Effective Strategies for Heterologous Expression of Plant Heterotrimeric G-protein γ Subunits without Gß Subunit Partners.

BACKGROUND: In plants, heterotrimeric G-protein (Gγ) subunits are diverse, and they have structural plasticity to provide functional selectivity to the heterotrimer. Although the Gß and Gγ subunits dimerize to function in the signaling pathway, the interaction mechanism of various Gγ subunits with the Gß subunit partners is still elusive. OBJECTIVE: To better understand the interaction mechanism, one approach is to separate the subunits for the re-assembly in vitro. Hence, developing a reliable method for achieving the efficient production and purification of these proteins has become necessary. METHODS: In this study, Gγ1 and Gγ2 proteins from Oryza sativa and Arabidopsis thaliana were successfully identified, cloned, expressed in bacteria, and purified as recombinant proteins with the fusion tags. Highly expressed recombinant Gγ subunits in E. coli were digested by proteases, which were also produced in the presented study. RESULTS: Preliminary structural characterization studies without the Gß partners showed that Gγ1 proteins have disordered structures with coiled-coil, α-helix extensions, and loops, whereas the Gγ2 protein has a more dominant ß-sheet and turns structure. Finally, computational analyses performed on Gγ genes have laid the foundation of new targets for biotechnological purposes. CONCLUSION: The proposed optimized expression and purification protocol can contribute to investigations on the Gßγ binding mechanism in plant G-protein signaling. The investigations on selective binding are critical to shed light on the role(s) of different plant Gγ subunit types in biological processes.

A chromosome-scale genome assembly of European hazel (Corylus avellana L.) reveals targets for crop improvement.

The European hazelnut (Corylus avellana L.) is a tree crop of economic importance worldwide, but especially for northern Turkey, where the majority of production takes place. Hazelnut production is currently challenged by environmental stresses, such as a recent outbreak of severe powdery mildew disease; furthermore, allergy to hazelnuts is an increasing health concern in some regions. In order to provide a foundation for using the available hazelnut genetic resources for crop improvement, we produced a fully assembled genome sequence and annotation for a hazelnut species, from C. avellana cv. 'Tombul', one of the most important Turkish varieties. A hybrid sequencing strategy, combining short reads, long reads and proximity ligation methods, enabled us to resolve heterozygous regions and produce a high-quality 370-Mb assembly that agrees closely with cytogenetic studies and genetic maps of the 11 C. avellana chromosomes, and covers 97.8% of the estimated genome size. The genome includes 27 270 high-confidence protein-coding genes, over 20 000 of which were functionally annotated based on homology with known plant proteins. We focused particularly on gene families encoding hazelnut allergens, and the Mildew resistance Locus O (MLO) proteins that are an important susceptibility factor for powdery mildew. The complete assembly enabled us to differentiate between members of these families and to identify homologues that may be important in mildew disease and hazelnut allergy. These findings provide examples of how the genome can be used to guide research and to develop effective strategies for crop improvement in C. avellana.

Identification and quantitation of genetically modified (GM) ingredients in maize, rice, soybean and wheat-containing retail foods and feeds in Turkey.

The cultivation area and diversity of genetically modified (GM) crop varieties worldwide is increasing rapidly. Taking Turkey as an example of a country with tight restrictions on the import and use of GM crops but limited resources for product monitoring, we developed a cost-effective 3-tier screening protocol, and tested 110 retail food products and 13 animal feeds available in 2016-2017 for GM ingredients. No evidence was found for the presence of GM wheat or rice in the foodstuffs tested; however, 6 feeds and 3 food products containing soybean and/or maize were positive for one or more GM elements. GM events present in positive samples were identified by event-specific PCR and quantified by real-time PCR. We also compared the results with previous surveys in Turkey. Overall, we observed consistent use of GM animal feeds; however, these were not labelled as GM at the point of sale. Occasional food products also tested positive for GM ingredients, usually at low concentrations that could be attributed to accidental contamination.

Atropa belladonna Expresses a microRNA (aba-miRNA-9497) Highly Homologous to Homo sapiens miRNA-378 (hsa-miRNA-378); both miRNAs target the 3'-Untranslated Region (3'-UTR) of the mRNA Encoding the Neurologically Relevant, Zinc-Finger Transcription Factor ZNF-691.

Recent advances in ethnobotanical and neurological research indicate that ingested plants from our diet may not only be a source of nutrition but also a source of biologically relevant nucleic-acid-encoded genetic information. A major source of RNA-encoded information from plants has been shown to be derived from small non-coding RNAs (sncRNAs) such as microRNAs (miRNAs) that can transfer information horizontally between plants and humans. In human hosts, the 3'-untranslated region (3'-UTR) of messenger RNAs (mRNAs) is targeted by these miRNAs to effectively down-regulate expression of that mRNA target in the host CNS. In this paper, we provide evidence that the Atropa belladonna aba-miRNA-9497 (miRBase conserved ID: bdi-miRNA-9497) is highly homologous to the CNS-abundant Homo sapiens miRNA-378 (hsa-miRNA-378) and both target the zinc-finger transcription factor ZNF-691 mRNA 3'-UTR to down-regulate ZNF-691 mRNA abundance. We speculate that the potent neurotoxic actions of the multiple tropane alkaloids of Atropa belladonna may be supplemented by the neuroregulatory actions of aba-miRNA-9497 on ZNF-691, and this may be followed by the modulation in the expression of ZNF-691-sensitive genes. This is the first example of a human brain-enriched transcription factor, ZNF-691, targeted and down-regulated by a naturally occurring plant microRNA, with potential to modulate gene expression in the human CNS and thus contribute to the neurotoxicological-and-psychoactive properties of the Atropa belladonna species of the deadly nightshade Solanaceae family.

Application of advanced X-ray methods in life sciences.

BACKGROUND: Synchrotron radiation (SR) sources provide diverse X-ray methods for the investigation of structure-function relationships in biological macromolecules. SCOPE OF REVIEW: Recent developments in SR sources and in the X-ray tools they offer for life sciences are reviewed. Specifically, advances in macromolecular crystallography, small angle X-ray solution scattering, X-ray absorption and fluorescence spectroscopy, and imaging are discussed with examples. MAJOR CONCLUSIONS: SR sources offer a range of X-ray techniques that can be used in a complementary fashion in studies of biological systems at a wide range of resolutions from atomic to cellular scale. Emerging applications of X-ray techniques include the characterization of disordered proteins, noncrystalline and nonequilibrium systems, elemental imaging of tissues, cells and organs, and detection of time-resolved changes in molecular structures. GENERAL SIGNIFICANCE: X-ray techniques are in the center of hybrid approaches that are used to gain insight into complex problems relating to biomolecular mechanisms, disease and possible therapeutic solutions. This article is part of a Special Issue entitled "Science for Life". Guest Editors: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Plant abiotic stress signaling.

Stress signaling is central to plants which--as immobile organisms--have to endure environmental fluctuations that constantly interfere with vigorous growth. As a result, plant-specific, elaborate mechanisms have evolved to perceive and respond to stress conditions. Currently, these stress responses are plausibly being revealed to involve crosstalks with energy signaling pathways as any growth-limiting factor alters plant's energy status. Among these, autophagy, conventionally regarded as the mechanism whereby plants recycle and remobilize nutrients in bulk, has frequently been associated with stress responses. With the recent discoveries, however, autophagy has attained a novel role in stress signaling. In this review, major elements of abitoic stress signaling are summarized along with autophagy pathway, and in the light of recent discoveries, a putative, state-of-art role of autophagy is discussed.