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Protein synthesis during fungal spore germination
ARCHIVES
OF
BIOCHEMISTRY
Protein V. Evidence
AND
137, 442-452 (1970)
RIOPHYSICS
Synthesis
During
Fungal
Spore
Germination
that the Ungerminated Conidiospores of Botryodiplodia Contain Messenger Ribonucleic Acid13* ROBERT Department
i\,I. BRAMBL3
of Plant Pathology,
Received
December
AND
JAMES
L. VAX- ETTEN
University of Nebraska,
10, 1969; accepted
theobromae
January
Lincoln,
Nebraska
21, 1970
Ungerminated conidiospores of Botryodiplodia theobromae were examined for the presence of mRNA. A comparison of the incorporation kinetics of ‘Gleucine and I%-uracil into pulse-labeled germinating spores indicated that a cycloheximidesensitive protein synthesis began immediately upon germination in the apparent absence of RNA synthesis; the rate of incorporation of ‘Gleucine increased sharply later in the germination sequence when an actinomycin D-sensitive ‘Guracil incorporation began. Polyribosomes characteristic of eukaryotic cells were detected in spores at all stages of germination, including the ungerminated spores. The mRNA in polyribosomes of ungerminated spores was probably functional because the ribosomal aggregates became associated in viva with 14C-labeled amino acids during the first 30 min of germination. Ribosome fractions enriched with polyribosomes were isolated from both ungerminated and germinated spores, and these fractions, when from germinated spores, stimulat.ed tested with a supernatant enzyme fraction appreciably more amino acid incorporation than did purified monoribosomes from the same spores. It is concluded that ungerminated spores of this fungus contain upon the onset of germination. mRNA which is translated immediately
processes and of the specific roles of the rapid transcriptional and translational functions that are characteristically resumed upon activation and subsequent growth. With a view toward defining these hypothetical control mechanisms, several investigators have studied the protein-synthesizing apparatus of fungal spores, in addition to bacterial spores, plant seeds, and animal embryos (see reviews l-5). Our previous studies have shown that ungerminated conidiospores of the mycelial fungus Botryodiplodia theobromae Pat. contained many of the known components of the protein-synthesizing apparatus [ribosomes (B), tRNA (7),4 aminoacyl-tRNA enzymes synthetases (8), and transfer (9)], and that in vitro, the component re-
In life cycles of plants, animals, and microorganisms, there exists a period of dormancy or latency, often characterized by quiescent metabolic functions and by the presence of morphologically and biochemically specialized cells which are capable of being activated for rapid growth and development. An important topic in developmental biology is the understanding of the general metabolic regulatory mechanisms of the dormancy and activation 1 Published with the approval of the Director as Paper No. 2748, Journal Series, Nebraska Agricultural Experiment Station. 2 This report is part of a dissertation by R. Brambl, submitted to the Graduate Faculty of the University of Nebraska in partial fulfillment of requirements for the Ph.D. degree. 3 Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.
4 Abbreviations: tRNA, transfer acid; mRNA, messenger ribonucleic ribosomal ribonucleic acid.
442
ribonucleic acid; rRNA,
MESSENGER
RIBONUCLEIC
actions or the overall reaction was active when tested with a synthetic mRNA, polyuridylic acid. Similar conclusions have been drawn by other investigators (10-13). In an early report, Henney and Storck (14) thoughtfully anticipated subsequent research in spore development studies by examining conidiospores and ascospores of Neurospora crassa for the presence of polyribosomes and hence mRNA. Although these structures were readily detected in extracts from germinating spores and hyphae, no evidence could be found that they existed in either type of ungerminated spore; these authors suggested that, unlike tRNA and rRKA, mRKA was not conserved in resting or dormant fungal spores. Studies of other spores by later workers provided similar conclusions (15, 16). Such evidence suggested that mRNA is not present in the latent spore and that perhaps a crucial step during spore germination is the synthesis of mRNA. This report presents evidence that ungerminated spores of B. theobrmae contain mRNA which is translated immediately upon the onset of germination during a period in which no discernible RNA synthesis is occurring. Recent reports from other laboratories have presented evidence that polyribosomes could be detected in extracts of Uromyces phaseoli uredospores (17) and that zoospores of Blastocladiella ewersonii (IS) and conidiospores of Perono(19) incorporated amino spora tabacina acids during germination in the presence of inhibitors of RNA synthesis. MATERIALS
AND
METHODS
Uuterials. Ribonuclease A (bovine pancreas, type I-A), phosphodiesterase (bovine spleen, type I), and ol-chymotrypsin were obtained from Sigma Chemical Co.; reconstituted ‘%-protein hydrolyzate (13 amino acids) was obtained from Schwarz BioResearch; and L-leucine-14C (UL, 263 &i/ rmole). uracil-2-14C (34.3 pCi/pmole), and uridine366.5 &i/pmole) were I%-triphosphate (UL, obtained from New England Nuclear Corp. Actinomycin D (Dactinomycin) was obtained from Merck Sharp and Dohme. The suppliers of other materials and the methods used for obtaining B. theobromae spores were similar to those previously described (G). The spores were germinated in a chemically defined medium (20) in 7. or g-liter
ACID
IN SPORES
443
batches in a MicroFerm (Model MF-114) Laboratory Fermentor (New Brunswick Scientific Co.); the concentration of the spores in the medium was 1 mg/ml (1.5 X lo5 spores/ml), the temperature was 34”, the paddle speed was 7OlX300 rpm, and the air flow rate was 16,000 cc/min. For the extraction of ribosomes, the spore suspensions were rapidly dumped over crushed ice and then collected on single Miracloth (Chicopee Mills, Inc.) sheets in ice-filled Biichner funnels; the packed spores were resuspended and washed repeatedly with iced water and, in t.he last, wash, with the appropriate extraction buffer. Labeled precursor incorporation assays. At various times during the spore-germination period, IO-ml aliquots of the spore suspension were removed from the fermentor vessel and incubated an additional 15 min at 34” in the presence of a I%labeled precursor of protein or RNA (0.05 &X/ml spore suspension). If the effects of an inhibitor of protein or RNA synthesis were to be measured, the incubation was begun 15 min earlier so that the spores were exposed to the inhibitor for 15 min before the addition of the labeled precursor. The incubation was stopped by the addition of 2 ml of 30% (w/v) trichloroacetic acid, and the assay mixtures were placed on ice for at least 1 hr. The spores were t,hen quantitat)ively transferred to Whatman GF/A glass filter discs (2.4 cm), washed wit,h 40 to 50 ml of water, dried, placed in counting vials containing 10 ml of counting solution [4 g of 2,5-diphenyloxazole, 50 mg of 1,4-bis-2-(&methyl5-phenyloxazolyl)-benzene and toluene to 1 liter], and counted in a liquid scintillation spectrometer. In graphs of these data, all values were corrected for zero-time incorporation. Preparation of ribosome and supernatant enzyme fractions. The procedures for obtaining ribosomes from ungerminated and germinated spores were identical. The spores were disrupted in a Braun MSK mechanical homogenizer (Bronwill Scientific Co.); 3-4 g of spores were combined with 27 g of l-mm glass beads and 8 ml of buffer A and shaken for 60 set at 4000 rpm with simultaneous chilling with CO*. The contents of each flask were combined with an additional 10 ml of buffer A, and the brei was cent,rifuged for 15 min at 2O,OOOg,30 min at 3O,oOOg, and 180 min at 105,OOOg.The final pellet surface was rinsed with 6 ml of buffer B, and the pellet was suspended in a minimlun amount (l-2 ml) of buffer B. This ribosomal suspension was then centrifuged for 15 min at 20,OOOg to remove aggregated and nonsuspended mat,erial; the upper three-fourths of the supernatant fluid (the semipurified ribosome fraction) were removed and used for subsequent ribosome analysis. For the densitygradient analyses, the ribosomal fraction was
444
BRAMBL
AND
