting rhizoid formation and therefore the lack of increase in plastid number as compared with the number in total darkness at 120 h. DISCUSSION
Hahn and Miller [5] reported that the plastids in spores of Polytrichum do not multiply in darkness, but we find that this is not exactly the case. Initial counts of plastid number are not reported Lq their paper. Rather remarkably, all the treatments they used seem to have yielded the same plastid number after 48 h of incubation. This included both continuous darkness and continuous light. Our own cultures in the light gave much higher numbers of plastids per spore or germling compared with those in the dark. We cannot account for this evident difference between their results and ours, unless it involves differences in handling of the spores. We have tried to copy their techniques using static liquid culture, and cannot use them to obtain reproducible results. But static liquid culture is not optimal for rapid, vigorous growth of Polytrichum sporelings. On the other hand our data are in basic agreement with those of Hahn and Miller [5] if one concentrates on the period between 48 and 96 h of incubation. Our results support the contention that the promotion of plastid replication by red light is far red reversible. It has been shown that a Polytrichum spore contains only one piastid directly after meiosis [ 11 ]. Because the mature spore contains 2--4 plastids, one or more replications can occur during ripening. If the mature spores are cultured in the light, the plastids replicate in essentially all spores [8]. If the mature spores are incubated in the dark the mean number of plastids increases but does not double. Therefore, some fraction of the initial population of plastids cannot multiply without light. By 48 h, all the plastids are in this category because replication is halted by that time in darkness. It is this circum. stance that allows for a successful demonstration of red--far red reversibility. because all the plastids nave become light-sensitive, and no further alteration in plastid number occurs in the dark, until rhizoids differentiate. This gives additional support to the concept derived from the results with inhibitors of 84
plastid replication in light and dark [ 8] that the chloroplasts of Polytrichum exist in two phases with respect to their ability to replicate: (1) a lightsensitive phase and (2) a light-insensitive phase. We do not k n o w the nature of the light response (presumably mediated by p h y t o c h r o m e ) t h a t occurs in the plastid. Because the plastids of the spore are chloroplasts [ 11 ] we are n o t dealing with the structural changes that require light in the greening of etioplasts [3]. But some parallel may exist with the p h y t o c h r o m e mediation of plastid development in the lag phase of greening in etioplasts [ 3], and with the p h y t o c h r o m e mediation of the stimulation of RNA and protein synthesis in developing plastids of higher plants [ 4]. Recently evidence derived from isolated plastids has been presented to argue t h a t "a p h y t o c h r o m e mechanism exists, if not inside the plastid, then on the plastid envelopes" [ 12]. Finally, we wish to point out that as cytodifferentiation occurs in the germling, the plastids that are in cells that participate in rhizoid formation divide themselves into many small plastids that have a light green color. Because this occurs in the darkness ~s well as in light, it is evident that the restraint on plastid division imposed by darkness depends on the state of diff:rentiation o f the cells that contain the plastids. As in Onoclea [ 10], p h y t o c h r o m e controls differ in the rhizoid as compared to other cells of the germling. ACKNOWLEDGMENT
This research was supported by a National Science Foundation grant, GB-25097. REFERENCES
1 H.L. Alder and E.B. Roessler, Intro6rction to Probability and Statistics. Freeman, San Francisco, 1960, p. 117. 2 N.T.J. Bailey, Statistical Methods in Biology, Wiley, New York, 1959, p. 38. 3 L. Bogorad, in T.W. Goodwm(Ed.), Biochemistry of Chloroplasts, Vol. II, Academic Press, New York, i967., p. 615. 4 N. Scott, R. Munns, D. Graham and R.M. Smillie, in N.K. Boar~man, A.W. Linnano and R.M. Smillie (Eds.), Autonomy and Biogenesis of Mitoehondria and Chloroplasts, North Holland, Amsterdam, 1971, p. 383. 5 L.W. Hahn and J.H. Miller, Physiol. Plant., 19 (1966) 134. 6 D.J. Paolillo Jr. and R.H. Jagels, Bryologist, 72 (1969) 444. 7 P.D. Voth, Bot. Gaz., 104 (1943) 591. 8 L.B. Kass and D.J. Paolillo Jr., Z. Pflanzenphysiol., (1974) (in press). 9 J. Boisard and R. Malcoste, Ph37siol. V~g., 8 (1970) 565. 10 J.H. Miller and P.M. Miller, Plant and Cell Physiol., 4 (1963) 65. 11 D.J. Paolillo Jr., Cytologia, 34 (1969) 133. 12 F.A.M. Wellburn and A.R. Wellburn, New Phytol., 72 (1973) 55.
85