Germination of bracken fern spores

Experimental Cell Research63 (1970) 341-352

GERMINATION

OF BRACKEN FERN SPORES

Regulation of Protein and RNA Synthesis During Initiation and Growth of the Rhizoid V. RAGHAVAN’ School of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia

SUMMARY Germination of the spores of bracken fern (Pteridium aquilinum) was accompanied by hydrolysis of storage proteins and increased accumulation of RNA. Incorporation of labeled precursors of protein and RNA into the spores did not begin until about 36 h after sowing. Addition of cycloheximide to the incubation medium reversibly inhibited initiation and growth of the rhizoid and incorporation of W-leucine into proteins of the spores. Actinomycin D inhibited elongation of the rhizoid, but not its initiation. RNA synthesis during elongation of the rhizoid was more susceptible to inhibition by actinomycin D than during its initiation. Protein synthesis during initiation and elongation of the rhizoid was not appreciably affected by the drug. Sedimentation profile of RNA on sucrose gradient showed that the only significant incorporation of *H-uridine during rhizoid initiation was into low molecular weight RNA. There was appreciable incorporation of the label into heavy molecules of RNA during elongation of the rhizoid and actinomycin D completely suppressed this incorporation, The results are discussed in the light of possible mechanism involving the existence of preformed messengersin the dormant spore which provide templates for the first proteins of germination.

In the life cycle of the fern, a dormant spore, upon contact with an appropriate medium, initiates a series of subtle and precisely-timed events which lead to the development of the gametophyte. The spore is able to germinate and develop in a simple mineral salt medium, independently of exogenous nutriment during the period when primary morphogenetic events such as protrusion of the rhizoid and protonema takes place. With a view toward correlating biochemical activity with morphogenetic events and to provide a framework for probing the control mechanisms that operate during germination, the regulation of RNA and protein synthesis during

initiation and growth of the rhizoid in the early stages of germination of the spores of bracken fern (Pteridium aquilinum) has been investigated. In this work, the use of cycloheximide, an inhibitor of protein synthesis has permitted a study of the requirement for protein synthesis, while actinomycin D, an inhibitor of DNA-dependent synthesis of RNA, has been employed to study the genomic control of developmental events during germination.

1 Present address: Department of Botany, Ohio State University, Columbus, Ohio 43210, USA.

Spores of Pteridium aquilinum kindly supplied by Dr Bruce R. Voeller (Rockefeller University, New

23 - 701801

MATERIAL AND METHODS Standard culture conditions

Exptl Cell Res 63

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V. Raghavan

York) were sterilized according to methods already described [16], except that after Clorox treatment, they were further treated with 0.1 % HgCI, for 10 set, and washed four times with water. The spores were then suspended in water and inoculated on the surface of a mineral salt medium [15]. In most of the experiments, 15 ml of the medium was dispended in 50 ml Erlenmeyer flasks and autoclaved for 15 min at 15 Ib/in2. In experiments using actinomycin D, volume of the medium in the flask was reduced to 5 ml. Each flask was inoculated with about 5-8 x lo5 spores (15 mg dry wt). For phenol extraction of RNA, about 3 x lo6 spores (60 mg dry wt) were sown on the surface of 30 ml medium contained in 125 ml flasks. Both cycloheximide and actinomycin D were cold-sterilized through Millipore filters and added to the medium.

Initiation and growth of rhizoid and protonema Although bracken fern spores germinate slowly in darkness [5], germination-is enhanced by irradiation with red light. The protocol for inducing synchronous initiation and growth of the rhizoid in the spores consisted of 12 h inbibition following inoculation, 3 h red light (first red light) and harvest at 72 h after sowing. For initiation and growth of protonemal cell, spores were given additional 2 h red light (second red light) at 72 h after sowing and harvested 10 h later. Except when irradiated, the flasks were covered with black cloth and kept in an incubator at 25 ? 1°C. Red light was administered from a 40 W fluorescent tube filtered through a sheet of red plexiglass (Rohm & Haas, no. 2444), giving ca 400 ergs/cm2/sec light. Temperature during irradiation was 26-27°C. Percentages of spores showing rhizoid initiation were based on counts of at least 100 viable spores. Spores were scored for initiation of the rhizoid when the exine was broken and the rhizoid was seen as a papillate projection.

