Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm-2; sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylis and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process.

The oxygen requirements during the three phases of photoinduced germination of Onoclea senisibilis L. spores were analyzed by temporarily applying nitrogen atmosphere. The dark preinduction phase, during which the spores imbibe water and establish sensitivity to irradiation, involves an oxidative process which can be reversibly inhibited and stimulated by nitrogen and air, respectively. The induction phase of germination is characterized by a pure photochemical reaction, independent of temperature and oxygen. The postinduction phase, when the photoproduct triggers dark processes eventually leading to the protrusion of the rhizoidal or protoneniatal cells, involves an oxidative process which occurs within the first 10 hours of this phase. This oxidative process differs in kinetic characteristics from that in the preinduction phase. Light induces or enhances germination of spores of many fern species (13,14,16,18). Photocontrolled fern spore germination may operationally be divided into at least three sequential phases: (a) a dark preinduction phase in which the spores imbibe water and establish sensitivity to irradiation; (b) an induction phase, when light induces germination maximally; and (c) the postinduction phase, in which the photoproduct triggers dark processes that ultimately terminate in the protrusion of the rhizoid and protonemata. Although many investigations have dealt with characterization of the quality and quantity of light required for the induction phase of germination, the nature of the preinduction and postinduction phases of germination in fern spores has been little investigated (6,9,15,20 In a series of preliminary experiments, Onoclea spore germination was found to be inhibited by N2 atmosphere; the spores, however, could recover the ability to germinate once they were transferred to air and irradiated with visible light. Anaerobiosis thus seemed to be a useful tool for the investigation of oxidative reactions involved in the germination processes. The present paper is, therefore, aimed at analyzing the oxidative requirements of the three phases of germination by temporary application of a N2 atmosphere. MATERIALS AND METHODSPlant Material and Germination Procedure. Onoclea sensibilis L. spores were collected, harvested, and stored as described earlier (19). The batch of spores used in the present investigation had a somewhat lower final percentage of germination (65.1 + 2.8) and a slightly slower time course of germination than the batch of spores used in the earlier experiments (19). This was ascribed to aging of the spores after collection. The general characteristics of the light requirements remained the same, however.Germination was scored 6 days after treatment by the protrusion method described earlier, unless stated otherwise. Counts of 400 spores from each replicate treatment were averaged to give the percentage of germination for a given treatment.Nitrogen Gas Experiments. Experiments requiring a N2 atmosphere were carried out using air-tight, glass, baby food jars (5-cm...

Germination of Oncwk seblis L. spores is controlied by light and temperature. Temperatures of 30 C can induce maximal germination in the dark to a level of 60 to 95% of that induced by a saturating dose of red light (0.38 joules/square meter) providing the spores are placed at the elevated temperature immediately after being sown. Maximum dark germination occurs with a minhmum exposure of 16 to 24 hours at 30 C, suggestn that the temperature treatment is required for the induction of germination rather than for the germination process per se. Interaction of temperature and light for induction of germination shows nonaddWive behavior. Germination induced by light and temperature applied consecutively never exceeded that which could be induced by a saturatin dose of red light alone. Imbibition of the spores at 25 C in the dark for 12 or more hours prior to incubation at 30 C results in a loss of thermosensitivity. Fern spore germination has been shown repeatedly to be stimulated by light (4,12 The objective of this study was to determine the relationship between induction of dark germination by high temperatures and the induction of germination by very short exposures to low intensity red radiation. The data presented suggest that the thermo-and photocontrol of Onoclea spores is mediated by two different mechanisms. February, 1975 and the spores were harvested and stored as described previously (15). The light requirements and characteristics for germination were comparable to those reported in Towill and Ikuma (15). MATERIALS AND METHODSGermination was scored 5 days after treatment by the protrusion method. Each experimental treatment was done in duplicate. Counts from two slides of 200 spores/slide from each of the replicate dishes were averaged to yield the per cent germination for a given treatment. Standard deviations of the mean were calculated for each value with N = 4.Temperature Experiments. Temperatures used in this study included 10, 18, 20, 25, 27, 30, 33, and 36 C and were attained using temperature-regulated incubation chambers which fluctuated ±0.5 C from the stated value. Spores which were kept at room temperature were placed in light-tight drawers in a dark room maintained at 25 ± 1 C.Approximately 5 mg of spores were sown on 10 ml of distilled H20 in a 5-cm Petri dish under a dim green safelight. The Petri dishes were placed in light-tight cylinders for transfer to the appropriate temperature.Duration and time of application of the temperature and light treatments are presented under "Results." Experiments were repeated at least three times. The data presented in the figures and tables represent the results from a single experiment, the trends of which are consistent with the data from replicate experiments.Experiments Using Osmotica and Agents Which Affect Membrane Properties. In experiments using osmotic agents or agents which affect membranes, 5 mg of spores were sown on the respective media under dim green light, and the spores maintained at room temperature in the dark for the t...

