Metal-binding sites in germinating fern spores (Onoclea sensibilis)

Summary During germination of the spore of the sensitive fernOnoclea sensibilis L. the nucleus migrates from a central position to the proximal face and then to one end of the ellipsoidal spore. An asymmetric cell division follows giving rise to a small cell which differentiates immediately into a rhizoid, and a large cell which divides further to give rise to the prothallus. The proximal face of the spore coat is differentiated from the remainder of the spore by its ability to bind nickel ions under certain conditions and by its staining with a sulfide-silver procedure which localizes heavy metals. The inner portion of the exine at the proximal face is differentiated from the outer part by its ability to stain with sulfide-silver at specific periods during germination. The exine at the proximal face also contains pore-like structures 50 nm in diameter which extend from the inner layer of the exine to the outer surface. Sulfide-silver staining material appears to be extruded through the pores at specific periods during germination. The percentage of spores showing nickel-binding and sulfide-silver stainability increases sharply during the first two to four hours of imbibition, then decreases sharply during the following two hours. This is followed by a second rise in staining at 8 to 12 hours of imbibition.The role of the ion-binding sites in the exine is discussed in relation to the stable polarity of the spore.Publishing prior to 1984 asAlix R. Bassel     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

Cell expansion in the filamentous gametophyte of the fernOnoclea sensibilis L.

Filamentous gametophytes of the fernO. sensibilis were exposed to paired combinations of light of different qualities, hormones and cations in the attempt to elucidate the underlying processes that regulate cell expansion. Simultaneous treatments with high-pH buffers or the auxin antagonistp-chlorophenoxyisobutyric acid abolished blue-light-mediated expansion but did not influence growth in red light. In contrast, the red-light response was preferentially altered by the ethylene absorbant KMnO₄ or the Ca²⁺ chelator ethyleneglycol-bis(-aminoethyl ether) N,N-tetraacetic acid. The Ca²⁺ ionophore A23187 caused a significant reduction in cell expansion under both blue and red irradiation. A marked promotion of expansion was mediated by high concentrations of indole-3-acetic acid, but this effect was dependent on the presence of low-pH buffers. The ethylene-generating agent 2-chloroethylphosphonic acid decreased the magnitudes of both photoresponses; this inhibition was further enhanced by high Ca²⁺ concentrations. These findings and those with other plants are interpreted in terms of two independent control mechanisms for cell expansion: 1) a blue light photoreceptor-auxin-hydrogen ion system, and 2) a phytochrome-ethylene-calcium ion system.Abbreviations APW-X artificial pond water (the associated number designates pH) - CEPA 2-chloroethylphosphonic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)N,N-tetraacetic acid - IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid     

Effects of cyanide, salicylhydroxamic acid, and temperature on respiration and germination of spores of the fern Sphaeropteris cooperi

During the first 96 h of culture, germinating spores of the fern Sphaeropteris cooperi (F. v. Muell.) Tryon showed a gradual rise in respiratory activity to a maximum of about 6.5 μl 0₂ h⁻¹ mg⁻¹ dry wt. This was followed by a transitory decline in rate, concluded by a second respiratory rise preceding the emergence of the rhizoid after 192 h of culture. Oxygen uptake during the first 120 h of germination was insensitive to 1 m M potassium cyanide (KCN) but was inhibited by 1 m M salicylhydroxamic acid (SHAM); however, beyond this time cyanide showed increasing inhibitory effectiveness whereas SHAM became less effective. Regardless of time of application, KCN had no effect on germination. Maximum inhibition of germination by SHAM was achieved if applied up to 120 h after culture initiation, after which spores became insensitive to SHAM. Heat treatment (50°C for 90 min) during the cyanide-resistant phase of respiration (0 h–120 h) induced cyanide-sensitive respiration and completely inhibited spore germination. Elevated temperatures had little effect if applied during the cyanide-sensitive phase (beyond 120 h). Temperature inhibited spores regained their ability to germinate if maintained in culture until the cyanide-resistant pathway was restored and then subjected to a second photoinductive light treatment. These results suggest the presence and possible involvement of the cyanide-resistant, alternative respiratory pathway during germination of Sphaeropteris cooperi spores.     

Sequential release of cellulose enzymes during germination ofTrichoderma reesei spores

The pattern of release of extracellular cellulase during the germination ofTrichoderma reesei spores has been examined. The four enzymes namely, filter paper degrading enzyme, Β-1,4 endoglucanase, Β-glucosidase and xylanase appear sequentially in the culture broth during germination of the spores. The order of enzyme appearance is not altered by the type of carbon source in the germination medium. Measureable quantities of filter paper degrading enzyme is detected only after the outgrowth has occurred. A possible mechanism of spore germination and induction of the enzymes by insoluble cellulose is suggested. An erratum to this article is available at .     

