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Synthesis of protein and RNA for initiation and growth of the protonema during germination of bracken fern spore
Experimental Cell Research 65 (1971) 401-407
SYNTHESIS GROWTH
OF PROTEIN
AND
OF THE PROTONEMA BRACKEN
RNA
FOR INITIATION
DURING FERN
GERMINATION
AND OF
SPORE1
V. RAGHAVAN Academic Faculty of Botany, The Ohio State University, Columbus, Ohio 43210, USA
SUMMARY Cycloheximide inhibited initiation and elongation of the protonemal cell during germination of the spores of bracken fern. Incorporation of W-leucine into protein was also profoundly affected by the drug. Concentration of actinomycin D sufficient to inhibit incorporation of 3Huridine into heavy RNA fractions of spores did not prevent initiation of the protonema, but inhibited its subsequent elongation. Protein synthesis during initiation and growth of protonema was not appreciably sensitive to actinomycin D. As in the case of rhizoid initiation, protein synthesis necessary for initiation of protonema during germination appears to involve preformed messenger RNA.
The first morphological landmark in the germination of bracken fern spore is the breakage of the exine and appearance of a papillate rhizoid. After the rhizoid initial has elongated to a few microns, the protonemal initial appears as a hemispherical projection at an angle roughly opposite the site of emergence of the rhizoid. Nuclear division preparatory to the formation of rhizoid and protonema probably takes place before rupture of the exine [l] and the spore is considered to be germinated when it has formed both rhizoid and protonema. In a previous study [6] synchronous germination of bracken fern spores and temporal separation of phases of initiation and elongation of the rhizoid and protonema during germination were achieved by giving 1 Papers from the Faculty of Botany, The Ohio State University, No. 784.
brief periods of red light to fully imbibed and actinospores. Using cycloheximide mycin D as selective inhibitors of protein and RNA synthesis, respectively, a correlation was established between macromolecule synthesis and initiation and elongation of the rhizoid. On the basis of the known effects of actinomycin D as an inhibitor of DNA-dependent RNA synthesis, it was suggested that protein synthesis necessary for rhizoid initiation occurred independent of concurrent genomic readout, probably on messenger RNA stored in the dormant spore. In the present study, experiments have been extended to the period of protonemal initiation and elongation, measuring the differential effects of cycloheximide and actinomycin D on protein and RNA synthesis to define the nature of the control mechanisms operating in the spores during the latter phase of germination. ExptI CeN Res 65
402
V. Raghavan
Fig. 1. (a) Spores of Pteridium aquilinum at 76 h after sowing, showing initiation of protonema; (b) at 84 h after sowing, showing elongate protonema. (pi, protonemal initial; p, elongate protonema; r, rhizoid).
MATERIAL
AND
METHODS
Spores of bracken fern (Pteridium aquilinum) kindly supplied by Dr B. R. Voeller (Rockefeller University, New York) were used for this study. The protocol for inducing protonemal initiation and growth consisted of irradiation with red light (400 ergs/cm2/ set) for 3 h at 12 h after sowing, and for 2 h at 72 h after sowing. Except when irradiated, the spores were incubated in the dark. Protonemal initials are first visible 2-3 h after the second exposure to red light Exptl Cell Res 65
and in the following 7-8 h they elongate to about 50-60 urn in length (fig. 1). For exuerimental uurposes. the period between 74-77 h after sowing is designated as the phase of initiation and that between 81-84 h after sowing, as the phase of elongation of the protonema. References in the text and in the figure captions to red light exposure of the spores apply to the 2 h red light given at 72 h after sowing. Chloroplast-studded spores with a hemispherical projection at an angle to the rhizoid were scored at 84 h after sowing for initiation of the protonema.
Protein and RNA synthesis for protonemal growth during spore germination
403
Fig. 2. Effect of cycloheximide (0.2 mg/l) on the initiation and elongation of protonema. The spores were exposed to the drug immediately after red light, and photographed 10 h later.
