Preferential digestion of chloroplast DNA in male gametangia during the late stage of gametogenesis in the anisogamous algaBryopsis maxima

Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).     

Comparative inhibitory effects of cytochalasins on the gametangial differentiation inAllomyces arbuscula

In contrast to cytochalasin D which selectively prevents differentiation of female gametangia, cytochalasins A, B and E also masculinizeAllomyces arbuscula by preventing septation between female and male gametangia, thereby allowing the dominant expression of the male characteristics.     

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.     

Actin stringently accumulated in the specifically positioned,differentiating female gametangia of Allomyces

Summary Female gametangia of the normal bisexual Allomyces species are richer in fluorescently probed (FITC) actin, independent of their apical or subapical positioning during differentiation on the fertile hyphae. The anti-actin, cytochalasin D, can selectively suppress male differentiation in both species.     

Untersuchungen zur Entwicklung des Phycomyceten Allomyces arbuscula

Polysomes were isolated both from growing gametophytes of Allomyces arbuscula and from gametangia prepared from mycelia at different periods during gametogenesis. Analysis of polysomes by sucrose gradients showed that ribosomes present in the gametangia monosome pool were shifted into polysomes. This shift was found to be correlated with gametangia differentiation. The ribosome distribution remained virtually unchanged during the early stage of gamete formation. In mature gametes and swarming zygotes a low level of polysomes was detected. Labeling of rRNA by ³²PO₄ demonstrated a de novo synthesis of monosomes throughout the period of gametangia differentiation. No incorporation of ³²PO₄ was found to be present in ribosomes prepared from gametangia after onset of gamete formation. On the basis of these labeling experiments it is concluded that radioactivity in polysomes extracted from mature gametes and swarming zygotes can be attributed in part to conserved mRNA.Synchronous formation of gametangia was induced by transferring the vegetative mycelia from growth medium into a low salt buffer. Under these conditions the incorporation of either ³²PO₄ or ³H-uridine into RNA, particularly into rRNA, was found to be markedly decreased. This obviously indicates a shutdown of RNA synthesis. rRNA from induced mycelia examined by polyacrylamide gel electrophoresis was found to be severely degraded. In contrast to this, rRNA isolated from ribosomes of developing gametangia and from gametes exhibited no degradation products. It is suggested that endonucleases cause rRNA hydrolysis in the hyphal cytoplasm during gametangia differentiation. Ribosomes compartmentalized in gametangia seem to be inaccessible to nucleases during the later process of gametogenesis.

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Abkürzungen MAK Methyl-Albumin-Kieselgur - PAA Polyacrylamid - stains all 4,5,4,5-Dibenzo-3,3-diäthyl-9-methylthiacarbocyaninbromid (Serva, Heidelberg)     

Effects of chloramphenicol on the mitochondrial respiratory chain in the wild strain and in a cytoplasmic chloramphenicol-resistant mutant of Tetrahymena pyriformis

The effects of chloramphenicol (CAP) on mitochondrial respiratory activity in the wild strain (ST) and in a cytoplasmic CAP-resistant mutant (STR1) of Tetrahymena pyriformis were studied by determining oxygen consumption, by spectrophotometry, and by cytochemistry. In the absence of CAP both strains had the same respiration capacity, and the low-temperature spectra of their isolated mitochondria were similar. Furthermore, the mitochondria of both strains showed a positive reaction with diaminobenzidine, denoting a similar cytochrome oxidase activity. However, when cells were grown in CAP for 24 or 48 h, the peaks of cytochrome oxidase and cytochromb b were almost absent in the wild type. In this type the oxygen consumption was greatly decreased, and the mitochondria were no longer stained by diaminobenzidine. In the mutant, the peaks of cytochrome oxidase and cytochrome b were decreased only; respiration was less affected than in the wild type, and cytochrome oxidase activity was still disclosed by the diaminobenzidine reaction. These results show that CAP inhibits the synthesis of two cytochromes (b and oxidase) which are partially translated into the mitochrondria of T. pyriformis. In the mutant, CAP reduces only the mitochondrial translation, resulting in reduced mitochondrial activity and reduced growth rate of the cell. These results are compared with the nucleo-mitochondrial regulation mechanisms discussed in our previous works.     

