Developmental stages during the germination of Mucor sporangiospores

EXPERIMENTAL MYCOLOGY 12, 47-59 (1988)

Developmental Stages during the Germination of Mucor Sporangiospores

CARMEN CANO*‘~ AND JOSE RUIZ-HERRERA*?~

“Institute de Znvestigacion en Biologia Experimental, Universidad de Guanajuato, and tDepartmento de Genetica y Biologia Molecular, Centro de Znvestigacion y Estudios Avanzados, ZPN, Mexico

Accepted for publication September 25, 1987

CANO, C., AND RUIZ-HERRERA, .I. 1988. Developmental stages during the germination of Mucor sporangiospores. Experimental Mycology 12, 47-59. Sporangiospores from Mucor rouxii or M. bacilliformis placed in an appropriate culture medium enter into a developmental growth program with three discrete phases: Ia, characterized by acquisition of a spherical shape by the spore; Ib. where a maximal spherical volume is obtained by the spore; and II, after germ tube or bud emergence. During spore germination protein and RNA content increased exponentially without a noticeable lag phase and with no change in rate during all the growth phases. Their inhibition stopped spore germination at discrete steps, suggesting that specific mRNAs and proteins were required at each phase. DNA biosynthesis started after a long lag phase, about 30 min before the onset of growth phase II. Its inhibition prevented the cells from entering phase II, but did not prevent nuclear division. It is suggested that Mucor spores are in the G2 phase of the cell cycle, with their genetic information “programmed” for spherical growth, and that DNA replication is required for the synthesis of specific mRNAs necessary for the onset of polarized growth, Q 1988

Academic Press, Inc. INDEX DESCRIPTORS: Mucor; spore germination; development; DNA synthesis; protein synthe-

sis; RNA synthesis.

Several Mucor species are characterized by their dimorphic behavior; i.e., they can display mycelial or yeast-like growth de- pending on the environmental conditions (Bartnicki-Garcia, 1963). However, an interesting observation is that regardless of the growth conditions, sporangiospores go through the same growth pattern in the initial stages of germination. Ellipsoidal or bacillus-like spores become spherical and grow isodiametrically before the emergence of germ tubes or buds, according to the environmental conditions (Bartnicki-Garcia et al., 1968). This initial phase of growth does not merely involve water uptake; active macromolecule biosynthesis, organelle formation, and development of a new cell wall (Bartnicki-Garcia et al., 1968) all take place. Incipient buds are not morphologi-

i Predoctoral fellow from CONACyT, Mexico. ’ Investigador National, Mexico.

tally different from emerging germ tubes (Lara and Bartnicki-Garcia, 1974) but they soon become spherical and grow isodiametrically, whereas germ tubes grow apicahy (Bartnicki-Garcia and Lippman, 1969).

This pattern of germination suggests that Mucor spores contain the information to grow isodiametrically, and it is not until a certain phase of their development has passed that they acquire the informative for polarized growth. This pattern of growth may then be maintained (hyphae) or only temporarily exhibited (yeasts). Our recent observations on the effects of ornitbine carboxylase inhibitors (Calvo-Men&z an Ruiz-Herrera, 1987) support this hypot sis and point out the role of polyamines in the onset of polarization mechanisms. Ac- cordingly, germination of Mucor ~spomn- giospores appears to be an attractive model for a developmental process. In this work we have studied germination of s~~ra~~~~-

41 0147-5975188 $3.00 Copyright Q 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

48 CAN0 AND RUIZ-HERRERA

spores from M. rouxii and M. bacilliformis with emphasis on their morphological changes, their patterns of macromolecule biosynthesis, and the effect of some inhibitors on further spore development. We have concluded that germination occurs in discrete steps, each requiring specific mR- NAs and proteins, and that DNA replication is required for the onset of polarized growth.