Since the protonemal initial is somewhat diffuse in appearance, one must be able to distinguish spores with incipient protonemal initials from the rest of the population in any treatment. In borderline cases, swelling of the spores, and presence of abundant chloroplasts in them as revealed by a fluorescent microscope has made it possible to make this distinction on cytological grounds. About 100 spores were scored in each case to determine the percentage of spores with protonema and average length of the protonema. Methods followed for aseptic culture of the spores, phenol extraction of RNA, and density gradient centrifugation are the same as described in detail in earlier papers [3, 4, 61 and so they will not be repeated here. In the homogenization of spores for phenol extraction of RNA, 1 ml of 1 % sodium pyrophosuhate was substituted for bentonite. For labeling of protein and RNA, W-leucine (spec. act. 311 mCi/ mM: Radiochemical Centre. Amersham) and SHuridine (spec. act. 16.3 and 22.8 Ci/mM;‘Radiochemica1 Centre, Amersham), respectively, were used; for further details, see the appropriate figure captions.
RESULTS Cycloheximide effect on initiation and growth of protonema If spores are incubated with different concentrations of cycloheximide immediately after exposure to red light, and examined 10 h later (84 h from sowing), concentrations
of the drug >0.2 mg/l were found to completely inhibit initiation of the protonemal cell (fig. 2; table 1). At progressively lower concentrations of cycloheximide, increasing number of spores escaped inhibition and formed protonema. Chloroplast formation was sparse in spores in which protonemal initiation was inhibited by the drug. Addition of cycloheximide (0.2 mg/l) up to 3 h Table 1. Effect of cycloheximide on the initiation and growth of the protonema Concentration of cycloheximide, !mdl)
Percentage of spores with protonemaa
Length of protonema, pm *SE.
0 0.03 0.05 0.07 0.09 0.10 0.20 0.50
83.6 55.4 40.0 18.6 8.0 9.8 1.9 0
35.6k2.1 32.9 k2.3 28.8 + 1.9 24.7 + 1.6 19.2k1.4 19.2k1.6 -
a Includes spores with protonema initial or elongate protonema. Measurements were made at 84 h after sowing. Exptl Cell Res 65
404 V. Raghavan Table 2. Effect of actinomycin D on the ini-
tiation and growth of the protonema
400
200
Concentration of actinomycin D, m/l
Percentage of spores with protonema’
Length of protonema, pm f S.E.
0 50 100 200 400
78.2 84.5 81.1 68.5 74.5
58.9k3.8 36.9k1.9 24.7F1.8 13.7k1.8 11.0*1.1
0 74
78
82
06
90
94
98
102 106 110.
Fig. 3. Abscissa: hours after sowing; ordinate: cpm in protein/54 x lo5 spores. Incorporation of W-leucine (0.1 nmole, 1 h) into protein of control ( l ) and cycloheximide-treated populations (0) of spores during initiation and growth of the protonema. Shaded portion indicates inhibition due to cycloheximide. At time indicated by the arrow, spores were washed in sterile distilled water and transferred to the basal medium. The stippled portion represents extent of recovery in the basal medium.
following exposure to red light prevented initiation of the protonema, but if the drug was added at later periods, an increasing number of spores escaped inhibition. This
a Includes spores with protonemal initial or elongate protonema. Measurements were made at 84 h after sowing.