always used immediately after preparation; when ribosomes were prepared for other purposes, the suspensions were stored at - 196”. The method for the preparation of the enzyme fraction has been described in detail previously (6,8). Buffer A was composed of sucrose, 0.4 M; Tris0.005 M; and 2-mercapCl, 0.05 M; MgC12.6HOH, toethanol, 0.005 M; buffer B was composed of Tris-Cl, 0.01 M; KCl, 0.015 M; and MgCl,.6HOH, 0.005 M. The pH of buffers A and B was adjusted to 8.5. Buffer C was composed of Tris-Cl, 0.02 M, pH 7.8; magnesium acetate, 0.01 M; KCl, 0.06 M; 2-mercaptoethanol, 0.02 M; reduced glutathione, 0.003 M; and spermine, 2 X lO+ M. Density-gradient centrifugation. The ribosome fractions were analyzed in lO-35% linear sucrose gradient columns prepared by hand-layering four layers of charcoal-clarified sucrose solutions (21) (100, 200, 300, and 400 g sucrose/liter; 5, 7, 7, and 7 ml/layer, respectively) diluted so that the buffer B concentration was uniform throughout the column; the gradient columns were allowed to equilibrate by diffusion for 7 to 12 hr at 4’. The ribosome sample t,o be layered onto the gradient column was diluted to a 0.5-ml volume with buffer B. Beckman SW 25.1 rotors were centrifuged at 23,000 rpm for 180 min under refrigeration. The gradient columns were fractionated and scanned photometrically with an ISCO Model 180 Density Gradient Fractionator (Instrumentation Specialties Co.). The columns were pumped through a flow cell with a l-cm light path at 3 ml/min; the absorbance of the contents was measured at 253.6 rn/* and simultaneously recorded on a constant-speed recorder (20 in/hr) as a fun&ion of column depth. In one set of experiments, 8-drop fractions were collected in glass liquid scintillation vials, diluted with 0.5 ml distilled water, and counted in a scintillation solvent for aqueous solutions (22). The ribosome sedimentation constants were determined by the “linear log” gradient method (23). Brome mosaic virus, tobacco mosaic virus, and southern bean mosaic virus were used as markers. Ribosomal fractions, enriched in polyribosomes, were prepared from both types of spores. Gradient columns of 5-207, sucrose (50, 108, 162, and 216 g sucrose/liter; 5, 7, 7, and 7 ml/layer, respectively, in buffer B) were overloaded with samples equivalent to 3.3 mg or 4.7 mg rRNA from the semipurified ribosomes prepared from ungerminated or germinated spores, respectively. After centrifuging for 200 min the depth of the monoribosome boundary in a representative tube was determined by lightscatt,ering to provide an estimate of the additional time needed (70 min) to pellet the polyribosomes. This technique permit,ted the polyribosomal material to be pelleted while leaving most of the
VAN
ETTEN
monoribosomal material in the supernatant fluid. The small pellets were suspended in a minimum amount of buffer C (0.14.2 ml) and centrifuged for 15 min at 20,OOOg. The supernatant fluid was withdrawn, and this ribosomai suspension was used in the in vitro amino acid-incorporation experiments. The purified monoribosome fractions were obtained by centrifugation of semipurified ribosomal suspensions through the usual IO-357, sucrose density gradients. While each of the tubes was photometrically scanned, the monoribosome fraction of the gradient was collected. These monoribosome fractions were concentrated by centrifugation for 210 min at 105,000g. The pellets were resuspended in buffer C, given a low-speed centrifugation, and the supernatant fluid was stored at -196”. Assay for template activity in spore ribosomes. The assay for amino acid incorporation was similar to that described previously (6), except that the magnesium acetate concentration was reduced to 8 rmoles/ml assay volume. The standard assay (in a final volume of 0.5 ml) also contained 1 PCi of reconstituted ‘Y-protein hydrolyzate (13 amino acids); 0.1 @mole each of the remaining (unlabeled) amino acids; 200 rg of tRNA prepared from germinated spores of B. theobromae (7) ; and 300 rg of enzyme fraction protein. IJnless otherwise indicated, the concentration of ribosomal material was equivalent to 150 /*g rRNA/assay. The glass filters, containing hot trichloroacetic acid-precipitable material, were treated as described previously (6) and counted in a liquid scintillation spectrometer. Other determinations. Protein was determined by the method of Lowry et al. (24) using bovine albumin as a standard. Ribonucleic acid concentration was measured by assuming that 1 mg RNA/ml HOH has an absorbance of 24 at 260 mF. All experiments described in this report were performed two or more times, and the examples cited represent typical results. RESULTS
General characteristics of spore germination. Under the specified germination conditions, B. theobromae conidiospores began to form germ tubes at 2.5 hr, and by 5 hr of germination, 93 % of the spores had germ tubes. During this time, the dry weight of the spores did not change appreciably, but by 9 hr, the dry weight increased about 1.5 times (Fig. 1). Pulse-labeling of whole spores during germination. The kinetics of uracil and leucine incorporation into the spores during
MESSENGER
RIBONUCLEIC
7” t
GERMINATION
t
012345678
9 GERMINATION
TIME (hours)
FIG. 1. Germination and dry weight curves of B. theobromae conidiospores germinated under conditions described in the text.
germination are shown in Fig. 2. The relative rates of incorporation of these precursors of protein and RXA would suggest that protein synthesis began immediately upon placing the spores in the germination medium and continued for a period in the apparent absence of KNA synthesis. For these pulse-labeling data to be valid, how ever, it must be established that the respective labeled precursors are entering the protein or RSA fractions. In other esperimcnts, spores were grown to mnximum germination in the continuous presence of either 14C-leucinr or 14C-uracil; after chemical fractionation of the spores (25), it ~-as determined that S9 7% of t’he 14Cleucine appeawd in the fraction \vhich would be expected to contain hydrolyzed protein (hot :! M ~VatOH), and 94% of the 14C-uracil entered the fraction that contained hydrolyzed nucleic acids (hot 10 ‘3%trichloroacetic acid). Additionally, germinated spores \vtw labeled with 14C-uracil and then incubated in 1 nr SaOH for 1S hr at 3T”, waslhed with cold 5 ‘3%trichloroacetic acid, and the radioactivity determined. As a result of t,his mild alkaline hydrolysis, the high quantity of isotope otSherwise incorporated w-as completely abolished; t,his
ACID
445
IN SPORES
fact indicates that the 14C-uracil entered the RSA of the spores and not the DNA. The substitution of 14C-lysine and l*Curidine for 14C-leucine and 14C-uracil showed that these respective precursors of protein and RKA were incorporated into germinating spores with similar kinetics. The finding that no 14C-uracil was incorporated into the spores during the earlier phases of germination was interpreted to mean that no RNA synthesis was occurring in a period in which protein was synthesized. There are also at least two alternative explanations: (1) the spores contained a pool of RNA precursors which were preferentially incorporated before the exogenous 14C-uracil, and (2) the spores were impermeable to the exogenous 14C-uracil during this stage of germination. At 1.25 hr of germination, when no discernible 14C-uracil was incorporated into the cellular RKA, the spore suspension was pulsed with 14C-uracil, rapidly collected by filtration, 1600/
604-1
I 0
I
2
GERMINATION
3
4
TIME (hours)
FIG. 2. Pulse-labeling experiment: Incorporation of ‘4C-leucine and ‘4C-uracil into germinating spores. The points plotted represent the amount of isotope incorporated during each incubation period, and the connecting lines reflect changes in the capacity for incorporation. The correct,ion for zero-time incorporation iu all these graphs was always less than 5 pmoles per assay.
446
BRAMBL I
1600
AND VAN ETTEN
r
chromatographed along with 14C-UTP on Whatman No. 1 paper in three solvent systems : 0.15 M (NH,)HCO,; NH,OH (coned.) : isobutyric acid: EDTA (0.1 M) : water (1: 66 : 1: 32, v/v/v/v) ; and n-butanol: acetic acid: water (4: 1: 5, v/v/v). In all three solvent systems, the radioactive fraction of the spore extract migrated at the same rate as the authentic 14C-UTP. These facts indicated that the spores were permeable to uracil in the early germination stages and that the uracil was rapidly metabolized to UTP, an immediate precursor of RNA. E$ects of antibiotics upon the precursor incorporation kinetics. Cycloheximide completely inhibited the incorporation of 14Cleucine into germinating spores (Fig. 3). Especially noteworthy was the fact, that the early leucine incorporation was sensitive to this antibiotic, thereby reinforcing the previous indication that this early burst of incorporation reflected genuine protein synthesis. If the conidiospores of B. theobromae were incubated in water (instead of the medium) under standard conditions, a few spores (less than 7%) eventually germinated after 5 or 6 hr, but the germ tubes did not grow more than four or five spore
'I
6001
i l
1
::
5oc-
d,/ i : ;' 8' i ; ;! i: ,i I' ; ,a
400-
LEUCINE ~cmtrol)
0J I 0
2
I
GERMINATION
3
I
4
5
TIME (hours)
FIG. 3. Pulse-labeling experiment: Incorporation of I%-leucine and 14C-uracil into germinating spores after a 15-min treatment with 20 pg/ml cycloheximide.
washed free of the medium with iced water, and then disrupted in the homogenizer in the presence of cold 5 % trichloroacetic acid.
