Numerical and structural changes in dictyosomes during zygospore germination ofClosterium ehrenbergii

Summary Numerical and structural changes in dictyosomes during the germination of zygospores inClosterium ehrenbergii were examined by electron microscopy. In the dormant mature zygospores, two parallel cisternac were seen which were derived from the disorganization of dictyosomes during the maturation of zygospores. After the induction of germination, the two parallel cisternae developed into dictyosomes with ten or eleven cisternae. The dictyosomes doubled in number by division every day for four days and reached, at the time of germination, a density of distribution similar to that found in the youngest zygospore. On the 4th day after the induction of germination, dictyosomes produced two kinds of vesicles which appear to be involved in the formation of new cell wall layers. The germination of the zygospore was effected by the escape of the cell covered with the new cell wall layers through the broken old cell wall layers.     

Factors Affecting Germination and the Mode of Germination of Zygospores of Choanephora cucurbitarum

Treatment of zygospores of Choanephora cucurbitarum with KMnO₄, NaClO or H₂O₂ effectively activated the spores and induced their germination. The optimum concentration of KMnO₄ for activation of zygospores was 0.25 to 0.5%. Zygospores were not able to germinate in darkness even after activation by KMnO₄. When zygospores from 40 to 50-day-old cultures were treated with 0.5% KMnO₄ solution for 60 min before incubation on water agar at 24% under light, about 50% germinated in 10 days. KMnO₄ treatment killed more than 99% of residual mycelial fragments, sporangiospores and sporangiola in the zygospore suspension. During germination disappearance of oil droplets in zygospores occurred prior to the cracking on zygospore wall. Both sporangial germination and mycelial germination were found. Moreover, sporangiole germination was observed for the first time.     

PHOTOPERIODIC ADAPTATIONS IN EUPATORIUM RUGOSUM

Racial differences based on flowering response to several photoperiods were detectable in two widely separated populations of white snakeroot, Eupatorium rugosum Houtt. The most favorable photoperiod for advanced flowering in Georgia stocks was 12 hr, for those from North Dakota, 14 hr. The difference in latitude between these populations was approximately 12° and represents a mean difference of 75 days in the frost-free season. Under noninductive photoperiod a 1-hr interruption of white light in the middle of 15 hr of darkness stimulated floral initiation in North Dakota plants, whereas the same application at the beginning or at the end of the dark period failed to produce flower buds. The effect of red light (660 mμ) for 10 min given in the middle of the long night was similar to white light on the northern strain, and was negated by far-red (730 mμ). Georgia stocks initiated flowering under 15 hr of darkness but were retarded by white light applied in the middle of the period, thus differing in basic response from North Dakota plants. Red light, in contrast to effects observed in North Dakota plants, retarded initiation of flower buds. This effect was offset by far-red light. When compared with other studies on long-day and short-day species our results suggest that photoperiodic adaptations related to latitudinal distribution occur in white snakeroot. The North Dakota strain showed correspondence to long-day types while short-day tendencies were exhibited by Georgia plants.     

Effect of light on abscisic acid content in photosensitive Scots pine (Pinus sylvestris L.) seed

Dormancy-breaking treatment of the photosensitive Scots pine (Pinus sylvestris L.) seed by white light incubation or a 15-min exposure to red light decreased the abscisic acid content prior to radicle protrusion. Incubation in the dark or exposure to red light followed by a 5-min far-red light irradiation did not cause as great a decrease in abscisic acid content nor was the dormancy relieved. The ability of the far-red light to keep the ABA level high and to prevent germination gradually disappeared as the length of the dark period between the red and far-red treatments was increased to 24 h. ABA was quantified on a gas chromatograph with an electron capture detector.     

Disappearance of the heteroplasmic state for chloroplast markers in zygospores of Chlamydomonas reinhardtii