suggests that some of the proteins required for initiation of the protonema are made during the first 3 h following irradiation. 14C-leucine incorporation into protein of the spores was sensitive to the presence of cycloheximide (0.2 mg/l) added after exposure to red light; incorporation was inhibited by 32% during the first 4 h of incubation and after 12 h inhibition was 86% of the control (fig. 3). To ascertain whether cyclo-
Fig. 4. Effect of actinomycin D (400 mg/l) on the initiation and growth of the protonema. The-spores mrere-exposed to the drug immediately after red light and photographed 10 h later. Exptl CeN Res 65
Protein and RNA synthesis for protonemal growth during spore germination heximide had totally incapacitated the synthetic ability of the spores, drug-treated spores were washed and transferred to a basal medium; normal protonemal growth and a step-up in the incorporation of the label into protein were resumed during the ensuing 12 h. These findings indicate a requirement for protein synthesis for initiation and growth of the protonema. Effects of actinomycin D Incubation of the spores with different concentrations of actinomycin D immediately following exposure to red light inhibited elongation of the protonema without having any effect on its initiation (table 2). Even at the highest concentration of the drug tested (400 mg/l), the spores were swollen with abundant chloroplasts or with incipient protonema, indicating that protonemal initiation was not affected by the drug (fig. 4). Initiation of protonema in the presence of actinomycin D introduced after exposure to red light suggests that transcription for this event probably occurred before the drug was applied. However, since irradiation of the spores with red light in the presence of actinomycin D did not prevent initiation of the protonema, transcription during irradiation appeared unlikely. Spores exposed to ac-
I 0
I 100
I
,
I
200
300
400
Fig. 5. Abscissa: actinomycin D (mg/l); ordinate: cum in RNA/6.8 x lo5 snores. Effect of different c¢rations of actinomycin D on the incorporation of 3H-UdR (2.5 pCi, 1 h, 16.3 Ci/mM) into RNA during 0, initiation (74-77 h after sowing) and 0, elongation (81-84 h after sowing) of the protonema. -
405
6o01
“L 0
100
200
300
400
Fig. 6. Abscism: actinomycin D (mg/l); ordinate: cpm in proteim6.6 x lo5 spores. Effect of different concentrations of actinomycin D on the incorporation of l*C-leucine (0.1 nmole, 1 h) into protein during 0, initiation (74-77 h after sowing) and 0, elongation (81-84 h after sowing) of the protonema.
tinomycin D formed normal protonema in about 48 h after transfer to the basal medium. Application of actinomycin D (200-400 mg/l) to the spores during the period of initiation of the protonema led to about 70% inhibition of 3H-uridine (3H-UdR) incorporation into RNA and 30 % inhibition of 14C-leucine incorporation into protein (figs 5, 6). Sucrose gradient centrifugation profile of RNA extracted from the spores during protonema initiation showed a good deal of radioactivity throughout the gradient, with considerable amounts of it in the 4s and lighter regions; in the presence of actinomycin D incorporation of label into heavy regions of the gradient was completely abolished and the remaining radioactivity was confined to low molecular weight RNA (fig. 7). The rapid increase in 3H-UdR incorporation into RNA which occurred during elongation of the protonema was very sensitive to actinomycin D; again, 14C-leucine incorporation into protein during this period was inhibited by actinomycin D to the same extent as before (figs 5, 6). Sedimentation pattern of RNA extracted from the spores during this period was qualitatively the same as that of RNA obtained during protonemal Exptl Cell Res 65
406
V. Raghavan
0.16
300
0.14
250
0.12
200
0.10
150
0.08
100
0.06
50
0.04
0 0
5
10
15
20
25
30
Figs 7, 8, Abscissa: fraction number, bottom of tube, left; ordinate: (lqft) l , optical density; (G&t) 0, cpm in
RNA. (a) Control; (b) actinomycin D. Fig. 7. Sedimentation pattern of RNA synthesized during protonema initiation and the effect on it of actinomycin D. Immediately after red light, about 1 g fresh weight of spores were aseptically transferred to 10 ml basal medium or basal medium containing 400 mg/l actinomycin D. At 76 h after sowing, cultures were pulsed with 5 /-cCi 3H-UdR (22.8 Ci/mM, 1 h) and samples collected for RNA extraction. The stippled portions in this and fig. 8 indicate the extent of radioactivity. 0.16
,
I
I
I
1
0.14
250
0.12
200
0.10
150
0.08
100
0.06
50
0.04
0
5
IO
0 15
20
25
30
0
5
10
15
20
25
30
Fig. 8. Sedimentation pattern of RNA synthesized during protonema elongation and the effect of actinomycin
D on it. About 1 g fresh weight of spores were aseptically transferred to 10 ml basal medium or basal medium containing 400 mg/l actinomycin D, at 7 h after red light (81 h from sowing). At 83 h after sowing, cultures were pulsed with 5 p Ci aH-UdR (22.8 Ci/mM) for 1 h.