This trichloroacetic
acid extract
was then
I4
i
5mr I
9
LEUCINE INCORPORATION
1200-
URACIL
lNCORPORATlOi’l
i
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i
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2
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MEDIUM
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4 GERMINATION
TIME
(hours)
FIG. 4. Pulse-labeling experiment: Incorporation of 14C-leucine and I%-uracil into spores incubated in water under standard conditions; also shown is the effect of a 15.min treatment with 20 pgg/ml cycloheximide upon the W-leucine incorporation pattern.
MESSENGER
RIBONUCLEIC
ACID
IN
SPORES
ACTINOMYCIN
I
2
4
3
GERMINATION
FIG. 5. Pulse-labeling germinating spores after
TIME
I (hours)
2
447
D
3
4
experiment: Incorporation of ‘Gleucine and 14C-uracil a 15-min treatment with 25 rg/ml actinomycin D.
diameters in 8 hr. The results of a pulselabeling experiment in which the spores were incubated in water and with cycloheximide are demonstrated in Fig. 4. It is evident that such spores incorporated r4Cleucine in a pattern which initially resembled the early pattern obtained under ordinary germination conditions, while essentially no incorporation of 14C-uracil could be detected. This leucine incorporation was abolished by cycloheximide. Incubation of the spores with actinomytin D for 15 min prior to the addition of 14C-uracil caused an inhibition of uracil incorporation without having any effect on the incorporation of leucine in the early stages of germination. The rapid increase in leucine incorporation, which occurred later in the germination sequence, was slightly depressed by actinomycin D (Fig. 5). In a separate experiment, the spores were incubated in the continuous presence of actinomycin D (25 pg/ml), and samples were taken at various times and pulselabeled with 14C-leucine and 14C-uracil. Again, the incorporation of leucine in the early stages of germination was unaffected by actinomycin D, while at 5 hr there was approximately a 70% inhibition of leucine incorporation (226 pmoles, treated spores: S24 pmoles, untreated spores). At this same
into
germination stage, the uracil incorporation was inhibited about SO% by actinomycin D (243 pmoles, treated spores: 1419 pmoles, untreated spores). It is possible, however, that only the germinating spores are permeable to the antibiotic. Therefore, these findings suggest that the early increase in protein synthesis was independent of new RNA synthesis, but dependent on preexisting mRNA, while the later protein synthesis was at least partially dependent on the synthesis of new RNA. When the spores were germinated continously in 2.5 pg/ml of actinomycin D, the germination rate was unaffected. However, after 8 or 9 hr of germination when the spores in the control incubation had profusely branched germ tubes, those spores treated with actinomycin D had germ tubes which were nearly devoid of branches. Density-gradient analyses of ribosolnal j+ractions. The density-gradient columnscanning profiles of centrifuged ribosome fractions from spores at 0, 1, 2, 3, 4, and 5 hr of germination are shown in Fig. 6. Polyribosomes were clearly detectable at all stages of germination, including the ungerminated spores. It is evident that by 4 hr of germination, there were more polyribosomes extracted from the spores than in previous stages. The sedimentation
448
BRAMBL
II hour SPORES
I
AND
VAN
ETTEN
2 hew
lhcurSPORES
SPORES
DEPTH IN COLUMN
3hour SPORES
II
4 hcu SPORES
0.4-
0.4-
0.3-
0.3-
n
5 hwr SPORES
ll
0.4
2 2 '0
03
Y 5
0.2
:
4% 0.1 BOTTOM :I
I: hL 0.2-
0.2
0 I-
0.1
DEPTH IN COLUMN
FIG. 6. Sedimentation patterns of semipurified ribosomes obtained from spores at indicated stages of germination. Direction of sedimentation is from right to left in all scanning patt,erns. Hibosome samples on each gradient column were equivalent to 266 fig of ribosomal RNA. The chase solution contained potassium acid phthalate, and the increase in OD at the bottom of the tube is due to this chase solution.
constants of the monoribosomal subunits were determined to be 37 S and 60 S; the monoribosomes, 81 S; the dimer and trimer polyribosomes, 123 S and 164 S. In order to determine if the relatively low population of polyribosomes extracted from the ungerminated spores was due to a selective RNase activity in these homogenates, a mixed experiment was performed in which purified ribosomes from germinated spores were mixed with a 30,OOOglowspeed supernatant fraction from the ungerminated spores. The crude extract from the un germinated spores had no deleterious effect upon the polyribosomal profile from the germinated spores.
Treatment of the ungerminated spore ribosomal fraction with 0.0,5 M EDTA caused a complete dissociation of the ribosomes into the two ribosomal subunits. The exonuclease spleen phosphodiesterase (3 pg/ml) had no apparent effect upon the polyribosomal profile. If the semipurified ribosomes from ungerminated or 5-hr germinated spores were treated with 5 pg/ml pancreatic ribonuclease for 15 min at 0” before density-gradient centrifugation, the polyribosomes were nearly abolished (Fig. 7). However, ribonuclease treatment invariablv caused the appearance of an apparent dimer of ribosomes which was seemingly resistant to
MESSENGER
I 0.4-
UNGERMINATED +RleONUUEASE
RIBONUCLEIC
ACID
IN
449
SPORES
n +WONUUEASE
1 BOTTOM1
u
TOP
DEPTH IN COLUMN
FIG. 7. Effect of 5 pg/ml pancreatic ribonuclease upon ribosomes from and germinated spores. Incubation conditions are given in text. Samples 200 pg ribosomal RNA. See Fig. 6 for control experiment curves.
ribonuclease. If the concentrations of ribonuclease were increased or decreased lo-fold, there was no change in the size of this dimer unit; other alterations which produced no change in the dimer peak included incubation of the ribosomes with ribonuclease for longer time periods at 30” and shifting the pH of the ribosomal suspension closer to the optimum for ribonuclease. However, a brief treatment of the ribosomes with cY-chymotrypsin (10 pg/ml) before the usual treatment with ribonuclease caused the dimer peak to disappear in the scanning patterns. To investigate this phenomenon further, monoribosomes from the ribosomal fractions of both types of spores were carefully isolated from preparative density-gradient columns, concentrated by high-speed centrifugation, and then recentrifuged through density-gradient columns. As a result, dimer ribosome peaks appeared. If it is assumed that the isolated monoribosome fractions were uncontaminated with dimer units, this experiment would suggest that the anomalous peaks which appeared after ribonuclease treatment were due not to incomplete degradation of the polyribosomes, but to spontaneous aggregation of liberated monoribosome units. It is important to note that no aggregates were
ungerminated equivalent to
detected which sedimented faster than the ribosome dimers. Because the ungerminated spores contained polyribosomes which were detectable by their physical properties, the following experiment was performed to test the functional activity of these ribosome units. The spores were suspended in the germination medium in the usual manner, and a lliter sample immediately withdrawn and incubated with reconstituted 14C-protein hydrolyzate (0.05 &X/ml medium) at 34” for 30 min. At the conclusion of the incubation, these labeled spores were rapidly chilled with large quantities of crushed ice, collected on a filter, and washed free of medium and extraneous labeled amino acids. These spores were then mixed with similarly prepared unlabeled carrier spores, and the spores were disrupted by the usual procedure; the ribosomal fraction was isolated by high-speed centrifugation, and aliquots of the suspended semipurified ribosomes were layered onto sucrose density-gradients, centrifuged, and scanned. During the scanning process, S-drop fractions were collected and analyzed for radioactivity. In Fig. 8, a plot of the radioactivity patterns superimposed on the optical scanning profile from the same gradient shows that the absorbance peaks of the
450
BRAMBL AND VAN ETTEN r :: :;
0.4
+RIEoNuCLEASE
200 175 0.3150 125 0.2-
'
(SdWs
each)
100
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; I -200 2 : : -175 : I 1 jl -150 f k ,; -125 o ', r *' -100 5 : 8
75
25
, m
35
I TOP
BOTTOM FRACTIONS
DEPTH IN COLUMN
FIG. 8. Sucrose density-gradient optical scanning pattern of semipurified ribosomes from spores germinated 30 min in presence of W-amino acid mixture. Also shown is the effect of 5 pg/ml pancreatic ribonuclease upon these patterns. The gradients were fractionated into S-drop samples for radioactivity determinations. Ribosomal samples were equivalent to 200pg of ribosomal RNA. The solid line represents the optical density and the dashed line, radioactivity.
