A high-quality genome of Eragrostis curvula grass provides insights into Poaceae evolution and supports new strategies to enhance forage quality.

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.

Temporal and spatial expression of genes involved in DNA methylation during reproductive development of sexual and apomictic Eragrostis curvula.

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.

Cholesterol depletion activates rapid internalization of submicron-sized acetylcholine receptor domains at the cell membrane.

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.

Steroids differentially inhibit the nicotinic acetylcholine receptor.

The effect of various natural and synthetic steroids on the function of the nicotinic acetylcholine receptor (AChR) was studied at the single-channel level. AChR channel kinetics was affected by some substitutions in the cyclopentaneperhydrophenantrene ring. Functionally relevant substitutions shortened channel open state duration, an effect that varied for different steroids. The presence of a polar group at C11 contributed to the inhibitory potency of the steroid. Among mono-hydroxylated steroids such as 11- and 17-OH progesterone, the highest potency was displayed by the former showing a level similar to that of the reference compound, hydrocortisone. When the effects were analyzed in terms of the octanol-water partition coefficient, a linear relationship was unexpectedly found between the hydrophilicity of the steroids and their inhibitory potency.

Nongenomic effects of steroids on the nicotinic acetylcholine receptor.

A fast signaling mode of natural and synthetic steroids is exerted on some ion channels and cell-surface receptors. This activity contrasts with their classic mode of action, via intracellular receptors. Early studies from our laboratory demonstrated that spin-labeled androstanol and cholestane interact with the nicotinic acetylcholine receptor (AChR) and that lipid mobility at the lipid belt surrounding the AChR is reduced relative to that of the bulk membrane lipid. The occurrence of discrete and independent sites for phospholipids and sterols, both accessible to fatty acids, was subsequently disclosed in the native membrane. Synthetic and natural glucocorticoids were found to act as noncompetitive inhibitors of AChR function. The influence of different substituent groups in the cyclepentane perhydrophenanthrene ring on the channel-shortening potency of various steroids has also been assayed in muscle-type AChR, and we found a certain selectivity of this effect. Some organochlorine pesticides are xenoestrogens, that is, environmental agents capable of disrupting endocrine system signaling. We determined their effects on the AChR membrane using novel fluorescence techniques.

Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics.

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.