initiation with a general increase in the amount of radioactivity in the different parts of the gradient (fig. 8). In contrast to the virtually complete inhibition of synthesis of heavy RNA during initiation of the protonema, radioactivity detectable in the heavy fractions of RNA extracted from spores treated with actinomycin D during elongation of the protonema was significantly above background. The significance of this observation will be discussed below. DISCUSSION The foregoing experiments demonstrate a requirement for protein synthesis in the iniExptl Cell Res 65
tiation and growth of the protonema during germination of the spores of Pteridium aquilinum. Since protein synthesis may be directed by DNA-dependent RNA synthesis, a connection was sought between synthesis of protein and RNA during this phase of spore germination. Although 3H-UdR was incorporated into all RNA fractions during emergence of the protonema, actinomycin D virtually eliminated the synthesis of RNA heavier than 4S-6 S without inhibiting initiation of the protonema. Since messenger properties to code for polypeptides of normal length are associated with RNA molecules heavier than 4S-6s [2], it appears that
Protein and RNA synthesis for protonemal growth during spore germination actinomycin D treatment during initiation of protonema inhibits the synthesis of RNA with potential template function. However, initiation of protonema is complicated by the simultaneous synthesis of heavy RNA required for rhizoid elongation, and by experimental design it was not possible to separate RNA involved in rhizoid elongation from that involved in initiation of the protonema. Protein synthesis during initiation of the protonema was only marginally inhibited by actinomycin D. Since synthesis of RNA with potential template function is inhibited by actinomycin D during the period when initiation of protonema proceeds normally, it is reasonable to assume that the bulk of the proteins necessary for initiation of the protonema is probably made on templates of stable mRNA already present in the dormant spore. Elongation of the protonema appears to be dependent upon the synthesis of DNAdependent RNA as demonstrated by the inhibition of this event by actinomycin D. However, judged from the effects of the drug on protein synthesis it seems that only a small portion of the protein synthesized during elongation of the protonema is directed by the simultaneous synthesis of actinomycin D-sensitive RNA. A similar control of protein and RNA synthesis during initiation and growth of the rhizoid has been described before [6]. In these experiments it was found that actinomycin D did not completely inhibit the synthesis of all of the heavy RNA formed during elongation of the protonema. As noted before, initiation and growth of the protonema are associated with the appearance of chloroplasts. Earlier studies on the game-
401
tophytes of this species have shown that a considerable portion of cellular RNA is localized in the chloroplasts [4]. Incorporation of 3H-UdR into RNA of chloroplasts was very heavy and it was only partially inhibited by actinomycin D [3, 51. Thus the fraction of heavy RNA insensitive to actinomycin D may represent an autonomous RNA replicating mechanism of the chloroplasts; investigations to resolve this question are currently under way. From the developmental point of view, germination of bracken fern spore presents a complex picture of regulatory control of macromolecule synthesis. It is interesting to note that proteins necessary for the initiation of the rhizoid and protonema are faithfully coded on preformed messenger RNA’s, although these events are separated by an interval of 36 h during which period the rhizoid elongates at the expense of proteins made on newly synthesized messengers. Control of protein synthesis during germination of the spores is thus by a selective utilization of preformed messengers which ensures formation of a sporeling with rhizoid and protonema, capable of functioning as an independent photosynthetic unit. This work was carried out at the School of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia. Appreciation is expressed to the University of Malaya for the facilities provided.
