Intergeneric fusion of Zygnemataceae protoplasts

In intergeneric fusion fromMougeotia andZygnema protoplasts, the fate of fusion products, as well as nuclei and chloroplasts, could be classified according to the number of protoplasts involved from the two algae. Stable elongation growth occurred only in products of groups involving one protoplast from one alga and several protoplasts from the other alga. The features of the elongating products were those of the alga more numerously represented. The different nuclei combined by fusion failed to co-exist. In the groups involving one protoplast from one alga and several from the other, the nucleus from the former degenerated in an early period and only the nuclei from the latter were maintained. Also, the different chloroplasts combined did not co-exist. The genus of the chloroplasts maintained coincided with that of the nuclei maintained. The chloroplasts from the other genus degenerated gradually. An early morphological change in the degenerating chloroplasts was seen in the quantity of starch grains. Later, the chloroplasts generally became rounded, In degeneratingZygnema chloroplasts, thylakoid stacking was prominent. Without collapse of the thylakoid or accumulation of plastoglobules, the degenerating chlorplasts showed rupture of the chloroplast envelope.     

Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts

Summary Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.Supported by National Research Council of Canada, Grant A6304     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

Summary Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.     

Rapid-growth responses of corn root segments: Effect of citrate-phosphate buffer on elongation

Summary Chloroplasts from the alga, Vaucheria dichotoma (L.) Ag., are taken up into protoplasts of carrot (Daucus carota L.) during polyethylene-glycol treatment. Since chloroplasts are found with equal frequency in uni- and multinucleate protoplasts, chloroplast uptake does not depend on protoplast fusion. However, higher frequencies of chloroplast uptake are observed when experimental conditions favor greater aggregation of protoplasts. The intracellular localization of chloroplasts is confirmed by electron microscopy, and it is shown that the chloroplasts, once within the protoplasts, are not surrounded by a limiting membrane of carrot origin.     

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.Abbreviations PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy Supported by National Research Council of Canada, Grant A6304     

Rapid degeneration of the protoplasm in artificially induced small cells ofMicrasterias crux melitensis

Summary Growing cells ofMicrasterias crux melitensis grew into asymmetric cells with small half cells in 0.2 M mannitol. Cells with small half cells could multiply in the normal culture medium. When mother half cells were less than half the size of the normal mother half cells small daughter half cells were formed. When the mother half cells were very small and had no chloroplasts, the protoplasts rapidly degenerated. Many autophagic vacuoles and lysosomes were found in the protoplasm of the degenerating cells. The rapid degeneration was assumed to be due to the activity of these autophagic lysosomes, whose induction is probably triggered by some factor produced by the shortage of protein precursors and energy sources.     

Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L.

Summary Hypocotyl protoplasts of 45 different genotypes of German winter oilseed rape Brassica napus L. (double zero quality: high in yield, seeds low in erucic acid and glucosinolate content) were regenerated to plants. Triazine/triazinone (tri)-tolerant chloroplasts of the Canadian spring oilseed rape variety OAC Triton were introduced into some winter oilseed rapes by means of protoplast fusion. X-ray irradiation was used to limit the transfer of nuclear DNA of Triton protoplasts and to promote the selective transfer of tri-tolerant chloroplasts. Regenerated cybrid plants survived a treatment rate of 1000 g/ha metribuzin. The presence and segregation of the tri-tolerant chloroplasts in winter oilseed rape plants, regenerated from fusion products and their progeny, was investigated by restriction fragment length polymorphism (RFLP). Our results indicate that chloroplast segregation was not completed in plants regnerated from fusion products derived from X-irradiated OAC Triton mesophyll protoplasts and German winter oilseed rape hypocotyl protoplasts. In regenerants and their progeny both chloroplast types can still be present. Chloroplasts derived from wintertype protoplasts can outcompete tritolerant chloroplasts during plant development. In some instances, even progeny plants not kept under selective conditions (metribuzin) lost tri-tolerant chloroplasts. A homogenous population of tri-tolerant chloroplasts was necessary to obtain stable tri-tolerant winter oilseed rape plants.     

Spiral cell wall growth in zygnemataceae

Using two species ofSpirogyra and one species ofZygnema, it was demonstrated on a quantitative basis that these algal filaments grow while twisting around their own axis. The sense of spiral growth of the cell wall inSpirogyra-1 was always left-handed being coincident with the sense of chloroplast helix. InSpirogyra-2, the growth vector of the cell wall was likewise left-handed in most cases, but there occurred right-handed growth also. InZygnema both left-handed and right-handed senses of spiral growth were found in nearly equal frequencies. Besides the natural cell wall growth, the effects of longitudinal tension and turgor pressure on elongation and twisting of the filaments were briefly studied. It was shown that the cell wall of Zygnemataceae exhibited mechanical anisotropy in helical direction.     

LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS OF PREFERENTIAL DESTRUCTION OF CHLOROPLAST AND MITOCHONDRIAL DNA AT EARLY MALE GAMETOGENESIS OF THE ANISOGAMOUS GREEN ALGA DERBESIA TENUISSIMA (CHLOROPHYTA)1

Gametogenesis in male and female gametophytes was studied by light microscopy and EM in the dioecious multinucleate green alga Derbesia tenuissima (Moris & De Notaris) P. Crouan & H. Crouan, where male and female gametes differ in size. Gametogenesis was divided into five stages: 32 h (stage 1), 24 h (stage 2), 16 h (stage 3), 8 h (stage 4), and 0.5 h (stage 5) before gamete release. At stage 1, the first sign of gametogenesis observed was the aggregation of gametophyte protoplasm to form putative gametangia. At stage 2, gametangia were separated from the vegetative protoplasm of gametophytes. Morphological changes of nuclei and organelles occurred at this early stage of male gametogenesis, and organelle DNA degenerated. At stage 3, male organelle DNA had completely degenerated, whereas in female gametangia, organelle DNA continued to exist in both chloroplasts and mitochondria. Gametogenesis was almost completed at stage 4 and fully at stage 5. Small male gametes had a DNA‐containing nucleus and a large mitochondrion and one or several degenerated chloroplasts. The mitochondria and plastids were devoid of DNA. The large female gametes had a nucleus and multiple organelles, all of which contained their own DNA. Thus, degeneration of chloroplast DNA along with morphological changes of organelles occurred at male gametogenesis in anisogamous green algae (Bryopsis and D. tenuissima), in contrast with previous studies in isogamous green algae (Chlamydomonas, Acetabularia caliculus, and Dictyosphaeria cavernosa) in which degeneration of chloroplast DNA occurred after zygote formation.     

The taxonomic significance of the mitotic spindle and nucleus-associated organelle ofEntomophaga aulicae

Summary Nuclei in protoplasts ofEntomophaga aulicae contain abundant condensed chromatin and a large central nucleolus. The metaphase spindle occupies a small eccentric area of the nucleus while the remainder of the nucleus is filled with condensed chromatin. Small portions of condensed chromatin are aligned along a broad metaphase plate and connected to the spindle poles by kinetochore microtubules. The nucleus associated organelle (NAO) is a solid barlike structure which lies at the spindle poles and is closely associated with the outer membrane of the nuclear envelope. Comparison of the nuclear characteristics ofE. aulicae with those of other members of theEntomophthorales supports the separation of theEntomophthoraceae from theBasidiobolaceae andAncylistaceae. Further comparison of details of nuclear division in theEntomophthoraceae, specifically NAO morphology, may be useful in helping to delineate evolutionary lines within the family.     

PHYLOGENY OF SPIROGYRA AND SIROGONIUM (ZYGNEMATOPHYCEAE) BASED ON RBCL SEQUENCE DATA1

DNA sequence data were obtained for the gene encoding the large subunit of RUBISCO (rbcL) from 26 strains of Spirogyra and seven of Sirogonium, using as outgroups 10 genera in the Zygnematales and Desmidiales (Closterium, Cosmarium, Cylindrocystis, Gonatozygon, Mesotaenium, Netrium, Penium, Zygnema, Zygnemopsis, Zygogonium). Sequence data were analyzed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI), with bootstrap replication (MP, ML) and posterior probabilities (BI) as measures of support. MP, ML, and BI analyses of the rbcL data strongly support a single clade containing Spirogyra and Sirogonium. The Spirogyra taxa are monophyletic, with the exception of Spirogyra maxima (Hassall) Wittrock, which is nested within a clade with Sirogonium and shares with them the characters of loosely spiraled chloroplasts (<1 complete turn per cell) and anisogamy of gametangial cells; S. maxima differs from Sirogonium in displaying well‐defined conjugation tubes rather than a tubeless connection involving bending (genuflection) of filaments. The ML and BI analyses place this Sirogonium/Spirogyra maxima clade sister to the remaining Spirogyra. Morphological differences among strains of Spirogyra grouped together on the basis of rbcL data, including laboratory strains derived from clonal cultures (Spirogyra communis, S. pratensis), indicate that some characters (filament width, chloroplast number) used in the traditional taxonomy of this group are poor measures of species identity. However, some characters such as replicate end walls and loose spiraling of chloroplasts may be synapomorphies for Spirogyra clades.     

UV-B radiation and selenium affected energy availability in green alga <Emphasis Type="Italic">Zygnema</Emphasis>

Green alga Zygnema was exposed to three concentrations of selenium and two levels of UV-B radiation. The combined effects of both treatments on energy availability; photochemical quantum yield and respiratory potential were studied. Our findings show that traces of selenium enhance metabolic process connected with photochemical quantum yield and mitochondrial respiration. Surprisingly, selenium does not diminish the effects of UV-B radiation; on the contrary, the combined action of UV-B radiation and traces of selenium leads to pronounced negative effects on photochemical quantum yield and the respiratory potential. Selenium is involved in the activation of energy resources in green alga Zygnema. The importance of selenium for activity of the mitochondria is possibly an evolutionary recollection from an endosymbiotic bacterium.     

Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.     