The role of calcium ions in phytochrome-mediated germination of spores of Onoclea sensibilis L.

Phytochrome is confirmed to be the photoreceptor pigment in the germination response of Onoclea sensibilis L. by demonstrating red-far-red (R-FR) photoreversibility. External Ca²⁺ is required for this response with a threshold at a submicromolar concentration. Ethylene glycol-bis(-amino-ethyl ether)-N,N,N,N-tetraacetic acid, La³⁺ and Co²⁺ reversibly inhibit germination. Lanthanum only inhibits germination when applied before or during irradiation, indicating that the external Ca²⁺ requirement is transient, although in the absence of Ca²⁺ the R-stimulated system remains maximally poised to accept the ion for over 4 h after irradiation. The ability to respond to Ca²⁺ 4.1 h after R-irradiation is not reversed by FR-irradiation, indicating that Ca²⁺ transport has been uncoupled from phytochrome. Barium and Sr²⁺, but not Mg²⁺ can substitute for Ca²⁺. Artificially increasing the concentration of intracellular free Ca²⁺ with the ionophore A 23187 stimulates germination in the dark. The Ca²⁺-calmodulin antagonists, trifluoperizine and chlorpromazine, reversibly inhibit germination. Calcium is required in phytochrome-mediated fern spore germination; it may be acting as a second messenger.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - R fed light     

Chloroplast DNA in Pinus monticola

Summary Restriction sites on the chloroplast genome of Pinus monticola have been mapped, and the gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, the genes for the photosystem II polypeptides psbA, psbD and psbC, and the 16S and 23S ribosomal RNA genes have been located. The genome lacks the large inverted repeat characteristic of most angiosperms. The gene order is similar to that found in P. radiata. The presence of dispersed repeated sequences is likely. Two structural features, lack of a large inverted repeat and the presence of dispersed repeats, may confer a degree of variability on the genome which will prove useful in studies of population structure.     

Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

Chloroplast development and the synthesis of chlorophyll a and b and chlorophyll protein complexes I and II in the dark in Tradescantia albiflora (Kunth)

Continued synthesis of chlorophyll a and chlorophyll b occurs in Tradescantia albiflora Kunth on transfer to darkness. This synthesis continues for several days and may result in a doubling of chlorophyll content per leaf. It is accompanied by continued cell division and development of normal chloroplast ultrastructure, including stacked thylakoids.     

Chloroplast DNA restriction site mapping and the phylogeny ofRanunculus (Ranunculaceae)

A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense and one species ofTrautvetteria. A total of 341 informative restriction site changes were detected. Parsimony jackknifing, bootstrapping and decay analysis were undertaken in order to evaluate the amount of support for the monophyletic groups. The results suggest that the analysed species ofRanunculus are divisible into two main clades. Only few of the traditional sections and subgenera ofRanunculus are monophyletic. The genusTrautvetteria is nested within a clade comprising, e.g.Ranunculus cymbalaria, R. andersonii, R. lapponicus andR. ficaria. SubgenusBatrachium lies within a larger clade containing, e.g.R. sceleratus andR. hyperboreus. Contractions of the inverted repeat due to parallel deletions of 200–300 bp close to the J_SB have occurred in many clades and the phylogenetic distribution of this size reduction was mapped among the species.     

Chloroplast DNA variation and phylogeny of theRanunculaceae

Restriction site mapping of chloroplast DNA from 31 species representing 26 genera of theRanunculaceae was performed using eleven restriction endonucleases. The chloroplast genome varies in length from approximately 152 to 160 kb. Length variants are frequent in theRanunculaceae and range from usually less than 300 bp to rarely 1.5 kb. The inverted repeat is extended into the large single copy (LSC) region by 4–4.5 kb inAnemone, Clematis, Clematopsis, Hepatica, Knowltonia, andPulsatilla. Several inversions are present in the LSC-region of the cpDNA in all these genera and inAdonis. The frequency of restriction site mutations varies within the chloroplast genome in theRanunculaceae between 4 and 32 mutations per kilobase, and is lowest in the inverted repeat and the regions containing the ATPase-genes and the genespsaA, psaB, psbA, rpoB, andrbcL. A total of 547 phylogenetically informative restriction sites was utilized in cladistic analyses of the family using Wagner, Dollo, and weighted parsimony. These three parsimony analyses result in different tree topologies. Four, six, and one equally most parsimonious trees were obtained with Wagner, Dollo, and weighted parsimony, respectively. The amount of support for the monophyletic groups was evaluated using bootstrapping and decay analysis. All three parsimony methods suggest thatHydrastis is the sister group to the remainder of theRanunculaceae, and that theAnemone-Clematis group, which shares several derived cpDNA rearrangements, is monophyletic. Only a few of the traditional groups in theRanunculaceae are supported by cpDNA restriction side data. Only Dollo parsimony provides support for the hypothesis thatThalictroideae andRanunculoideae are monophyletic.     

Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

Effects of caffeine on germination and differentiation in spores of the fern, Onoclea sensibilis

During spore germination in the fern, Onoclea sensibilis L., the nucleus moves from a central position to one end, and an asymmetrical cell division partitions the spore into two cells of greatly unequal size. The smaller cell differentiates directly into a rhizoid, whereas the larger cell and its derivatives give rise to the prothallus. In the presence of 5 mM caffeine, the nuclei of most of the spores undergo mitotic replication, whereas cell wall formation is blocked. Multinucleate single cells are produced, which are capable of growth, but no rhizoid differentiation occurs. In some cases a partial cell wall is produced, but the nucleus moves through the discontinuity back to the center of the spore, and the enucleate, incompletely partitioned small “cell” fails to differentiate into a rhizoid. In less than 1% of the spores a broad protuberance, whose wall is yellow-brown, is formed in a multinucleate single cell. The color, staining reaction to ruthenium red, and ultrastructural appearance of the protuberance resemble that of the rhizoid wall. It appears that infrequently in the caffeine-treated spores, a feature which is characteristic of rhizoids is expressed, in the absence of asymmetric cell division, in a cell which otherwise is unable to produce a rhizoid. The results are interpreted to mean that the spore has a highly localized, persistent differentiated region. For rhizoid differentiation to occur, a nucleus must be confined in that region – a confinement which normally is accomplished by the geometrically asymmetric first cell division of germination.     

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole     

Chloroplast DNA inheritance in Populus

Summary The inheritance of chloroplast (cp) DNA was examined in F₁ hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F₁ offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.     

Effect of red light and gibberellic acid on lipid metabolism in germinating spores of the fern Anemia phyllitidis

The spores of the fern Anemia phyllitidis contain abundant quantities of lipid as reserve material. Germination of these spores can be induced either by red light or, even in the dark, by gibberellic acid. The effects of both these factors on lipid degradation, lipase and isocitrate lyase activities, and on the fatty acid composition have been studied in the course of the germination process. During germination in darkness with gibberellic acid, the fatty acid composition remained similar to that in the ungerminated spore. In contrast, when spores were germinated in red light, α-linolenic acid was synthesized. Little activity of lipase and isocitrate lyase could be detected in the dry spore. Red light or gibberellic acid affected a dramatic increase of the activities of these enzymes. Lipid breakdown and lipase activity were more active in red light, however. Permanent stimuli were necessary for growth and complete lipid degradation. Induction of germination simultaneously with both factors revealed an additivity of the effects of red light and gibberellic acid.     

Nicotiana chloroplast genome III. Chloroplast DNA evolution

Summary Nicotiana chloroplast genomes exhibit a high degree of diversity and a general similarity as revealed by restriction enzyme analysis. This property can be measured accurately by restriction enzymes which generate over 20 fragments. However, the restriction enzymes which generate a small number (about 10) of fragments are extremely useful not only in constructing the restriction maps but also in establishing the sequence of ct-DNA evolution. By using a single enzyme, Sma I, a elimination and sequential gain of its recognition sites during the course of ct-DNA evolution is clearly demonstrated. Thus, a sequence of ct-DNA evolution for many Nicotiana species is formulated. The observed changes are all clustered in one region to form a hot spot in the circular molecule of ct-DNA. The mechanisms involved for such alterations are mostly point mutations but inversion and deficiency are also indicated. Since there is a close correlation between the ct-DNA evolution and speciation in Nicotiana a high degree of cooperation and coordination betwen organellar and nuclear genomes is evident.     

Chloroplast development and the synthesis of chlorophyll and protochlorophyllide in Zostera transferred to darkness

Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night.Abbreviations Chl chlorophyll - T _o time of transfer to darkness     

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

AIMS: To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. METHODS AND RESULTS: Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. CONCLUSIONS: This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.     

Chloroplast DNA restriction analysis and the infrageneric grouping ofAllium (Alliaceae)

The utility of chloroplast DNA variation for checking a recently proposed infrageneric classification of the genusAllium was tested. cpDNA restriction patterns of 49 species representing the main subgenera, sections, and subsections of the existing classification were compared. 363 different fragments generated by 4 restriction enzymes were identified and analysed by UPGMA clustering. The resulting phenogram largely confirms the subgeneric classification based on an integration of morphological and other methods.