REFERENCES 1. Bell, P R, Sci progr 58 (1970) 27. 2. Monier, R, Navno, S, Hayes, D, Hayes, F & Gros, F, J mol biol 5 (1962) 311. 3. Raghavan, V, Planta 81 (1968) 35. 4. - Physiol plant 21 (1968) 1020. 5. - Am j bot 55 (1968) 767. 6. - Exptl cell res 63 (1970) 341. Received October 5, 1970
Exptl
Cell Res 65
SYNTHESIS GROWTH
OF PROTEIN
AND
OF THE PROTONEMA BRACKEN
RNA
FOR INITIATION
DURING FERN
GERMINATION
AND OF
SPORE1
V. RAGHAVAN Academic Faculty of Botany, The Ohio State University, Columbus, Ohio 43210, USA
SUMMARY Cycloheximide inhibited initiation and elongation of the protonemal cell during germination of the spores of bracken fern. Incorporation of W-leucine into protein was also profoundly affected by the drug. Concentration of actinomycin D sufficient to inhibit incorporation of 3Huridine into heavy RNA fractions of spores did not prevent initiation of the protonema, but inhibited its subsequent elongation. Protein synthesis during initiation and growth of protonema was not appreciably sensitive to actinomycin D. As in the case of rhizoid initiation, protein synthesis necessary for initiation of protonema during germination appears to involve preformed messenger RNA.
The first morphological landmark in the germination of bracken fern spore is the breakage of the exine and appearance of a papillate rhizoid. After the rhizoid initial has elongated to a few microns, the protonemal initial appears as a hemispherical projection at an angle roughly opposite the site of emergence of the rhizoid. Nuclear division preparatory to the formation of rhizoid and protonema probably takes place before rupture of the exine [l] and the spore is considered to be germinated when it has formed both rhizoid and protonema. In a previous study [6] synchronous germination of bracken fern spores and temporal separation of phases of initiation and elongation of the rhizoid and protonema during germination were achieved by giving 1 Papers from the Faculty of Botany, The Ohio State University, No. 784.
brief periods of red light to fully imbibed and actinospores. Using cycloheximide mycin D as selective inhibitors of protein and RNA synthesis, respectively, a correlation was established between macromolecule synthesis and initiation and elongation of the rhizoid. On the basis of the known effects of actinomycin D as an inhibitor of DNA-dependent RNA synthesis, it was suggested that protein synthesis necessary for rhizoid initiation occurred independent of concurrent genomic readout, probably on messenger RNA stored in the dormant spore. In the present study, experiments have been extended to the period of protonemal initiation and elongation, measuring the differential effects of cycloheximide and actinomycin D on protein and RNA synthesis to define the nature of the control mechanisms operating in the spores during the latter phase of germination. ExptI CeN Res 65
402
V. Raghavan
Fig. 1. (a) Spores of Pteridium aquilinum at 76 h after sowing, showing initiation of protonema; (b) at 84 h after sowing, showing elongate protonema. (pi, protonemal initial; p, elongate protonema; r, rhizoid).
MATERIAL
AND
METHODS
Spores of bracken fern (Pteridium aquilinum) kindly supplied by Dr B. R. Voeller (Rockefeller University, New York) were used for this study. The protocol for inducing protonemal initiation and growth consisted of irradiation with red light (400 ergs/cm2/ set) for 3 h at 12 h after sowing, and for 2 h at 72 h after sowing. Except when irradiated, the spores were incubated in the dark. Protonemal initials are first visible 2-3 h after the second exposure to red light Exptl Cell Res 65
and in the following 7-8 h they elongate to about 50-60 urn in length (fig. 1). For exuerimental uurposes. the period between 74-77 h after sowing is designated as the phase of initiation and that between 81-84 h after sowing, as the phase of elongation of the protonema. References in the text and in the figure captions to red light exposure of the spores apply to the 2 h red light given at 72 h after sowing. Chloroplast-studded spores with a hemispherical projection at an angle to the rhizoid were scored at 84 h after sowing for initiation of the protonema.
Protein and RNA synthesis for protonemal growth during spore germination
403
Fig. 2. Effect of cycloheximide (0.2 mg/l) on the initiation and elongation of protonema. The spores were exposed to the drug immediately after red light, and photographed 10 h later.