Determination

of protein and RNA

For protein determination, the spores were homogenized in 10 ml 5 % trichloroacetic acid (TCA) in a mortar and allowed to precipitate in the cold for about 12 h. The precipitate was filtered, washed with additional 10 ml-T@ and carefully scraped off the filter paper. Protein content of the precipitate was determined by the Folin-phenol method [12], using bovine serum albumin as the standard. For determination of RNA, spores were homogenized in 0.05 M Tris-HCl buffer (PH 7.4) containing 0.01 m MgC& and 0.005 M mercaptoethanol. After about 12 h in the cold, RNA content of the homogenate was determined by a modification of the method of Smillie & Krotkov ([20]; see also [15]). RNA was hydrolyzed from lipid-free pellets with 0.3 N KOH at 37°C for 16 h. After extraction. the mixture was chilled and acidified with perchlorid acid (PCA) to give a final concentration of 0.3 N and centrifuged: RNA content of the supernatant was estimated by referring absorbancy differences Exptl Cell Res 63

between 260 and 290 nm to a standard curve prepared with similarly treated yeast RNA.

Incorporation precursors

of radioactive

Rate of protein synthesis was estimated by means of 14C-leucine incorporation (spec. act. 311. mCi/mM) into TCA-insoluble material. Incorporation was measured by adding 0.1 nmole (ca 55 000 cpm) laCleucine to the flask and incubating for 1 h during the terminal hour of a specified period. The spores were homogenized in TCA after three washings in water and four washings in 10 ml each 0.05 % “V-leucine. Aliquots of the homogenate were pipetted onto glass fibre filter discs (2.1 cm diameter; Reeve Angel 934 AH or Whatman GF/A) and washed five times with TCA-leucine mixture (0.05 % 12C-leucine in 5 % TCA). The discs were dried, placed in counting vials containing 5 ml scintillation fluid [lo0 g naphthalene, 5 g 2.5-diohenvloxazol (PPO), 1 1 dioxane] and cot&ted in a -liquid scintillation counter to 1 % standard error. For total uptake determination, 1 ml aliquots of the homogenate were mixed with 10 ml scintillation fluid and counted. RNA was labeled by adding 2.5 &i of aH-uridine (SH-UdR: spec. act. 16.3-22.8 Ci/mM) to the flasks and incubating for 1 h. The spores’were washed three times with water and four times with 10 ml each of non-radioactive uridine (100 mg/l). They were homogenized in Tris-HCl buffer and RNA extracted according to the method previously outlined. To determine the amount of SH-UdR incorporated into RNA, the acidified KOH extract was neutralized with KOH and the precipitated KCIO, removed by centrifugation. Aliquots (0.4 ml) of the supernatant were mixed with 10 ml scintillation fluid and counted.

Sedimentation analysis The specific conditions used to label RNA for sedimentation analysis will be found in the appropriate figure legends. RNA was extracted from the spores by the cold phenol method [ll]. About 0.5-1.0 g fresh weight of the tissue was homogenized in a Ten Broeck homogenizer with 5 ml 0.01 M Tris-HCl buffer (pH 7.6) containing 0.001 M MgCI,, 0.01 M KCI, 10 ml redistilled phenol, 0.5 ml 0.5 % bentonite and 0.2 ml 25 % sodium laurvl sulfate. The homogenate was allowed to stand fo; 1 h at room temperature with occasional stirring and centrifuged in the cold at 3 500 g for 20 min. The supernaiant water layer was treated with 0.25 mg DNase for 30 min at 4°C mixed with additional 5 ml phenol and recentrifuged at 20000 g for 10 min. RNA was precipitated from the aqueous layer by the addition of potassium acetate to a final concentration of 2% and 3 vol of 95 % ethanol. The mixture was allowed to stand overnight at 4°C and centrifuged at 20000 g for 10 min. The pellet was dissolved in 2 ml TrisHCl buffer and recentrifuged at 20 000 g for 10 min to remove the debris. RNA was precipitated as before with potassium acetate-ethanol in the cold for l-2 h. The precipitation procedure was repeated

Germination of bracken fern spores

343

Fig. 1. Stages in the germination of the spores of bracken fern. Time in hours after sowing is indicated at the top left-hand comer of each figure. Spores at 0 time were photographed immediately after sterilization. Note polarization of the contents and breakage of the exine at 36 h; formation of rhizoid initial at 48 h; elongating rhizoids at 60 h and 12 h and protonema at 84 h. Exptl Cd Res 63

