RESUMEN
Barley is one of the most important crops in the world. Barley is used as both food and feed and is important for malt production. Demands for malting quality differ among countries and customs. Malting quality is a complex characteristic involving barley genetics, the environmental conditions during barley growth, and the technological parameters of the malting process. In this study, the hypothesis was that there were no differences between two groups of barley varieties with different but defined malting qualities, which was tested using RNA sequencing during selected stages of malting. In total, 919 differentially transcribed genes between the two barley groups were identified and annotated. Differentially expressed genes (DEGs) were primarily assigned to gene ontology (GO) terms of oxidation-reduction process - oxidoreductase activity, response to stress, carbohydrate metabolic process, and proteolysis - hydrolase activity, and metal ion binding. Genes connected with the plasma membrane and its integral components also play important roles in malting quality. DEG profiles of selected genes in the three malting stages indicate a complex character of malting quality. Many single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels) were identified. SNPs and indels with the best quality were used for primer design. After optimization and validation, five molecular markers were developed for use in barley breeding.
Asunto(s)
Grano Comestible/genética , Perfilación de la Expresión Génica , Hordeum/genética , Transcriptoma/genética , Mapeo Cromosómico , Productos Agrícolas , Grano Comestible/crecimiento & desarrollo , Hordeum/metabolismo , Fenotipo , Fitomejoramiento , Sitios de Carácter Cuantitativo/genética , Plantones/genética , Plantones/crecimiento & desarrolloRESUMEN
The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4-DNA binding domain (FOXO4-DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4-DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4-DBD upon binding to the DNA. This was further confirmed by using the time-resolved tryptophan fluorescence anisotropy decay measurements which revealed profound reduction of segmental dynamics of FOXO4-DBD upon the complex formation. Alanine scanning of amino acid residues engaged in polar contacts with the DNA showed that certain non-specific contacts with the DNA backbone are very important for both the binding affinity and the binding specificity of FOXO4-DBD.
Asunto(s)
ADN/química , Factores de Transcripción/química , Sitios de Unión , Proteínas de Ciclo Celular , Factores de Transcripción Forkhead , Humanos , Cinética , Modelos Moleculares , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1.
Asunto(s)
Proteínas 14-3-3/metabolismo , Saccharomyces cerevisiae/enzimología , Trehalasa/metabolismo , Activación Enzimática , Mutación , Fosforilación , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trehalasa/genéticaRESUMEN
The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4.14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4.