Nuclear degeneration in the myxomycetePhysarella oblonga

Summary Nuclear degeneration is a distinct and normal process occurring during sporogenesis in the myxomycete,Physarella oblonga. About 30% of the nuclei present in the developing sporangium abort. Nuclei that undergo degeneration are first surrounded by vacuoles. The membranes of the vacuoles and nuclear envelope are 75 thick. As degeneration progresses the vacuoles fuse resulting in a nucleus surrounded by a single, narrow vacuole. The membrane next to the nucleus degenerates while the membrane adjacent to the sporangial cytoplasm thickens to 100 . Lysosomes then fuse with the vacuole, and digestion of the nucleus begins. The undigested remains of nuclei (residual bodies) are excreted from the protoplast. Acid phosphatase is localized in the lysosomes and degenerating nuclei.A portion of this work was conducted at Department of Botany and Plant Pathology, Iowa State University, Ames.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

Phylogenetic position of Zygogonium ericetorum (Zygnematophyceae,Charophyta) from a high alpine habitat and ultrastructural characterization of unusual aplanospores

Zygogonium ericetorum, the type species of the genus, was studied from a natural population collected in Mt. Schönwieskopf, Tyrol, Austria. Generic concepts of Zygogonium and Zygnema were tested with atpB, psbC, and rbcL gene sequence analysis, which showed a sister relationship between Z. ericetorum and Mesotaenium, in an early branching clade sister to a grouping of Zygnema and several other filamentous and unicellular zygnematalean taxa. A variety of light, confocal, transmission electron microscopy, and cytochemical techniques provided new data on the variable chloroplast shape of Z. ericetorum, and its aplanospore structure and development, which has been previously considered taxonomically important but has been ambiguously interpreted. Zygogonium can be distinguished from other zygnematophytes (particularly Zygnema), based on the combination of two characters: (i) irregular, compressed plate‐like chloroplasts and (ii) residual cytoplasmic content left in sporangia outside of the fully developed aplanospores or zygospores. The presence of a sporangial wall that separates the spores from the parent cell should be excluded from the definition of Zygogonium, because it is also observed in Zygnema. Similarly, the ecological characterization of Zygogonium as acidophilic is not unique to the genus. The names of 18 species currently belonging to Zygogonium are here changed to Zygnema, because of incompatibility with this new proposed Zygogonium concept. In the species transferred to Zygnema, chloroplasts are typically stellate in three‐dimensions, and the entire content of fertile cells is transformed into the spore, so there is no cytoplasmic residue.     

Organellar segregation,rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli

Summary Cauliflower protoplasts were fused to determine the effect of protoplast source and pretreatment on organellar segregation in fusion products. Mitochondrial and chloroplast type were determined for over 250 calli from eight fusions between iodoacetate-treated or -irradiated leaf or hypocotyl protoplasts with fertile or Ogura cytoplasms. Organelles in fusion-derived calli were identified with five mitochondrial probes and one chloroplast probe. Mitochondrial and chloroplast segregation were independent but biased. Most calli had B. oleracea chloroplasts, but more calli had Ogura mitochondria than B. oleracea ones. Neither protoplast source nor pretreatment alone affected organelle segregation. However, iodoacetate treatment of hypocotyl protoplasts reduced their mitochondrial contribution to the fusion products although it did not affect chloroplast segregation. Over half of the calli had mitochondrial genomes distinct from those of either fusion partner; many of these contained the complete mitochondrial genome of one partner along with some mitochondrial DNA from the other. Out of 258 calli, 83 showed evidence of mitochondrial recombination, most commonly by formation of a novel 11-kb PstI fragment near the atp9 region.     

Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement

We characterized four meiotic mutants of the fission yeast Schizosaccharomyces pombe by live observation of nuclear movement. Nuclei were stained with either the DNA-specific fluorescent dye Hoechst 33342 or jellyfish green fluorescent protein (GFP) fused with the N-terminal portion of DNA polymerase α. We first followed nuclear dynamics in wild-type cells to determine the temporal sequence of meiotic events: nuclear fusion in the conjugated zygote is immediately followed by oscillatory nuclear movements that continue for 146 min; then, after coming to rest, the nucleus remains in the center of the cell for 26 min before the first meiotic division. Next we examined nuclear dynamics in four meiotic mutants: mei1 (also called mat2), mei4, dhc1, and taz1. Mei1 and mei4 both arrest during meiotic prophase; our observations, however, show that the timing of mei1 arrest is quite different from that of mei4: the mei1 mutant arrests after nuclear fusion but before starting the oscillatory nuclear movements, while the mei4 mutant arrests after the nucleus has completed the oscillatory movements but before the first meiotic division. We also show examples of the dynamic phenotypes of dhc1 and taz1, both of which complete meiosis but exhibit impaired nuclear movement and reduced frequencies of homologous recombination: the dhc1 mutant exhibits no nuclear movement after nuclear fusion, while the taz1 mutant exhibits severely impaired nuclear movement after nuclear fusion. Received: 28 October 1999; in revised form: 10 December 1999 / Accepted: 13 December 1999