Lack of correlation between pollen aperture number and environmental factors in pansies (Viola L., sect. Melanium Ging.) - pollen heteromorphism re-examined.

The development of different pollen morphs by one specimen - pollen heteromorphism - occurs in ca. one-third species of the genus Viola. Melanium section species (pansies) stand out in producing the widest range of apertures among Viola species. Aperture number decreases with elevation increase, and faster germination of five-aperturate pollen as compared with three-aperturate has previously been postulated. We re-examined pollen heteromorphism in the context of its viability, and made correlations with elevation (>1500 m a.s.l. versus <1500 m a.s.l.), soil type (metalliferous versus non-metalliferous; MET versus NMET) and chromosome number based on selected study criteria of ca. 20% karyologically and morphologically strongly differentiated but genetically closely related pansies. A total of 79% of analysed species were heteromorphic, forming three- to six-colp or ate pollen per individual flower. Mean aperture number and pollen viability were not affected by soil type (MET versus NMET). Mean aperture number was also not influenced by elevation or species chromosome number. Positive correlations were established between aperture number and pollen viability, negative between pollen viability and elevation (increasing altitude of 100 m decreased pollen viability by 0.4%) and lack of correlation between chromosome number and pollen viability. The varied frequencies of different pollen morphs among species are not under the general pressure of ecological conditions, as previously postulated for the species of Melanium section. Rather, this trait in pansies, similar to other floral characters (e.g. long, curved nectar spur, 'landing platform', posterior petals with nectar guides), is adaptive but dependent on the breeding system (inbreeding versus outbreeding) of the individual species.

No evidence of contemporary interploidy gene flow between the closely related European woodland violets Viola reichenbachiana and V. riviniana (sect. Viola, Violaceae).

Viola reichenbachiana (2n = 4x = 20) and V. riviniana (2n = 8x = 40) are closely related species widely distributed in Europe, often sharing the same habitat throughout their overlapping ranges. It has been suggested in numerous studies that their high intraspecific morphological variability and plasticity might have been further increased by interspecific hybridisation in contact zones, given the sympatry of the species and the incomplete sterility of their hybrid. The aims of this study were to: (i) confirm that V. reichenbachiana and V. riviniana have one 4x genome in common, and (ii) determine the impact of hybridisation and introgression on genetic variation of these two species in selected European populations. For our study, we used 31 Viola populations from four European countries, which were analysed using AFLP and sequencing of a variable plastid intergenic spacer, trnH-psbA. Our analysis revealed that V. reichenbachiana exhibited larger haplotype diversity, having three species-specific haplotypes versus one in V. riviniana. The relationships among haplotypes suggest transfer of common haplotypes into V. riviniana from both V. reichenbachiana and hypothetically the other, now extinct, parental species. AFLP analysis showed low overall genetic diversity of both species, with V. riviniana showing higher among-population diversity. None of the morphologically designated hybrid populations had additive AFLP polymorphisms that would have indicated recent hybridisation. Also, kinship coefficients between both species did not indicate gene flow. V. riviniana showed significant population subdivision and significant isolation by distance, in contrast to V. reichenbachiana. The results indicate lack of gene flow between species, high influence of selfing on genetic variability, as well as probably only localised introgression toward V. riviniana.

Morphological versus genetic diversity of Viola reichenbachiana and V. riviniana (sect. Viola, Violaceae) from soils differing in heavy metal content.

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

Violets of the section Melanium, their colonization by arbuscular mycorrhizal fungi and their occurrence on heavy metal heaps.

Violets of the sections Melanium were examined for their colonization by arbuscular mycorrhizal fungi (AMF). Heartsease (Viola tricolor) from several heavy metal soils was AMF-positive at many sites but not at extreme biomes. The zinc violets Viola lutea ssp. westfalica (blue zinc violet) and ssp. calaminaria (yellow zinc violet) were always AMF-positive on heavy metal soils as their natural habitats. As shown for the blue form, zinc violets germinate independently of AMF and can be grown in non-polluted garden soils. Thus the zinc violets are obligatorily neither mycotrophs nor metalophytes. The alpine V. lutea, likely ancestor of the zinc violets, was at best poorly colonized by AMF. As determined by atomic absorption spectrometry, the contents of Zn and Pb were lower in AMF colonized plants than in the heavy metal soils from where the samples had been taken. AMF might prevent the uptake of toxic levels of heavy metals into the plant organs. Dithizone staining indicated a differential deposition of heavy metals in tissues of heartsease. Leaf hairs were particularly rich in heavy metals, indicating that part of the excess of heavy metals is sequestered into these cells.

