Ecological and evolutionary drivers of phenotypic and genetic variation in the European crabapple [Malus sylvestris (L.) Mill.], a wild relative of the cultivated apple.

BACKGROUND AND AIMS: Studying the relationship between phenotypic and genetic variation in populations distributed across environmental gradients can help us to understand the ecological and evolutionary processes involved in population divergence. We investigated the patterns of genetic and phenotypic diversity in the European crabapple, Malus sylvestris, a wild relative of the cultivated apple (Malus domestica) that occurs naturally across Europe in areas subjected to different climatic conditions, to test for divergence among populations. METHODS: Growth rates and traits related to carbon uptake in seedlings collected across Europe were measured in controlled conditions and associated with the genetic status of the seedlings, which was assessed using 13 microsatellite loci and the Bayesian clustering method. Isolation-by-distance, isolation-by-climate and isolation-by-adaptation patterns, which can explain genetic and phenotypic differentiation among M. sylvestris populations, were also tested. KEY RESULTS: A total of 11.6 % of seedlings were introgressed by M. domestica, indicating that crop-wild gene flow is ongoing in Europe. The remaining seedlings (88.4 %) belonged to seven M. sylvestris populations. Significant phenotypic trait variation among M. sylvestris populations was observed. We did not observe significant isolation by adaptation; however, the significant association between genetic variation and the climate during the Last Glacial Maximum suggests that there has been local adaptation of M. sylvestris to past climates. CONCLUSIONS: This study provides insight into the phenotypic and genetic differentiation among populations of a wild relative of the cultivated apple. This might help us to make better use of its diversity and provide options for mitigating the impact of climate change on the cultivated apple through breeding.

Contrasted patterns of selection since maize domestication on duplicated genes encoding a starch pathway enzyme.

Maize domestication from teosinte (Zea mays ssp. parviglumis) was accompanied by an increase of kernel size in landraces. Subsequent breeding has led to a diversification of kernel size and starch content among major groups of inbred lines. We aim at investigating the effect of domestication on duplicated genes encoding a key enzyme of the starch pathway, the ADP-glucose pyrophosphorylase (AGPase). Three pairs of paralogs encode the AGPase small (SSU) and large (LSU) subunits mainly expressed in the endosperm, the embryo and the leaf. We first validated the putative sequence of LSU(leaf) through a comparative expression assay of the six genes. Second, we investigated the patterns of molecular evolution on a 2 kb coding region homologous among the six genes in three panels: teosintes, landraces, and inbred lines. We corrected for demographic effects by relying on empirical distributions built from 580 previously sequenced ESTs. We found contrasted patterns of selection among duplicates: three genes exhibit patterns of directional selection during domestication (SSU(end), LSU(emb)) or breeding (LSU(leaf)), two exhibit patterns consistent with diversifying (SSU(leaf)) and balancing selection (SSU(emb)) accompanying maize breeding. While patterns of linkage disequilibrium did not reveal sign of coevolution between genes expressed in the same organ, we detected an excess of non-synonymous substitutions in the small subunit functional domains highlighting their role in AGPase evolution. Our results offer a different picture on AGPase evolution than the one depicted at the Angiosperm level and reveal how genetic redundancy can provide flexibility in the response to selection.

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.

Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ.

After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron-dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine-receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000-Mr protein binding to the inner face of the subsynaptic membrane.

Interaction of a Golgi-associated kinesin-like protein with Rab6.

Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.

Effects of temperature, lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmarata.

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the alpha-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with N-ethylmaleimide and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins (6.0 +/- 0.6 MSL molecules per alpha-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving 2.2 +/- 0.4 MSL molecules per alpha-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with N-[14C] ethylmaleimide suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (rotational correlation time tau C greater than 1 ms). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane (100 less than or equal to tau c less than or equal to 120 microseconds). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-proteins ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor and rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored (tau c greater than or equal to 1 ms).

Study of the transverse diffusion of spin labeled phospholipids in biological membranes. I. Human red bloods cells.

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the beta acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22 degrees C with sonicated spin-labed phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the beta chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0 degrees C allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creaging a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37 degrees C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: use of phosphatidylcholine exchange protein.

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated "inner membrane+matrix" particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22 degrees C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidycholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at 0 degrees C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incorporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0 degrees C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25 degrees C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).

Establishment of a human malignant fibrous histiocytoma cell line, COMA. Characterization By conventional cytogenetics, comparative genomic hybridization, and multiplex fluorescence In situ hybridization.

The human COMA cell line has been established from a storiform pleomorphic malignant fibrous histiocytoma (MFH). As expected for this tumor type, a very complex karyotype was observed after R-banding analysis. An extensive analysis by 24-color painting, comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH) was performed. Twelve complex marker chromosomes recurrently observed were clearly identified; among them, three were systematically present in all analyzed metaphases. Amplifications detected by CGH were refined by FISH with probes specific for various candidate loci. A significant aneuploidy and numerous micronuclei were observed, which could be related to the anomalies of centriole numbers detected in a proportion of cells. Such an analysis, performed on a series of MFH cell lines, would allow the delineation of the genomic alterations specific for the oncogenesis or progression of this complex tumor type or both.

Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii.

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg2+ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.

Rat mesencephalic neurons in culture exhibit different morphological traits in the presence of media conditioned on mesencephalic or striatal astroglia.