344

V. Raghavan

0

12

24

36

48

60

72

a4

Fig. 2. Abscissa: hours after sowing; ordinate: (left) ,ng protein/57 x lo5 spores; (right) pg RNA/6.8 x 10” spores. Changes in protein (0) and RNA (0) content of the spores during germination. Stippled portions indicate periods of red light treatment. twice and the final precipitate obtained was dissolved in 0.5 ml of the buffer and layered atop 4.5 ml of a linear (5-20 %) sucrose gradient. The tubes were centrifuged in the SW 5OL rotor of the Spinco model L ultracentrifuge for 12 h at 24000 rpm. Fractions were collected dropwise from the tube directly into scintillation vials. They were diluted with 0.5 ml distilled water and used for optical density measurements at 260 nm and for scintillation counting. For the latter purpose, samples were dried and mixed with 5 ml scintillation fluid and counted until a minimum total of 3 000 counts were recorded for each vial.

Chemicals Yeast RNA and RNase-free DNase were purchased from Worthington Biochemical Corporation, Freehold, N.J.; RNasefree sucrose for density gradient and bovine serum albumin from Mann Research Laboratories, New York; scintillation chemicals from E. Merck AG, Darmstadt, and radioisotopes as specified above from The Radiochemical Centre, Amersham. Actinomycin D was generously supplied by Dr Walter B. Gall of Merck, Sharpe & Dohme Inc., Rahway, N.J. and cycloheximide by Dr Harry L. Vincent of Upjohn International Inc., Kalamazoo, Mich. All other chemicals were of reagent grade.

Statistical analysis All experiments described in the text have been repeated once or twice with essentially the same results. Statistical methods follow Snedecor [22].

RESULTS Morphogenetic events during germination

Within a few hours after the spores are sown in the medium, they begin to imbibe water and swell. As imbibition continues after the first Exptl Cell Res 63

red light, a clear localized cytoplasmic area appears on one side of the spore due to a polarized distribution of the spore contents. The first indication of morphological differentiation appears at about 36-48 h after sowing when the exine breaks at the site of the presumptive rhizoid. The rhizoid appears as a rudimentary protrusion at about 48-60 h after sowing and during the ensuing 12-24 h it attains a length of more than 50 pm. When the spores are irradiated with additional red light at 72 h, rapid formation of the chloroplasts and protrusion of the protonemal cell are detectable in the next 10 h (fig. 1). Changes in protein and RNA content of the spores during germination

As seen from fig. 2, the dry spore has a relatively high protein content. Starting from about 24 h after sowing there is a decreasein the protein content and it dominates the pattern of protein changesin the spore for the next 48 h. However, after the second red light and the subsequent initiation of the protonemal initial, a small increase in protein content occurs. RNA content of the spore shows a slow increase 36 h from sowing until initiation of the protonema. These results are sufficiently similar to the histochemical observations on the spores of Matteuccia [6] to assumethat hydrolysis of the storage proteins and increased RNA accumulation are distinctive features of germination of fern spores. Incorporation of radioactive precursors into protein and RNA

Fig. 3 illustrates the pattern of 14C-leucine and SH-uridine incorporation into protein and RNA, respectively, as a function of the stage in germination. There was no detectable incorporation of the amino acid into protein during the first 36 h after sowing, but the rate of incorporation rose slowly thereafter. Since there was no net accumula-

Germination of bracken fern spores 345

tion of protein during germination, the pattern of increasing incorporation of the label implies that breakdown of the preexisting proteins and their resynthesis were nevertheless occurring. The pattern of incorporation of aH-UdR into RNA also followed a trend somewhat similar to that described for l*Cleucine, with little, if any, incorporation during O-24 h from sowing. Since the amount of amino acid available in the pool may determine the rate of protein synthesis, the rates of entry of exogenous leucine into the pool during germination were measured. It was also pertinent to determine whether there were stage-specific changes in the permeability of the spores to leucine. As seen from fig. 4, the amount of feucine in the pool remained relatively constant during the entire period studied, but the percentage of absorbed leucine incorporated into protein increased with the progress of germination. From these data it appears that variability in the size of the pool and the low permeability of the spores to the precursor are not important limitations in the incorporation of the isotope into protein. In the following experiments, increased 3HUdR uptake into the spores with increasing time from sowing and generalized depression 400

Fig. 4. Abscissa: hours after sowing; ordinate: (left) cpm in soluble pool/&4 x lo5 spores; (right) percentage of absorbed W-feucine incorporated into nroGin. Rate of uptake of T.Xeucine (0.1 nmole, 1 h) into soluble pools and of incorporation into protein as a function of the time after sowing the spores. Pool incorporation ( l ) is the total uptake of ITleucine minus inchoation into protein. Protein incorporation (0) represents z*C-leucine incorporated into protein expressed as a percentage of the absorbed label. Stippled portions indicate periods of red light treatment.