In the investigations reported here, the length of zygospore incubation or “maturation” prior to the induction of meiosis was found to affect the inheritance pattern of chloroplast genes. The frequency of zygospores transmitting chloroplast alleles from both parents drops with increasing zygospore age following mating, while the frequencies of zygospores homoplasmic for maternal or paternal chloroplast alleles increase correspondingly. Since there is a negligible reduction in viability, zygospores which are initially biparental appear to become pure for the chloroplast genes from one or the other parent prior to the occurrence of cell division. These results are amplified in crosses of mt⁺ cells which have been irradiated with ultraviolet (uv) light or grown in the presence of the base analog, 5-fluorodeoxyuridine, which also perturbs maternal inheritance. Low doses of uv irradiation, applied to zygospores derived from crosses in which the maternal parent was also irradiated prior to mating, increase the biparental zygospore frequency while reducing the proportion of maternal zygospores. This indicates that at least some maternal zygospore clones are actually derived from zygospores which still contain both parental chloroplast genomes prior to the induction of germination. Thus, a subclass of zygospores must contain paternal chloroplast genomes which are either eliminated upon germination or are not expressed in the resulting zygospore clone. Tetrad analysis of biparental zygospores derived from uv-irradiated mt⁺ gametes demonstrates that the frequency of maternal chloroplast alleles in biparental zygospores decreases as they age. One result is an increase in the proportion of meiotic products homoplasmic for all paternal markers. The increased segregation of homoplasmic daughter cells during the meiotic divisions may result from a reduction in chloroplast ploidy by elimination of maternal genomes. Alternatively, it may reflect an altered ratio of maternal:paternal genomes due to continuous rounds of pairing and gene conversion between heterologous chloroplast DNAs leading to genetic drift within the DNA population of the organelle.     

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.     

ALTERED ZYGOSPORE WALL ULTRASTRUCTURE CORRELATES WITH REDUCED ABIOTIC STRESS RESISTANCE IN A MUTANT STRAIN OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

The zygospore of Chlamydomonas is a diploid resting stage that provides protection from environmental extremes. The remarkable abiotic stress resistance of the zygospore can be explained, in part, by the presence of a massive wall that includes a sporopollenin‐containing surface layer ( Van Winkle‐Swift and Rickoll 1997 ). A Chlamydomonas monoica Strehlow zygospore‐specific mutant strain (D19) was obtained previously by screening for loss of chloroform resistance in zygospore populations derived from self‐mating of post‐mutagenesis clones. Exposure of D19 zygospores to solar UV radiation or germicidal radiation also resulted in a pronounced decrease in survival of D19 zygospores relative to wildtype zygospore survival. Similarly, resistance to NaCl‐induced osmotic shock was reduced in D19 zygospores, especially when exposed to very high (e.g., 20% w/v) salt concentrations. Mature zygospores of C. monoica exhibit a UV‐induced blue surface autofluorescence that may indicate the presence of phenolic wall components. The intensity of zygospore autofluorescence was significantly reduced in D19 zygospores. As revealed by TEM, the surface layer of mature homozygous D19 zygospores was disrupted, suggesting a defect in wall assembly. Zygospore‐specific chloroform sensitivity, UV sensitivity, and reduced autofluorescence cosegregated in tetrads derived from D19 heterozygotes (i.e., if a progeny clone from a cross involving D19 and a normal strain was found to be chloroform sensitive, it was always also UV sensitive and showed reduced autofluorescence), indicating that all three characteristics were the consequence of the same Mendelian mutation.     

Flowering responses to light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant

Flowering response and plant form of photomorphogenic mutants (hy1, hy2, hy3, hy4 and hy5) of Arabidopsis thaliana (L.), a long-day plant, were examined in long and short days. There were only slight differences among genotypes including Landsberg wild type with respect to the flowering time under long days. The effect of 1 h light-(night)-breaks of far-red, red, blue and white light given in the middle of the dark period of plants grown under short days, was studied. Effects of far-red light applied at the end or the beginning of the main photoperiod on flowering and plant form were also examined. The light-breaks with all the above mentioned light qualities promoted floral initiation of all the genotypes including the wild type in terms of both the flowering time and the number of rosette leaves. In general, far-red light was most effective. It is possible to classify the hy-mutants into 3 groups by their responses to light-breaks under short day conditions: (a) Mutants hy2 and hy3, which have a reduced number of rosette leaves, and flower early. Red light is as effective as far-red light. The wavelength of light-breaks is relatively unimportant for flowering response. (b) Mutants hy4, hy5 and Landsberg wild type, which have a greater number of rosette leaves, and flower relatively late. The effectiveness of light-breaks is in the following order, far-red, blue, and red light, which is in reverse order to the transformation of phytochrome to the P_fr form. (c) Mutant hy1, which behaves anomalously with respect to relations between flowering time and number of rosette leaves; late flowering with reduced number of rosette leaves. Red, blue and far-red light are effective, but white light is ineffective for reducing the number of rosette leaves. When far-red light was given in the middle of the night or at the end of the main photoperiod, it markedly reduced the number of rosette leaves compared to those grown under short days for all the genotypes, while when applied at the beginning of the main photoperiod far-red light did not affect the number of rosette leaves. Different effects on the plant form dependent on the time of treatment with far-red light-breaks are also discussed.     