Embryonic rat mesencephalic neurons were plated at low density in a chemically defined medium (CDM) or in CDM conditioned on either mesencephalic or striatal astrocytes (CM Gmes and CM Gstr). It was found that "axon-like" neurites, in general long with few branching points, could be initiated in CM Gmes and CM Gstr, whereas "dendrite-like" neurites (shorter and with a high branching capacity) were preferentially initiated in CM Gmes. The effects of CM Gmes and CM Gstr on the morphology of mesencephalic neurons were abolished by protein denaturating treatments. Comparisons with basic FGF, laminin, or fibronectin demonstrated that these three molecules were also able to modify the morphological traits of the neurons. However the different morphologies observed in CM Gmes and CM Gstr could not be explained only by the presence of these proteins in the conditioned media. Our results therefore indicate that different factors may regulate the initiation of different categories of neurites and that in contrast to several molecules able to promote neurite elongation these "initiation" factors may show important regional specificity.

[Importance of protein-protein interactions for the structural integrity of membrane framents from Torpedo marmorata electric organ]. / Importance des interactions protéine-protéine dans les maintien de la structure des fragments excitables de l'organe électrique de Torpedo marmorata.

Electron spin resonance and electron microscopy were used to show that alkaline extraction of a 43,000 dalton protein from torpedo membrane induces structural destabilization of the membrane and rotational diffusion of the cholinergic receptor protein.

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids.

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.

The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody. An electron-spin-resonance study.

Five derivatives of Naja nigricollis toxin alpha, spin-labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion-exchange and high-pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin-specific immunoglobulin (M alpha 1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M alpha 1. The data obtained give the following indications. In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 had no residual mobility and reflects the toxin rotation. Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M alpha 1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27-bound nitroxide remains immobilized upon binding of either AcChR or M alpha 1. The change in nitroxide immobilization observed upon AcChR or M alpha 1 binding correlates well with the variation of nitroxide accessibility to a water-soluble paramagnetic N2+i ion. Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR. All the data indicate that AcChR and M alpha 1 bind at two topographically distinct sites on the toxin surface.

Fluidity of the lipids next to the acetylcholine receptor protein of torpedo membrane fragments. Use of amphiphilic reversible spin-labels.

Choline esters of spin-labeled fatty acids (long-chain acylcholines) were used to probe the hydrophobic environment of the acetylcholine receptor protein in membrane fragments from Torpedo marmorata. These spin-labels competitively inhibit the binding of [3H]acetylcholine to the receptor site. Their inhibition constants (KI) were close to 200 nM. At the high membrane concentration required for electron spin resonance (ESR) experiments, the apparent inhibition constants (KIapp) differed from KI determined by using dilute membrane concentration. This is due to the amphiphilic character of long-chain acylcholine. For most spin-labels used, only difference ESR spectroscopy provided reliable spectra corresponding to receptor-bound spin-labeled acylcholines. Acetylcholine receptor agonists and antagonists displaced the acylcholine from the receptor sites, whereas choline had only a weak effect. This produced a modification in the ESR spectra of the bound acylcholines and provided evidence that the acylcholines bound to the receptor sites in a specific manner. The interpretation of the spectra of receptor-bound spin-labels favored a strong barrier to the motion of the probe when attached to the middle of the acyl chain. However, when the probe was close to the methyl terminal of a stearoylcholine molecule a much greater fluidity was found. Short-range spin-spin interactions were created between spin-labels bound to the receptor site and spin-labels in a fluid phase. This indicates that lipids next to the receptor protein are not completely immobilized in spite of the semicrystalline organization of the proteins in the postsynaptic region.

In vitro regulation of neuronal morphogenesis and polarity by astrocyte-derived factors.

Mesencephalic neurons were cultured from 2 to 5 days in mesencephalic (CM Gmes) or striatal (CM Gstr) astrocyte conditioned media or in the soluble (S100) and insoluble (P100) fractions prepared from these media by ultracentrifugation. CM Gmes as well as all soluble fractions induced dendritic and axonal elongation, whereas CM Gstr and the insoluble fractions promoted axonal growth only. The study of the shape of the neuronal cell bodies and the measurement of their adhesion to the substratum revealed that axons elongated under low adhesion conditions, but that dendrite growth was highly dependent upon adhesion and spreading of the neuronal soma. This different dependency of axonal and dendritic elongation upon spreading is explained by a model in which we consider the respective viscosities of axons and dendrites. From these observations and speculations we propose that axons and dendrites have different modes of elongation and that the primary effect of the astrocyte-derived factors capable of regulating neuronal polarity is to modify the adhesion of the neurons to their culture substratum.

Further studies on the role of astroglia in brain neurons maturation and morphogenesis.

Using in vitro cultures of dissociated brain neurons and astrocytes, we have compared the morphologies of mesencephalic and striatal neurons cultured for two days on mesencephalic and striatal astrocytes in the four possible combinations. From these comparisons, it appears that: 1. Neurons grown on co-regionalized (homotopic) astrocytes have more primary neurites and branching points than neurons grown on heterotopic astrocytes. 2. The total neuritic length is only slightly affected by the type of co-culture. 3. The branched arborization which develop faster on homotopic astrocytes present several dendritic features. Following these morphological observations, we have been able to demonstrate: 1. That mesencephalic astrocytes (but not striatal astrocytes) secrete trypsin sensitive factors different from laminin and FGF that increase the number of primary neurites and branching points but have no or little effect on total neuritic length. 2. That mesencephalic astrocytes (but not striatal astrocytes) present at their surface a 190 KD glycoprotein specifically recognized by the fucose-specific lectin UEA.