polyribosomes were associated with radioactivity from the 14C-amino acids. The deleterious effect of ribonuclease upon both the absorbance and radioactivity profiles is also demonstrated in Fig. 8. Therefore, the polyribosomes detected in the ungerminated spores had the capacity to become functionally associated in vivo with labeled amino acids during the first 30 min of germination. Cell-free amino acid incorporation assay. In order to test whether ribosomes extracted from ungerminated spores possessed template activity, the polyribosome-enriched fraction and purified monoribosomes were assayed with a supernatant enzyme fraction and tRNA obtained from 5-hr germinated spores. The data in Table I show that the polyribosome fraction in both germinated and ungerminated spores had the capacity to stimulate amino acid incorporation into hot trichloroacetic acid-precipitable material above the amount of incorporation with monoribosomes. If the amount of polyribosomal RNA added was halved or doubled, the resulting amount of 14C-amino acid incorporation was correspondingly reduced or increased. The deletion of tRNA
reduced incorporation by about 32% in both cases, while the deletion of the enzyme fraction or the polyribsomal material severely reduced incorporation. Treatment with ribonuclease reduced incorporation by about 62 % in both cases, and puromycin reduced incorporation by about 50 % ; as might be expected, chloramphenicol essentially had no effect upon either assay system. DISCUSSION The results of several early studies (1416) suggested that ungerminated fungal spores may be deficient in mRNA; one implication of such a suggestion was that this defect could participate in the regulation of dormancy and germination. This hypothesis is not supported by the evidence presented in this report; instead, results of the experiments described argue persuasively that the ungerminated conidiospores of B. theobromae do contain mRNA, that this mRNA is immediately functional upon initiation of germination, and that this mRNA apparently contains enough information to allow the formation of a germ tube.
MESSENGER TABLE
RIBONUCLEIC
I
TI,;MPL.\T~S ACTIVITY FROM UNGERMINATED
OF RIBOSOM~L FRICTIONS AND GERMINATED SPORES OF B. theobromae IN THE PRESENCE nw ENZYMEFR~CTION FROM THE GERMIN.4TEDsPORES
Assay
Ribosomal
material
Counts/ min incorporated per assaya
system
1
Deletions
or additions
1972 1208 186 155 1720 1647 1201 943 576 273 1329 872 134 407
Polyribosomesh Monoribosome9 ~2 polyribosomes 3s polyribosomes
II
Polyribosomes Polyribosomes Polyribosomes Polyribosomes Polyribosomes Polyribosomes
!
- polyribosomes - tRNA - SKOenzyme fraction +ribonuclease (30 rd -l-puromycin (50 NT) +chloramphenicol (50 !A +cycloheximide (50 Pd
II
756 458 952 579 19611173 583 377 I
a All values are corrected for zero-time incorporation. b The terms “polyribosomes” and “monoriboused here refer to the polyribosomesomes” enriched ribosomal fraction and to purified monoribosomes, respectively, the preparations of which are described in the text. Ribosomal material in each assay tube was equivalent to 150 lg of ribosomal RNA unless otherwise indicated.
In the germinating spores of this organism, the precursor incorporation kinetics indicate that resumption of protein and RNA syntheses are temporally separate and distinct events. The fact that a cycloheximide-sensitive protein synthesis is begun immediately upon initiation of germination, but in the absence of any detectable RNA synthesis, clearly suggests that these latent spores contained mRNA which is responsible for the protein synthesis required for development in the first
ACID
IN SPORES
451
phases of germination. This interpretation of the evidence necessarily assumes that no internal pools of RNA precursors selectively depress exogenous precursor uptake during the first X.5 hr of germination and that the cells are permeable to the labeled precursor during the same period. The validity of the latter assumption is shown by the fact that by 1.2.5-1.5 hr of germination, when no uracil was incorporated, the 14C-uracil was not only taken up by the spores, but was converted to UTP, an immediate precursor of RNA. We have no evidence that hypothetical endogenous pools of RNA precursors in the spores could influence uptake of exogenous precursors. Further evidence that the initial protein synthesis is supported by a preexisting mRNA is provided by the results of treatment of the germinating spores with actinomycin D. If the synthesis of protein early in germination required simultaneous synthesis of mRNA, the actinomycin D treatment should have at least partially inhibited the incorporation of 14C-leucine provided ungerminated spores are permeable to this compound. The protein synthesis early in germination appears to be independent of concurrent RNA synthesis. Density-gradient analyses of spore extracts clearly indicated that discrete classes of polyribosomes exist in all the germination stages examined, including the ungerminated spores. The ribosomes were readily dissociable by EDTA treatment, and the polyribosomal material was sensitive to pancreatic ribonuclease. After treatment, with ribonuclease, however, dimers of ribosomes were prominent in the scanning patterns. A similar effect was noted, but not discussed, by Henney and Storck (14) in their study of N. crassa. Our evidence would suggest that this ribosome dimer is caused not by incomplete digestion of the polyribosomes but by spontaneous aggregation of monoribosome units. However, Mackintosh and Bell (26) recently reported the appearance of these dimers in sea urchin egg ribosomes treated with ribonuclease, and they ascribed considerably more significance to the phenomenon of this dimer ribosome peak.
452
BRAMBL
AND
That the mRNA of polyribosomes of ungerminated spores is apparently functional and probably supports the protein synthesis in the early stages of germination was demonstrated by the ability of the polyribosomes to become associated in viva with 14C-amino acids during the first 30 min of germination. Furthermore, polyribosome-enriched ribosome fractions isolated from both germinated and ungerminated spores had the capacity to stimulate amino acid incorporation at levels somewhat above the incorporation stimulated by purified monoribosomes. Although the level of incorporation in the germinated spore polyribosome fraction was higher than its counterpart from the ungerminated spores, the amino acid incorporation systems from both spore stages had characteristics expected of an authentic amino acid incorporating system. The conclusion that mRNA is conserved in ungerminated spores is supported by recent reports from other laboratories; Staples et al. (17) have presented evidence that polyribosomes exist in ungerminated U. phaseoli uredospores, and Lovett (18) and Hollomon (19), each using selective metabolic inhibitors, have shown that B. emersonii and P. tabmina spores do not require simultaneous RNA synthesis to support the protein synthesis necessary for germination. Dunkle et al. (27) have presented similar evidence that urediospores of Puccinia grawzinis tritici may germinate in the presence of RNA synthesis inhibitors. Therefore, the results of these several studies, when included with other information available about protein synthesis in fungal spores, suggest that many of the known macromolecular components, enzymes, and informational molecules of the protein synthesis apparatus are present in the ungerminated spores and can be extracted in an active form. ACKNOWLEDGMENTS The authors are indebted to Dr. Myron K. Brakke and Mrs. Nancy Van Pelt for generously providing the marker viruses and useful advice about the density-gradient centrifugation. Mr. Don Merlo, Mrs. Sandra Swanda, and Mr. Rex Koski helped provide us with spore material. This investigation was supported in part by funds from the Research Council of the University of
VAN
ETTEN
Nebraska National
and from PHS grant Xnst.itutes of Health.