of l*C-leucine and SH-UdR uptake upon treatment of the spores with inhibitors were noted, but radioactivity in the pools remained relatively constant in spores sampled at different times during germination, and in the control and inhibitor-treated series, indicating no differences in the availability of intracellular pools of amino acids or nucleotides under the different conditions of treatment. In order to conserve space, these data are not presented in the text, but the general conclusion derived from fig. 4 is applicable in all cases. Inhibition of initiation and growth of rhizoid by cycloheximide

300

200

100

I 0

84

Fig. 3. Abscissa: hours after sowing; ordinate: cpm/ 6.4 x lo6 (protein) or 5.1 x 106 (RNA) spores. Rate of incorporation of 14C-leucine (0.1 nm mole, 1 h) and sH-UdR (2.5 ,&i, 1 h, 22.7 Ci/mM) into protein ( 0) and RNA ( l ), respectively, during germination of the spores. Stippled portions indicate periods of red light treatment.

The possible relation between macromolecule synthesis and rhizoid initiation and growth during germination of the spores was studied using inhibitors of protein and RNA synthesis. When spores were incubated in different concentrations (O.~l~.l mg/l) of cycloheximide added immediately after the first red light and examined 57 h later (total 72 h after sowing), it was found that the drug inhibited initiation of the rhizoid or its subExptl Cell Res 63

346

V. Raghauan

Table 1. Effect of addjtion of cyc~o~e~i~ide (0.07 mg/l) at different times after the first red light on the initiation and growth of the rhizoid Time after sowine (hours) 16 I7 19 23 26 36 41 44 48 60 Control

Percentage of spores with rhizoid or with ruptured exine 0 x 10.4 15.3 20.5 41.8 66.0 86.4 84.5 86.4

Length of the rhizoid, pm + S.E. 11.01tl.l ll.Ort1.3 17.8 + 1.8 24.71-2.1 34222.6 41.4k2.6 45.2k3.3 65.8 & 3.3

a Red light (3 h) was administered 12 h after sowing. Measurements were made 72 h after sowing.

Fig. 5. Effect of cycloheximide (0.07 mg/l) and ac-

tinomycin D (100 mg/l> on the initiation of the rhizoid. Both inhibitors were added immediately after the first red fight, and spores were photographed 33 h later (48 h after sowing). (a> ~clohe~mide-treated spores; note that the exine is still intact and no rhizoid initiation has occurred. (b) Actinomycin Dtreated spores; note formation of rhizoid initials.

sequent growth or both. Initiation and growth of the rhizoid were only slightly affected by the lower concentrations of the drug tested. When incubated in 0.~7-0.05 mg/l cycloheximide, less than 50 % of the sporesformed barely noticeable rhizoids, while at concentrations >0.07 mg/l, the exine had failed to rupture resulting in complete inhibition of initiation and growth of the rhizoid (fig. 5). Cyclohe~mide-induced arrest of initiation and growth of the rhizoid was fully reversible upon transfer of the spores to the basal medium. In another experiment an attempt was made to determine the time after sowing Exptl Cell Res 63

when proteins necessaryfor initiation of the rhizoid were made. For this purpose, the cultures were supplied with 0.07 mgfl cycloheximide at different times after the first red light. Results presented in table 1 show that cycloheximide given any time up to 23 h after sowing inhibited rhizoid initiation in Table 2. Effect of additjo~ of cyclohe~i~ide (0.07 mgjl) at different times to 48 h old spores on the subsequent growth of the rhizoid Time after addition of cvcloheximideQ

Length of rhizoid, @m+S.E. at time of addition of cycloheximide

Length of rhizoid, ,umtS.E. at 72 h after sowing

x 6.8 +0.9 10.0+ 1.0 18.4k1.6 25.1* 2.0 32.8 rf: 1.9 46.4k2.6 -

0 0 7.8rt:l.O 14.6k1.2 21.8kl.O 32.Ok1.9 41.Ok2.0 49.6k2.1 68.2k2.6

0

1 : 8 12 15 18 Control

a Red light (3 h) was administered 12 h after sowing, and spores were allowed to remain in the dark for 33 h before cycloheximide was added.