MATERIALS AND METHODS

Fungal strains and growth conditions. M. rouxii IM-80 (ATCC-24905) and M. bacilliformis NRRL-2346 (ATCC-12830) were used in this study. Spores were obtained from Roux bottles containing solid YPG medium (Bartnicki-Garcia and Nickerson, 1962) with 2 or 0.5% glucose inoculated with M. rouxii or M. bacilliformis, respectively, and incubated for 6 days at 28°C (M. rouxii) or 8 days at 24°C (M. bacilliformis). Spores were washed three times with ster- ile distilled water by centrifugation and used before 4 days of storage at 4°C. The behavior of spores used in this range was similar. Spores were counted with a hemo- cytometer and inoculated (5 x lo5 per ml) in Erlenmeyer flasks containing the syn- thetic liquid medium described by Bart- nicki-Garcia and Nickerson (1962) except that 0.625% casamino acids (Difco) were used as nitrogen source and the medium was supplemented with 2 or 0.5% glucose for growth of M. rouxii or M. bacilliformis, respectively. Flasks were shaken in a water bath at 28°C for M. rouxii or 24°C for M. bacilliformis unless otherwise stated. Aer- obiosis was provided with a stream of ster- ile air, whereas anaerobiosis was maintained by sparging with a mixture of N,:CO, (70:30, v/v) (20-30 mUmin). Sam- ples of different volumes were withdrawn at intervals and used for analyses.

Determination of spore morphology, volume, and water content. Samples obtained from the cultures were treated with an equal volume of 0.4% glutaraldehyde in 0.1

M cacodylate buffer, pH 7.0, and centrifuged. The cells were resuspended in one- tenth of the original volume of water. Sam- ples were observed and photographed in a Zeiss laboval 2a-fl microscope. Negatives were observed with a calibrated dissecting microscope and the size of at least 100 spores per sample was measured. To calcu- late the volume, ungerminated spores were considered as ellipsoids of revolution.

To measure water content, cell samples were centrifuged in graduated centrifuge tubes, and excess water was eliminated by placing the tubes upside down over paper towels. Samples were weighed and dried at 105°C to constant weight. Intercellular water content was measured with blue dextran (Pharmacia) .

Chemical methods. We employed chemical determinations of proteins, RNA, and DNA to follow their accumulation. Cells were collected by filtration through Milli- pore filters (5-pm pore diameter), washed three times with distilled water, and frozen until analyzed. For protein and RNA determination, samples were treated at 0°C with acetone and ethyl ether as described by Storck and Mot-t-ill (1977). RNA was extracted with perchloric acid (PCA)3 as described by these authors and measured by the orcinol method with ribose as the standard (Schneider, 1957). RNA concentration was calculated from the ribose values con- sidering the average nucleotide molecular weight. Protein in the PCA-insoluble frac- tion was measured by Lowry’s method using serum albumin as standard. DNA was measured as described by Cattolico and Gibbs (1975) using calf thymus DNA (Sigma) as standard, calibrated by its absor- bance at 260 nm, assuming that 1 mg/ml solution of DNA has an absorbancy of 22 (l-cm light path). Protein, RNA, and DNA content were expressed in grams per either 1013 or lOI spores.

3 Abbreviations used: PCA, perchloric acid; DAPI, 4,6-diamine-2-phenyl indole.

Mucor SPORE GERMINATION 4

Nuclei staining. After several methods were tried, the best results were obtained by use of 4,6-diamine-2-phenyl indole (DAPI) @later, 1976). Cells were fixed in 70% ethanol for 30 min at 22”C, washed twice with water by centrifugation, stained for 10 min at 22°C with 0.5 pg/ml DAPI (M. rouxii) or 0.25 pg/ml DAPI (M. bacilliformis), and washed three times with water by centrifugation. These concentrations of stain were optimal and selectively stained the nuclei on a nonfluorescent background. The concentration of DAPI recommended for Candida albicans and Saccharomyces cerevisiae (5 pg/ml; Slater, 1976) stained other cell structures and left some background. Stained cells were mixed with the same volume of 0.5 pglml diphenylamine (as a screen), resuspended in 0.5% agar, and observed and photographed in an epi- fluorescence microscope (Zeiss laboval 2a- fl) with a set of blue filters (2XK-P490 plus B229 and G247/G245). Nuclei were counted in photographs of at least 100 cells.