Al08057
of the
REFERENCES 1. VAN ETTEN, J. L., Phytopathology 69, 1060 (1969). 2. HALVORSON, H. O., VARY, J. C., AND STEINBERG, W., Ann. Rev. Microbial. 20,169 (1966). 3. KORNBERC, A., SPUDICH, J. A., NELSON, D. L., AND DEUTSCHER, M. P., Ann. Rev. Biothem. 37, 51 (1968). 4. NEMER, M., Progr. Nucl. Acid Res. Mol. Biol. 7, 243 (1967). 5. TYLER, A., Develop. Biol. Suppl. 1, 170 (1967). 6. VAN ETTEN, J. L., Arch. Biochem. Biophys. 136, 13 (1968). 7. VAN ETTEN, J. L., KOSKI, R., AND EL-OLEMY, M., J. Bacterial. 100, 1182 (1969). 8. VAN ETTEN, J. L., AND BRAMBL, R. M., J. Bacterial. 96, 1042 (1968). 9. VAN ETTEN, J. L., AND BRAMRL, R. M., Phytopathology 69, 1894 (1969). 10. STAPLES, R. C., AND BEDIGIAN, D., Contrib. Boyce Thompson Inst. 23, 345 (1967). 11. YANIV, Z., AND STAPLES, R. C., Co&rib. Boyce I’hompson Inst. 24, 103 (1968). 12. HORIKOSHI, K., AND IKEDA, Y., Biochim. Biophys. Acta 166, 505 (1968). 13. RADO, T. A., AND COCHRANE, V. W., Fed. Proc. 28, 890 (1969). 14. HENNEY, H. R., AND STORCK, R., Proc. Nat. Acad. Sci. U.S.A. 61, 1050 (1964). 15. HORIKOSHI, K., OTHTAKA, Y., AND IKEDA, Y., Agr. Biol. Chem. 29, 724 (1965). 16. SCHMOYER, I. R., AND LOVETT, J. S., Bacterial. Proc. 1968, 30 (1968). 17. STAPLES, R. C., BEDIGIAN, D., AND WILLIAMS, P. H., Phytopathology 68, 151 (1968). 18. LOVETT, 3. S., J. Bacterial. 96, 962 (1968). 19. HOLLOMON, D. W., J. Gen. Microbial. 66, 267 (1969). 20. BRAMRL, R. M., Ph.D. Thesis, University of Nebraska, Lincoln (1969). 21. BRAKKE, M. K., in “Methods in Virology” (K. Maramorosch and H. Xoprowski, eds.), Vol. 2, p. 119. Academic Press, New York (1967). 22. BRUNO, G. A., AND CHRISTIAN, J. E., Anal. Chem. 33, 1216 (1961). 23. BRAKKE, M. K., Anal. Biochem. (in press). 24. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 25. GOTTLIEB, D., AND VAN ETTEN, J. L., J. Bucteriol. 88, 114 (1964). 26. MACKINTOSH, F. R., AND BELL, E., J. Mol. BioZ. 41, 365 (1969). 27. DUNKLE, L. D.,MAHESH~ARI, R., AND ALLEN, P. J., Scienre 163, 481 (1969).
OF
BIOCHEMISTRY
Protein V. Evidence
AND
137, 442-452 (1970)
RIOPHYSICS
Synthesis
During
Fungal
Spore
Germination
that the Ungerminated Conidiospores of Botryodiplodia Contain Messenger Ribonucleic Acid13* ROBERT Department
i\,I. BRAMBL3
of Plant Pathology,
Received
December
AND
JAMES
L. VAX- ETTEN
University of Nebraska,
10, 1969; accepted
theobromae
January
Lincoln,
Nebraska
21, 1970
Ungerminated conidiospores of Botryodiplodia theobromae were examined for the presence of mRNA. A comparison of the incorporation kinetics of ‘Gleucine and I%-uracil into pulse-labeled germinating spores indicated that a cycloheximidesensitive protein synthesis began immediately upon germination in the apparent absence of RNA synthesis; the rate of incorporation of ‘Gleucine increased sharply later in the germination sequence when an actinomycin D-sensitive ‘Guracil incorporation began. Polyribosomes characteristic of eukaryotic cells were detected in spores at all stages of germination, including the ungerminated spores. The mRNA in polyribosomes of ungerminated spores was probably functional because the ribosomal aggregates became associated in viva with 14C-labeled amino acids during the first 30 min of germination. Ribosome fractions enriched with polyribosomes were isolated from both ungerminated and germinated spores, and these fractions, when from germinated spores, stimulat.ed tested with a supernatant enzyme fraction appreciably more amino acid incorporation than did purified monoribosomes from the same spores. It is concluded that ungerminated spores of this fungus contain upon the onset of germination. mRNA which is translated immediately
processes and of the specific roles of the rapid transcriptional and translational functions that are characteristically resumed upon activation and subsequent growth. With a view toward defining these hypothetical control mechanisms, several investigators have studied the protein-synthesizing apparatus of fungal spores, in addition to bacterial spores, plant seeds, and animal embryos (see reviews l-5). Our previous studies have shown that ungerminated conidiospores of the mycelial fungus Botryodiplodia theobromae Pat. contained many of the known components of the protein-synthesizing apparatus [ribosomes (B), tRNA (7),4 aminoacyl-tRNA enzymes synthetases (8), and transfer (9)], and that in vitro, the component re-
In life cycles of plants, animals, and microorganisms, there exists a period of dormancy or latency, often characterized by quiescent metabolic functions and by the presence of morphologically and biochemically specialized cells which are capable of being activated for rapid growth and development. An important topic in developmental biology is the understanding of the general metabolic regulatory mechanisms of the dormancy and activation 1 Published with the approval of the Director as Paper No. 2748, Journal Series, Nebraska Agricultural Experiment Station. 2 This report is part of a dissertation by R. Brambl, submitted to the Graduate Faculty of the University of Nebraska in partial fulfillment of requirements for the Ph.D. degree. 3 Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305.
4 Abbreviations: tRNA, transfer acid; mRNA, messenger ribonucleic ribosomal ribonucleic acid.
442
ribonucleic acid; rRNA,
MESSENGER
RIBONUCLEIC
actions or the overall reaction was active when tested with a synthetic mRNA, polyuridylic acid. Similar conclusions have been drawn by other investigators (10-13). In an early report, Henney and Storck (14) thoughtfully anticipated subsequent research in spore development studies by examining conidiospores and ascospores of Neurospora crassa for the presence of polyribosomes and hence mRNA. Although these structures were readily detected in extracts from germinating spores and hyphae, no evidence could be found that they existed in either type of ungerminated spore; these authors suggested that, unlike tRNA and rRKA, mRKA was not conserved in resting or dormant fungal spores. Studies of other spores by later workers provided similar conclusions (15, 16). Such evidence suggested that mRNA is not present in the latent spore and that perhaps a crucial step during spore germination is the synthesis of mRNA. This report presents evidence that ungerminated spores of B. theobrmae contain mRNA which is translated immediately upon the onset of germination during a period in which no discernible RNA synthesis is occurring. Recent reports from other laboratories have presented evidence that polyribosomes could be detected in extracts of Uromyces phaseoli uredospores (17) and that zoospores of Blastocladiella ewersonii (IS) and conidiospores of Perono(19) incorporated amino spora tabacina acids during germination in the presence of inhibitors of RNA synthesis. MATERIALS
AND
METHODS
Uuterials. Ribonuclease A (bovine pancreas, type I-A), phosphodiesterase (bovine spleen, type I), and ol-chymotrypsin were obtained from Sigma Chemical Co.; reconstituted ‘%-protein hydrolyzate (13 amino acids) was obtained from Schwarz BioResearch; and L-leucine-14C (UL, 263 &i/ rmole). uracil-2-14C (34.3 pCi/pmole), and uridine366.5 &i/pmole) were I%-triphosphate (UL, obtained from New England Nuclear Corp. Actinomycin D (Dactinomycin) was obtained from Merck Sharp and Dohme. The suppliers of other materials and the methods used for obtaining B. theobromae spores were similar to those previously described (G). The spores were germinated in a chemically defined medium (20) in 7. or g-liter
ACID
IN SPORES
443
batches in a MicroFerm (Model MF-114) Laboratory Fermentor (New Brunswick Scientific Co.); the concentration of the spores in the medium was 1 mg/ml (1.5 X lo5 spores/ml), the temperature was 34”, the paddle speed was 7OlX300 rpm, and the air flow rate was 16,000 cc/min. For the extraction of ribosomes, the spore suspensions were rapidly dumped over crushed ice and then collected on single Miracloth (Chicopee Mills, Inc.) sheets in ice-filled Biichner funnels; the packed spores were resuspended and washed repeatedly with iced water and, in t.he last, wash, with the appropriate extraction buffer. Labeled precursor incorporation assays. At various times during the spore-germination period, IO-ml aliquots of the spore suspension were removed from the fermentor vessel and incubated an additional 15 min at 34” in the presence of a I%labeled precursor of protein or RNA (0.05 &X/ml spore suspension). If the effects of an inhibitor of protein or RNA synthesis were to be measured, the incubation was begun 15 min earlier so that the spores were exposed to the inhibitor for 15 min before the addition of the labeled precursor. The incubation was stopped by the addition of 2 ml of 30% (w/v) trichloroacetic acid, and the assay mixtures were placed on ice for at least 1 hr. The spores were t,hen quantitat)ively transferred to Whatman GF/A glass filter discs (2.4 cm), washed wit,h 40 to 50 ml of water, dried, placed in counting vials containing 10 ml of counting solution [4 g of 2,5-diphenyloxazole, 50 mg of 1,4-bis-2-(&methyl5-phenyloxazolyl)-benzene and toluene to 1 liter], and counted in a liquid scintillation spectrometer. In graphs of these data, all values were corrected for zero-time incorporation. Preparation of ribosome and supernatant enzyme fractions. The procedures for obtaining ribosomes from ungerminated and germinated spores were identical. The spores were disrupted in a Braun MSK mechanical homogenizer (Bronwill Scientific Co.); 3-4 g of spores were combined with 27 g of l-mm glass beads and 8 ml of buffer A and shaken for 60 set at 4000 rpm with simultaneous chilling with CO*. The contents of each flask were combined with an additional 10 ml of buffer A, and the brei was cent,rifuged for 15 min at 2O,OOOg,30 min at 3O,oOOg, and 180 min at 105,OOOg.The final pellet surface was rinsed with 6 ml of buffer B, and the pellet was suspended in a minimlun amount (l-2 ml) of buffer B. This ribosomal suspension was then centrifuged for 15 min at 20,OOOg to remove aggregated and nonsuspended mat,erial; the upper three-fourths of the supernatant fluid (the semipurified ribosome fraction) were removed and used for subsequent ribosome analysis. For the densitygradient analyses, the ribosomal fraction was