Germination of bracken fern spores 347

36 40 h4 48 52 56 60 64 68 72

6 Fig. 6. Abscissa: hours after sowing; ordinate:

cpm in protein/55 x lo5 spores. Incorporation of W-leucine (0.1 nmole, 1 h) into protein of control (0) and cycloheximide-treated populations (0) of spores during initiation and growth of the rhizoid. 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 the extent of recovery from inhibition. Fig. 7. Abscism: actinomycin D (mg/l); ordinate: cpm in RNA/6.7 x 106spores. Effect of different concentrations of actinomycin D on the incorporation of 8H-UdR (2.5 yCi, 1 h, 16.3 Ci/mM) into RNA during rhizoid initiation (3348 h after sowing, 0) and rhizoid elongation (48-72 h after sowing, l ), respectively. Fig. 8. Abscissa: actinomycin D (mg/l); ordinate: cpm in protein/7.1-7.5 x lo5 spores. Effect of different concentrations of actinomycin D on the incorporation of W-leucine (0.1 nmole, 1 h) into protein during rhizoid initiation (33-48 h after sowing, 0) and rhizoid elongation (48-72 h after sowing, l ), respectively.

virtually all of the spores when examined at 72 h after sowing. If the drug was applied between 26 and 41 h after sowing, about 15-40 % of the spores appeared to have ruptured the exine and had visible rhizoid initials, while addition of the drug at 44 h after sowing had little effect on the percentage of spores with visible rhizoids. By this means it was possible to delineate a sensitive period between 23 and 41 h after sowing (as well as before 23 h) when proteins essential for rhizoid initiation were synthesized. The progressive decrease in length of the rhizoid with increasing periods of exposure of the spores to cycloheximide as seen in table 1 shows a requirement for continued protein synthesis for its sustained elongation. This was also confirmed in an experiment where 48 h old spores which had already initiated rhizoids were given cycloheximide at different times and examined at 72 h (table 2). Exposure of the spores to cycloheximide starting at 48 h prevented elonga-

tion of the rhizoid completely, while introduction of the drug at later periods permitted elongation of the rhizoid to a few microns more than its original length before inhibition setin. Sinceaddition of cycloheximide any time after 48 h did not lead to elongation of the rhizoid as fully as in the control, the data indicate that there is no sensitive period when proteins necessary for rhizoid elongation are synthesized. Fig. 6 shows that cycloheximide (0.07 mg/l) added at 36 h after sowing reversibly inhibited 14C-leucine incorporation into TCAinsoluble material of the spores sampled at different times during initiation and growth of the rhizoid. Significant decreasein incorporation of the isotope was observed 4 h after addition of the inhibitor and inhibition was about 75 % of the control after 24 h. If the spores were washed in sterile distilled water at this stage and transferred to the basal medium, 14C-leucine incorporation was resumed in about 4 h. Exptl

Cell Res 63

348 V. Raghavan Table 3. Effect of pretreatment of the spores suppressedbut the exine had ruptured at the with actinomycin D (ZOOmgll)on thesubsequent site of the presumptive rhizoid indicating that events preparatory to initiation of the growth of the rhizoids rhizoid were unaffected by the drug (fig. 5). Length of rhizoid, pm + This was also the case when actinomycin D S.E. after pretreatment in was administered immediately after sowing Time of pretreatment Actinomycin in actinomycin Da Basal and cultures examined at 72 h, thus eliminatmedium D (hours) ing the possibility that rhizoid initiation did not occur prior to light exposure. The addi4 43.8 & 3.2 26.Ok2.5 tion of actinomycin D at different times to 10 46.6 f 3.2 26.Ok3.2 48 h old spores affected subsequent elonga22 47.9 5 3.0 13.721.1 32 53.4 4 2.3 8.2kO.5 tion of the rhizoid much the sameway as did cycloheximide, suggesting that there was no a Actinomycin D was added immediateiy after the restricted period of sensitivity when actinofirst red light (15 h after sowing) and at specified mycin D-sensitive RNA for rhizoid elongatimes, spores were washed several times in distilled water before being transferred to the basal medium. tion was synthesized. The eventual recovery Controls consisted of spores pretreated in the basal medium for the same lengths of time and transferred of actinomycin D-treated sporesfrom inhibito freshbasalmedium. tion upon their transfer to the basal medium argues against the possibility that the biological effects of the drug on the spores are Effects of actjnomycin D on the manifestations of its toxic side effects. initiation and growth of the rhizoid Since actinomycin D is a relatively bulky Results described above indicate that im- molecule and the spores have a thick exine, pairment of the capacity of germinating spo- it is pertinent to determine whether failure res to form rhizoids is due to a block in the of the drug to inhibit rhizoid initiation is due synthesis of proteins essential for this event. to its failure to penetrate the spores. This According to the present ideas in molecular was investigated by determining the ability biology, protein synthesis may be accom- of spores treated with actinomycin D (100 panied by production of specific RNA under mg/l) for different periods after the first red the influence of the nucleus. Results of expe- light to resume rhizoid elongation upon riments on the effects of actinomycin D on transfer to the basal medium. Measurements the spores indicate that DNA-dependent syn- of lengths of rhizoids were made at 40 h thesis of RNA is not probably required for after sowing. Results (table 3) showed that initiation of the rhizoid. When spores were there was significant decrease in length of incubated in different concentrations of ac- the rhizoid in spores transferred to the basal tinomycin D (20-100 mg/l) immediately after medium even after 4 h in actinomycin D, the first red light and examined 57 h later, while nearly 80% inhibition of rhizoid elonthe drug appeared to inhibit primarily the gation occurred after 22 h (37 h from sowing) elongation rather than the i~tiation of the in the drug. It thus appears that actinomycin rhizoid. Even in the lowest concentration of D begins to penetrate the spores long before the drug tested, there was significant inhibi- any measurable synthesis of RNA begins tion of elongation of the rhizoid. When spores (seefig. 3) and that lack of penetration of the were treated with 100 mgfl actinomycin D, drug is not a factor in the observed lack of elongation of the rhizoid was completely inhibition of initiation of the rhizoid. Exptt Cell Res 63