Electron microscopic observations. Cells in growth medium were mixed with an equal volume of 4% glutaraldehyde in 0.1 it4 cacodylate buffer, pH 7.2 (previously taken to the sample temperature). After 15 min, samples were centrifuged, resuspended in

2% glutaraldehyde for 2 h, centrifuged, washed five times with cacodylate buffer, postfixed with 2% osmium tetroxide for 3 washed three times with cacodylate buffer, and washed twice with distilled water. Cells were stained for 2 h with 2% uranyl acetate, washed with water, dehydrated in a graded series of ethanol, treated twice with propyl- ene oxide, and embedded in Poly- sections were stained with 2.5% ~ra~~i acetate and lead citrate. Specimens were examined and photographed with a Jeol 108s electron microscope at 60 keV.

RESULTS

Physical Parameters during the Germination of the Sporangiospores from M. rouxii and M. baciiliformis

Sporangiospores from both M. rouxii and M. baciiliformis have an ellipsoidal morphology, but their respective sizes are very different (Table 1). M. bacilliformis spores contain lower amounts of protein, R and DNA (Table 1). This last important ference was first noticed by Storck an Morrill (1977). Our quantitative data somewhat different from those obtain these authors, possibly due to the differen% methods used, the different calibration

TABLE 1 Physical and Chemical Parameters of Ungerminated Mucor Sporangiospores”

Species

Mucor roux2 Mucor bacilliformis

Long axis (w-4

4.15 F 0.2 2.0 rt 1.0

Physical parameters

Short axis (w)

2.80 t 0.13 0.78 +- 0.20

Chemical parameters (g per 1Ol4 spores)

--___- Volume b-m”;

17.04 5.0

Mucor vouxii Mucor bacilliformis

DNA

5.06 ” 1.63 1.95 ct 0.58

RNA

92.8 k 0.02 36.3 ‘- 2.3

Proteifi

939 t 141 78 t I.0

- a Physical parameters were measured for 500 spores obtained in five different experiments, DNA data for M.

rouxii come from 19 different spore batches, and for M. bacilliformis, from 4 batches; RNA and protein were measured in spores from 6 different batches for both species. For methods see the text.


standards employed, and the way data are expressed. Lin and Inderlied (Inderlied et al., 1985) have described a higher content of DNA in M. rucemOSuS sporangiospores.

When placed in an appropriate culture medium, spores germinate in two stages. First, spores grow isodiametrically (spherical growth or phase I) and after reaching a critical stage they emit one or more germ tubes in aerobic conditions or one or more buds in anaerobic conditions with high CO, tensions (polarized growth or phase II; Bart- nicki-Garcia et al., 1968). Upon closer examination we observed that spherical growth in M. rouxii can be divided into two phases: one where the spore morphology changes from ellipsoidal to spherical without a noticeable change in the long axis (phase Ia), and a second one where the spherical spore increases in size (phase Ib). Support for this concept was provided by use of inhibitors (see below). The several stages in germination and the average times at which they occur under the standard conditions used are shown schematically in Fig. 1.

Volume increase of the spores of M. rouxii during the phase of spherical growth in aerobiosis was exponential up to 656 2 17 pm3. Under these conditions no noticeable further increase in volume occurred after the appearance of germ tubes. On the other hand, under anaerobiosis the increase in spore volume was exponential only up to

FIG. 1. Diagrammatic representation of the stages of germination of Mucor rouxii spores in aerobiosis or anaerobiosis. Average times (in h) under our standard conditions are indicated. Phase 0, dormant spore; phase la, spherical spore: phase Ib, “swollen” spore; phase II. after germ tube or bud emergence.

200 pm3 and the mother cell continued increasing in volume during the whole budding process (Fig. 2). Similar results were obtained with M. bacilliformis. Bacillus- like spores of this species, with a volume of 5.0 pm3, reached a final volume similar to that of M. rouxii before development of germ tubes or buds. This value represents an approximate 130-fold increase in volume.

