Physiological and Morphological Correlation of Rhizopusstolonifer Spore Germination1
JAMES L. VAN ETTEN, LEE A. BULLA, JR., AND GRANT ST. JULIAN
Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503, and Northern RegionalResearch Laboratory, Agricultural Research Service, Peoria, Illinois 61604
Received for publication 29 October 1973
Sporangiospores of Rhizopus stolonifer were examined at various stages ofgermination by scanning electron and phase-contrast microscopy. These observa-tions were correlated with changes in spore dry weight, spore volume, respiration,and syntheses of ribonucleic acid, deoxyribonucleic acid, and protein duringgermination.
One of the impressive morphological develop-ments in the life cycle of a mycelial fungus is theconversion of a dormant spore into an activelygrowing mycelium. The initiation of spore ger-mination leads to rapid increases in the rates ofrespiration and in protein and nucleic acidsyntheses. Our previous studies have focused onthe isolation and characterization of the compo-nents involved in protein, ribonucleic acid(RNA), and deoxyribonucleic acid (DNA) syn-theses from ungerminated and germinatedspores of the fungi Rhizopus stolonifer andBotryodiplodia theobromae (5, 7, 14). Ultimate-ly, we want to relate biochemical events withmorphological changes. In this report, the se-quential morphological changes of R. stolonifersporangiospores during germination are de-picted by phase-contrast and scanning electronmicroscopy; correlation is made with the syn-theses of protein, RNA, and DNA as well aswith alterations in the rate of respiration.
MATERIALS AND METHODS
Organism and cultural conditions. R. stolonifer(mating strain +) was obtained from W. Gauger,Botany Department, Univ. of Nebraska, Lincoln,Neb. The techniques for growth, harvesting, andgermination of sporangiospores were identical to thosepreviously described (18) except that the spores weregrown in a medium containing glucose, 20 g; aspara-gine, 2 g; KH2PO4, 0.5 g; MgSO4.7H2O, 0.26 g; agar,20 g; and water to 1 liter. The spores were germinatedat a concentration of 1.0 mg/ml in the same mediumwithout agar.
Analytical measurements. Total RNA was deter-mined by the orcinol procedure (17), and total protein
'Journal series no. 3555, Nebraska Agricultural Experi-ment Station. Research reported was conducted under projectno. 21-17.
was measured by the method of Lowry et al. (13) afterchemical fractionation of the cells (8). Total DNA wasdetermined by the absorbance of the isolated DNA at260 nm (4).
Polarographic measurements of oxygen consump-tion by germinating spores were made with Clark-type oxygen electrodes as previously described (5)except that the temperature was maintained at 29 C.
Labeled precursor incorporation assays. At vari-ous times during the germination period, 5-ml sam-ples of the spore suspension were removed and incu-bated an additional 15 min at 29 C in the presence of["4C]uracil or ["C]leucine (0.05 pCi of spore suspen-sion per ml). At the end of this period, 2 ml of 30%(wt/vol) trichloroacetic acid was added, and the assaymixtures were stored on ice for at least 1 h. The sporeswere transferred to glass fiber disks and processed asdescribed previously (1). The time at which DNAsynthesis was initiated was determined by adding 7.5'UCi of [4C ]guanine to 25 ml of the spore suspensionand incubating for 1 h. The labeled spores werediluted with 1 liter of unlabeled spores, the DNA wasisolated from them and centrifuged to equilibrium onCsCl gradients, and the radioactivity was determinedas described previously (4).
Microscopy. The techniques for observing speci-mens by phase-contrast and scanning electron micros-copy were modified from those described earlier (3,19). For phase-contrast microscopy, mounting slideswere prepared by spreading a thin film of 1% Nobleagar evenly over the surface of glass microscope slides.A small amount (about 0.05 ml) of a cell suspensionwas placed on the solidified agar surface and coveredwith a cover slip. Cells were photographed on Pana-tomic-X film through Neofluar phase optics of a WLZeiss microscope.