Germination of bracken fern spores 349

0

5

10

15

20

25

30 a

b

Fig. 9. Abscissa: fraction number, bottom of the tube at left; ordinate: (left) optical density (0); (right) cpm in RNA ( 0). (a) control; (b) actinomycin D.

Sedimentation patterns of RNA synthesized during rhizoid initiation in the presence or absence of actinomycin D (100 mg/l). Immediately after the first red fight, spores from 15 flasks each were filtered aseptically and transferred to 10 ml basal medium or basal medium containing actinomycin D. At 47 h after sowing, cultures were pulsed with 10 /.Ki aH-UdR (22.8 Ci/mM, 1 h) and samples collected for RNA extraction. The stippled areas in this and in the next figure indicate the extent of radioactivity.

Inhibition

of RNA synthesis

Inhibition

of protein synthesis

The effects of actinomycin D on the incor- The protocol for experiments designed to poration of 3H-UdR into RNA of the study the effect of actinomycin D on protein spores sampled during rhizoid initiation and synthesis in germinating spores was the same elongation are shown in fig. 7. In one experi- as that described in the preceding section, ment different concentrations of actinomycin except that the isotope used was 14C-leucine. D were added to the medium immediately As seen from fig. 8, during both rhizoid after the first red light. Thirty-three hours initiation and elongation, the highest conlater (48 h after sowing) 3H-UdR incorpora- centration of actinomycin D tested inhibited tion into RNA was reduced by only 25 % of protein synthesis to only about 25% of the the control in the highest concentration of the control. drug tested. In another experiment, actinomycin D was added to the incubation me- Characterization of RNA synthesized dium 48 h after sowing, when the rhizoids On the basis of the known effects of actinowere mostly elongating and the rate of 3H- mycin D as an inhibitor of messengerRNA UdR incorporation into RNA determined synthesis, it is tempting to conclude from 24 h later. The results (fig. 7) showed that theseresults that when initiation of the rhizoid the inhibitory effect of actinomycin D on the proceedsnormally in the presenceof the drug, incorporation of 3H-UdR into RNA in- continued protein synthesis necessaryfor this creased with increasing concentration of the event is programmed by preexisting mesdrug, and at a concentration of 100 mg/l, sengers.On the other hand, elongation of the rhizoid which is inhibited by actinomycin D about 80% of RNA synthesis was inhibited. It should be noted that 48-72 h old spores may require sustained synthesis of new meshave synthesized 2-3 times as much RNA as senger RNA. Since relatively high concentrao-48 h old spores, although the same con- tions of actinomycin D did not inhibit all of centration of actinomycin D was more effec- RNA synthesizedin the spores during rhizoid tive in the former than in the latter; the pe- initiation, it is necessary to determine the riod of contact of the spores with the drug types of RNA synthesized in the presence of the inhibitor to permit firm conclusions on was longer in the latter than in the former. Exptl Cell Res 63

350

V. Rughavan

0.14 0.12 0.10 -

f

i

0.03 0.06 -

0

5

10

15

20

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30 a

b

Fig. IO. Abscissa: and ordinate as for Fig. 9. (a) control; (b) actinomycin D.

