University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Plant Pathology Plant Pathology Department 2-1974 Physiological and Morphological Correlation of Rhizopus stolonifer Spore Germination James L. Van Etten University of Nebraska-Lincoln, [email protected] Lee Bulla Jr. University of Nebraska-Lincoln Grant St. Julian University of Nebraska-Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/plantpathpapers Part of the Plant Pathology Commons Van Etten, James L.; Bulla, Lee Jr.; and St. Julian, Grant, "Physiological and Morphological Correlation of Rhizopus stolonifer Spore Germination" (1974). Papers in Plant Pathology. 144. https://digitalcommons.unl.edu/plantpathpapers/144 This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. JOURNALOF BACTERIOLOGY, Feb. 1974, p. 882-887 Val. 117, No. 2 Copyright O 1974 American Society for Microbiology Printed in U.S.A. Physiological and Morphological Correlation of Rhizopus stolonifer 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 Regional Research Laboratory, Agricultural Research Service, Peoria, Illinois 61604 Received for publication 29 October 1973 Sporangiospores of Rhizopus stolonifer were examined at various stages of germination 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 during germination. One of the impressive morphological develop- was measured by the method of Lowry et al. (13) after ments in the life cycle of a mycelial fungus is the chemical fractionation of the cells (8).Total DNA was conversion of a dormant spore into an actively determined by the absorbance of the isolated DNA at growing mycelium. The initiation of spore ger- 260 nm (4). Polarographic measurements of oxygen consump- mination leads to rapid increases in the rates of tion by germinating spores were made with Clark- respiration and in protein and nucleic acid type oxygen electrodes as previously described (5) syntheses. Our previous studies have focused on except that the temperature was maintained at 29 C. the isolation and characterization of the compo- Labeled precursor incorporation assays. At vari- nents involved in protein, ribonucleic acid ous times during the germination period, 5-ml sam- (RNA),and deoxyribonucleic acid (DNA)syn- ples of the spore suspension were removed and incu- theses from ungerminated and germinated bated an additional 15 min at 29 C in the presence of spores of the fungi Rhizopus stolonifer and ["C]uracil or ["C]leucine (0.05 pCi of spore suspen- Botryodiplodia theobromae (5, 7, 14). Ultimate- sion per ml). At the end of this period, 2 ml of 30% (wtlvol) trichloroacetic acid was added, and the assay ly, we want to relate biochemical events with mixtures were stored on ice for at least 1 h. The spores morphological changes. In this report, the se- were transferred to glass fiber disks and processed as quential morphological changes of R. stolonifer described previously (1). The time at which DNA sporangiospores during germination are de- synthesis was initiated was determined by adding 7.5 picted by phase-contrast and scanning electron pCi of ["Clguanine to 25 ml of the spore suspension microscopy; correlation is made with the syn- and incubating for 1 h. The labeled spores were theses of protein, RNA, and DNA as well as diluted with 1 liter of unlabeled spores, the DNA was with alterations in the rate of respiration. isolated from them and centrifuged to equilibrium on CsCl gradients, and the radioactivity was determined as described previously (4). MATERIALS AND METHODS Microscopy. The techniques for observing speci- Organism and cultural conditions. R. stolonifer mens by phas.e-contrast and scanning electronmicros- (mating strain +) was obtained from W. Gauger, copy were modified from those described earlier (3, Botany Department, Univ. of Nebraska, Lincoln, 19). For phase-contrast microscopy, mounting slides Neb. The techniques for growth, harvesting, and were prepared by spreading a thin film of 1% Noble germination of sporangiospores were identical to those agar evenly over the surface of glass microscope slides. previously described (18) except that the spores were A smhll amount (about 0.05 ml) of a cell suspension grown in a medium containing glucose, 20 g; aspara- was placed on the solidified agar surface and covered with a cover slip. Cells were photographed on Pana- gine, 2 g; KH,PO,, 0.5 g; MgSO,.7H,O, 0.26 g; agar, 20 g; and water to 1 liter. The spores were germinated tomic-X film through Neofluar phase optics of a WL at a concentration of 1.0 mglml in the same medium Zeiss microscope. without agar. Squares (10 by 10 mm) cut from glass microscope Analytical measurements. Total RNA was deter- slides were placed on aluminum specimen stubs for mined by the orcinol procedure (17), and total protein scanning electron microscopy. About 0.05 ml of a diluted