Historical biogeography of olive domestication (Olea europaea L.) as revealed by geometrical morphometry applied to biological and archaeological material

Aim This study intends to improve our understanding of historical biogeography of olive domestication in the Mediterranean Basin, particularly in the north-western area. Location Investigations were performed simultaneously on olive stones from extant wild populations, extant cultivated varieties from various Mediterranean countries, and archaeological assemblages of Spanish, French and Italian settlements. Methods A combination of morphometrics (traditional and geometrical) allowed us to study both the size and shape of endocarp structure. Concerning shape, a size-standardized method coupled with fitted polynomial regression analysis was performed. Results We found morphological criteria for discriminating between wild and cultivated olive cultivars, and established patterns of morphological variation of olive material according to the geographical origin (for extant material) and to the age of the olive forms (for archaeological material). Levels of morphological convergences and divergences between wild olive populations and cultivated varieties are presented as evidence. Main conclusions Morphological changes of endocarps of olive under domestication at both geographical and chronological scales provide new criteria for the identification of olive cultivars. They allow to determine the origins of cultivated forms created and/or introduced in the north-western Mediterranean regions and to understand how human migrations affected the rest of the Western Mediterranean regions. A model of diffusion of olive cultivation is proposed. It shows evidence of an indigenous origin of the domestication process, which is currently recognized in the north-western area since the Bronze Age.     

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

Summary A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (–)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.Abbreviations PAA phenylalkylamine - DHP dihydropyridine - FDA fluorescein diacetate     

Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells

Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [³H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [³H]leucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20°C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.     

The 5'' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.     

Regeneration of fertile plants from protoplasts of sunflower (Helianthus annuus L.)

Summary Sunflower hypocotyl protoplasts (Helianthus annuus L.) from 5 PIONEER genotypes (PT024, SMF3, EMIL, HA300*PT024, VK5F) and 1 public line (RHa 274) formed colonies at frequencies of up to 60% when plated in 0.25ml agarose beads in a modified L4 medium (Lenée and Chupeau 1986) containing 3mg/l NAA, 1mg/l BA and 0.1mg/l 2,4-D, and 1000mg/l casamino acids. Protoplast-derived colonies grew slowly into calli. Organogenesis was obtained from callus of PT024 on a MS medium containing NAA and BA at 1mg/l and GA at 0.1mg/l. Freshly excised shoots were induced to root by an IAA treatment. Regenerated plants were transferred to the greenhouse and seed was harvested within 7 months of the initial protoplast isolation.Abbreviations BA 6-benzylaminopurine - NAA -naphtaleneacetic acid - GA gibberellic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog mineral elements - B5 Gamborg mineral elements     

Studies on plant regeneration from protoplasts in the genus Helianthus

Summary Protoplasts were produced from 7-day-old hypocotyls of two cultivated sunflower genotypes and three wild sunflowers. When included in agarose droplets and cultured in TL medium supplemented with 0.1 mg/l 2,4-dichlorophenoxyacetic acid, the protoplasts gave rise to loose colonies and to embryoids. After two months the small calli emerging from the agarose were transferred to a regeneration medium on which they grew and began to differentiate. A second transfer to the same medium 40 days later induced shoot formation on one callus of H. petiolaris. Several shoots were successfully rooted and transferred to soil where they flowered. This is the first documented report, in the genus Helianthus. of regeneration from protoplasts to fully soil-adapted plants.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - GA₃ gibberellic acid - BAP 6-benzyl-aminopurine - EDTA ethylenediamine-tetraacetic acid     

Chromosomal Transmission Bias in Laboratory Hybrids between Wild Strains of the Two European Subspecies of House Mice

Laboratory crosses between wild strains of the two European house mouse subspecies Mus musculus domesticus (2n = 34) and M. m. musculus (2n = 40) were performed to analyze the selective processes involved in the non-introgression of centromeric regions of Robertsonian (Rb) fusions in the Danish hybrid zone. The chromosomal analysis of 226 backcross progeny from 22 reciprocal crosses showed that the segregation of the three Rb fusions present did not significantly differ from Mendelian expectations. However, a significant negative correlation was found between Rb transmission rates and the average litter sizes of the F(1) pairs. Among the different models of selection discussed, the most likely one supported the existence of two opposing selective factors resulting in an overall compensation of chromosomal types in the backcross progeny. A two-phase selective process involving embryo competition was postulated with non-Rb carriers being favored during pre-implantation but disadvantaged after implantation. Such balanced selective pressures acting on musculus non-Rb centromeres are compatible with the steep slope and off-centered position of the chromosomal cline observed in the Danish hybrid zone. These results suggested that these selective factors may be more related to centromere origin (musculus or domesticus) than to centromere structure (Rb or non-Rb).     

Isolation and Characterization of Vacuoles from Melilotus alba Mesophyll

Methods for the preparation of protoplasts and vacuoles from mesophyll tissues of sweet clover (Melilotus alba Desr.) are described. Vacuoles are obtained using a new procedure which involves lysis of the plasmalemma during a brief centrifugation of protoplasts through a diethylaminoethyl dextran layer. This method combines the release of vacuoles and their purification in one step. The contamination of vacuole preparations was found to be low, as judged by enzymic markers and microscopic inspection. The method described is rapid and gives a good yield of vacuoles without causing changes in osmotic pressure. Several hydrolases were found to be located in vacuoles from sweet clover, which were also examined for their amino acid content.     

Photoregulation of the incorporation of guaiacyl units into lignins

When fed with [¹⁴C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled.When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaCl₂) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.Abbreviations S syringyl residue - G guaiacyl residue This is paper No. 4 in a series. For paper No. 3 see Grand and Ranjeva, in press