The water content of the native M. rouxii spores was 58%, whereas that from the fully swollen spores was 80%.

Protein and RNA Synthesis during Sporangiospore Germination

When M. rouxii spores were placed in an adequate culture medium, the content of protein and RNA increased exponentially in either aerobiosis or anaerobiosis without an apparent lag phase (Figs. 3 and 4). No change in the rate of accumulation of either macromolecule occurred during germ tube

I /’ e 4 6 8 10 I2 14 16 18 20

TIME OF INCUBATION (h)

FIG. 2. Volume increase of M. rouxii sporangiospores during germination. Samples of cells grown for variable periods of time either aerobically or anaero- bically were removed, treated with glutaraldehyde, centrifuged, washed, and photographed as described under Materials and Methods. Cell size of at least 100 spores per sample was measured and the volumes were calculated. Cell volume of aerobic spores (0); cell volume of anaerobic spores (A), mother cells only; percentage aerobic cells with germ tubes (0); percentage anaerobic-budding cells (A).


1 2 3 4 5 TIME OF INCUBATION (h)

FIG. 3. Protein and RNA synthesis during aerobic germination of M. rouxii sporangiospores. Cells were grown in defined medium. At intervals samples were removed, cells were harvested and processed, and RNA and protein were measured as described under Materials and Methods. A smaller aliquot was fixed with glutaraldehyde to determine the germination stage. RNA concentration in grams per lOI spores (0); protein concentration in grams per 1013 spores (@); percentage of cells with germ tubes (x).

or bud emergence. Similar results were obtained with M. bacilliformis (not shown).

We measured the effect of cycloheximide and actinomycin D on protein and RNA biosynthesis. At the selected concentrations, cycloheximide inhibited protein biosynthesis in germlings of both Mucor species by more than 98% during the period of examination (5 h). Using the techniques described under Materials and Methods, it was observed that actinomycin D inhibited RNA accumulation by ca. 95%, but did not inhibit protein synthesis in Mucor germlings, for at least 3 h. We tested the effect of both inhibitors on the germination of sporangiospores from M. rouxii. Spores were incubated aerobically, and at intervals culture samples were removed, an inhibitor was added to the sample, and the incubation was continued. After 5 h the cell morphology in each culture was scored (Table

TIME OF INCUBATION (hl

FIG. 4. Protein and RNA synthesis during the anaerobic germination of M. rouxii sporangiospores. Cells were grown in defined medium under an atmo- sphere of N,:CO, (70:30, v/v). At intervals samples were removed and treated as described in Fig 3. RNA concentration in grams per lOI spores (0); protein concentration in grams per lOI spores (0); percentage budding cells (x).

2). At this time 80% of the untreated control cells were in growth phase II. Cyclohexi- mide added at the time of inoculation completely blocked spherical growth of the spores. Tripp and Paznokas (1982) also reported that cycloheximide completely blocked germination of M. racemosus rangiospores. Addition of the inhibitor 30 minutes allowed completion of phase 1a but prevented phase Ib of growth. This growth phase occurred only if cycloheximide was added after 90 minutes of incubation. Even when added to 4-h-old cultur cycloheximide inhibited phase II of grow by 87%. Addition of actinomycin D at the time of inoculation allowed the phase Ta of growth to proceed in 50% of the spore pop- ulation. Orlowski and Sypherd (1978) reported that actinomycin D inhibited germ tube emergence but allowed the completion of the swelling stage of M. racemosus spo-


TABLE 2 Effect of Cycloheximide and Actinomycin D on

Germination of Sporangiospores from Mucor rouxii”

