Seasonal changes in the germination responses of Hordeum maritimum and H. murinum seeds in relation to salinity,temperature and after‐ripening time

Abstract

The effects of temperature and salinity (NaCl) on germination of Hordeum maritimum With, (halophyte) and H. murinum L. (glycophyte) seeds were investigated. Dehulled caryopses were used for monthly germination trials, starting from November (120 days of after‐ripening in darkness at 20±1°C). Trials were continued for one year. Differences in germination response between the two species were observed, confirming that H. Maritimum is better adapted to high salinity levels and to variations in external temperature than H. murinum. H. maritimum showed a germination control mechanism related to after‐ripening time and based on seed dormancy break/resumption. At higher temperature (30°C), thermodormancy was also recorded. No germination strategies were observed in H. murinum that is relatively insensitive to the combined effects of temperature and salinity. Thus, in virtually all treatments, H. murinum exhibited a higher germination rate compared with H. maritimum, as early as 72 h after imbibition, suggesting that dormancy, both in the presence or absence of salt, is totally abolished by early fall or at the latest in winter.     

Combining Molecular Data with Classical Morphology for Uncultured Phagotrophic Euglenids (Excavata): A Single‐Cell Approach

Phagotrophic euglenids are one of the most diverse and important forms of heterotrophic flagellates in sediment systems, and are key to understanding the evolution of photosynthetic euglenids and ‘primary osmotrophs’, yet relatively little is known about their biodiversity and phylogenetic relationships. A wealth of light microscopy‐based information is available, but little progress has been made in associating this with molecular sequence data. We established a protocol to obtain light microscopy data and molecular data from single euglenid cells isolated from environmental samples. Individual cells from freshwater and marine benthic samples were isolated and rinsed by micropipetting, documented using high‐resolution photomicroscopy, then subjected to single‐cell nested PCR using taxon‐specific primers in combination with universal eukaryotic primers, generating > 75% or full‐length SSU rDNA sequences. As a proof‐of‐principle eight individuals were characterised and subjected to phylogenetic analyses. Many of these cells were identified as Anisonema or Dinema, and grouped with existing sequences assigned to these taxa, and with a ‘Peranema sp.’ sequence that we could now clearly demonstrate was misidentified or misannotated. Another cell is Heteronema c.f. exaratum, the first ‘skidding heteronemid’ for which sequence data are available. This is not closely related to Heteronema scaphurum, and intriguingly, branches as the sister group to primary osmotrophs. A cell similar to Ploeotia vitrea (the type of this genus), shows no particular phylogenetic affinity to Ploeotia costata, the best studied Ploeotia species. Our experimental protocol provides a useful starting point for future analyses on euglenid biodiversity (including environmental sequence surveys), and their evolution and systematics.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and alkylation of aza‐glycinyl dipeptide building blocks

Aza‐glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza‐glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by‐products. Employing N,N′‐disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza‐glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza‐glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza‐propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross‐couplings, dipolar cycloadditions and intramolecular exo‐dig cycloadditions to furnish a variety of azapeptide building blocks. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

α‐N‐Protected dipeptide acids: a simple and efficient synthesis via the easily accessible mixed anhydride method using free amino acids in DMSO and tetrabutylammonium hydroxide

The importance of dipeptides both in medicinal and pharmacological fields is well documented and many efforts have been made to find simple and efficient methods for their synthesis. For this reason, we have investigated the synthesis of α‐N‐protected dipeptide acids by reacting the easily accessible mixed anhydride of α‐N‐protected amino acids with free amino acids under different reaction conditions. The combination of TBA‐OH and DMSO has been found to be the best to overcome the low solubility of amino acids in organic solvents. Under these experimental conditions, the homogeneous phase condensation reaction occurs rapidly and without detectable epimerization. The present method is also applicable to side‐chain unprotected Tyr, Trp, Glu, and Asp but not Lys. This latter residue is able to engage two molecules of mixed anhydride giving the corresponding isotripeptide. Moreover, the applicability of this protocol for the synthesis of tri‐ and tetrapeptides has been tested. This approach reduces the need for protecting groups, is cost effective, scalable, and yields dipeptide acids that can be used as building blocks in the synthesis of larger peptides. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

Stable peptides have been explored as epitope mimics for protein–protein and protein–nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a β‐hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1‐naphthylalanine or 2‐naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12‐residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1‐naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2‐naphthylalanine appears most similar to a substituted phenylalanine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Peptides and peptidoaldehydes as substrates for the Pictet–Spengler reaction

The Pictet–Spengler (PS) reaction was performed with various types of substrates: H‐Trp‐OMe and dipeptides with N‐terminal Trp as arylethylamine components and Z‐protected amino aldehydes and peptidoaldehydes as carbonyl components. We found that the C‐terminal part of Trp derivatives did not have any influence on the stereoselectivity of the reaction and the results are the same for simple esters of Trp and dipeptides. On the contrary, the selectivity of the PS reaction with peptidoaldehydes with L configuration of the C‐terminus residue is totally different from that obtained with simple L‐amino aldehydes. It allows us to obtain cis stereoisomers, which cannot be isolated from the reaction with amino aldehydes. But the utility of the peptidoaldehydes as substrates for the PS reaction is reduced by the side formation of enamides which decrease the yield of cyclization. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.