Control of Flowering of Xanthium pensylvanicum by Red and Far-red Light

A study was made of the effects of various durations, intensities and combinations of red and far-red light interruptions on the flowering responses of Xanthium pensylvanicum Wallr. A dual response to treatments of far-red light was observed. In short dark periods, far-red light alone did not greatly affect flowering but was able to overcome the inhibition of flowering caused by red light. In dark periods longer than 15 hours, far-red inhibited flowering and added to rather than overcame the inhibition by red light. The dark period length required for far-red inhibition remained the same whether far-red was given at the start or at the eighth hour of darkness.

In 48-hour dark periods Xanthium showed 3 responses to additions of red and far-red light breaks: A) response to red light; B) response to far-red light; and C) response to red followed by far-red light. Red light given any time in the first 30 hours of darkness overcame the inhibitory effect of far-red light given at either the start or the eighth hour of darkness. Red light given later than the thirtieth hour did not overcome the far-red effect.

Approximately the same energy of red light was required to overcome the inhibitory effect of far-red at the second hour of darkness as was required to produce maximum red light inhibition at the eighth hour. Although far-red light was most inhibitory when given early in a long dark period, approximately the same energy of far-red light was required to saturate the far-red response at the fourth, eighth and sixteenth hours.

The results are discussed in relation to other reports of far-red inhibition of flowering in short-day plants.

     

Natural populations of the Closterium ehrenbergii (Desmidiales, Chlorophyta) species complex in Nepal

Several natural populations of the Closterium ehrenbergii Meneghini ex Ralfs species complex were collected in Nepal, in October–December 1982. Water temperature and pH were also recorded. Clonal isolates from these populations were identified to one of four mating groups (H, I, J and M) by test crossing with standard mating-type strains of known mating groups. Groups H and M have smooth walled zygospores, while Groups I and J have scrobiculated zygospore walls. Several undetermined isolates were found in some population samples. In contrast to the previously reported population samples from Nepal, especially from dried soil samples, some of these populations appeared to be rather heavily loaded with mutations that are deleterious to the sexual cycle (i.e. sexual compatibility, zygospore formation and germination). By genetic analysis, a zygote maturation-defective mutation (zym) was detected. One reason for such a heavy genetic load was suggested to be that most population samples had been maintained exclusively by asexual reproduction for a long period in large lakes and nearby ponds, or left-over vegetative populations in paddy fields after other members entered into dormancy through sexual reproduction. The significance of studying such mutations at sexual gene loci is discussed in the light of speciation problems in microalgae.     

Comparative analysis of zygospore transcripts during early germination in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has a haplontic life cycle, and forms diploid zygotes for reproduction. The zygospore, a sporulating zygote, begins germination in response to light signals, generating haploid progenies and inducing several cell-biological events; e.g., DNA synthesis and meiotic division, successively. Their regulatory mechanisms remain largely unknown, so we focused on the early stages of germination and analyzed the dynamics of gene expression associated with the germination process. The gene expression levels of zygospores at 1 and 6 h after light exposure were analyzed by a next-generation sequencing platform, the 454 GS Junior. At 6 h, the photosynthesis pathway, including its antenna proteins and two methionine metabolism-related genes (methionine synthase and sulfite reductase), were up-regulated compared to 1 h after light exposure. Meanwhile, three uncharacterized genes that contained an antibiotic biosynthesis monooxygenase domain and an HSP20/alpha crystallin family protein were specifically expressed at 1 h after light exposure. These gene expressions were also verified by quantitative real-time PCR analysis. These results suggest that the photosynthesis and methionine synthesis pathways, both of which occur in the chloroplast, are activated in zygospores at around 6 h after light exposure, and that some polyketides and/or a small heat shock protein may be related to the initiation of zygospore germination.     

Variations of the Surface Sculpture and Cell Wall Ultrastructure of the Zygospores in Chlamydomonas geitleri (Chlorophyta)

The superficial cell wall ornamentation in the zygospores of the alga Chlamydomonas geitleri Ettl (Chlorophyta) is formed by thickenings of the cell wall which are shaped into a network of anastomosing ribs, sometimes with local wart-like protuberances. Clearly different sculpture patterns (given by presence, arrangement and/or morphological modification of sculpture elements) were accompanied by many transient forms. Sculpture variations occurred even in clonal cultures. In the zygospore cell wall of C. geitleri, the inner, outer and middle layer can be distinguished from the morphological point of view. The relatively thin outer (sporopollenin) layer covers the whole surface of the zygospore wall. The thicker inner layer adhering to the zygospore protoplast forms, either solely or together with the middle layer (possessing a fine meshwork substructure), variously shaped thickening of the zygospore cell wall. Discussed are the ultrastructural morphology of the cell wall in Chlamydomonas zygospores, the striking similarity of the cell wall ultrastructure of zygospores in C. geitleri to the ultrastructure of the cell wall of vegetative cells in some green algae (subfamily Scotiellocystoideae), as well as the extensive morphological variability of the zygospore wall sculpture in C geitleri and its species specificity.     