Time of Maximal phase of development

inhibitor after 5 h (percentage)

addition Cycloheximide Actinomycin D

0 0 la (50%) 0.5 la (100%) - 1.0 la (100%) Ib (100%) 1.5 Ib (100%) - 2.0 Ib (100%) tb (100%) 2.5 lb (100%) - 3.0 Ib (100%) 11 (60%) 3.5 II (2%) - 4.0 II (10%) 11 (70%)

a Spores were inoculated in minimal medium. At the indicated times, aliquots (10 ml) were transferred to 50 ml Erlenmeyer flasks and either cycloheximide (50 pg per ml) or actinomycin D (200 p,g per ml) was added. After 5 h of incubation, aliquots of each flask were withdrawn, glutaraldehyde was added, and morphology was scored as described under Materials and Meth- ods. At this time 80% of control spores with no addition were in phase Il. Phase 0 corresponds to spores with the original oval morphology; la, “round” cells; Ib, “swollen” cells; phase 11, spores with one or more germination tubes. In the control, DNA increase was noticed after 3.5 h and emission of germ tubes started after 4 h of incubation.

rangiospores. The difference with our results may be due to a slow escape of spores from the inhibitory action of actinomycin D. It should be noted that our observations were made after 5 h of incubation. Addition of actinomycin D after 1 or 2 h allowed completion of phase Ib, but prevented spores from proceeding into phase II. Ad- dition after 3 h barely inhibited (25%) phase II of growth. Accordingly, it is proposed that growth of the sporangiospores of M. rouxii may occur in the form of a specific developmental program, each step requiring different populations of mRNAs and proteins. Messenger RNAs, but not proteins, required specifically for phase Ia of growth, are mostly present in the original sporangiospore, whereas proteins required for phase Ia are synthesized during the first 30 minutes of incubation. At least some

mRNAs specific for phase Ib are not present in the quiescent spore and are synthesized within 1 h of growth; synthesis of proteins specific for this phase occurs later; mRNAs for phase II of growth are synthesized between 3 and 4 h, but proteins re- quire a longer time to be synthesized. These times, of course, are relative and can be applied only to the particular conditions we employed. The implications, on the other hand, are general.

DNA Biosynthesis during Germination of Sporangiospores

In contrast to the synthesis of RNA and protein, the synthesis of DNA exhibited a lag phase during spore germination, in both Mucor species, under either aerobiosis or anaerobiosis. Data for M. rouxii appear in Fig. 5. After this lag, synthesis of DNA started about 30-45 minutes before the on-

r I

I 3 5

I: I’ I’ dI

,A 3 6 9 12 15 18

TIME OF INCUBATION (h)

FIG. 5. DNA biosynthesis during germination of M. rouxii sporangiospores. Spores were grown in defined medium either aerobically or under a stream of N,:CO,. At intervals samples were removed, cells were harvested and treated as described under Mate- rials and Methods, and the DNA was measured (cir- cles). Small samples were also removed and treated with glutaraldehyde to determine germination stage (triangles). DNA content in aerobic cells (grams per 10“’ spores) (0); DNA content in anaerobic cells (grams per 1V4 spores) (0); aerobic cells with germ tubes (A); anaerobic budding cells (A).


set of phase II of growth and proceeded exponentially for at least a few hours.

We studied the effect of inhibition of DNA biosynthesis on spore germination. Storck and Morrill(1977) reported that germination of M. bacilliformis spores is inhibited at 30°C. Spores go through phase I of growth, but do not emit buds or germ tubes. We confirmed these results and found that at 30°C spores synthesize proteins and RNA, although less efficiently than at 25”C, but do not synthesize DNA. Also they do not go beyond phase Ib of growth (Table 3). Under these conditions synthesis of orni- thine decarboxylase is prevented (Calvo- Mendez et al., 1987). It may be hypothe- sized that incubation at high temperature inactivates particularly sensitive proteins necessary for DNA duplication. Addition of hydroxyurea to spores of both Mucor species prevented DNA, but not protein or RNA, accumulation (at least for 8 h; Table 4). In its presence, spores completed phases Ia and Ib of growth and a variable proportion (30-55%) entered phase II of growth. However, further incubation proved that phase II of growth did not con- tinue; instead, cell volume increased to high ,values (ca. 7000 p,m3) as if cell shape

