Evidence of post-pollination barriers among three colour morphs of the deceptive orchid Dactylorhiza sambucina (L.) Soó

Floral-colour polymorphism in rewardless orchids has been hypothesized to be maintained by means of naïve insects, which after visiting a flower without reward will tend to fly elsewhere, looking for a flower of a different colour. In this study, levels of male and female reproductive success were monitored in Southern Italy populations of the deceptive orchid Dactylorhiza sambucina, through field observations over 3 years. These populations were characterized by the presence of a rare pink morph which is sympatric with the more frequent yellow and red morphs. In addition, final plant fertility was evaluated through percentages of embryo-containing seeds produced in both natural conditions and hand-pollination experiments. Results showed that pollinator preferences were independent of the morph frequencies and thus do not promote the predicted negative frequency-dependent selection. Although yellow and pink morphs showed significantly higher male reproductive success (RS), we found comparable levels of female RS, which suggest that pollinator behaviour cannot be the main mechanism which maintains this polymorphism. Interestingly, we found different percentages of embryo-containing seeds in fruits set under natural conditions as well as in those obtained from experimental crosses. In particular, pink morph showed a very low intrinsic fertility. Moreover, fertility in intra- was higher than in inter-morph crosses. To our knowledge, this is the first study pointing out the occurrence in the orchid family of post-pollination reproductive barriers. Findings are discussed in light of present hypothesis on the evolution and maintenance of colour polymorphism in deceptive orchids and other angiosperms.     

Effects of geometric isomerism in dinuclear platinum antitumor complexes on aquation reactions in the presence of perchlorate, acetate and phosphate

The aquation and subsequent reactions of the dinuclear Pt antitumor complexes [{trans-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1,1/t,t) and [{cis-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1,1/c,c) in 15 mM perchlorate, acetate or phosphate solutions were followed at 298 K by [(1)H,(15)N] HSQC 2D NMR spectroscopy. Rate and equilibrium constants for the initial reversible aquation and the subsequent reversible reaction with phosphate or acetate are reported. The rate constant for the first aquation step is two-fold lower for 1,1/c,c than 1,1/t,t but the anation rate constants are similar so that the equilibrium lies further towards the chloro form for the 1,1/c,c compound. A pK (a) value of 6.01+/-0.03 was determined for the diaquated species [{cis-Pt(NH(3))(2)(H(2)O)}(2)(mu-NH(2)(CH(2))(6)NH(2))](4+) (1,1/c,c-3) which is 0.4 units higher than that of the 1,1/t,t compound. The rate constants for the binding of acetate and phosphate to 1,1/t,t are similar, but the rate constant for the reverse reaction is close to ten-fold higher in the case of phosphate so that equilibrium conditions are attained more rapidly (12 h compared with 64 h). On the other hand, for 1,1/c,c the rate constants for the forward and reverse reactions with acetate and phosphate are quite similar so that equilibrium conditions are reached very slowly (80-100 h) and a greater proportion of phosphate-bound species are present. The reduced lability of the bound phosphate for 1,1/c,c is attributed to the formation of a macrochelate phosphate-bridged species which was characterized by (31)P NMR and ESI-MS. The speciation profiles of 1,1/t,t and 1,1/c,c under physiological conditions are explored.     

Effects of geometric isomerism in dinuclear antitumor platinum complexes on their interactions with N-acetyl-L-methionine