444
BRAMBL
AND
always used immediately after preparation; when ribosomes were prepared for other purposes, the suspensions were stored at - 196”. The method for the preparation of the enzyme fraction has been described in detail previously (6,8). Buffer A was composed of sucrose, 0.4 M; Tris0.005 M; and 2-mercapCl, 0.05 M; MgC12.6HOH, toethanol, 0.005 M; buffer B was composed of Tris-Cl, 0.01 M; KCl, 0.015 M; and MgCl,.6HOH, 0.005 M. The pH of buffers A and B was adjusted to 8.5. Buffer C was composed of Tris-Cl, 0.02 M, pH 7.8; magnesium acetate, 0.01 M; KCl, 0.06 M; 2-mercaptoethanol, 0.02 M; reduced glutathione, 0.003 M; and spermine, 2 X lO+ M. Density-gradient centrifugation. The ribosome fractions were analyzed in lO-35% linear sucrose gradient columns prepared by hand-layering four layers of charcoal-clarified sucrose solutions (21) (100, 200, 300, and 400 g sucrose/liter; 5, 7, 7, and 7 ml/layer, respectively) diluted so that the buffer B concentration was uniform throughout the column; the gradient columns were allowed to equilibrate by diffusion for 7 to 12 hr at 4’. The ribosome sample t,o be layered onto the gradient column was diluted to a 0.5-ml volume with buffer B. Beckman SW 25.1 rotors were centrifuged at 23,000 rpm for 180 min under refrigeration. The gradient columns were fractionated and scanned photometrically with an ISCO Model 180 Density Gradient Fractionator (Instrumentation Specialties Co.). The columns were pumped through a flow cell with a l-cm light path at 3 ml/min; the absorbance of the contents was measured at 253.6 rn/* and simultaneously recorded on a constant-speed recorder (20 in/hr) as a fun&ion of column depth. In one set of experiments, 8-drop fractions were collected in glass liquid scintillation vials, diluted with 0.5 ml distilled water, and counted in a scintillation solvent for aqueous solutions (22). The ribosome sedimentation constants were determined by the “linear log” gradient method (23). Brome mosaic virus, tobacco mosaic virus, and southern bean mosaic virus were used as markers. Ribosomal fractions, enriched in polyribosomes, were prepared from both types of spores. Gradient columns of 5-207, sucrose (50, 108, 162, and 216 g sucrose/liter; 5, 7, 7, and 7 ml/layer, respectively, in buffer B) were overloaded with samples equivalent to 3.3 mg or 4.7 mg rRNA from the semipurified ribosomes prepared from ungerminated or germinated spores, respectively. After centrifuging for 200 min the depth of the monoribosome boundary in a representative tube was determined by lightscatt,ering to provide an estimate of the additional time needed (70 min) to pellet the polyribosomes. This technique permit,ted the polyribosomal material to be pelleted while leaving most of the
VAN
ETTEN
monoribosomal material in the supernatant fluid. The small pellets were suspended in a minimum amount of buffer C (0.14.2 ml) and centrifuged for 15 min at 20,OOOg. The supernatant fluid was withdrawn, and this ribosomai suspension was used in the in vitro amino acid-incorporation experiments. The purified monoribosome fractions were obtained by centrifugation of semipurified ribosomal suspensions through the usual IO-357, sucrose density gradients. While each of the tubes was photometrically scanned, the monoribosome fraction of the gradient was collected. These monoribosome fractions were concentrated by centrifugation for 210 min at 105,000g. The pellets were resuspended in buffer C, given a low-speed centrifugation, and the supernatant fluid was stored at -196”. Assay for template activity in spore ribosomes. The assay for amino acid incorporation was similar to that described previously (6), except that the magnesium acetate concentration was reduced to 8 rmoles/ml assay volume. The standard assay (in a final volume of 0.5 ml) also contained 1 PCi of reconstituted ‘Y-protein hydrolyzate (13 amino acids); 0.1 @mole each of the remaining (unlabeled) amino acids; 200 rg of tRNA prepared from germinated spores of B. theobromae (7) ; and 300 rg of enzyme fraction protein. IJnless otherwise indicated, the concentration of ribosomal material was equivalent to 150 /*g rRNA/assay. The glass filters, containing hot trichloroacetic acid-precipitable material, were treated as described previously (6) and counted in a liquid scintillation spectrometer. Other determinations. Protein was determined by the method of Lowry et al. (24) using bovine albumin as a standard. Ribonucleic acid concentration was measured by assuming that 1 mg RNA/ml HOH has an absorbance of 24 at 260 mF. All experiments described in this report were performed two or more times, and the examples cited represent typical results. RESULTS
General characteristics of spore germination. Under the specified germination conditions, B. theobromae conidiospores began to form germ tubes at 2.5 hr, and by 5 hr of germination, 93 % of the spores had germ tubes. During this time, the dry weight of the spores did not change appreciably, but by 9 hr, the dry weight increased about 1.5 times (Fig. 1). Pulse-labeling of whole spores during germination. The kinetics of uracil and leucine incorporation into the spores during
MESSENGER
RIBONUCLEIC
7” t
GERMINATION
t
012345678
9 GERMINATION
TIME (hours)
FIG. 1. Germination and dry weight curves of B. theobromae conidiospores germinated under conditions described in the text.
germination are shown in Fig. 2. The relative rates of incorporation of these precursors of protein and RXA would suggest that protein synthesis began immediately upon placing the spores in the germination medium and continued for a period in the apparent absence of KNA synthesis. For these pulse-labeling data to be valid, how ever, it must be established that the respective labeled precursors are entering the protein or RSA fractions. In other esperimcnts, spores were grown to mnximum germination in the continuous presence of either 14C-leucinr or 14C-uracil; after chemical fractionation of the spores (25), it ~-as determined that S9 7% of t’he 14Cleucine appeawd in the fraction \vhich would be expected to contain hydrolyzed protein (hot :! M ~VatOH), and 94% of the 14C-uracil entered the fraction that contained hydrolyzed nucleic acids (hot 10 ‘3%trichloroacetic acid). Additionally, germinated spores \vtw labeled with 14C-uracil and then incubated in 1 nr SaOH for 1S hr at 3T”, waslhed with cold 5 ‘3%trichloroacetic acid, and the radioactivity determined. As a result of t,his mild alkaline hydrolysis, the high quantity of isotope otSherwise incorporated w-as completely abolished; t,his
ACID
445
IN SPORES
fact indicates that the 14C-uracil entered the RSA of the spores and not the DNA. The substitution of 14C-lysine and l*Curidine for 14C-leucine and 14C-uracil showed that these respective precursors of protein and RKA were incorporated into germinating spores with similar kinetics. The finding that no 14C-uracil was incorporated into the spores during the earlier phases of germination was interpreted to mean that no RNA synthesis was occurring in a period in which protein was synthesized. There are also at least two alternative explanations: (1) the spores contained a pool of RNA precursors which were preferentially incorporated before the exogenous 14C-uracil, and (2) the spores were impermeable to the exogenous 14C-uracil during this stage of germination. At 1.25 hr of germination, when no discernible 14C-uracil was incorporated into the cellular RKA, the spore suspension was pulsed with 14C-uracil, rapidly collected by filtration, 1600/
604-1
I 0
I
2
GERMINATION
3
4
TIME (hours)
FIG. 2. Pulse-labeling experiment: Incorporation of ‘4C-leucine and ‘4C-uracil into germinating spores. The points plotted represent the amount of isotope incorporated during each incubation period, and the connecting lines reflect changes in the capacity for incorporation. The correct,ion for zero-time incorporation iu all these graphs was always less than 5 pmoles per assay.