The effects of cytochalasin B and colchicine on the morphogenesis of mitochondria in Drosophila hydei during meiosis and early spermiogenesis

Summary Morphogenesis of mitochondria in male germ cells in cultivated cytocysts begins in early prophase I at which time mitochondria thicken and become ordered along the spindle apparatus during meiosis. At the end of the second meiotic division they aggregate to form the Nebenkern.In the presence of colchicine or cytochalasin B mitochondria are able to begin differentiation, although the correct course of meiosis is not guaranteed. In medium supplemented with colchicine they undergo normal thickening but do not aggregate, in a pattern known from untreated cultures. This may indicate that microtubules are involved in the aggregation process of mitochondria as colchicine is known to inhibit microtubule formation. Moreover, in cell cultures treated with cytochalasin B mitochondrial aggregation does occur; it is concluded that microfilaments, which are sensitive to cytochalasin B, do not play a detectable role in the aggregation of mitochondria.     

Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Protoplast fusion-derived Ogura male sterile cauliflower with cold tolerance

Cauliflower (Brassica oleracea L. ssp. botrytis) protoplasts with Ogura male sterile and fertile B. oleracea cytoplasms were fused, producing plants with an array of organellar types. Plants with Ogura mitochondria were male sterile; those with B. oleracea chloroplasts were cold tolerant. In some fusions, unfused parental protoplasts were eliminated by double inactivation with iodoacetate and gamma-irradiation; in others, fused protoplasts were physically isolated by micromanipulation or by cell sorting. Double inactivation fusions produced the most plants, including many which were male sterile, female fertile, cold tolerant and diploid.Abbreviations IA iodoacetate - FDA fluorescein diacetate - CMS cytoplasmic male sterility - mtDNA mitochondrial DNA     

Vegetative growth and reproduction of Fossombronia brasiliensis Steph.: the influence of photoperiod,temperature and inorganic nitrogen source

Abstract

Monoclonal cultures of Fossombronia brasiliensis were grown with different photoperiods, temperatures and inorganic nitrogen sources. The subsequent vegetative growth and production of gametangia is described. A quantitative analysis of the numbers of antheridia and archegonia and their relative proportions is given. At 18°C, F. brasiliensis was found to be a short-day plant, with a critical night length of between six and twelve hours, while at 10°C it exhibited quantitative SD plant properties. Plants produced more male gametangia at 18°C and more female gametangia at 10°C. Nitrogen as nitrate consistently produced more gametangia than the ammonium source.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710     

Life history of Japanese Stypocaulon durum (Sphacelariales, Phaeophyceae)

Phenology, morphology, life history and responses to different temperature and photoperiod conditions were studied in Japanese Stypocaulon durum (Ruprecht) Okamura. Erect thalli of the species were collected year-round, but the mature thalli forming either uniloc-ular sporangia or two different types of plurilocular structures (evidently gametangia) on separate thalli were found only in winter. ln culture, an isomorphic life history is suggested for the species, alternating between a sporophyte forming unilocular sporangia and gam-etophytes forming plurilocular macro- (female) and micro- (male) gametangia. Contents of unilocular sporangia were not released, but germinated in situ, developing into erect thalli forming plurilocular gametangia. Macrogametangia released aplanogametes (oospores), but male gametangia appeared to be non-functional, although flagellated cells were once formed in the loc-uli. This is the first report of plurilocular gametangia in the species. Although the species grew well and matured under considerably lower temperature conditions than European Stypocaulon scoparium (L.) Sauvageau, its temperature requirements showed similarity to northwestern Atlantic Stypocaulon species. This supports the notion that northwestern Atlantic Stypocaulon is conspecific with S. durum.     

Genetically confirmed interbreeding between western Bonelli’s warbler ( Phylloscopus bonelli ) and wood warbler ( P. sibilatrix )

During May and June 2003, a mixed breeding pair consisting of a male western Bonelli’s warbler (Phylloscopus bonelli) and a female wood warbler (P. sibilatrix) successfully raised six young in a broad-leafed forest near Jünkerath, western Germany, an area far outside the regular breeding range of P. bonelli. The identity of the adult birds was confirmed by song, calls, colouration, and wing measurements. Based on blood samples taken from both parents and three juveniles, the potential interbreeding was analysed by molecular methods. Sequences of the mitochondrial cytochrome b gene differed between male and female by 8.7% and confirmed the identification of both parents. Sequences of the mitochondrial cytochrome b gene revealed that the male was a western Bonelli's and the female a wood warbler. The hybridisation and the parentage of male and female were corroborated by multilocus DNA fingerprinting. This is the first documented and genetically proven hybridisation event between these two warbler species.     

