Novel effects of cholesterol (Chol) on nicotinic acetylcholine receptor (AChR) cell-surface stability, internalization and function are reported. AChRs are shown to occur in the form of submicron-sized (240-280 nm) domains that remain stable at the cell-surface membrane of CHO-K1/A5 cells over a period of hours. Acute (30 min, 37 degrees C) exposure to methyl-beta-cyclodextrin (CDx), commonly used as a diagnostic tool of endocytic mechanisms, is shown here to enhance AChR internalization kinetics in the receptor-expressing clonal cell line. This treatment drastically reduced ( approximately 50%) the number of receptor domains by accelerating the rate of endocytosis (t(1/2) decreased from 1.5-0.5 h). In addition, Chol depletion produced ion channel gain-of-function of the remaining cell-surface AChR, whereas Chol enrichment had the opposite effect. Fluorescence measurements under conditions of direct excitation of the probe Laurdan and of Förster-type resonance energy transfer (FRET) using the intrinsic protein fluorescence as donor both indicated an increase in membrane fluidity in the bulk membrane and in the immediate environment of the AChR protein upon Chol depletion. Homeostatic control of Chol content at the plasmalemma may thus modulate cell-surface organization and stability of receptor domains, and fine tune receptor channel function to temporarily compensate for acute AChR loss from the cell surface.

To overcome environmental stress, plants develop physiological responses that are triggered by genetic or epigenetic changes, some of which involve DNA methylation. It has been proposed that apomixis, the formation of asexual seeds without meiosis, occurs through the temporal or spatial deregulation of the sexual process mediated by genetic and epigenetic factors influenced by the environment. Here, we explored whether there was a link between the occurrence of apomixis and various factors that generate stress, including drought stress, in vitro culture, and intraspecific hybridization. For this purpose, we monitored the embryo sacs of different weeping lovegrass (Eragrostis curvula [Schrad.] Nees) genotypes after the plants were subjected to these stress conditions. Progeny tests based on molecular markers and genome methylation status were analyzed following the stress treatment. When grown in the greenhouse, the cultivar Tanganyika INTA generated less than 2% of its progeny by sexual reproduction. Plants of this cultivar subjected to different stresses showed an increase of sexual embryo sacs, demonstrating an increased expression of sexuality compared to control plants. Plants of the cv. Tanganyika USDA did not demonstrate the ability to generate sexual embryo sacs under any conditions and is therefore classified as a fully apomictic cultivar. We found that this change in the prevalence of sexuality was correlated with genetic and epigenetic changes analyzed by MSAP and AFLPs profiles. Our results demonstrate that different stress conditions can alter the expression of sexual reproduction in facultative tetraploid apomictic cultivars and when the stress stops the reproductive mode shift back to the apomixis original level. These data together with previous observations allow us to generate a hypothetical model of the regulation of apomixis in weeping lovegrass in which the genetic/s region/s that condition apomixis, is/are affected by ploidy, and is/are subjected to epigenetic control.

The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alphaM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alphaM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alphaC418A and alphaT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alphaT422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by approximately 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alphaM4 in AChR channel gating mechanism.

A long-standing goal in plant breeding has been the ability to confer apomixis to agriculturally relevant species, which would require a deeper comprehension of the molecular basis of apomictic regulatory mechanisms. Eragrostis curvula (Schrad.) Nees is a perennial grass that includes both sexual and apomictic cytotypes. The availability of a reference transcriptome for this species would constitute a very important tool toward the identification of genes controlling key steps of the apomictic pathway. Here, we used Roche/454 sequencing technologies to generate reads from inflorescences of E. curvula apomictic and sexual genotypes that were de novo assembled into a reference transcriptome. Near 90% of the 49568 assembled isotigs showed sequence similarity to sequences deposited in the public databases. A gene ontology analysis categorized 27448 isotigs into at least one of the three main GO categories. We identified 11475 SSRs, and several of them were assayed in E curvula germoplasm using SSR-based primers, providing a valuable set of molecular markers that could allow direct allele selection. The differential contribution to each library of the spliced forms of several transcripts revealed the existence of several isotigs produced via alternative splicing of single genes. The reference transcriptome presented and validated in this work will be useful for the identification of a wide range of gene(s) related to agronomic traits of E. curvula, including those controlling key steps of the apomictic pathway in this species, allowing the extrapolation of the findings to other plant species.