had “frozen” and the cells had continued ’ phase Ib of growth (Fig. 6). The effect hydroxyurea was more clearly seen in bacilliformis where spores entered phase Ib, but germ tube emergence was completely inhibited. Cells continued to grow spher- ically and reached large volumes (Fig. 7). These swollen cells were highly fragile, but addition of 0.5 M sorbitol prevented cell breakage. Van Etten et al. (1976) obs no effect of hydroxyurea on the ger tion of spores from Rhizopus s~o~oni~r and Botryodiplodia theobromae. This discrep- ancy with our data may be due to the lower concentration of inhibitor (10 mm used by these authors. The concentration of hydroxyurea we used is slightly higher than that used to synchronize Saccharomyces cerevisiae cells at the onset of the S phase of the cell cycle (Eorincz and Miller, 1982). The effects of growth at 30°C and hydroxyurea were both completely reversible for at least 12 h. Transfer of M. bac~~l~ormis sporangiospores to 25°C or removal of hydroxyurea from sorbitol-containing cultures with both Mucor species restored phase II of growth. The morphology of t tubes, however, was irregular and took some time to become normal.

TABLE 3 Macromolecule Biosynthesis during Germination of Mucor bncilliformis Sporangiospores at

Two Temperatures” -

Time of Spores in Spore Macromolecule concentration

Temperature incubation phase II volume (g x lOI spores)

of growth (“C) 04 (percentage) bm3) Protein RNA DNA

25 0 0 N.D.b 18 35 1.53 2 0 N.D. 172 48 1.73

14 50 654 7060 209 9.53 18 96 N.D. N.D. N.D. 16.20

30 0 0 N.D. 78 35 P.53 2 0 N.D. 130 43 1.53

14 0 57.9 614 98 I .66 18 0 61.6 746 150 I .46

a Spores of M. badijormis were inoculated in minimal medium and incubated under aerobiosis at 25 or 30°C. At intervals, aliquots were taken, and parameters for the spores were measured as described under Materials and Methods.

b N.D., Not determined.


TABLE 4 Effect of Hydroxyurea on Macromolecule Biosynthesis by Sporangiospores from M. roux+

Hydroxyurea (mM)

Time of incubation

6-4 Protein

Macromolecule concentration (g X 1014 spores)

RNA DNA

0 0 960 106 6.6 8 11200 1030 24.3

110 8 23260 1120 6.3

a Spores were incubated in minimal medium with or without 110 n&I hydroxyurea. After 8 h, samples were analyzed as described under Materials and Methods.

Nuclear Division during Sporangiospore Germination

As previously described by Storck and Merrill (1977), we observed that most spores of M. rouxii contain a single nucleus, although a small proportion contain two or three nuclei (Table 5, Fig. 8). When spores were inoculated into growth medium, the number of uninucleated spores de- creased and those with two or more nuclei increased, well before synthesis of DNA started. Between 3 and 4 h of incubation, when DNA synthesis was initiated, the average nuclei per spore was about 2 (Table 5). Similar data were obtained with M. bacilliformis. In this organism the completion of nuclear division prior to DNA synthesis was more clearly shown by incubation at 30°C (Table 6). Nuclear division occurred although DNA synthesis was halted. This phenomenon was also observed in M. roaxii cells treated with hydroxyurea. After 8 h of exposure to the drug, the cells pre- sented more than one large nuclear mass as revealed by both DAPI staining (Table 6, Fig. 9) and electron microscopy (not shown). After 18-24 h cells contained a large number of smaller fluorescent bodies (Fig. 10). By electron microscopy, however, these multiple fluorescent bodies observed after long periods of incubation with hydroxyurea were not correlated with nuclei (Fig. 11). Apparently the cells enter into a process of autophagy (Figs. 11 and

12) and DNA may be taken into newly formed vacuoles.