446
BRAMBL I
1600
AND VAN ETTEN
r
chromatographed along with 14C-UTP on Whatman No. 1 paper in three solvent systems : 0.15 M (NH,)HCO,; NH,OH (coned.) : isobutyric acid: EDTA (0.1 M) : water (1: 66 : 1: 32, v/v/v/v) ; and n-butanol: acetic acid: water (4: 1: 5, v/v/v). In all three solvent systems, the radioactive fraction of the spore extract migrated at the same rate as the authentic 14C-UTP. These facts indicated that the spores were permeable to uracil in the early germination stages and that the uracil was rapidly metabolized to UTP, an immediate precursor of RNA. E$ects of antibiotics upon the precursor incorporation kinetics. Cycloheximide completely inhibited the incorporation of 14Cleucine into germinating spores (Fig. 3). Especially noteworthy was the fact, that the early leucine incorporation was sensitive to this antibiotic, thereby reinforcing the previous indication that this early burst of incorporation reflected genuine protein synthesis. If the conidiospores of B. theobromae were incubated in water (instead of the medium) under standard conditions, a few spores (less than 7%) eventually germinated after 5 or 6 hr, but the germ tubes did not grow more than four or five spore
'I
6001
i l
1
::
5oc-
d,/ i : ;' 8' i ; ;! i: ,i I' ; ,a
400-
LEUCINE ~cmtrol)
0J I 0
2
I
GERMINATION
3
I
4
5
TIME (hours)
FIG. 3. Pulse-labeling experiment: Incorporation of I%-leucine and 14C-uracil into germinating spores after a 15-min treatment with 20 pg/ml cycloheximide.
washed free of the medium with iced water, and then disrupted in the homogenizer in the presence of cold 5 % trichloroacetic acid.
This trichloroacetic
acid extract
was then
I4
i
5mr I
9
LEUCINE INCORPORATION
1200-
URACIL
lNCORPORATlOi’l
i
ii 400cc z p2 300-
I
8 b Y zoo-
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f MEDIUM
i
,’
d s
!
7400u z
0
I
2
3
MEDIUM
;’ .’
i
4 GERMINATION
TIME
(hours)
FIG. 4. Pulse-labeling experiment: Incorporation of 14C-leucine and I%-uracil into spores incubated in water under standard conditions; also shown is the effect of a 15.min treatment with 20 pgg/ml cycloheximide upon the W-leucine incorporation pattern.
MESSENGER
RIBONUCLEIC
ACID
IN
SPORES
ACTINOMYCIN
I
2
4
3
GERMINATION
FIG. 5. Pulse-labeling germinating spores after
TIME
I (hours)
2
447
D
3
4
experiment: Incorporation of ‘Gleucine and 14C-uracil a 15-min treatment with 25 rg/ml actinomycin D.
diameters in 8 hr. The results of a pulselabeling experiment in which the spores were incubated in water and with cycloheximide are demonstrated in Fig. 4. It is evident that such spores incorporated r4Cleucine in a pattern which initially resembled the early pattern obtained under ordinary germination conditions, while essentially no incorporation of 14C-uracil could be detected. This leucine incorporation was abolished by cycloheximide. Incubation of the spores with actinomytin D for 15 min prior to the addition of 14C-uracil caused an inhibition of uracil incorporation without having any effect on the incorporation of leucine in the early stages of germination. The rapid increase in leucine incorporation, which occurred later in the germination sequence, was slightly depressed by actinomycin D (Fig. 5). In a separate experiment, the spores were incubated in the continuous presence of actinomycin D (25 pg/ml), and samples were taken at various times and pulselabeled with 14C-leucine and 14C-uracil. Again, the incorporation of leucine in the early stages of germination was unaffected by actinomycin D, while at 5 hr there was approximately a 70% inhibition of leucine incorporation (226 pmoles, treated spores: S24 pmoles, untreated spores). At this same
into
germination stage, the uracil incorporation was inhibited about SO% by actinomycin D (243 pmoles, treated spores: 1419 pmoles, untreated spores). It is possible, however, that only the germinating spores are permeable to the antibiotic. Therefore, these findings suggest that the early increase in protein synthesis was independent of new RNA synthesis, but dependent on preexisting mRNA, while the later protein synthesis was at least partially dependent on the synthesis of new RNA. When the spores were germinated continously in 2.5 pg/ml of actinomycin D, the germination rate was unaffected. However, after 8 or 9 hr of germination when the spores in the control incubation had profusely branched germ tubes, those spores treated with actinomycin D had germ tubes which were nearly devoid of branches. Density-gradient analyses of ribosolnal j+ractions. The density-gradient columnscanning profiles of centrifuged ribosome fractions from spores at 0, 1, 2, 3, 4, and 5 hr of germination are shown in Fig. 6. Polyribosomes were clearly detectable at all stages of germination, including the ungerminated spores. It is evident that by 4 hr of germination, there were more polyribosomes extracted from the spores than in previous stages. The sedimentation
448
BRAMBL
II hour SPORES
I
AND
VAN
ETTEN
2 hew
lhcurSPORES
SPORES
DEPTH IN COLUMN
3hour SPORES
II
4 hcu SPORES
0.4-
0.4-
0.3-
0.3-
n
5 hwr SPORES
ll
0.4
2 2 '0
03
Y 5
0.2
:
4% 0.1 BOTTOM :I
I: hL 0.2-
0.2
0 I-
0.1
DEPTH IN COLUMN
FIG. 6. Sedimentation patterns of semipurified ribosomes obtained from spores at indicated stages of germination. Direction of sedimentation is from right to left in all scanning patt,erns. Hibosome samples on each gradient column were equivalent to 266 fig of ribosomal RNA. The chase solution contained potassium acid phthalate, and the increase in OD at the bottom of the tube is due to this chase solution.
constants of the monoribosomal subunits were determined to be 37 S and 60 S; the monoribosomes, 81 S; the dimer and trimer polyribosomes, 123 S and 164 S. In order to determine if the relatively low population of polyribosomes extracted from the ungerminated spores was due to a selective RNase activity in these homogenates, a mixed experiment was performed in which purified ribosomes from germinated spores were mixed with a 30,OOOglowspeed supernatant fraction from the ungerminated spores. The crude extract from the un germinated spores had no deleterious effect upon the polyribosomal profile from the germinated spores.
Treatment of the ungerminated spore ribosomal fraction with 0.0,5 M EDTA caused a complete dissociation of the ribosomes into the two ribosomal subunits. The exonuclease spleen phosphodiesterase (3 pg/ml) had no apparent effect upon the polyribosomal profile. If the semipurified ribosomes from ungerminated or 5-hr germinated spores were treated with 5 pg/ml pancreatic ribonuclease for 15 min at 0” before density-gradient centrifugation, the polyribosomes were nearly abolished (Fig. 7). However, ribonuclease treatment invariablv caused the appearance of an apparent dimer of ribosomes which was seemingly resistant to
MESSENGER
I 0.4-
UNGERMINATED +RleONUUEASE
RIBONUCLEIC
ACID
IN
449
SPORES
n +WONUUEASE
1 BOTTOM1
u
TOP
DEPTH IN COLUMN
FIG. 7. Effect of 5 pg/ml pancreatic ribonuclease upon ribosomes from and germinated spores. Incubation conditions are given in text. Samples 200 pg ribosomal RNA. See Fig. 6 for control experiment curves.