Eragrostis curvula includes biotypes reproducing through obligate and facultative apomixis or, rarely, full sexuality. We previously generated a “tetraploid-dihaploid-tetraploid” series of plants consisting of a tetraploid apomictic plant (T), a sexual dihaploid plant (D) and a tetraploid artificial colchiploid (C). Initially, plant C was nearly 100% sexual. However, its capacity to form non-reduced embryo sacs dramatically increased over a four year period (2003–2007) to reach levels of 85–90%. Here, we confirmed high rates of apomixis in plant C, and used AFLPs and MSAPs to characterize the genetic and epigenetic variation observed in this plant in 2007 as compared to 2003. Of the polymorphic sequences, some had no coding potential whereas others were homologous to retrotransposons and/or protein-coding-like sequences. Our results suggest that in this particular plant system increased apomixis expression is concurrent with genetic and epigenetic modifications, possibly involving transposable elements.

Recent reports in model plant species have highlighted a role for DNA methylation pathways in the regulation of the somatic-to-reproductive transition in the ovule, suggesting that apomixis (asexual reproduction through seeds) likely relies on RdDM downregulation. Our aim was therefore to explore this hypothesis by characterizing genes involved in DNA methylation in the apomictic grass Eragrostis curvula. We explored floral transcriptomes to identify homologs of three candidate genes, for which mutations in Arabidopsis and maize mimic apomixis (AtAGO9/ZmAGO104, AtCMT3/ZmDMT102/ZmDMT105, and AtDDM1/ZmCHR106), and compared both their spatial and temporal expression patterns during reproduction in sexual and apomictic genotypes. Quantitative expression analyses revealed contrasting expression patterns for the three genes in apomictic vs sexual plants. In situ hybridization corroborated these results for two candidates, EcAGO104 and EcDMT102, and revealed an unexpected ectopic pattern for the AGO gene during germ line differentiation in apomicts. Although our data partially support previous results obtained in sexual plant models, they suggest that rather than an RdDM breakdown in the ovule, altered localization of AtAGO9/ZmAGO104 expression is required for achieving diplospory in E. curvula. The differences in the RdDM machinery acquired during plant evolution might have promoted the emergence of the numerous apomictic paths observed in plants.

A bright yellow color is an important quality criterion for pasta making. Yellow color depends on the amount of carotenoid pigments in grain, which is the result of the balance between pigment synthesis and degradation by lipoxygenases (LPX). The organization of genes coding for lipoxygenases in the tetraploid wheat genome is not completely understood. Here, we report the screening of a durum wheat BAC library with barley probes to characterize the physical distribution of Lpx genes. PCR characterization and BAC fingerprinting of the positive clones suggests that Lpx-B1.1 and Lpx-B3 are less than 103-kb apart, whereas Lpx-B1.2 is further apart from them. In the A genome a partially deleted copy of Lpx-1 (Lpx-A1_like) was found, colocalizing within a 42 kbp region with Lpx-A3, confirming that in both genomes these two genes are close to each other.The knowledge of the physical location of these two genes is important to understand the evolution of this family but also has practical implications since closely linked genes are difficult to separate by recombination. This may limit the number of Lpx allele combinations that can be obtained and affect the selection of optimal Lpx allele combinations for pasta quality improvement.

The Poaceae constitute a taxon of flowering plants (grasses) that cover almost all Earth’s inhabitable range and comprises some of the genera most commonly used for human and animal nutrition. Many of these crops have been sequenced, like rice, Brachypodium, maize and, more recently, wheat. Some important members are still considered orphan crops, lacking a sequenced genome, but having important traits that make them attractive for sequencing. Among these traits is apomixis, clonal reproduction by seeds, present in some members of the Poaceae like Eragrostis curvula . A de novo , high-quality genome assembly and annotation for E . curvula have been obtained by sequencing 602 Mb of a diploid genotype using a strategy that combined long-read length sequencing with chromosome conformation capture. The scaffold N50 for this assembly was 43.41 Mb and the annotation yielded 56,469 genes. The availability of this genome assembly has allowed us to identify regions associated with forage quality and to develop strategies to sequence and assemble the complex tetraploid genotypes which harbor the apomixis control region(s). Understanding and subsequently manipulating the genetic drivers underlying apomixis could revolutionize agriculture.