Condition optimization for electroporation transfection in horse skeletal muscle satellite cells.

Satellite cells are an important cellular model for studying muscle growth and development and mammalian locomotion-related molecular mechanisms. In this study, we investigated the effects of voltage, pulse duration, and DNA dosage on horse skeletal muscle satellite cells' electroporation transfection efficiency using the eukaryotic expression plasmid Td Tomato-C1 (5.5 kb) encoding the red fluorescent protein gene mainly based on fluorescence-positive cell rate and cell survival rate. By comparison of different voltages, pulse durations, and DNA doses, horse skeletal muscle satellite cells have nearly 80% transfection efficiency under the condition of voltage 120 V, DNA dosage 7 µg/ml, and pulse duration 30 ms. This optimized electroporation condition would facilitate the application of horse skeletal muscle satellite cells in genetic studies of muscle function and related diseases.

Common protein-coding variants influence the racing phenotype in galloping racehorse breeds.

Selection for system-wide morphological, physiological, and metabolic adaptations has led to extreme athletic phenotypes among geographically diverse horse breeds. Here, we identify genes contributing to exercise adaptation in racehorses by applying genomics approaches for racing performance, an end-point athletic phenotype. Using an integrative genomics strategy to first combine population genomics results with skeletal muscle exercise and training transcriptomic data, followed by whole-genome resequencing of Asian horses, we identify protein-coding variants in genes of interest in galloping racehorse breeds (Arabian, Mongolian and Thoroughbred). A core set of genes, G6PC2, HDAC9, KTN1, MYLK2, NTM, SLC16A1 and SYNDIG1, with central roles in muscle, metabolism, and neurobiology, are key drivers of the racing phenotype. Although racing potential is a multifactorial trait, the genomic architecture shaping the common athletic phenotype in horse populations bred for racing provides evidence for the influence of protein-coding variants in fundamental exercise-relevant genes. Variation in these genes may therefore be exploited for genetic improvement of horse populations towards specific types of racing.

MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

Myostatin (MSTN), a member of the transforming growth factor-ß superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an analysis of the differentially expressed genes was carried out. It was revealed that MSTN regulatory networks mainly involve genes related to muscle function and cell-cycle regulation, and signaling pathways, such as Notch, MAPK, and WNT. Subsequent real-time PCR in equine satellite cells and immunohistochemistry on newborn and adult muscle also verified the MSTN regulatory network found in RNA sequencing analysis. The results of this study provide new insight into the regulatory mechanism of equine MSTN.

The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs).

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro.

Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.

Arabidopsis thaliana circadian clock is regulated by the small GTPase LIP1.

BACKGROUND: At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative-feedback loops and generate a basic approximately 24 hr oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. RESULTS: The lip1-1 (light insensitive period 1) mutant isolated from the model plant Arabidopsis thaliana displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. In wild-type plants, period length shortens with increasing light fluence rates and the phase of rhythms can be shifted by light pulses administered to dark-adapted plants. In contrast, in lip1-1, period length is nearly insensitive to light intensity and significantly larger phase shifts (delays) can be induced during the subjective night. The mutant also displays elevated photomorphogenic responses to red and blue light, which cannot be explained by the circadian defect, suggesting distinct functions for LIP1 in the circadian light input and photomorphogenesis. The LIP1 gene encodes a functional, plant-specific atypical small GTPase, and therefore we postulate that it acts similarly to ZEITLUPE at postranscriptional level. CONCLUSIONS: LIP1 represents the first small GTPase implicated in the circadian system of plants. LIP1 plays a unique negative role in controlling circadian light input and is required for precise entrainment of the plant clock.

Characterization of three Rop GTPase genes of alfalfa (Medicago sativa L.).

Three cDNA clones coding for Medicago sativa Rop GTPases have been isolated. The represented genes could be assigned to various linkage groups by genetic mapping. They were expressed in all investigated plant organs, although at different level. Relative gene expression patterns in response to Sinorhizobium infection of roots as well as during somatic embryogenesis indicated their differential participation in these processes. DNA sequences coding for altogether six different Medicago sp. Rop GTPases could be identified in sequence databases. Based on their homology to each other and to their Arabidopsis counterparts, a unified nomenclature is suggested for Medicago Rop GTPases.

Nitric oxide is required for, and promotes auxin-mediated activation of, cell division and embryogenic cell formation but does not influence cell cycle progression in alfalfa cell cultures.

It is now well established that nitric oxide (NO) serves as a signaling molecule in plant cells. In this paper experimental data are presented which indicate that NO can stimulate the activation of cell division and embryogenic cell formation in leaf protoplast-derived cells of alfalfa in the presence of auxin. It was found that various NO-releasing compounds promoted auxin-dependent division (as shown by incorporation of bromodeoxyuridine) of leaf protoplast-derived alfalfa cells. In contrast, application of NO scavenger or NO synthesis inhibitor inhibited the same process. Both the promotion and the inhibition of cell cycle activation correlated with the amount and activity of the cognate alfalfa p34cdc2 protein Medsa;CDKA;1,2. The effect of l-NG-monomethyl-L-arginine (L-NMMA) was transient, and protoplast-derived cells spending more than 3 days in culture become insensitive to the inhibitor as far as cell cycle progression was concerned. L-NMMA had no effect on the cell cycle parameters of cycling suspension-cultured cells, but had a moderate transient inhibitory effect on cells re-entering the cell cycle following phosphate starvation. Cycling cultured cells, however, could respond to NO, as indicated by the sodium nitroprusside (SNP)- and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-dependent accumulation of the ferritin protein. Based on these observations, it is hypothesized that L-NMMA-sensitive generation of NO is involved in the activation, but not the progression of the plant cell division cycle. In addition, SNP promoted and L-NMMA delayed the exogenous auxin [2,4-dichlorophenoxyacetic acid (2,4-D)] concentration-dependent formation of embryogenic cell clusters expressing the MsSERK1 gene; this further supports a link between auxin- and NO-dependent signaling pathways in plant cells.