cell suspension was spread over the mounting 'Journal series no. 3555, Nebraska Agricultural Experi- surface, dried, and coated with aluminum to a thick- ment Station. Research reported was conducted under project ness of 15 nm. Specimens were examined in a Cam- no. 21-17. bridge Stereoscan Mark I1 scanning electron micro- 882 R. STOLONIFER GERMINATION scope at an accelerating voltage of 20 kV; the final aperture was 200 pm, and the beam specimen angle was 45". RESULTS Germ tube formation, oxygen uptake, and dry weight at various times during germination are shown in Fig. 1. Sporangiospores began to swell between 1 and 2 h (TI and T2),and germ tube emergence was first observed at Ts,, to T,. By T,, 90% of the spores had formed germ tubes and the dry weight of the cultures had increased about three- to fourfold. During the swelling FIG.2. A, Incorporation of leucine into protein process the spore diameter increased from 8.8 to during 15-min pulse periods (0)and total protein about 15 bm. Assuming that the spores are content (bar); B, incorpomtion of umcil into RNA spherical, their volume increased about fivefold during 15-min pulse periods (0)and total RNA during swelling. The rate of O2uptake increased content (bar) of the spores at various stages of approximately 50% from Toto To., and, there- germination. The total RNA or protein was deter- after, continued to increase linearly until it had mined from 100-ml samples of the spore suspension at increased 10- to 12-fold at T,; the initial Q(02) the time periods indicated. The time at which germ was 13 and increased to 86 at T,.,. The rate of O2 tubes were initially observed is indicated by the uptake usually remained constant during the arrows. remainder of the germination process. mination process. All three types of RNA, The incorporation of leucine into protein and ribosomal, transfer, and messenger, initiate uracil into RNA at various stages of spore synthesis within this time period (J. R. Roheim, germination is demonstrated in Fig. 2. The R. H. Knight, and J. L. Van Etten, unpublished kinetics of incorporation revealed that protein data). The rates of leucine and uracil incorpora- and RNA syntheses began within the first 30 tion decreased at the time of germ tube emer- min and rapidly increased throughout the ger- gence (Ts.,). This reduction was probably the result of increases in the endogenous pools of protein and RNA precursors rather than de- creases in their synthetic rates because total protein and total RNA continued to increase throughout the germination process. Similar kinetics were observed for the incorporation of either [14C]guanine or ["Cladenosine. How- ever, the reduced incorporation rate observed after T,., was not as pronounced with adenosine as it was with uracil or guanine. Separate experiments indicated that a high percentage of the labeled precursors was actually incorpo- rated into protein or RNA. Spores were incu- bated for 2 h in the presence of either [14C]leu- cine or ["Cluracil; after chemical fractionation of the spores (a), 90% of [14C]leucine appeared in the fraction that contained hydrolyzed pro- tein and 91% of [14C]uracil appeared in the fraction that contained hydrolyzed nucleic acid. In addition, if the spores labeled with ["C ]uracil were incubated in 1 M KOH for 18 h at 37 C, all of the radioactivity was completely solubilized. Therefore, the [14C]uracilentered the RNA and TIME.HR. not the DNA of the spores. FIG. 1. Percentage of germination (0).rate of The changes in DNA content and the incorpo- oxygen uptake (A),and dry weight of spores (O),at ration of [14C]guanine into DNA during spore various time periods during germination of R. stolo- germination are given in Table 1. Guanine was nifer. used to monitor DNA synthesis because R. 884 VAN ETTEN, BULLA, AND ST. JULIAN J. BACTERIOL. TABLE1. Changes in DNA content and the opment presents an interesting aspect of cellu- incorporation of ["Clguanine into DNA during spore lar differentiation. The sequence of certain germination of R. stolonifer biochemical events and alterations in overall spore shape during germination and germ tube Germination Spore suspensiona Counts per min per time (h, (mg of DNN~~ml) A,,; of^^^ per h' development of R. stolonifer are outlined in Fig. 3. The events from spore through germ tube formation are time framed and are delineated as follows: (i) onset of RNA and protein synthesis and increased O2 uptake by To.,; (ii) onset of 2,546 DNA synthesis with enhanced RNA and protein 0.16 4,000 syntheses, increases in rate of O2uptake, spore 5 0.23 6 0.28 volume, and initial increase in spore dry weight by T2; (iii) further increases in O2uptake, dry a Values are not absolute because inherent with the weight, and syntheses of DNA, RNA, and extraction process is an approximate 50% loss ofDNA.