An evaluation of mitochondrial heterosis and in vitro mitochondrial complementation in wheat,barley and maize

Summary Two families each of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and maize (Zea mays L.) were studied for mitochondrial heterosis and in vitro mitochondrial complementation. Inbred parents and their hybrids were compared for seedling heights and rate of oxygen uptake by the whole tissue to find out if the hybrids showed greater growth and respiratory activity at the seedling stage. Further comparisons were made by isolating mitochondria from the seedling tissues and measuring their ADP0 ratio, respiratory control ratio and cytochrome c oxidase activity for mitochondrial heterosis. Mixtures of parental mitochondria were similarly compared with parental and hybrid mitochondria for in vitro mitochondrial complementation. No evidence for mitochondrial heterosis or in vitro mitochondrial complementation was found, nor any correlation between the different mitochondrial parameters, seedling heights or rates of oxygen uptake by seedling tissue. The suggested use of mitochondrial heterosis and in vitro mitochondrial complementation for plant breeding is discussed.Data for this paper is taken from the author's dissertation written as a part of Ph.D. degree requirements at the Biology Department, Texas A & M University, College Station, Texas     

AN ELECTRON MICROSCOPE STUDY OF GAMETE RELEASE AND SETTLING IN THE COMPLANATE FORM OF SCYTOSIPHON (SCYTOSIPHONACEAE,PHAEOPHYTA)1

This paper describes the ultrastructural characteristics of gametes and their liberation from the gametangia in Scytosiphon sp., a brown alga showing only slight sexual differentiation. Both male and female gametes are released initially into a central cavity which forms in the gametangial sorus by the extensive dissolution of the internal cell walls. Scytosiphon sp. gametes possess structural features in common with the zoids of other species of brown algae. Gamete fine structure is discussed in relation to cell function. After release from the gametangial sorus, female gametes can be distinguished from males by the presence of a large number of Golgi-derived vesicles with electron dense cores. It is possible that these vesicles contain the sex attractant compound. When gametes settle they become spherical, the flagella and eyespot are withdrawn into the cell and adhesive material, apparently originating from the activity of the Golgi body, appears on the surface of the cell.     

Obligatory female philopatry affects genetic population structure in the ant Proformica longiseta

Evolution of sociality has instigated many changes in the biology of social insects. Particularly, evolution towards complex social systems in ants affects how individuals move in space, usually by making females philopatric. Proformica longisetais well-suited for studying the effects of female philopatry, because female sexuals are wingless and do not actively disperse. We studied genetic population structure in P. longiseta in local scale both as genetic viscosity within one subpopulation, and as differentiation between closely (0.1–1.5 km) located subpopulations, by using nuclear (microsatellites) and mitochondrial (SSCP) markers. Dependent colony founding by splitting old nests is the only known nest founding strategy in P. longiseta. However, no genetic viscosity was detected at the nuclear markers within the subpopulation studied, possibly due to the dynamic nature of P. longiseta populations. The extreme female philopatry showed as strong structure between closely located subpopulations in the mitochondrial genome, but there was no isolation by distance showing that the differentiation pattern was random. Genetic structure in the nuclear genome was much weaker, and there was an indication of isolation by distance. This suggests that male dispersal is strong but not totally free across the area. Finally, non-dispersing P. longiseta females necessarily mate locally raising the possibility of inbreeding, but inbreeding coefficients showed that mating is random. Received 10 January 2006; revised 13 April 2006; accepted 20 April 2006.     

Le corps paranucléaire des gamètes géants d'Allomyces javanicus traité à l'acide borique

Summary The gametes ofAllomyces javanicus exhibit a nuclear cap essentially made of ribonucleoprotein with sulphydryl groups.When dipped in phosphate buffered solutions of boric acid M/20–M/400, the male and female gametangia ofAllomyces liberate giant male and female gametes. Such giant gametes exhibit either a single regular nuclear cap (with 2 or 3 nuclei) or a massive and irregular basophilic formation (with 4–10 nuclei).Boric acid may interfere with ribonucleic acid, essential constituent of the nuclear cap, during the cleavage of the gamete-units in the gametangia.     