Sedimentationpatterns of RNA synthesizedduring rhizoid elongation in the presenceor absenceof actinomy& D (100 mgil). The samedescription as for fig. 10, except that sporeswere transferredfrom individual flasks to the basal medium or basal medium with actinomycin D at 48 h after sowing. A 1 h pulse of WJJdR (10 &i, 22.8 Ci/mM) was given at 71 h after sowing.

the role of preformed messengersin protein synthesis. This is important because even a small fraction of an active messenger can program the synthesis of a great deal of proteins and can have important developmental effects during germination. This point was examined by a study of the sedimentation of RNA synthesized in the spores during initiation and elongation of the rhizoid and the effect of the inhibitor thereof. Typical sucrosegradient centrifugation profiles of RNA extracted from spores grown in the basal medium and in medium containing actinomycin D during rhizoid initiation are given in fig. 9. It is seenthat distribution of material according to optical density at 260 nm is the usual one with peaks corresponding somewhat in their placement on the gradient to 23S, 165 and 4s RNA. However, labeled RNA was found almost exclusively in 4s and lighter regions and counts in the relatively heavier fractions were barely above background. Addition of actinomycin D inhibited to some extent incorporation of 3H-UdR into the lighter fractions. Sedimentation data for RNA extracted from the spores during rhizoid elongation are plotted in fig. 10. During elongation of the rhizoid, labeled RNA was found throughout Expti CeNRes 63

the gradient with considerable amounts of it in 4s and lighter regions. Synthesis of heavy RNA was significant as represented by the peaks roughly coincident with two optical density peaks at 23s and 16s. Actinomycin D completely inhibited incorporation of 3HUdR into heavier fractions and the only detectable label was in the 4s and lighter regions where RNA synthesis proceeded at a lower rate than in control. These findings suggest that elongation of the rhizoid is dependent upon the synthesis of actinomycin D-sensitive heavy RNA molecules about 48-72 h after sowing. However, it is uncertain to what extent the heavy new RNA is messenger RNA and to what extent it is ribosomal. DISCUSSION The most pertinent aspect of this study bears upon the control of transcription and translation processes during initiation and growth of the rhizoid in germinating bracken fern spores. From the reversible block to initiation and elongation of the rhizoid caused by cycloheximide, an inhibitor of protein synthesis at the level of aminoacyl transfer to nascent polypeptides [lg, 191,it appears that the early events of germination are initiated

Germination of bracken fern spores 351

by the synthesis of proteins which are relatively specific for them. Experiments with actinomycin D have demonstrated that even relatively high concentrations of the drug are ineffective in preventing initiation of the rhizoid, while low concentrations inhibit its growth in length. The well-known effects of actinomycin D as an inhibitor of DNAdependent synthesis of RNA [17] have led to theassumptionthat if an organ differentiates normally after actinomycin D treatment, genomic readout necessaryfor initiation of the organ has occurred before application of the drug. By analogy it implies that while initiation of the rhizoid can proceed independently of DNA-primed RNA synthesis, its subsequent growth may be expected to require continued synthesis of such RNA. When viewed in this light, some aspects of the metabolic activity of RNA and protein during germination are noteworthy. The implication from the results of experiments on the effects of actinomycin D on RNA synthesis during rhizoid initiation and elongation is very strong that only a small fraction of RNA synthesized during rhizoid initiation has potential template activity, while rhizoid elongation is dependent upon concurrent synthesis of template-active RNA. A second noteworthy feature is that incorporation of amino acid into protein is relatively insensitive to actinomycin D during the period when initiation of the rhizoid proceeds normally in the presence of the drug. That bulk of protein synthesis can take place without parallel DNA-dependent RNA synthesis suggests that spores are supplied with suitably preformed messengers which contain a program of information necessary to code for the first proteins of germination. Since actinomycin D inhibits elongation of the rhizoid, concomitant synthesis of proteins which are at least in part coded on actinomycin D-sensitive RNA templates is