Dark reactions in the flowering ofLemna perpusilla 6746

Summary This study concerns the effects of red and far-red light on flowering in the short day plantLemna perpusilla 6746. The critical day length for maximum flowering was found to be 10 hours. Exposure to red light near the middle of the dark period inhibited flowering, and the time of maximum sensitivity to red light occurred 9 hours after the beginning of dark periods of either 14 or 17 hours. The inhibition by red light was not reversible by far-red light, which also inhibited flowering, especially when given early in the dark period. Flowering inhibited by exposure to far-red light at the beginning of the dark period could be restored by subsequent exposure to red light. It appears that two photoperiodic partial processes in some plants may be controlled by the red, far-red reversible pigment system.With 5 Figures in the Text     

Effects of Light, pH, Temperature, and Crowding on Megaspore Germination and Sporophyte Formation in Marsilea

Megaspore germination and sporophyte formation of Marsilea vestitaH. and G. was studied under different light, pH, temperature,and crowding conditions during a 6 d experimental period. Maximumgermination and sporophyte development occurs under relativelylow light intensity. Darkness and high light intensity inhibitsporophyte development. Selected wavelengths of light (red,far-red, green, blue) and darkness reduce both megaspore germinationand sporophyte development as compared to white light. Megasporesand sporophytes show maximum development at 25 ?C in light,whereas their development is reduced at all temperatures indarkness. The optimum pH range for megaspore germination ispH 7–8 and that for sporophyte development is pH 7. Sporocarpcontents alter the pH of unbuffered acidic and basic media towarda more optimal growth condition (pH 6–8) for megasporegermination. Megaspore germination and sporophyte developmentvary inversely with conditions of crowding. Root and leaf growthon developed sporophytes is very similar in most treatments.     

The characteristics of light in floral induction in vitro of Cichorium intybus. The possible role of phytochrome

The action of light in the initiation of floral buds in vitro has been studied using monochromatic light qualities on root explants of a long day plant, Cichorium intybus L. cv. Witloof. Red light (660 nm, 0.30 W m^-2) promotes flowering, while far-red (730 nm, 0.31 W m^-2) and irradiation with combined red + far-red (0.20 + 0.41 W m^-2) have no effect. In short day conditions floral response can be obtained in two ways: 1) by interrupting the dark period with 5 brief irradiations of red light (0.45 W m^-2, 12 min) at regular intervals, although these are counteracted by far-red irradiations of equal intensity and duration; 2) by interrupting the long night with 5 h red light applied during the second third of the night, while at the beginning or at the end it is ineffective. Red light efficiency appears to depend on the photosynthetic activity of the tissues, so that flowering increases with increasing intensity of white light and is suppressed if no white light is supplied. The reproductive development is determined by the coordination of proper irradiation conditions with sufficient sensitivity of the perceiving meristematic cells. The period of highest sensitivity to environmental light conditions in the life cycle of a Cichorium root explant occurs between the 8th and the 16th day after the start of the culture. The data strongly suggest that phytochrome is involved in flower induction of Cichorium in vitro.     

Photoperiodic Responses of Brassica campestris cv. Ceres

The photoperiodic responses of Brassica campestris L. cv. Ceres were investigated to determine the suitability of this plant for further studies on the spectral require ments for floral initiation. This is a long-day plant, sensitive to one inductive photocycle on the fourth day from germination. The flowering response increased with the length and intensity of a single period of supplementary light used to extend an 8-hour daylength and was greatest at 25°C. Application of nitrates retarded floral initiation by about two days under short day conditions, but did not affect the re sponse to one long day. Gibberellic acid induced earlier floral initiation under short day conditions. The photoperiodic response was little affected by omitting the main light period immediately before or after the supplementary light, as long as the intensity of supplementary light was greater than 5000 lux. Short interruptions (5–10 minutes) of a single 16-hour dark period with high energy red or far-red radiation did not promote flowering. When given continuously during a single 16-hour dark period, far-red radiation was more effective in flower promotion than an equal energy of red.     