ribonuclease. If the concentrations of ribonuclease were increased or decreased lo-fold, there was no change in the size of this dimer unit; other alterations which produced no change in the dimer peak included incubation of the ribosomes with ribonuclease for longer time periods at 30” and shifting the pH of the ribosomal suspension closer to the optimum for ribonuclease. However, a brief treatment of the ribosomes with cY-chymotrypsin (10 pg/ml) before the usual treatment with ribonuclease caused the dimer peak to disappear in the scanning patterns. To investigate this phenomenon further, monoribosomes from the ribosomal fractions of both types of spores were carefully isolated from preparative density-gradient columns, concentrated by high-speed centrifugation, and then recentrifuged through density-gradient columns. As a result, dimer ribosome peaks appeared. If it is assumed that the isolated monoribosome fractions were uncontaminated with dimer units, this experiment would suggest that the anomalous peaks which appeared after ribonuclease treatment were due not to incomplete degradation of the polyribosomes, but to spontaneous aggregation of liberated monoribosome units. It is important to note that no aggregates were
ungerminated equivalent to
detected which sedimented faster than the ribosome dimers. Because the ungerminated spores contained polyribosomes which were detectable by their physical properties, the following experiment was performed to test the functional activity of these ribosome units. The spores were suspended in the germination medium in the usual manner, and a lliter sample immediately withdrawn and incubated with reconstituted 14C-protein hydrolyzate (0.05 &X/ml medium) at 34” for 30 min. At the conclusion of the incubation, these labeled spores were rapidly chilled with large quantities of crushed ice, collected on a filter, and washed free of medium and extraneous labeled amino acids. These spores were then mixed with similarly prepared unlabeled carrier spores, and the spores were disrupted by the usual procedure; the ribosomal fraction was isolated by high-speed centrifugation, and aliquots of the suspended semipurified ribosomes were layered onto sucrose density-gradients, centrifuged, and scanned. During the scanning process, S-drop fractions were collected and analyzed for radioactivity. In Fig. 8, a plot of the radioactivity patterns superimposed on the optical scanning profile from the same gradient shows that the absorbance peaks of the
450
BRAMBL AND VAN ETTEN r :: :;
0.4
+RIEoNuCLEASE
200 175 0.3150 125 0.2-
'
(SdWs
each)
100
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75
25
, m
35
I TOP
BOTTOM FRACTIONS
DEPTH IN COLUMN
FIG. 8. Sucrose density-gradient optical scanning pattern of semipurified ribosomes from spores germinated 30 min in presence of W-amino acid mixture. Also shown is the effect of 5 pg/ml pancreatic ribonuclease upon these patterns. The gradients were fractionated into S-drop samples for radioactivity determinations. Ribosomal samples were equivalent to 200pg of ribosomal RNA. The solid line represents the optical density and the dashed line, radioactivity.
polyribosomes were associated with radioactivity from the 14C-amino acids. The deleterious effect of ribonuclease upon both the absorbance and radioactivity profiles is also demonstrated in Fig. 8. Therefore, the polyribosomes detected in the ungerminated spores had the capacity to become functionally associated in vivo with labeled amino acids during the first 30 min of germination. Cell-free amino acid incorporation assay. In order to test whether ribosomes extracted from ungerminated spores possessed template activity, the polyribosome-enriched fraction and purified monoribosomes were assayed with a supernatant enzyme fraction and tRNA obtained from 5-hr germinated spores. The data in Table I show that the polyribosome fraction in both germinated and ungerminated spores had the capacity to stimulate amino acid incorporation into hot trichloroacetic acid-precipitable material above the amount of incorporation with monoribosomes. If the amount of polyribosomal RNA added was halved or doubled, the resulting amount of 14C-amino acid incorporation was correspondingly reduced or increased. The deletion of tRNA
reduced incorporation by about 32% in both cases, while the deletion of the enzyme fraction or the polyribsomal material severely reduced incorporation. Treatment with ribonuclease reduced incorporation by about 62 % in both cases, and puromycin reduced incorporation by about 50 % ; as might be expected, chloramphenicol essentially had no effect upon either assay system. DISCUSSION The results of several early studies (1416) suggested that ungerminated fungal spores may be deficient in mRNA; one implication of such a suggestion was that this defect could participate in the regulation of dormancy and germination. This hypothesis is not supported by the evidence presented in this report; instead, results of the experiments described argue persuasively that the ungerminated conidiospores of B. theobromae do contain mRNA, that this mRNA is immediately functional upon initiation of germination, and that this mRNA apparently contains enough information to allow the formation of a germ tube.
MESSENGER TABLE
RIBONUCLEIC
I
TI,;MPL.\T~S ACTIVITY FROM UNGERMINATED
OF RIBOSOM~L FRICTIONS AND GERMINATED SPORES OF B. theobromae IN THE PRESENCE nw ENZYMEFR~CTION FROM THE GERMIN.4TEDsPORES
Assay
Ribosomal
material
Counts/ min incorporated per assaya
system
1
Deletions
or additions
1972 1208 186 155 1720 1647 1201 943 576 273 1329 872 134 407
Polyribosomesh Monoribosome9 ~2 polyribosomes 3s polyribosomes
II
Polyribosomes Polyribosomes Polyribosomes Polyribosomes Polyribosomes Polyribosomes
!
- polyribosomes - tRNA - SKOenzyme fraction +ribonuclease (30 rd -l-puromycin (50 NT) +chloramphenicol (50 !A +cycloheximide (50 Pd
II
756 458 952 579 19611173 583 377 I
a All values are corrected for zero-time incorporation. b The terms “polyribosomes” and “monoriboused here refer to the polyribosomesomes” enriched ribosomal fraction and to purified monoribosomes, respectively, the preparations of which are described in the text. Ribosomal material in each assay tube was equivalent to 150 lg of ribosomal RNA unless otherwise indicated.
In the germinating spores of this organism, the precursor incorporation kinetics indicate that resumption of protein and RNA syntheses are temporally separate and distinct events. The fact that a cycloheximide-sensitive protein synthesis is begun immediately upon initiation of germination, but in the absence of any detectable RNA synthesis, clearly suggests that these latent spores contained mRNA which is responsible for the protein synthesis required for development in the first
ACID
IN SPORES
451
phases of germination. This interpretation of the evidence necessarily assumes that no internal pools of RNA precursors selectively depress exogenous precursor uptake during the first X.5 hr of germination and that the cells are permeable to the labeled precursor during the same period. The validity of the latter assumption is shown by the fact that by 1.2.5-1.5 hr of germination, when no uracil was incorporated, the 14C-uracil was not only taken up by the spores, but was converted to UTP, an immediate precursor of RNA. We have no evidence that hypothetical endogenous pools of RNA precursors in the spores could influence uptake of exogenous precursors. Further evidence that the initial protein synthesis is supported by a preexisting mRNA is provided by the results of treatment of the germinating spores with actinomycin D. If the synthesis of protein early in germination required simultaneous synthesis of mRNA, the actinomycin D treatment should have at least partially inhibited the incorporation of 14C-leucine provided ungerminated spores are permeable to this compound. The protein synthesis early in germination appears to be independent of concurrent RNA synthesis. Density-gradient analyses of spore extracts clearly indicated that discrete classes of polyribosomes exist in all the germination stages examined, including the ungerminated spores. The ribosomes were readily dissociable by EDTA treatment, and the polyribosomal material was sensitive to pancreatic ribonuclease. After treatment, with ribonuclease, however, dimers of ribosomes were prominent in the scanning patterns. A similar effect was noted, but not discussed, by Henney and Storck (14) in their study of N. crassa. Our evidence would suggest that this ribosome dimer is caused not by incomplete digestion of the polyribosomes but by spontaneous aggregation of monoribosome units. However, Mackintosh and Bell (26) recently reported the appearance of these dimers in sea urchin egg ribosomes treated with ribonuclease, and they ascribed considerably more significance to the phenomenon of this dimer ribosome peak.
452
BRAMBL
AND
That the mRNA of polyribosomes of ungerminated spores is apparently functional and probably supports the protein synthesis in the early stages of germination was demonstrated by the ability of the polyribosomes to become associated in viva with 14C-amino acids during the first 30 min of germination. Furthermore, polyribosome-enriched ribosome fractions isolated from both germinated and ungerminated spores had the capacity to stimulate amino acid incorporation at levels somewhat above the incorporation stimulated by purified monoribosomes. Although the level of incorporation in the germinated spore polyribosome fraction was higher than its counterpart from the ungerminated spores, the amino acid incorporation systems from both spore stages had characteristics expected of an authentic amino acid incorporating system. The conclusion that mRNA is conserved in ungerminated spores is supported by recent reports from other laboratories; Staples et al. (17) have presented evidence that polyribosomes exist in ungerminated U. phaseoli uredospores, and Lovett (18) and Hollomon (19), each using selective metabolic inhibitors, have shown that B. emersonii and P. tabmina spores do not require simultaneous RNA synthesis to support the protein synthesis necessary for germination. Dunkle et al. (27) have presented similar evidence that urediospores of Puccinia grawzinis tritici may germinate in the presence of RNA synthesis inhibitors. Therefore, the results of these several studies, when included with other information available about protein synthesis in fungal spores, suggest that many of the known macromolecular components, enzymes, and informational molecules of the protein synthesis apparatus are present in the ungerminated spores and can be extracted in an active form. ACKNOWLEDGMENTS The authors are indebted to Dr. Myron K. Brakke and Mrs. Nancy Van Pelt for generously providing the marker viruses and useful advice about the density-gradient centrifugation. Mr. Don Merlo, Mrs. Sandra Swanda, and Mr. Rex Koski helped provide us with spore material. This investigation was supported in part by funds from the Research Council of the University of
VAN
ETTEN
Nebraska National
and from PHS grant Xnst.itutes of Health.
Al08057
of the
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