DISCUSSION

Mucor spores placed into an appropriate culture medium enter rapidly into a high metabolic state, increasing their size atI& exponential rate up to 20 times their or*gi- nal volume for M. rouxii and about 130 times for M. bacilliformis. Tripp and Paznokas (198 1) described a critical volume of 130 pm3 at the time of germ tube emergence for M. racemosus. Whether the difference with M. rouxii and M. bacill is due to intrinsic properties of the s or to the different media employed remains to be determined. A proportion of this increase in volume was due to water uptake since its percentage increased from 53 to 80% during the swelling period, which is a factor of 53-fold, whereas RNA and protein content of the spores increased by a fac@r of about 10 in this growth phase for @: rouxii. This isodiametric phase of growth, denominated spherical growth, precedes a second phase of polarized growth (Bart- nicki-Garcia et al., 1968).

During the spherical phase of growth. RNA and protein content also increased exponentially without a noticeable lag phase. No change in the rate of RNA or protein increase was observed when spherical growth ceased and polarized growth was initiated. Similar results were obtained in


FIGS. 6-9. Morphological changes of Mucor spores induced by hydroxyurea, and nuclear staining with DAPI. FIG. 6. Morphological alterations of M. rouxii spores treated with hydroxyurea. Cells were incubated for 24 h. FIG. 7. Morphological alteration of M. bacilliformis spores incubated with hydroxyurea. Cells were incubated for 36 h. Notice that cells are almost spherical in contrast to M. rouxii cells. FIG. 8. Nuclear staining with DAPI of M. rouxii sporangiospores. FIG. 9. DAPI-nuclear staining of M. rouxii spores incubated for 24 h with hydroxyurea. Magnification bar for Figs. 6 and I, 20 pm; and for Figs. 8 and 9, 5 pm.


Time of incubation

O-4

TABLE 5 Nuclear Division during Germination of M. rouxii Sporangiospores”

1

93.0 85.3 60.4 47.7

9.8

Spores with nuclei (percentage) Spores in growth

Average nuclei phase II 2 3 4 5 6 per spore (percentage)

6.0 1.0 0 0 0 1.08 0 12.5 2.2 0 0 0 1.17 29.7 8.3 1.6 0 0 1.51 0 33.6 10.6 6.9 1.2 0 1.80 0 39.9 25.4 18.6 3.8 2.5 2.74 10

a Data are averages from four different experiments. Nuclei in spores grown in minimal medium were stained as described under Materials and Methods.

M. racemosus (Orlowski and Sypherd, 1978; Linz and Orlowski, 1982). Also chitin biosynthesis during germination of M. rouxii sporangiospores occurred without a noticeable lag phase (Bartnicki-Garcia and Lippman, 1977).

Inhibition of protein and RNA biosynthesis revealed that spore germination can be divided in discrete steps that we have called la, corresponding to the rounding up of the ellipsoidal spore; Ib, further increase in volume to a critical size; and II, emission of germ tubes or buds. Inhibition of protein or RNA biosynthesis at certain periods of the germination process can stop growth at the different discrete steps described above, suggesting that specific mRNAs and proteins may be required at each defined

growth phase. From our results it is evident that protein but not RNA biosynthesis is required to initiate spherical growth. Linz and Orlowski (1982) have described the presence of mRNA and polyribosomes in M. racemosus sporangiospores. Proteins synthesized during the first period of spore germination utilized the stored mRNA as template (Linz and Orlowski, 1982). More- over, mRNA extracted from spores was translated in vitro (Linz and Orlowski, 1982). Spores of Allomyces macrogynus (Smith and Burke, 1979), B. theobromae (Van Etten, 1972) and R. stolonifer (Van Etten and Freer, 1978) also contain mRNA and polyribosomes translatable into proteins during germination.

Inhibition of RNA biosynthesis even at

TABLE 6 Nuclear Division in Sporangiospores of Mucor rouxii and M. bacilliformis under Conditions of Inhibition of

DNA Biosynthesis”

Time of incubation

(h) DNA

Mucor rouxii Mucor bacilliformis

Nuclei per spore DNA Nuclei per spore

0 6.3 3 - 6 - 8 6.3

13 - 24 6.0

1.00 1.8 1.05 - 1.75 1.17 - 1.77 1.38

1.73 2.13 2.02 - 1.95 Uncountable

Uncountable - -

a Spores of M. rouxii were incubated at 28°C in the presence of 110 nuW hydroxyurea; spores of M. bacilliformis were incubated at 30°C. At intervals DNA and the number of nuclei were measured as described under Materials and Methods. DNA is expressed in grams per lOI spores.