Since the protonemal initial is somewhat diffuse in appearance, one must be able to distinguish spores with incipient protonemal initials from the rest of the population in any treatment. In borderline cases, swelling of the spores, and presence of abundant chloroplasts in them as revealed by a fluorescent microscope has made it possible to make this distinction on cytological grounds. About 100 spores were scored in each case to determine the percentage of spores with protonema and average length of the protonema. Methods followed for aseptic culture of the spores, phenol extraction of RNA, and density gradient centrifugation are the same as described in detail in earlier papers [3, 4, 61 and so they will not be repeated here. In the homogenization of spores for phenol extraction of RNA, 1 ml of 1 % sodium pyrophosuhate was substituted for bentonite. For labeling of protein and RNA, W-leucine (spec. act. 311 mCi/ mM: Radiochemical Centre. Amersham) and SHuridine (spec. act. 16.3 and 22.8 Ci/mM;‘Radiochemica1 Centre, Amersham), respectively, were used; for further details, see the appropriate figure captions.
RESULTS Cycloheximide effect on initiation and growth of protonema If spores are incubated with different concentrations of cycloheximide immediately after exposure to red light, and examined 10 h later (84 h from sowing), concentrations
of the drug >0.2 mg/l were found to completely inhibit initiation of the protonemal cell (fig. 2; table 1). At progressively lower concentrations of cycloheximide, increasing number of spores escaped inhibition and formed protonema. Chloroplast formation was sparse in spores in which protonemal initiation was inhibited by the drug. Addition of cycloheximide (0.2 mg/l) up to 3 h Table 1. Effect of cycloheximide on the initiation and growth of the protonema Concentration of cycloheximide, !mdl)
Percentage of spores with protonemaa
Length of protonema, pm *SE.
0 0.03 0.05 0.07 0.09 0.10 0.20 0.50
83.6 55.4 40.0 18.6 8.0 9.8 1.9 0
35.6k2.1 32.9 k2.3 28.8 + 1.9 24.7 + 1.6 19.2k1.4 19.2k1.6 -
a Includes spores with protonema initial or elongate protonema. Measurements were made at 84 h after sowing. Exptl Cell Res 65
404 V. Raghavan Table 2. Effect of actinomycin D on the ini-
tiation and growth of the protonema
400
200
Concentration of actinomycin D, m/l
Percentage of spores with protonema’
Length of protonema, pm f S.E.
0 50 100 200 400
78.2 84.5 81.1 68.5 74.5
58.9k3.8 36.9k1.9 24.7F1.8 13.7k1.8 11.0*1.1
0 74
78
82
06
90
94
98
102 106 110.
Fig. 3. Abscissa: hours after sowing; ordinate: cpm in protein/54 x lo5 spores. Incorporation of W-leucine (0.1 nmole, 1 h) into protein of control ( l ) and cycloheximide-treated populations (0) of spores during initiation and growth of the protonema. Shaded portion indicates inhibition due to cycloheximide. At time indicated by the arrow, spores were washed in sterile distilled water and transferred to the basal medium. The stippled portion represents extent of recovery in the basal medium.
following exposure to red light prevented initiation of the protonema, but if the drug was added at later periods, an increasing number of spores escaped inhibition. This
a Includes spores with protonemal initial or elongate protonema. Measurements were made at 84 h after sowing.
