Genome-Wide Identification and Characterization of Wheat 14-3-3 Genes Unravels the Role of TaGRF6-A in Salt Stress Tolerance by Binding MYB Transcription Factor.

14-3-3 proteins are a large multigenic family of general regulatory factors (GRF) ubiquitously found in eukaryotes and play vital roles in the regulation of plant growth, development, and response to stress stimuli. However, so far, no comprehensive investigation has been performed in the hexaploid wheat. In the present study, A total of 17 potential 14-3-3 gene family members were identified from the Chinese Spring whole-genome sequencing database. The phylogenetic comparison with six 14-3-3 families revealed that the majority of wheat 14-3-3 genes might have evolved as an independent branch and grouped into ε and non-ε group using the phylogenetic comparison. Analysis of gene structure and motif indicated that 14-3-3 protein family members have relatively conserved exon/intron arrangement and motif composition. Physical mapping showed that wheat 14-3-3 genes are mainly distributed on chromosomes 2, 3, 4, and 7. Moreover, most 14-3-3 members in wheat exhibited significantly down-regulated expression in response to alkaline stress. VIGS assay and protein-protein interaction analysis further confirmed that TaGRF6-A positively regulated slat stress tolerance by interacting with a MYB transcription factor, TaMYB64. Taken together, our findings provide fundamental information on the involvement of the wheat 14-3-3 family in salt stress and further investigating their molecular mechanism.

14-3-3 gene family in spotted sea bass (Lateolabrax maculatus): Genome-wide identification, phylogenetic analysis and expression profiles after salinity stress.

The tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation (14-3-3) proteins are a group of highly conserved homologous and heterologous proteins involved in a wild range of physiological processes, including the regulation of many molecular phenomena under different environmental salinities. In this study, we identified eleven 14-3-3 genes from the spotted sea bass (Lateolabrax maculatus) genome and transcriptomic databases and verified their identities by conducting phylogenetic, syntenic and gene structure analyses. The spotted sea bass 14-3-3 genes are highly conserved based on sequence alignment, conserved domains and motifs, and tertiary structural feature. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 14-3-3 genes in gill of spotted sea bass under normal physiological conditions indicated that the expression level of 14-3-3 zeta was the highest among tested genes, followed by 14-3-3 theta. Furthermore, expression profiles of 14-3-3 genes in gill tissue (in vivo and in vitro) indicated that the 14-3-3 zeta and 14-3-3 theta genes were significantly induced by different environmental salinities in spotted sea bass, suggesting their potential involvement in response to salinity challenge. Our findings may lay the foundation for future functional studies on the 14-3-3 gene family in euryhaline teleosts.

De novo sequencing and analysis of the lily pollen transcriptome: an open access data source for an orphan plant species.

Pollen grains of Lilium longiflorum are a long-established model system for pollen germination and tube tip growth. Due to their size, protein content and almost synchronous germination in synthetic media, they provide a simple system for physiological measurements as well as sufficient material for biochemical studies like protein purifications, enzyme assays, organelle isolation or determination of metabolites during germination and pollen tube elongation. Despite recent progresses in molecular biology techniques, sequence information of expressed proteins or transcripts in lily pollen is still scarce. Using a next generation sequencing strategy (RNAseq), the lily pollen transcriptome was investigated resulting in more than 50 million high quality reads with a length of 90 base pairs. Sequenced transcripts were assembled and annotated, and finally visualized with MAPMAN software tools and compared with other RNAseq or genome data including Arabidopsis pollen, Lilium vegetative tissues and the Amborella trichopoda genome. All lily pollen sequence data are provided as open access files with suitable tools to search sequences of interest.

Identification and characterization of the 14-3-3 gene family in Hevea brasiliensis.

The 14-3-3 proteins are a family of conserved phospho-specific binding proteins involved in diverse physiological processes. Although the genome-wide analysis of this family has been carried out in certain plant species, little is known about 14-3-3 protein genes in rubber tree (Hevea brasiliensis). In this study, we identified 10 14-3-3 protein genes (designated as HbGF14a to HbGF14j) in the latest rubber tree genome. A phylogenetic tree was constructed and found to demonstrate that HbGF14s can be divided into two major groups. Tissue-specific expression profiles showed that 10 HbGF14 were expressed in at least one of the tissues, which suggested that HbGF14s participated in numerous cellular processes. The 10 HbGF14s responded to jasmonic acid (JA) and ethylene (ET) treatment, which suggested that these HbGF14s were involved in response to JA and ET signaling. The target of HbGF14c protein was related to small rubber particle protein, a major rubber particle protein that is involved in rubber biosynthesis. These findings suggested that 14-3-3 proteins may be involved in the regulation of natural rubber biosynthesis.

Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B.

Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m(2)) and a 2.7-fold (at 800 J/m(2)) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo.

