GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. IV. INVOLVEMENT OF PHOTOSYNTHESIS IN OVERCOMING ETHYLENE INHIBITION OF SPORE GERMINATION

The inhibitory effects of ethylene on spore germination were investigated. In darkness spore germination was completely inhibited by 10 μ1 · 1⁻¹ ethylene. Light partially overcame this inhibition, and the effect of continuous irradiation with white fluorescent light saturated at about 450 μW · cm⁻². Monochromatic red, blue and far-red light were effective in overcoming ethylene inhibition, whereas green was not. Short periodic exposures to red or far-red light were not sufficient to overcome ethylene inhibition. This suggested that phytochrome was not involved. The photosynthetic inhibitor DCMU blocked the effect of light. Infrared gas analysis showed that photosynthesis saturated at about 450 μW · cm⁻² in white light. Red, blue and far-red light were more efficient photosynthetically than green light; DCMU blocked photosynthesis. Normalized curves of photosynthesis and germination vs. light intensity showed a similar dependence on light energy. It was concluded that light appears to overcome the inhibitory effects of ethylene through some process dependent on photosynthesis.     

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.     

SPORE GERMINATION AND DEVELOPMENT OF THE YOUNG GAMETOPHYTE OF THE OSTRICH FERN (MATTEUCCIA STRUTHIOPTERIS)

Light is required for the germination of spores of Matteuccia struthiopteris. Histochemical studies show that dormant spores contain no starch, but have an abundance of storage protein granules. Starch accumulates in the numerous chloroplasts of the spore on exposure to light and becomes gradually more extensive. Protein granules disappear as germination progresses. Following this, the centrally located nucleus migrates toward the proximal spore face. Concomitant with the nuclear migration, an increase of cytoplasmic RNA surrounding the nucleus occurs. An equal nuclear division and unequal cell division give rise to a 2-celled gametophyte consisting of a large prothallial cell and smaller rhizoidal cell. A new peripheral wall forms around the entire protoplast at the time of nuclear migration, while a transverse wall forms after nuclear division. The rhizoid emerges through the split raphe along the proximal spore face; it is rich in cytoplasmic RNA but contains very few chloroplasts and little starch. Electron microscopy of the 2-celled stage revealed a greater concentration of mitochondria, Golgi bodies, and a more extensive endoplasmic reticulum in the rhizoid than was found in the prothallial cell, which, however, was far richer in chloroplasts and lipid bodies. As the rhizoid elongates and becomes more vacuolated, cytoplasmic RNA decreases as cytoplasmic protein increases. The rhizoid undergoes no cell divisions, while the prothallial cell retains the potential for further cell division. The possible significance of the distribution of storage products, cell organelles, and other cell components were considered in relation to the non-equational cell division and differentiation of the 2 cells.     

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.     

PHOTOMORPHOGENESIS IN PTERIS VITTATA III. PROTECTIVE ACTION OF ETHANOL ON BLUE-LIGHT-INDUCED INHIBITION OF SPORE GERMINATION

In the fern Pteris vittata, the low-energy blue-light-induced inhibition of phytochrome dependent spore germination was protected by 0.1 mol ethanol. This protective action was observed only with ethanol in a sampling of 9 alcohols. Ethanol does not induce dark germination of spores and may act directly on the inhibition induced by blue light. It takes about a 15 times more intense dose of blue light for the induction of 50% inhibition of spore germination in 0.1 mol ethanol containing media compared with control media. The protective action of ethanol was apparent within 2 days after treatment and reached a maximum level at the 4th day. The effect of ethanol continues for about 32 hours after withdrawal of the ethanol. This action of ethanol was also observed in the case of far-red light irradiation but the mode of action of ethanol may be different from the case of blue light irradiation.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. II. INHIBITION OF CELL DIVISION

Ethylene, a natural product of sensitive fern (Onoclea sensibilis L.) gametophytes, has been demonstrated to inhibit cell division in light-grown prothallia. When plants were grown on Knop's solution plus 1 % sucrose under 300 ft-c or more of white light, all ethylene concentrations from 1–1000 μl/liter reduced the rate of increase of cell number by about one-half. The over-all rate of increase of cell number was regulated by various environmental and chemical factors, but regardless of the rate established in control cultures, ethylene treatment of 1–1000 μl/liter produced a relative 50 % depression of cell number. Ethylene was specific for inhibition of cell division and was not a general inhibitor of growth. The ethylene inhibition did not result from a reduction of photosynthesis or energy supply. Further demonstration of ethylene as the active gaseous component resulted when cultures were grown in small enclosed containers with an ethylene absorbent, mercuric perchlorate, and consequently the cell number of gametophytes was restored to the level of unenclosed controls.     

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.     

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.     

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.     

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.