In this study, the reactions of N-acetyl-L-methionine (AcMet) with [{trans-PtCl(NH₃)₂}₂-μ-H₂N(CH₂)₆NH₂](NO₃)₂ (BBR3005: 1,1/t,t 1) and its cis analog [{cis-PtCl(NH₃)₂}₂-μ-{H₂N(CH₂)₆NH₂}]Cl₂ (1,1/c,c 2) were analyzed to determine the rate and reaction profile of chloride substitution by methionine sulfur. The reactions were studied in PBS buffer at 37°C by a combination of multinuclear (¹⁹⁵Pt, {¹H-¹⁵N} HSQC) magnetic resonance (NMR) spectroscopy and electrospray ionization time of flight mass spectrometry (ESITOFMS). The diamine linker of the 1,1/t,t trans complex was released as a result of the trans influence of the coordinated sulfur atom, producing trans-[PtCl(AcMet)(NH₃)₂]⁺ (III) and trans-[Pt(AcMet)₂(NH₃)₂]²⁺ (IV). In contrast the cis geometry of the dinuclear compound maintained the diamine bridge intact and a number of novel dinuclear platinum compounds obtained by stepwise substitution of sulfur on both platinum centers were identified. These include (charges omitted for clarity): [{cis-PtCl(NH₃)₂}-μ-NH₂(CH₂)₆NH₂-{cis-Pt(AcMet)(NH₃)₂}] (V); [{cis-Pt(AcMet)(NH₃)₂}₂-μ-NH₂(CH₂)₆NH₂] (VI); [{cis-PtCl(NH₃)₂}-μ-NH₂(CH₂)₆NH₂-{PtCl(AcMet)NH₃] (VII); [{PtCl(AcMet)(NH₃)}₂-μ-NH₂(CH₂)₆NH₂] (VIII); [{trans-Pt(AcMet)₂(NH₃)}-μ-NH₂(CH₂)₆NH₂-{PtCl(AcMet)(NH₃)] (IX) and the fully substituted [{trans-Pt(AcMet)₂(NH₃)}₂-μ-{NH₂(CH₂)₆NH₂] (X). For both compounds the reactions with methionine were slower than those with glutathione (Inorg Chem 2003, 42:5498–5506). Further, the 1,1/c,c geometry resulted in slower reaction than the trans isomer, because of steric hindrance of the bridge, as observed previously in reactions with DNA and model nucleotides.     

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO₃ precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ ¹³C and δ ¹⁸ O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ ¹³C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ ¹³C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria.     

High water users can be drought tolerant: using physiological traits for green roof plant selection

Background and aims

Green roofs are often installed to reduce urban stormwater runoff. To optimally achieve this, green roof plants need to use water when available, but reduce transpiration when limited to ensure survival. Succulent species commonly planted on green roofs do not achieve this. Water availability on green roofs is analogous to natural shallow-soil habitats including rock outcrops. We aimed to determine whether granite outcrop species could improve green roof performance by evaluating water use strategies under contrasting water availability.

Methods

Physiological and morphological responses of 12 granite outcrop species with different life-forms (monocots, herbs and shrubs) and a common green roof succulent were compared in well watered (WW) and water deficit (WD) treatments.

Key results

Granite outcrop species showed a variety of water-use strategies. Unlike the green roof succulent all of the granite outcrop species showed plasticity in water use. Monocot and herb species showed high water use under WW but also high water status under WD. This was achieved by large reductions in transpiration under WD. Maintenance of water status was also related to high root mass fraction.

Conclusions

By developing a conceptual model using physiological traits we were able to select species suitable for green roofs. The ideal species for green roofs were high water users which were also drought tolerant.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Effects of (−)-epicatechin and derivatives on nitric oxide mediated induction of mitochondrial proteins

Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and likely contributes to the development of cardiovascular diseases (CVD). The stimulation of mitochondrial function and/or biogenesis is seen as a means to improve the bioenergetic and metabolic status of cells and thus, reduce CVD. In this study we examined the capacity of the flavanol (?)-epicatechin and two novel derivatives to enhance mitochondrial function and protein levels in cultured bovine coronary artery endothelial cells. As nitric oxide production by endothelial cells is suspected in mediating mitochondria effects (including biogenesis), we also examined the dependence of responses on this molecule using an inhibitor of nitric oxide synthase. Results indicate that the flavanol (?)-epicatechin and derivatives are capable of stimulating mitochondrial function as assessed by citrate synthase activity as well as induction of structural (porin, mitofilin) and oxidative phosporylation protein levels (complex I and II). Effects were blocked by the use of the chemical inhibitor of the synthase thus, evidencing a role for nitric oxide in mediating these effects. The results observed indicate that the three agents are effective in enhancing mitochondria function and protein content. The effects noted for (?)-epicatechin may serve to explain the healthy effects on cardiometabolic risk ascribed to the consumption of cocoa products.