14-3-3 CSF levels in sporadic Creutzfeldt-Jakob disease differ across molecular subtypes.

BACKGROUND: The 14-3-3 protein is a physiological cellular protein expressed in various tissues, and its release to CSF reflects extensive neuronal damage as in Creutzfeldt-Jakob disease (CJD), but also in other neurological diseases. 14-3-3 protein in CSF in the proper clinical context is a reliable diagnostic tool for sporadic CJD. However, the sensitivity varies across molecular CJD subtypes. OBJECTIVE: We determined the level of the 14-3-3 protein in CSF from 70 sporadic CJD patients with distinct molecular subtypes using an improved enzyme-linked immunosorbent assay (ELISA) protocol technique. RESULTS: The 14-3-3 levels varied markedly across various molecular subtypes. The most elevated levels of 14-3-3 protein were observed in the frequently occurring and classical subtypes, whereas the levels were significantly lower in the subtypes with long disease duration and atypical clinical presentation. PRNP codon 129 genotype, PrP(sc) isotype, disease stage and clinical subtype influenced the 14-3-3 level and the test sensitivity. CONCLUSIONS: The 14-3-3 protein levels differ across molecular subtypes and might be used for their early pre-mortem identification when the codon 129 genotype is known, especially for the less common molecular subtypes such as MV2 and MM2.

Expression analysis and tissue distribution of two 14-3-3 proteins in silkworm (Bombyx mori).

14-3-3 proteins, which have been identified in a wide variety of eukaryotes, are highly conserved acidic proteins. In this study, we identified two genes in silkworm that encode 14-3-3 proteins (Bm14-3-3zeta and Bm14-3-3epsilon). Category of two 14-3-3 proteins was identified according to phylogenetic analysis. Bm14-3-3zeta shared 90% identity with that in Drosophila, while Bm14-3-3epsilon shared 86% identity with that in Drosophila. According to Western blot and real time PCR analysis, the Bm14-3-3zeta expression levels are higher than Bm14-3-3epsilon in seven tissues and in four silkworm developmental stages examined. Bm14-3-3zeta was expressed during every stage of silkworm and in every tissue of the fifth instar larvae that was examined, but Bm14-3-3epsilon expression was not detected in eggs or heads of the fifth instar larvae. Both 14-3-3 proteins were highly expressed in silk glands. These results suggest that Bm14-3-3zeta expression is universal and continuous, while Bm14-3-3epsilon expression is tissue and stage-specific. Based on tissue expression patterns and the known functions of 14-3-3 proteins, it may be that both 14-3-3 proteins are involved in the regulation of gene expression in silkworm silk glands.

Structural basis for protein-protein interactions in the 14-3-3 protein family.

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.

Identification of a gene from the arbuscular mycorrhizal fungus Glomus intraradices encoding for a 14-3-3 protein that is up-regulated by drought stress during the AM symbiosis.

In the present study, a 14-3-3 protein-encoding gene from Glomus intraradices has been identified after differential hybridization of a cDNA library constructed from the fungus growing in vitro and subjected to drought stress by addition of 25% PEG 6000. Subsequently, we have studied its expression pattern under drought stress in vitro and also when forming natural symbioses with different host plants. The results obtained suggest that Gi14-3-3 gene may be involved in the protection that the arbuscular mycorrhizal (AM) symbiosis confers to the host plant against drought stress. Our findings provide new evidences that the contribution of AM fungi to the enhanced drought tolerance of the host plant can be mediated by a group of proteins (the 14-3-3) that regulate both signaling pathways and also effector proteins involved in the final plant responses.

Selective regulation of 14-3-3eta in primary culture of cerebral cortical neurons and astrocytes during development.

The 14-3-3 proteins exist predominantly in the brain and may play regulatory roles in cellular processes of growth, differentiation, survival, and apoptosis. The biological functions, however, of the various 14-3-3 isoforms (beta, epsilon, eta, gamma, and zeta) in the brain remain unclear. We have reported previously upregulation of 14-3-3gamma in ischemic astrocytes. In the present study, we report selective regulation of 14-3-3eta in cultured cerebral cortical neurons and astrocytes during in vitro development. In cultured neurons, gene expression levels of 14-3-3eta increase with culture age (0-10 days). Brain-derived neurotrophic factor and neurotrophin-3 upregulate 14-3-3eta gene expression. In cultured astrocytes, 14-3-3eta is downregulated with culture age (1-5 weeks). The gene expression level of 14-3-3eta is not affected by scratch injury in astrocytes or by ischemia in neurons. These data suggest a possible role of 14-3-3eta in growth and differentiation of neurons and astrocytes, indicating an intricate mechanism governing coordinated and well-controlled developmental events in the brain to ensure normal neural functions.