suggests that some of the proteins required for initiation of the protonema are made during the first 3 h following irradiation. 14C-leucine incorporation into protein of the spores was sensitive to the presence of cycloheximide (0.2 mg/l) added after exposure to red light; incorporation was inhibited by 32% during the first 4 h of incubation and after 12 h inhibition was 86% of the control (fig. 3). To ascertain whether cyclo-
Fig. 4. Effect of actinomycin D (400 mg/l) on the initiation and growth of the protonema. The-spores mrere-exposed to the drug immediately after red light and photographed 10 h later. Exptl CeN Res 65
Protein and RNA synthesis for protonemal growth during spore germination heximide had totally incapacitated the synthetic ability of the spores, drug-treated spores were washed and transferred to a basal medium; normal protonemal growth and a step-up in the incorporation of the label into protein were resumed during the ensuing 12 h. These findings indicate a requirement for protein synthesis for initiation and growth of the protonema. Effects of actinomycin D Incubation of the spores with different concentrations of actinomycin D immediately following exposure to red light inhibited elongation of the protonema without having any effect on its initiation (table 2). Even at the highest concentration of the drug tested (400 mg/l), the spores were swollen with abundant chloroplasts or with incipient protonema, indicating that protonemal initiation was not affected by the drug (fig. 4). Initiation of protonema in the presence of actinomycin D introduced after exposure to red light suggests that transcription for this event probably occurred before the drug was applied. However, since irradiation of the spores with red light in the presence of actinomycin D did not prevent initiation of the protonema, transcription during irradiation appeared unlikely. Spores exposed to ac-
I 0
I 100
I
,
I
200
300
400
Fig. 5. Abscissa: actinomycin D (mg/l); ordinate: cum in RNA/6.8 x lo5 snores. Effect of different c¢rations of actinomycin D on the incorporation of 3H-UdR (2.5 pCi, 1 h, 16.3 Ci/mM) into RNA during 0, initiation (74-77 h after sowing) and 0, elongation (81-84 h after sowing) of the protonema. -
405
6o01
“L 0
100
200
300
400
Fig. 6. Abscism: actinomycin D (mg/l); ordinate: cpm in proteim6.6 x lo5 spores. Effect of different concentrations of actinomycin D on the incorporation of l*C-leucine (0.1 nmole, 1 h) into protein during 0, initiation (74-77 h after sowing) and 0, elongation (81-84 h after sowing) of the protonema.
tinomycin D formed normal protonema in about 48 h after transfer to the basal medium. Application of actinomycin D (200-400 mg/l) to the spores during the period of initiation of the protonema led to about 70% inhibition of 3H-uridine (3H-UdR) incorporation into RNA and 30 % inhibition of 14C-leucine incorporation into protein (figs 5, 6). Sucrose gradient centrifugation profile of RNA extracted from the spores during protonema initiation showed a good deal of radioactivity throughout the gradient, with considerable amounts of it in the 4s and lighter regions; in the presence of actinomycin D incorporation of label into heavy regions of the gradient was completely abolished and the remaining radioactivity was confined to low molecular weight RNA (fig. 7). The rapid increase in 3H-UdR incorporation into RNA which occurred during elongation of the protonema was very sensitive to actinomycin D; again, 14C-leucine incorporation into protein during this period was inhibited by actinomycin D to the same extent as before (figs 5, 6). Sedimentation pattern of RNA extracted from the spores during this period was qualitatively the same as that of RNA obtained during protonemal Exptl Cell Res 65
406
V. Raghavan
0.16
300
0.14
250
0.12
200
0.10
150
0.08
100
0.06
50
0.04
0 0
5
10
15
20
25
30
Figs 7, 8, Abscissa: fraction number, bottom of tube, left; ordinate: (lqft) l , optical density; (G&t) 0, cpm in
RNA. (a) Control; (b) actinomycin D. Fig. 7. Sedimentation pattern of RNA synthesized during protonema initiation and the effect on it of actinomycin D. Immediately after red light, about 1 g fresh weight of spores were aseptically transferred to 10 ml basal medium or basal medium containing 400 mg/l actinomycin D. At 76 h after sowing, cultures were pulsed with 5 /-cCi 3H-UdR (22.8 Ci/mM, 1 h) and samples collected for RNA extraction. The stippled portions in this and fig. 8 indicate the extent of radioactivity. 0.16
,
I
I
I
1
0.14
250
0.12
200
0.10
150
0.08
100
0.06
50
0.04
0
5
IO
0 15
20
25
30
0
5
10
15
20
25
30
Fig. 8. Sedimentation pattern of RNA synthesized during protonema elongation and the effect of actinomycin
D on it. About 1 g fresh weight of spores were aseptically transferred to 10 ml basal medium or basal medium containing 400 mg/l actinomycin D, at 7 h after red light (81 h from sowing). At 83 h after sowing, cultures were pulsed with 5 p Ci aH-UdR (22.8 Ci/mM) for 1 h.