necessaryfor this process. This view has the support from brief experiments using 5fluorouracil, which has been shown to inhibit the synthesis of ribosomal and soluble RNA in certain plants [8, lo]. Although this compound inhibited rhizoid elongation and RNA synthesis to the same extent as actinomycin D, it was nearly three times more effective than the latter in arresting protein synthesis. This implies that only that fraction of protein whose synthesis is inhibited by actinomycin D is associated with elongation of the rhizoid, while additional protein sensitive to 5-fluorouracil may be structural protein whose synthesis is not dependent upon continued synthesis of DNA-primed RNA. Interpretation of spore germination based on control of transcription of preexisting messengersand synthesis of new messengers is rendered more reasonable from sedimentation data of RNA extracted from control and actinomycin D-treated spores. During initiation of the rhizoid, incorporation of “H-UdR was confined almost exclusively to soluble RNA. In the absenceof base analysis it is difficult to determine whether this is due to the synthesis of low molecular weight RNA, or due to the addition or turnover of cytidylate and adenylate residues to the end of an already existing transfer RNA as observed in other systems [l, 7, 91. It has been demonstrated in bacterial systems that messenger properties necessary to code for polypeptides of normal length reside in RNA molecules sedimenting with a coefficient higher than 6S-10s [14]. Since fragments of RNA no greater than 4S are made during rhizoid initiation, and since all RNA’s, including low molecular weight RNA’s are synthesized on DNA template [23, 251it is tempting to conclude that activation of low molecular weight RNA (by synthesis or turnover) using preprogrammed Exptl Cd Res 63

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messenger RNA independent of parallel gene activation is the main event associated with rhizoid initiation. Elongation of the rhizoid is accompanied by synthesis of heavy RNA and additional incorporation of the label into 4s material. The inhibition of rhizoid elongation and of synthesis of RNA sedimenting above 6S-10s by actinomycin D suggestsa dependence of rhizoid elongation on newly synthesized messenger RNA. However, it should be emphasized that before they can be considered to fulfil the role of biological messengers, RNA fractions should be shown to be capable of directing protein synthesis in vitro. Recent studies on the germination of seeds of higher plants [3, 13, 241 and on the development of eggs of invertebrates [9, 211 and amphibians [2, 41 have convincingly demonstrated the existence of preformed, template-active messenger RNA’s which carry the codes for the synthesis of the first proteins to initiate development. A logical extension of the present data would suggest that germination of bracken fern spores also falls into this general pattern.

3. Chen, D, Sarid, S & Katchalski, E, Proc natl acad sci US 60 (1968) 902. 4. Davidson, E H, Crippa, M, Kramer, F R & Mirsky, A E, Ibid 56 (1966) 856. 5. Davis, B D, Thesis, Purdue University (1965). 6. Gantt, E & Amott, H J, Am j bot 52 (1965) 82. 7. GliSin, V R & GliSin, M V, Proc natl acad sci US 52 (1964) 1548. 8. Gressel, J & Galun, E, Biochem biophys res comm 24 (1966) 162. 9. Gross, P R, Malkin, L I & Moyers, W A, Proc natl acad sci US 51 (1964) 407. 10. Key, J L, Plant physiol 41 (1966) 1257. 11. Kirby, K S, Biochem j 64 (1956) 405. 12. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 13. Marcus, A & Feeley, J, Proc natl acad sci US 51 (1964) 1075. 14. Monier, R, Naono, S, Hayes, D, Hayes, F & Gros, F, J mol biol 5 (1962) 311. 15. Raghavan, V, Am j bot 52 (1965) 900. 16. - Physiol plantarum 21 (1968) 1020. 17. Reich, E & Goldberg, I H, Progress in nucleic acid research and molecular biology (ed J N Davidson & W E Cohn) vol. 3, p. 184 (1964). 18. Siegel, M R & Sisler, H D, Biochim biophys acta 87 (1964) 70. 19. - Ibid 87 (1964) 83. 20. Smillie, R M & Krotkov, G, Canad j bot 38 (1960) 31. 21. Smith, K D, J exptl zoo1 164 (1967) 393. 22. Snedecor, G W, Statistical methods. Iowa State College Press, Ames (1956). 23. Spiegelman, S, Informational macromolecules (ed H J Vogel, V Bryson & J 0 Lampen) p. 27. Academic Press, New York (1963). 24. Waters, L C & Dure, L S, J mol biol 19 (1966) 1. 25. Watson, J D, Molecular biology of the gene. Benjamin, New York (1965).

REFERENCES 1. Brown, D D & Littna, E, J mol biol 8 (1964) 688. 2. - Ibid 20 (1966) 81.

Exptl Cell Res 63

Received May 19, 1970