Light has been implicated in the control of plant growth and development by regulation of enzyme synthesis, or enzyme activity, or both (14,22). In view of the observations that protein synthesis is required for the early phase of dark germination of spores of the bracken fern (11,12) and that one of the early events after the induction of germination by light in fern spores is hydrolysis of storage products (6, 11), it would appear that protein synthesis may play an important role in the control of photoinduced germination of fern spores.Investigations with a nitrogen atmosphere (18) by anaerobiosis, the recovery process is slow, thus implicating the synthesis of a substance or substances (possibly enzymes) required for the germination process. Edwards and Miller (2) also showed that during the first 8 to 10 hr of illumination germination is inhibited by ethylene, suggesting the involvement of nucleic acid synthesis during this period of germination.Involvement of protein and RNA synthesis in the germination of Onoclea spores was investigated using inhibitors. As indicated in the results, of the five inhibitors tested, cycloheximide inhibited germination completely in 2 hr, and this inhibition was found to be reversed after the removal of the inhibitor. This paper examines the requirement for protein synthesis during the pre-and postinduction phases of Onoclea spore germination by means of the temporary application of CH.MATERIALS AND METHODS Plant Material. Spores used in experiments on the effect of CH2 on germination were from the same batch employed in the previously reported experiments on anaerobiosis (18). Spores of this batch required 6 hr of dark presoaking to develop maximal photosensitivity, and gave the final germination of 65.1 + 2.8%. For these experiments, sterilization of the spores was not necessary, because possible fungal and bacterial contamination hardly altered the results.A second batch of spores was employed for the experiments on incorporation of the radioactivity. These spores were harvested from sporophylls collected in March 1972 from the same location as reported previously (17) and required 24 hr of dark presoaking to develop maximal sensitivity to irradiation. The final germination for this batch was 82.8 + 3.0%. Maximal inhibition of germination by CH was 90 to 97% for both batches of spores. For the incorporation experiments, spores were surface-sterilized as follows: a small spatulaful of spores (2-4 mg) was treated first with 5 ml of 2% (v/v) Clorox (prepared from a commercial solution) in a 5-cm Petri dish and allowed to stand with occasional agitation for 5 min. They were then treated with 5 ml of 0.1% (w/v) HgCl2 for 2 min and washed 4 times with sterile distilled H20. Aftre the final washing, 10 ml of sterile distilled H20 were added to the Petri dishes. To check for bacterial and fungal contamination, samples of spores were plated onto bacto-nutrient agar after surface sterilization. Experimental runs that showed contamination by this testing procedure were disca...

Boundary membranes were recovered from glyoxysomes, transition peroxisomes, and leaf-type peroxisomes purified from cotyledons of sunflower (Helianthus annuus 1.) at three stages of postgerminative growth. After membranes were washed in 100 mM Na2C03 (pH 11.5), integral peroxisome membrane proteins (PMPs) were solubilized in buffered aminocaproic acid/dodecyl maltoside (0.63 ~/1.5%) and analyzed by nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Six prominent nondenatured PMP complexes and 10 prominent SDS-denatured polypeptides were identified in the membranes of the three types of peroxisomes. A nondenatured complex of approximately 140 kD, composed mainly of 24.5-kD polypeptides, decreased temporally, independently of seedling exposure to white, blue, or red light; only far-red light seemed to prevent its decrease. PMP complexes of approximately 120 and 70 kD, in contrast, were present at all stages and changed in polypeptide content. It remains to be determined whether these data reflect changes within in vivo complexes or within complexes formed following/during detergent solubilization. Conversion of glyoxysomes to leaf-type peroxisomes in white or red light after a 2-d dark period was accompanied by the appearance of three SDS-denatured PMPs: 27.5, 28, and 47 kD. The former two became part of the PMPlZO and 70 complexes, as well as part of a new PMP130 complex that also possessed the PMP47. Growth of seedlings in blue or far-red light did not promote the appearance of PMPs 27.5 or 28. Blue light promoted the appearance of PMP47, and far-red light seemed to prevent its appearance. Chlorophyll likely is not the photoreceptor involved in accumulation of PMPs because the PMP composition is distinctly different in seedlings irradiated with red or blue light of comparable fluence rates. Several lines of evidence indicate that the synthesis and acquisition of membrane and all matrix proteins are not coupled. The data provide evidence for a change in PMP composition when sunflower or any other oilseed glyoxysomes are converted to leaf-type peroxisomes and suggest that the change is regulated by both photobiological and temporal mechanisms.Germinated oilseeds are an excellent system in which to study the developmental biogenesis of peroxisomes. Following germination, oil stored in cotyledons are converted mostly