Increased genetic diversity of Viola tricolor L. (Violaceae) in metal-polluted environments.

Changes in DNA sequences affecting cryptic intraspecific variability are very important mechanisms of plant microevolutionary processes, initiating species diversification. In polluted environments, intra- and interpopulation changes at the molecular level proceed rapidly and lead to the formation of new ecotypes in a relatively short time. We used ISSR PCR fingerprinting data to analyze the genetic diversity and genetic structure of seven populations of Viola tricolor: four growing on soil contaminated with heavy metals (Zn, Pb, Cd; waste heaps) and three from control soil. The populations from the polluted sites showed higher genetic polymorphism (%(poly)=84%) and gene diversity (H(T)=0.1709) than the control populations (%(poly)=75% and H(T)=0.1448). The number of private markers we detected within metallicolous (MET) populations was more than double that found within non-metallicolous (NON) populations (15 vs. 7). The STRUCTURE and UPGMA analyses showed clear genetic differences between the NON and MET populations. Based on broad analyses of the genetic parameters, we conclude that the effect of these polluted environments on the genetic diversity of the MET populations, separating them from the NON populations, is evidence of microevolutionary processes at species level, leading to species divergence and the emergence of local ecotypes better adapted to their different environments.

Distinct but overlapping epitopes for the interaction of a CC-chemokine with CCR1, CCR3 and CCR5.

Chemokines play an important role in inflammation. The mechanism via which they bind to more than one receptor and activate them is not well understood. The chemokines are thought to interact with their receptors via two distinct sites, one necessary for binding and the other for activation of signal transduction. In this study we have used alanine scanning mutagenesis to identify residues on RANTES that specifically interact with its receptors CCR1, CCR3, and CCR5 for binding and activation. Residues within a potential receptor binding site known as the N-loop (residues 12-20) and near the N-terminus of RANTES were individually mutated to alanine. The results of this study show that, within the N-loop, the side chain of R17 is necessary for RANTES binding to CCR1, F12 for binding to CCR3, and F12 and I15 for binding to CCR5, thus forming distinct but overlapping binding epitopes. In addition, our finding that P2 is necessary for binding to CCR5 is the first to show that a residue near the N-terminus of a CC-chemokine is involved in binding to a receptor. We have also found that P2, D6, and T7 near the N-terminus are involved in activating signal transduction via CCR1, P2 and Y3 via CCR3, and Y3 and D6 via CCR5. These results indicate that RANTES interacts with each of its receptors in a distinct and specific manner and provide further evidence to support the two-site model of interaction between chemokines and their receptors.

Polarography of conjugated unsaturated lipids.

Conjugated fat-soluble vitamins, methylenic interrupted and conjugated fatty acids were polarographically investigated in both basic and neutral solvents. The half-wave potentials of all-trans-retinol, 13-cis-retinol, all-trans-retinyl acetate, all-trans-retinal, and Vitamin D(2) and D(3) were related to the number of double bonds in conjugation and their geometrical configuration. A minimum of three double bonds in conjugation and their geometrical configuration. A minimum of three double bonds in conjugation was required before reduction took place at the cathode, and as the number of conjugated bonds increased in the lipid compounds, the initial reduction wave took place at a lower half-wave potential.Investigation of conjugated double bonds in triglycerides and in alkali-isomerized linolenic and arachidonic acids gave reduction waves the half-wave potentials of which were related to the number of double bonds in conjugation. In both basic and neutral solvents there was a minimum of three double bonds in conjugation necessary to obtain a reduction wave at the dropping mercury electrode. Ultraviolet absorption curves of the prolonged reduction of polyunsaturated conjugated fatty acids indicate a step-wise reduction of each end of the polyunsaturated conjugated double bonds.In neutral solvent the log of the conjugated double bonds versus the half-wave potential (versus mercury pool anode) gave a linear equation, E(1)=2.98-1.6 log C. A proposed mechanism for the step-wise reduction of conjugated lipids is presented and discussed.