Time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas reinhardii

Contradictory data have been published concerning the time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas. In the present study, adjacent ultrathin sections of Chlamydomonas reinhardii zygotes of various ages were examined with the electron microscope. These sections clearly reveal that nuclear fusion precedes chloroplast fusion.     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

Brassica carinata resynthesized by protoplast fusion

Brassica carinata (bbcc) was resynthesized by protoplast fusion betweenB. nigra (bb) andB. oleracea (cc). In two fusion experiments 64 hybrid plants were obtained and identified to be true hybrids by isoenzyme analysis, nuclear DNA content, chromosome number, and intermediate morphology. Of these plants 56% were normal amphidiploids with 2n=34 chromosomes and a DNA content equivalent to that of naturalB. carinata. The remaining plants were polyploid, morphologically abnormal, and infertile. The majority of the hybrids contained both chloroplasts and mitochondria fromB. nigra, but some plants combined chloroplast and mitochondria from the different progenitors. Hybrids with a DNA content equivalent to that ofB. carinata had a wide range of male fertility (4–98%), but consistently low female fertility. Only a few selfed seed were produced, but these germinated and grew into vigorous plants.Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No. 296-92     

Ammonium generation by nitrogen-starved cultures of Chlamydomonas reinhardii

Ammonium (NH ₄ ⁺ ) assimilation by Chlamydomonas reinhardii was inhibited when cultures were incubated with methionine sulphoximine (MSO). Methionine sulphoximine inhibited glutamine synthetase acitvity in vitro in extracts from wild-type (2192) and mutant (CC419) cultures. Mutant cultures were insensitive to MSO inhibition in vivo. Nitrogen-starved, wild-type cultures excreted ammonium when they were incubated with MSO in light or in darkness. Ammonium generation was stimulated by glutamine, inhibited by CO₂ and stoichiometrically related to loss of protein. Notrogen replete cultures treated with MSO excreted ammonium in light but little was excreted in darkness. Ammonium excretion in darkness, in the presence of MSO, was enhanced by either a period of nitrogen deprivation or by the addition of acetate. Nitrogen deprivation also diminished the lag before ammonium excretion commenced.Abbreviation MSO methionine sulphoximine     

Isolation and characterization of prolyl hydroxylase from Chlamydomonas reinhardii

Prolyl hydroxylase, which is responsible for the hydroxylation of peptidyl proline residues, has been isolated and purified from the green alga Chlamydomonas reinhardii. The enzyme, which appears to be loosely associated with microsomal membranes, was released into solution by sonication in the presence of detergent. Purification was achieved by ion-exchange chromatography followed by affinity chromatography using the immobilized substrate poly-L-proline. Apart from its differing substrate specificity the enzyme appears to possess similar molecular characteristics to prolyl hydroxylase isolated from animal tissues: the active enzyme is a tetramer of about 240–250 kDa and nonidentical monomers of 65 and 60 kDa. The monomers are capsule shaped having a dimension of 12×7 nm.Abbreviations Da dalton - DEAE diethylaminoethyl - DTT dithiothreitol - Hepes 4-(2-hydroxymethyl)-1-piperazine ethanesulfonic acid - -KGA -ketoglutarate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Extraction by lithium chloride of hydroxyproline-rich glycoproteins from intact cells of Chlamydomonas reinhardii

A procedure has been developed to isolate and analyse the cell-wall glycoproteins of Chlamydomonas reinhardii. Under appropriate conditions, cell-wall glycoproteins can be quantitatively extracted from intact cells by aqueous LiCl. Although proteins and glycoproteins, which are presumably not related to the cell wall, are coextracted with the cell-wall subunits, these components can be readily identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as demonstrated by comparative analysis of LiCl-extracts from wild-type cells and the cell-wall-deficient mutant CW-15. Apart from the high-molecular-weight cell-wall components, two glycoproteins with apparent molecular weights (M_rs) of 36000 and 66000 were found to be present in LiCl-extracts of wild-type cells but absent in LiCl-extracts from the cell-wall-less mutant. Pulse-labeling experiments with [³H]proline and [³⁵S]methionine revealed that the LiCl-extracts contained — in addition to the well-known cell-wall subunits — proteins of lower molecular weight, which are also preferentially labeled with [³H]proline. Protein components with M_rs of 68000, 44000, 36000, 26000 and 22000 were found to be more strongly labeled with [³H]proline than with [³⁵S]methionine, whereas protein components with M_rs of 57000 and 52000 were more prominent after labeling with [³⁵S]methionine. The portion of cell-wall subunits within the total amount of proteins extracted by LiCl was calculated to be at least 10% on the basis of the amount of hydroxyproline. Self-assembly of cell walls could be demonstrated after dialysis against water of a mixture of crude LiCl-extract and purified, insoluble, inner wall layers. Cell-wall glycoproteins could be enriched by gel exclusion chromatography of crude LiCl-extracts on Sepharose CL-4B columns equilibrated with 1 mol l^-1 LiCl.Abbreviations EDTA ethylenediaminetetraacetic-acid - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff's reagent - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

Utilization of nitrate,nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. l-Methionine-d,l-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.Abbreviations DCMU N-(3,4-dichlorophenyl)N,N-di-methyl-urea - PCCP Carbonylcyanid-p-trifluoromethoxy-phenylhydrazone - Mops 2-(N-morpholino)propanesulfonic acid - MSX l-Methionine-d,l-sulfoximine     

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion

Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.     

Sexual agglutination in Chlamydomonas eugametos before and after cell fusion

A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt⁺ or mt^-). It is shown that before mt⁺ and mt^- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt^- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt⁺ gametes constituting the outer surface of the clumps with the mt^- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt⁺ flagellar tips then deactivate and swimming is resumed. The original mt^- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt^- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt^+/- mating type plus or minus - FTA flagellar tip activation     

Variability of mitochondrial population in Chlamydomonas reinhardii

Several details have been published cocerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).     

Plant regeneration from protoplast fusion inPassiflora spp.

Summary Protoplasts were isolated from leaf explants ofPassiflora edulis var.flavicarpa (the yellow passion fruit) and from cell suspensions of fivePassiflora species. Chemical fusion was performed using polyethylene glycol and the microcolonies obtained were transferred to growth medium to produce calli. Electrophoresis of soluble proteins and analysis of isoenzymes from calli produced from the fusion experiments were performed to select somatic hybrids. Specific polypeptide bands allowed the identification of somatic hybrids betweenP. edulis var.flavicarpa (+)P. alata, P. edulis var.flavicarpa (+)P. amethystina, P. edulis var.flavicarpa (+)P. cincinnata, P. edulis var.flavicarpa (+)P. giberti andP. edulis var.flavicarpa (+)P. coccinea. An average of 3 to 5% hybrid calli were obtained. With the exception of theP. edulis var.flavicarpa (+)P. coccinea, whole plants were recovered from all hybrids. These somatic hybrids showed 4n=36 chromosomes, which represents a further evidence of their hybridity.     

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T.␣atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 g/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in␣vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 g/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T.␣atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.This work was presented as an oral lecture in section ‘Agriculture, Soil, Forest Microbiology’ at the BioMicroWorld2005 conference.     

Further studies on the genetics ofCephalosporium acremonium using protoplast fusion methods

Summary Genetic analysis of protoplast fusants inCephalosporium acremonium can be carried out by two complementary approaches: analysis of stable haploid segregants and analysis of unstable heterozygotes of uncertain ploidy. However, segregation may be distorted by physiological as well as genetic phenomena, i.e., cross-feeding, syntrophic growth, allele viability, clonal effects, or parental genome segregation.