Squares (10 by 10 mm) cut from glass microscopeslides were placed on aluminum specimen stubs forscanning electron microscopy. About 0.05 ml of adiluted cell suspension was spread over the mountingsurface, dried, and coated with aluminum to a thick-ness of 15 nm. Specimens were examined in a Cam-bridge Stereoscan Mark II scanning electron micro-
scope at an accelerating voltage of 20 kV; the finalaperture was 200 Mm, and the beam specimen anglewas 45°.
RESULTSGerm tube formation, oxygen uptake, and dry
weight at various times during germination areshown in Fig. 1. Sporangiospores began to swellbetween 1 and 2 h (T1 and T2), and germ tubeemergence was first observed at T3.5 to T4. ByT., 90% of the spores had formed germ tubesand the dry weight of the cultures had increasedabout three- to fourfold. During the swellingprocess the spore diameter increased from 8.8 toabout 15 um. Assuming that the spores arespherical, their volume increased about fivefoldduring swelling. The rate of 02 uptake increasedapproximately 50% from To to To., and, there-after, continued to increase linearly until it hadincreased 10- to 12-fold at T,; the initial Q(02)was 13 and increased to 86 at T,.,. The rate of 02uptake usually remained constant during theremainder of the germination process.The incorporation of leucine into protein and
uracil into RNA at various stages of sporegermination is demonstrated in Fig. 2. Thekinetics of incorporation revealed that proteinand RNA syntheses began within the first 30min and rapidly increased throughout the ger-
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FIG. 1. Percentage of germination (0), rate ofoxygen uptake (A), and dry weight of spores (0), atvarious time periods during germination of R. stolo-nifer.
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FIG. 2. A, Incorporation of leucine into proteinduring 15-min pulse periods (-) and total proteincontent (bar); B, incorporation of uracil into RNAduring 15-min pulse periods (0) and total RNAcontent (bar) of the spores at various stages ofgermination. The total RNA or protein was deter-mined from 100-ml samples of the spore suspension atthe time periods indicated. The time at which germtubes were initially observed is indicated by thearrows.
mination process. All three types of RNA,ribosomal, transfer, and messenger, initiatesynthesis within this time period (J. R. Roheim,R. H. Knight, and J. L. Van Etten, unpublisheddata). The rates of leucine and uracil incorpora-tion decreased at the time of germ tube emer-gence (T3-4). This reduction was probably theresult of increases in the endogenous pools ofprotein and RNA precursors rather than de-creases in their synthetic rates because totalprotein and total RNA continued to increasethroughout the germination process. Similarkinetics were observed for the incorporation ofeither [14C]guanine or ["4C]adenosine. How-ever, the reduced incorporation rate observedafter T3.4 was not as pronounced with adenosineas it was with uracil or guanine. Separateexperiments indicated that a high percentage ofthe labeled precursors was actually incorpo-rated into protein or RNA. Spores were incu-bated for 2 h in the presence of either ["4C]leu-cine or ["4C]uracil; after chemical fractionationof the spores (8), 90% of ["4C]leucine appearedin the fraction that contained hydrolyzed pro-tein and 91% of ["4C]uracil appeared in thefraction that contained hydrolyzed nucleic acid.In addition, if the spores labeled with [I4C luracilwere incubated in 1 M KOH for 18 h at 37 C, allof the radioactivity was completely solubilized.Therefore, the ["4C]uracil entered the RNA andnot the DNA of the spores.The changes in DNA content and the incorpo-
ration of ["C]guanine into DNA during sporegermination are given in Table 1. Guanine wasused to monitor DNA synthesis because R.
a Values are not absolute because inherent with theextraction process is an approximate 50% loss of DNA.