Reproductive traits in the tropical moss Octoblepharum albidum Hedw. differ between rainforest and coastal sites

Abstract

Octoblepharum albidum Hedw. is an autoicous moss commonly occurring in tropical savannas, dry forests, rainforests and coastal habitats. It frequently reproduces by spores and asexual structures (gemmae and protonemata or buds at leaf tips), making it a good model for understanding how reproductive traits change with respect to habitat type. Our aims were to characterize the different life-history traits in O. albidum relative to sexual and asexual cycles and to detect variations in reproductive performance among the different habitats, trade-offs among these traits, and relationships among reproductive traits and plant length. We studied colonies from two Atlantic rainforests and two coastal sites in north-eastern Brazil. Shoots in the coastal sites, compared to those of the forest sites, had higher numbers of sporophytes, male and female branches per shoot, male gametangia per sexual branch, and longer setae. Numbers of female gametangia per sexual branch did not differ between forest and coastal sites. A male-biased sex ratio of branches and gametangia occurred in all sites. Compared to gemmae, sporophytes and protonemata or buds were more likely to be found on longer shoots than on shorter ones, but this relationship was only applicable to forest sites. The abundant production of gemmae and protonemata or buds at leaf tips, and sporophytes (spores) in O. albidum are important components in explaining colonization success and maintenance in this tropical moss. Longer sporophytic setae in addition to a higher reproductive performance (especially for number of male gametangia and sporophytes per shoot) may favour spore dispersal and colonization in plants of coastal sites.     

Growth and reproductive responses of Laminaria longicruris (Laminariales,Phaeophyta) to nutrient enrichment

A series of comparative culture experiments were conducted in order to determine responses of Laminaria longicruris male and female gametophytes and juvenile sporophytes to several temperatures (5, 10, 15, 20 °C), light levels (10, 35, 75 µmol m^–2 s^–1) and media nitrogen concentrations (0, 20, 100 µM ammonium-nitrogen). Responses were measured as numbers of male and female gametophytes producing gametangia and number of sporophytes produced following fertilization. Both male and female gametogenesis was reduced at 5 and 20 °C versus 10 and 15 °C. At 20 °C gametogenesis inhibition was greater with higher levels of ammonium-nitrogen concentration (100 µM). Sporophyte production was more sensitive to light, temperature and nitrogen concentration than gametogenesis. Production of sporophytes was inhibited completely at 20 °C. At lower temperatures, increasingly higher nutrient concentrations produced greater inhibition of production of sporophytes.     

Sperm competitive advantage of a rare mitochondrial haplogroup linked to differential expression of mitochondrial oxidative phosphorylation genes

Maternal inheritance of mitochondria creates a sex‐specific selective sieve through which mitochondrial mutations harmful to males but not females accumulate and contribute to sexual differences in longevity and disease susceptibility. Because eggs and sperm are under disruptive selection, sperm are predicted to be particularly vulnerable to the genetic load generated by maternal inheritance, yet evidence for mitochondrial involvement in male fertility is limited and controversial. Here, we exploit the coexistence of two divergent mitochondrial haplogroups (A and B2) in a Neotropical arachnid to investigate the role of mitochondria in sperm competition. DNA profiling demonstrated that B2‐carrying males sired more than three times as many offspring in sperm competition experiments than A males, and this B2 competitive advantage cannot be explained by female mitochondrial haplogroup or male nuclear genetic background. RNA‐Seq of testicular tissues implicates differential expression of mitochondrial oxidative phosphorylation (OXPHOS) genes in the B2 competitive advantage, including a 22‐fold upregulation of atp8 in B2 males. Previous comparative genomic analyses have revealed functionally significant amino acid substitutions in differentially expressed genes, indicating that the mitochondrial haplogroups differ not only in expression but also in DNA sequence and protein functioning. However, mitochondrial haplogroup had no effect on sperm number or sperm viability, and, when females were mated to a single male, neither male haplogroup, female haplogroup nor the interaction between male/female haplogroup significantly affected female reproductive success. Our findings therefore suggest that mitochondrial effects on male reproduction may often go undetected in noncompetitive contexts and may prove more important in nature than is currently appreciated.