initiation with a general increase in the amount of radioactivity in the different parts of the gradient (fig. 8). In contrast to the virtually complete inhibition of synthesis of heavy RNA during initiation of the protonema, radioactivity detectable in the heavy fractions of RNA extracted from spores treated with actinomycin D during elongation of the protonema was significantly above background. The significance of this observation will be discussed below. DISCUSSION The foregoing experiments demonstrate a requirement for protein synthesis in the iniExptl Cell Res 65
tiation and growth of the protonema during germination of the spores of Pteridium aquilinum. Since protein synthesis may be directed by DNA-dependent RNA synthesis, a connection was sought between synthesis of protein and RNA during this phase of spore germination. Although 3H-UdR was incorporated into all RNA fractions during emergence of the protonema, actinomycin D virtually eliminated the synthesis of RNA heavier than 4S-6 S without inhibiting initiation of the protonema. Since messenger properties to code for polypeptides of normal length are associated with RNA molecules heavier than 4S-6s [2], it appears that
Protein and RNA synthesis for protonemal growth during spore germination actinomycin D treatment during initiation of protonema inhibits the synthesis of RNA with potential template function. However, initiation of protonema is complicated by the simultaneous synthesis of heavy RNA required for rhizoid elongation, and by experimental design it was not possible to separate RNA involved in rhizoid elongation from that involved in initiation of the protonema. Protein synthesis during initiation of the protonema was only marginally inhibited by actinomycin D. Since synthesis of RNA with potential template function is inhibited by actinomycin D during the period when initiation of protonema proceeds normally, it is reasonable to assume that the bulk of the proteins necessary for initiation of the protonema is probably made on templates of stable mRNA already present in the dormant spore. Elongation of the protonema appears to be dependent upon the synthesis of DNAdependent RNA as demonstrated by the inhibition of this event by actinomycin D. However, judged from the effects of the drug on protein synthesis it seems that only a small portion of the protein synthesized during elongation of the protonema is directed by the simultaneous synthesis of actinomycin D-sensitive RNA. A similar control of protein and RNA synthesis during initiation and growth of the rhizoid has been described before [6]. In these experiments it was found that actinomycin D did not completely inhibit the synthesis of all of the heavy RNA formed during elongation of the protonema. As noted before, initiation and growth of the protonema are associated with the appearance of chloroplasts. Earlier studies on the game-
401
tophytes of this species have shown that a considerable portion of cellular RNA is localized in the chloroplasts [4]. Incorporation of 3H-UdR into RNA of chloroplasts was very heavy and it was only partially inhibited by actinomycin D [3, 51. Thus the fraction of heavy RNA insensitive to actinomycin D may represent an autonomous RNA replicating mechanism of the chloroplasts; investigations to resolve this question are currently under way. From the developmental point of view, germination of bracken fern spore presents a complex picture of regulatory control of macromolecule synthesis. It is interesting to note that proteins necessary for the initiation of the rhizoid and protonema are faithfully coded on preformed messenger RNA’s, although these events are separated by an interval of 36 h during which period the rhizoid elongates at the expense of proteins made on newly synthesized messengers. Control of protein synthesis during germination of the spores is thus by a selective utilization of preformed messengers which ensures formation of a sporeling with rhizoid and protonema, capable of functioning as an independent photosynthetic unit. This work was carried out at the School of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia. Appreciation is expressed to the University of Malaya for the facilities provided.
REFERENCES 1. Bell, P R, Sci progr 58 (1970) 27. 2. Monier, R, Navno, S, Hayes, D, Hayes, F & Gros, F, J mol biol 5 (1962) 311. 3. Raghavan, V, Planta 81 (1968) 35. 4. - Physiol plant 21 (1968) 1020. 5. - Am j bot 55 (1968) 767. 6. - Exptl cell res 63 (1970) 341. Received October 5, 1970
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