Photoreversibility of flower initiation in Lemna perpusilla as affected by the light quality of the main light period

Reversible floral responses of Lemna perpusilla to red and far-redlights appeared only at the beginning of the inductive darkperiod when the 8 hr photoperiod consisted of white or red light.When blue or far-red light was given during the 8 hr photoperiod,the far-red given at the beginning of the dark period scarcelyinhibited flowering; red/far-red reversibility newly appearedat the middle of the dark period. This indicates that the photoregulationsystem in the flowering of L. perpusilla can be converted fromthe Pharbitis type to the Xanthium type by changing the lightquality of the main photoperiod from white or red to blue orto far-red, which is known to be effective for the so-calledhigh-energy photoreaction of photomorphogenesis. (Received July 2, 1975; )     

Photocontrol of germination in Amaranthus caudatus

Summary Germination of Amaranthus caudatus is inhibited by light, far-red being the most effective part of the spectrum. At temperatures of 25° and below there is a low final germination percentage under continuous far-red whereas above 25° there is only a delaying effect. In the presence of a saturating concentration of gibberellic acid (GA₃) at 25° seeds germinate under continuous far-red although they are delayed. At 25° seeds exposed to 48 hr far-red fail to germinate when transferred to darkness. This induced dormancy can be broken by a single short exposure to red light given at any time after the far-red illumination. This effect of short red can be reversed by a subsequent short period of far-red indicating that the seeds are phytochrome controlled. Although most seeds have escaped from the reversing effect of short far-red after an intervening dark period of 5 hours, germination is greatly reduced by continuous far-red at this time. Results of exposing seeds to varying periods of far-red before and after dark imbibition are interpreted in terms of a continual production of phytochrome in its active P _fr form and a requirement for P _fr action over a long period of time. Effects of intermittent and continuous low intensity far-red on the inhibition of germination provides further evidence for a low energy photoreaction involving phytochrome. Effects on Germination Index of continuous illumination with various light sources maintaining different P _fr /P _total ratios have been investigated. The results suggest that the proportion of phytochrome in the P _fr form is the most important factor in the regulation of germination. A scheme for the phytochrome control of germination in Amaranthus caudatus is presented and possible explanations for the dependence on P _fr /P _total ratio are discussed.Holder of a Science Research Council Studentship.     

Photocontrol of seed germination in the hemiparasite Buchnera hispida (Scrophulariaceae)

Abstract Buchnera hispida, a facultative root parasite of grasses and graminaceous crops, has a light requirement for germination. Studies were carried out on the effects of varying photoperiods with or without preceding dark incubation, on seed germination. Buchnera seeds showed long-day behaviour, since they germinated at all photoperiods including continuous light, and longer photoperiods were more effective in triggering seed germination than shorter photoperiods. Also, effects of red and far-red light indicated that the phytochrome system is operative in the light-induced germination of Buchnera. Although dark incubation in water before illumination was not absolutely necessary for germination, it caused the seeds to respond more rapidly to light. The longer the time of the dark incubation the more responsive the seeds were to photoperiod except when 15 min light was given. The effectiveness of a preceding dark incubation in making Buchnera seeds sensitive to rapid light action was completely inhibited at 4°C. This is in agreement with the hypothesis that a reaction partner of the far-red absorbing form of phytochrome is produced during dark incubation of Buchnera seeds. Such an intermediate has also been reported in some positively photoblastic seeds of non-parasitic flowering plants.     

MATURATION OF ALGAL ZYGOTES: ALTERNATIVE EXPERIMENTAL APPROACHES FOR CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)1

Young zygotes from crosses of Chlamydomonas reinhardtii Dang. mutant and wild-type strains were incubated, in the presence or absence of light and/or nitrogen to determine whether continuation of conditions inducing gamete formation permits zygospore formation without loss of viability. Different culture media, continuous illumination vs. dark incubation and various durations of the maturation period were tested, for effect on zygospore germination efficiency, zygospore “burst size” and zoospore viability. Following either the routine maturation procedure of dark incubation on standard minimal medium, or following a new procedure of incubation under continuous illumination on N-free medium, zygospore formation can be ensured and high germination efficiencies obtained within 3 days after mating. Tetrad analysis indicates meiosis occurs normally whether zygotes have been matured in the presence or absence of light or nitrogen. Preliminary data suggest an effect of increased maturation time on the transmission of cytoplasmic genes, if a N-free continuous illumination maturation protocol is followed. Two experimental approaches for the maturation of C. reinhardtii zygotes are suggested and advantages of each are discussed.