FIGS. 10-12. Morphological changes of Mucov rouxii spores induced by hydroxyurea. FIG. 10. Nuclear staining of M. rouxii sporangiospores grown for 24 h in the presence of hydroxyurea. Fros. 11 and 12. Sections of cells grown with hydroxyurea for 18 h. Notice the thickening and banding of the cell wall (CW). Multiple vacuolar bodies (V) may be involved in endocytic processes. In Fig. 32 a severely damaged nucleus is apparent and the nuciear membrane has separated (stars). M, mitochon- dria; N, nucleus; Nu. nucleolus. Magnification bar: 5 (Fig. LO), 2 (Fig. ll), and 1 p.m (Fig. 12).

57


late stages of spherical growth prevented emergence of germ tubes or buds, suggesting that dormant spores do not contain at least some of the mRNAs specific for the onset of growth phase II. Data from Or- lowski and Sypherd (1978) on M. racemosus sustain this hypothesis. Accordingly, transcription and further translation of mRNA into “phase II specific proteins” seems to be a necessary step for the initiation of polarized growth. Freer et al. (1978) have reported that specific proteins are synthesized during germ tube emission of R. stolonifer.

Biosynthesis of DNA during spore germination contrasted with that of proteins and RNA. It showed a long lag phase before increasing in amount at an exponential rate. Initiation of DNA biosynthesis occurred about 30-45 minutes before the onset of growth phase II. This delay in DNA biosynthesis during spore germination has been described in several fungi (see review by Van Etten et al., 1976).

Evidence for a correlation between DNA biosynthesis and growth polarization in Mucor was obtained by inhibiting DNA replication at elevated temperature (only M. bacilliformis), or with hydroxyurea. M. bacilliformis spores incubated at 30°C went through the spherical growth phase, but did not form germ tubes. When hydroxyurea was added to either Mucor species, the spores completed spherical growth to reach very large volumes, but did not produce germination tubes. In the case of M. rouxii a significant number of spores started growth phase II, producing very short germ tubes, and then continued growth to a large size while maintaining their original shape. It is possible that hydroxyurea did not inhibit DNA biosynthesis completely in all spores and that some of them started replication. M. rouxii spores are far from homo- geneous, and nuclear staining revealed that some contain compact round nuclei, whereas others show dumbbell-shaped or very long nuclei suggesting that they have

started nuclear division. In any case, it is clear that the start of growth phase II requires DNA replication. Since this growth phase requires mRNA biosynthesis, a plau- sible explanation is that some DNA regions coding for RNA specific for polarization are not accessible for transcription. Their accessibility would depend on the replication of DNA. Lin and Inderlied (Inderlied et al., 1985) have suggested a relationship between DNA synthesis and nuclear division in the yeast to hyphal transition in M. rucemosus since an increase in both parameters precedes the morphological change.

An interesting observation was that nuclear division preceded initiation of DNA biosynthesis. Even when the latter was blocked by high temperature or hydroxyurea, nuclear division still occurred. Ac- cordingly, it may be concluded that spores from both Mucor species examined here are in the premitotic phase (G2) of the cell cycle. Similar results have been described for spores from Phycornyces blakesleeanus (Van Assche and Carlier, 1973).

ACKNOWLEDGMENTS

Part of this work was supported by financial help from DIGICySA, of the Secretaria de Education Pub- lica, and the Consejo National de Ciencia y Tecnolo- gia, Mexico. Thanks are given to Dr. Alfonso Carabez- Trejo (University of Mexico) and Mr. Armando Obre- gon Herrera for their help with electron microscopic studies.

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Developmental stages during the germination of Mucor sporangiospores