PHOTOMORPHOGENESIS IN PTERIS VITTATA: I. PHYTOCHROME-MEDIATED SPORE GERMINATION AND BLUE LIGHT INTERACTION

1. Spores of the fern Pteris vittata did not germinate under totaldark conditions, while an exposure of the spores to continuouswhite light brought about germination. The germination was mosteffectively induced by red light and somewhat by green and far-red,but not at all by blue light. The sensitivity of spores to redlight increased and leveled off about 4 days after sowing at27–28. The promoting effect of red light could be broughtabout by a single exposure of low intensity. Far-red light givenimmediately after red light almost completely reversed the redlight effect, and the photoresponse to red and far-red lightwas repeatedly reversible. The photoreversibility was lost duringan intervening darkness between red and far-red irradiations,and 50% of the initial reversibility was lost after about 6hr of darkness at 27–28. These observations suggest thatthe phytochrome system controls the germination of the fernspore.
2. When the imbibed spores were briefly exposed to a low-energyblue light immediately before or after red irradiation, theirgermination was completely inhibited. The blue light-inducedinhibition was never reversed by brief red irradiation givenimmediately after the blue light. The escape reaction of redlight-induced germination as indicated by blue light given aftervarious periods of intervening darkness was also observed, andits rate was very similar to that determined by using far-redlight. Spores exposed to blue light required 3 days' incubationin darkness at 27–28 to recover their sensitivity tored light. The recovery in darkness of this red sensitivitywas temperature-dependent. It is thus suggested that an unknownbluelight absorbing pigment may be involved in the inhibitionof phytochrome-mediated spore germination.

(Received August 21, 1967; )     

RHIZOID CELL DIFFERENTIATION IN THE FERN GAMETOPHYTE OF PTERIS VITTATA

A series of events in the differentiation of rhizoid cells in the gametophyte of Pteris vittata L. is described. Differential in vivo staining reactions make it possible to trace a sequence of stages from pre-mitotic nucleocytoplasmic staining (corona stage), through the events of mitosis, to formation of an internally segmented rhizoid cell which then grows out from the parent thallus cell. When the standard ribonuclease test is employed, basophilic protoplasmic staining due to pyronin is prevented in developmental stages up through early formation of the rhizoid cell. Staining in these stages is therefore assumed to be due to ribonucleic acid associated with protein synthesis. A new developmental phase starts just prior to the protrusion growth of this cell when an intensely pyroninophilic material appears at the membrane of the rhizoid cell nucleus and along cytoplasmic strands radiating to the peripheral regions of the cell. The staining of this newly synthesized material cannot be prevented by ribonuclease treatment, and it shows strong positive tests for protein and polysaccharide. As long as the rhizoid continues to grow, this material remains concentrated in the growing tip region. Material showing similar staining reactions is also found in the cells of the meristematic notch area. Although thallus cell walls do not stain, the rhizoid cell wall adsorbs basic stain, in some cases, metachromatically. Finally, it is suggested on the basis of observations reported here that the sometimes neglected role of the nucleus in theories of unequal cell division-differentiation should be re-examined.     

INHIBITION OF FERN SPORE GERMINATION BY LIPOPHILIC SOLVENTS

Several alcohols and other solvents inhibit germination of spores of the fern, Onoclea sensibilis L. The inhibition is reversible when spores are transferred to solvent-free media. The effectiveness of different solvents, measured by the concentration needed to inhibit germination by 50%, increases with their lipid solubility. The activity of straight-chain alcohols from methanol through n-heptanol is highly correlated with lipid solubility, whereas the correlation is weaker for several other solvents. The results indicate that some lipophilic site in the spore is important in germination.     

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.     

3H-URIDINE INCORPORATION IN THE PREMITOTIC STAGE OF RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA L.

The premitotic rhizoid stage (corona stage) of P. vittata gametophytes was pulsed in radioactive uridine for 5, 15, or 30 min and the data analyzed quantitatively by autoradiography. After 5 min, only the nucleoplasmic compartment is labelled significantly, suggesting that this short pulse is insufficient time for labelled precursor to be fixed in the nucleolus or to be transported from the nucleus to the cytoplasm. Since after 15 and 30 min, all compartments are labelled, with the greatest proportional increase over the nucleolus, it is concluded that cytoplasmic labelling is nuclear in origin and that nucleolar RNA activity is relatively high in this stage.     

AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING SECONDARY RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA

The pre- and postmitotic stages of rhizoid cell differentiation in Pteris vittata L. were pulsed in radioactive uridine for 30 min and the data analyzed quantitatively by autoradiography. The total grain count rises from 594 in the nondifferentiating cell to 1,369 in the corona stage; increases to 2,745 in the internally segmented rhizoid cell (IRC) and to 2,779 in the wheel; and then drops to 1,306 in the protrusion and to 1,261 in the early rhizoid. The significant increase in nucleolar labeling in the corona reflects an increase in ribosomal RNA synthesis for mitosis-associated syntheses. The two-fold increase in grains over the cellular compartments of the IRC and wheel reflects an increase in RNA synthesis in these postmitotic stages. The similarity of labeling profiles between these stages suggests that they are not far enough separated to show a difference in their roles. The decline in total grain counts in the protrusion and early rhizoid stages to half the maximum levels suggests that an overall decrease in RNA synthesis is attended by a shift from a differentiating phase to a steady-state growth system.     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

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.     

SPORE GERMINATION AND CARBON METABOLISM IN FUSARIUM SOLANI. II. ENDOGENOUS RESPIRATION IN RELATION TO GERMINATION

Cochrane , V. W., Jean C. Cochrane , C. b . Collins , and F. G. Serafin . (Wesleyan U., Middletown, Conn.) Spore germination and carbon metabolism in Fusarium solani. II. Endogenous respiration in relation to germination. Amer. Jour. Bot. 50(8): 806–814. Illus. 1963.—Endogenous oxygen uptake by ungerminated macroconidia of Fusarium solani f. phaseoli is more than doubled by exogenous ammonium ion and is increased about 7-fold by germination. The respiratory quotient is halved by the provision of ammonia, which has essentially no effect on the endogenous formation of carbon dioxide. Stimulation by azide and 2,4-dinitrophenol suggests that the supply of phosphate acceptors regulates the rate of endogenous respiration. Mercurials poison the endogenous respiration, cyanide accelerates it, and iodoacetate, arsenite, fluoride, and chelating agents have little effect. Respiration is little affected by changes in pH, external phosphate, oxygen concentration, and spore density, within the limits tested. Spore lipid concentration is increased by cultivation in a high-glucose medium, but this variation in lipid content of spores docs not affect the endogenous Qo₂, nor does a high lipid content abolish the requirement for exogenous carbon for germination. Lipid utilization during endogenous respiration accounts for 37% of the loss in dry weight; lipid is also utilized during germination, but such utilization amounts to only about 5% of the carbon requirement. Neither mannitol nor nitrogenous compounds are significant substrates of endogenous respiration. The oxidation of the exogenous substrates tested does not measurably affect the concomitant rate of endogenous respiration. It is proposed that endogenous respiration can contribute to the synthetic processes of spore germination, although quantitatively insufficient to support germination without exogenous carbon. It is also questioned whether the respiratory quotient is an adequate index of the substrate of endogenous respiration.