GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

AN AUTOMATED METHOD FOR QUANTIFYING THE L-ALANINE TRIGGER OF BACILLUS SUBTILIS SPORE GERMINATION AND COMPETITIVE INHIBITION BY D-ALANINE

We developed an automated method for studying the germination kinetics of Bacillus subtilis spores using a microtiter plate (MP) reader. Phosphate buffer supplemented with L-alanine was used to isolate the germination phase as determined by decrease in optical density (OD₆₃₀). Using a standard 96-well MP, L-alanine triggered germination kinetics were measured by automatic OD measurement every 3 min until the maximum OD₆₃₀ change (OD₆₃₀) was determined. When OD₆₃₀ values were plotted against L-alanine concentration on a double reciprocal plot, a straight line (R²= 0.98) was produced. The addition of D-alanine to the medium demonstrated classical competitive inhibition on double reciprocal plots. A 3-dimensional representation of the untransformed data showed the response surface nature of competitive inhibition. The method automates the tedious task of determining loss of refractility associated with spore germination under defined conditions so that inhibitors to germination can be studied. Since 96 OD₆₃₀ determinations can be done simultaneously in small volumes (200 μL) extensive data can be generated about inhibitors using relatively small spore crops in a single, short (1.4 h) incubation.     

SPORE GERMINATION AND EARLY DEVELOPMENT OF THE GAMETOPHYTE OF SCHIZAEA PUSILLA

During germination of the spore of Schizaea pusilla, the first division of the protoplast was perpendicular to the polar axis and resulted in the formation of the rhizoid. The next division parallel to the polar axis of the spore gave rise to the protonemal initial. Following this “Vittaria”-type germination, the protonema that developed was characterized by an extensive branching to produce uniseriate filaments and rhizoidophores.     

INDUCTION OF SPORE GERMINATION IN SCHIZAEA PUSILLA (SCHIZAEACEAE)

Requirements for spore germination in the rare and native New Jersey fern, Schizaea pusilla Pursh., were examined. Spores did not germinate in darkness and gibberellins (GA) did not induce germination in the dark. However, a dark pretreatment promoted germination in a subsequent light treatment and low temperatures during the dark pretreatment greatly enhanced germination in culture. Three wks of dark pretreatment were required for maximum germination. GA₃ promoted germination in red light more effectively than GA₄₊₇. Greater than ten days of continuous illumination was necessary for germination. Spores given red light reached half-maximum germination six days earlier than spores under white light. Red light promoted germination while blue light did not. Far-red light alone could stimulate germination and enhanced the promotive effect of red light; typical phytochrome photoreversibility was not observed. Blue light reduced the effect of red light.     

ULTRASTRUCTURAL CHARACTERISTICS OF SPORE GERMINATION IN THE MOSS DAWSONIA SUPERBA

The spore wall of Dawsonia superba has characteristics that, in many respects, are similar to those of other mosses except for the exine, which is layered in Dawsonia. Imbibed spores have a well-developed endoplasmic reticulum with dilated cisternae that are associated with vesicles at the periphery of the cell. Ribosomes on the surface of the vesicles suggest that many vesicles originate from the endoplasmic reticulum. Two types of protein storage bodies are observed: membrane bound protein bodies with a homogeneous matrix which gradually becomes vesicular, and densely stained and non-membrane bound bodies consisting of crystalline arrays of fibrils. As in spores of higher plants, the protein reserves disappear during germination and may be converted to starch and other materials needed for development of the gametophyte.     

ON THE MECHANISMS OF LIGHT-INDUCED GERMINATION INHIBITION OF PHACELIA TANACETIFOLLV

Germination of seed of Phacelia tanacetifolia is inhibited by several mechanisms. In addition to physical restraints imposed by the seed coats, the seed contains a water-soluble inhibitor which is independent of light or temperature for its activity. Available evidence also points to the presence of 1 or more light-activated inhibitors which are not easily leached from the seed. The blue-light-activated inhibition can be negated by high oxygen tensions or mechanical abrasion of the micropylar end of the seed. The suppression of germination by far-red or red light can be negated by abrasion but is only partially reversed by oxygen. Combinations of abrasion and high oxygen tensions negate both light-induced and temperature-induced inhibitions of germination.     

AUTOINHIBITION OF SPORE GERMINATION IN NOSTOC SPONGIAEFORME (CYANOPHYCEAE)1

Medium in which cultures of Nostoc spongiaeforme Ag. have sporulated contains one or more substances which inhbit the germination of spores (akinetes) of this organism. Germination of spores occurs rapidly in the absence of these substances, but is virtually completely suppressed in their presence.     

CO-REQUIREMENT FOR CALCIUM AND POTASSIUM IN THE GERMINATION OF SPORES OF THE FERN ONOCLEA SENSIBILIS

Spores of Onoclea sensibilis L. do not germinate on distilled H₂O if they are pretreated for sufficient time with dilute NaClO solution. However, spores will germinate, after NaClO pretreatment, on a simple mineral medium containing the major and trace elements. Complete germination after pretreatment also is obtained on a solution containing only Ca²⁺ and K⁺ as the cations, but neither ion by itself is sufficient. Rb⁺, but not Li⁺ or Na⁺, can replace K⁺. Hypochlorite-treated spores do not require the continuous presence of Ca²⁺ and K⁺ to germinate; exposure during the first 4 hr of culture, with the remainder of the time on distilled H₂O, is sufficient. Extraction of spores with ethylene glycol bis(aminoethyl ether) tetraacetic acid [EGTA] makes their germination dependent on Ca²⁺, as reported by other workers, but it does not produce a co-requirement for K⁺. Colorimetric analysis with arsenazo III confirms that Ca²⁺ is extracted from Onoclea spores by NaClO. Extractable Ca²⁺ amounts to about 78 nmol/mg spore dry wt. Of this amount, 31% is contained in the perispore. The perispore comprises 13% of the total spore dry wt.