' Absorbancy at 260 nm.c Germination time represents the conclusion of the
1-h pulse period.
stolonifer spores do not readily incorporate thy-midine into DNA, presumably due to the ab-sence of thymidine kinase (9). DNA synthesisbegan between T1 and T2 and increased duringthe remainder of the germination process.The scanning electron and phase-contrast
(inserts) micrographs in Fig. 4 disclose thesequence of morphological change from a dor-mant spore through germ tube formation. Thesurface of an ungerminated (T.) sporangiosporeof R. stolonifer appeared striated in light optics(Fig. 4A, insert) and in the scanning microscopeexhibited prominent cylindrical ridges. Previ-ous observations by scanning microscopy (6)have indicated that such surface structures arereliable characteristics for grouping species.Figure 4B shows a swollen spore at T2 ,,. Char-acteristic of 90 to 95% of all spores examined atthis period is their triangular shape. By T, (Fig.4C), the spore appeared elongated with noapparent change in its surface configuration.Slight collapse of the spore suggests than aninternal modification within the spore decreasedits turgidity during treatment for electron mi-croscopic examination. Germ tube emergence(TM.) and extension (T,) are depicted in Fig.4D and E, respectively. The tip of the germtube appeared distinctly different from the wallbehind the apical dome (see Fig. 4D). A similarappearance was noted during germination ofpycnidiospores of B. theobromae (19).
DISCUSSIONAs was true for Saccharomyces cerevisiae (15,
16) and B. theobromae (4, 18, 19), correlation ofdifferential modification in surface topographyof R. stolonifer sporangiospores with variousphysiological activities during germ tube devel-
opment presents an interesting aspect of cellu-lar differentiation. The sequence of certainbiochemical events and alterations in overallspore shape during germination and germ tubedevelopment of R. stolonifer are outlined in Fig.3. The events from spore through germ tubeformation are time framed and are delineated asfollows: (i) onset of RNA and protein synthesisand increased 02 uptake by To.,; (ii) onset ofDNA synthesis with enhanced RNA and proteinsyntheses, increases in rate of 02 uptake, sporevolume, and initial increase in spore dry weightby T2; (iii) further increases in 02 uptake, dryweight, and syntheses of DNA, RNA, andprotein at T2-6; (iv) visual appearance of germtubes and germ tube elongation at T3.5 to T6.Whereas protein synthesis precedes RNA syn-thesis during germination of S. cerevisiae asco-spores (15) and B. theobromae pycnidiospores(1), RNA synthesis and protein synthesis areconcomitant in R. stolonifer and begin immedi-ately upon initiation of sporangiospore germina-tion.An intriguing morphological feature of R.
stolonifer germination is the contrast betweenthe extended germ tube and its parent sporangi-ospore. In no instance during our observationsdid we see any change in spore surface striationof the sporangiospores except at the point ofgerm tube emergence. Apparently, initial cellu-lar differentiation is distinctly internal andperhaps unrelated to the physiology of the outerspore wall. Previous investigations have demon-strated that the germ tube wall of certainRhizopus species is formed by a continuation ofan inner wall layer of the spore (2, 10-12).During the initial stages of germ tube develop-ment, specific physiological activities mayoccur at the site of emergence and may bedisplayed by the formation of a smooth tip. Thesubtle difference between the germ tube tipsurface and the remaining portion of the ex-tended germ tube also may be a reflection ofrelative rates of metabolic activity. Furtherstudies are being done on the biochemical and
tFIG. 4. Scanning electron micrographs of R. stolonifer spores at various stages of germination with corre-
sponding phase contrast micrographs (x2,375) inserted. A, Ungerminated spore, To; B, swollen spore, T2.5;C, elongated spore, T3; D, germ tube emergence, T73.,; and E, germ tube elongation, T6.
physiological events of germination and germtube formation in this organism.
ACKNOWLEDGMENTS
The authors are indebted to L. D. Dunkle, MerrileeRutledge, and F. L. Baker for their technical assistance. Thisinvestigation was supported in part by Public Health Servicegrant AI-108057 from the National Institute of Allergy andInfectious Diseases.
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