The correlation between transcription levels (abundance of individual mRNAs) and translation (level of corresponding proteins in the proteome) is modest. This confirms that gene expression regulation at the translation stage is crucial. Given the significance of investigations into translationally active mRNA as an indicator of translation effectiveness, researchers require reliable and structured functional sources of information on translation, such as plant translatomic maps that resembling transcription maps. The establishment of a standardized experimental and computational approach is required to generate translatomic maps. In this article, we explore possibilities for researchers to generate translatomic maps for plants. We present a comprehensive overview of experimental methods used to obtain translationally active mRNA fractions, taking into account their advantages and limitations. In addition, we discuss methodological approaches that enable obtaining a complete mRNA pool, sequencing methods for qualitative and quantitative evaluation of various mRNA fractions, as well as bioinformatic methods for multivariate analysis and indicators for assessing mRNA translational activity. This manuscript calls for the scientific community to develop translatomic maps of plant objects as a means to increase the utilization of translatomic data in both fundamental plant biology and practical applications.

In this work, the authors investigated the possibility of modifying the fatty acid (FA) composition of tomato leaves (Solanum lycopersicum L.) by introducing into its genome the gene of the cyanobacterial Δ9 acyl-lipid desaturase (Synechococcus vulcanus C.). To obtain transgenic tomato plants expressing cyanobacterial desaturase (desC) and to evaluate the effect of this desaturase on the FA composition of total lipids, vectors carrying the gene desC were constructed. The gene sequence was fused with leader sequences that ensure localization of the protein product in chloroplasts, endoplasmic reticulum (ER), or cytoplasmic membrane. The resulting vectors were used to transform the A. tumefaciens AGL0 strain for subsequent agrobacterium-mediated transformation of tomato plants. FA analysis of leaves showed that, when desC is localized in the cytoplasmic membrane or ER membrane, an increase in the content of C16:1 and C16:2 fatty acids is observed for some lines. For these lines, an increase in the relative representation of the transcript desC compared to transgenic lines that did not show changes in FA composition is shown. The obtained data can be used to develop strategies for targeted modification of the fatty acid composition of tomatoes.

The large body of data on plant gene expression accumulated through comparative studies directs the efforts of researchers to study the subtle mechanisms of the influence of target genes and, as a result, to develop relatively simple and, at the same time, effective approaches to understand the physiological role of protein products of a gene. Numerous studies have convincingly demonstrated the effectiveness of transient expression strategies for characterizing plant gene functions. The objectives of the review were (1) to review the advantages and limitations of various plant transient expression systems and methods used to elucidate the roles of gene products, (2) to summarize current knowledge on the use of transient expression approaches to understand the subtle mechanisms underlying gene function, and (3) to describe advances in efficient transient expression of plant genes. The review discusses the main and critical steps of each of the methods of transient gene expression in plants, their areas of application, and the main results obtained using plant objects and their contribution to our knowledge of the subtle mechanisms of gene functions underlying plant growth and development, including elucidation of the mechanisms regulating complex metabolic pathways.

Arabidopsis thaliana (L.) Heynh. is one of the major model organisms used in different areas of science: plant physiology and biochemistry, developmental biology, genetic engineering, genome editing, etc. These model plants possess the following advantages: short life cycle, simple cultivation, sequenced and rather well annotated genome, and numerous available reports concerning transcriptome, proteome, metabolic pathways, and mutations. The technique of A. thaliana cultivation under laboratory conditions is an important aspect of investigations dealing with this plant as a model. Choice of the growing mode depends on the goal of investigation as well as on quantity and type of required biomaterial. The aim of this work is to review the techniques of A. thaliana cultivation and their applicability to different tasks.

Additional file 2. R program and examples. The program code of RNAdeNoise in R language, examples of the use.

Various regulatory codes contained in mRNA can determine the fate of any transcript during translation. To search for similar regulatory codes and study their impact on translational efficiency, we have developed the JetGene Internet resource ( https://jetgene.bioset.org/ ). It contains CDS, cDNA, 5'‑UTR, and 3'‑UTR sequences from six major groups of living organisms, including plants. This internet resource has a user-friendly interface, puts together a wide range of tools for comparative analysis of nucleotide sequences and allows one (1) to estimate variations of length, nucleotide composition, and codon frequency, to analyze GC-content, CpG-islands, start codon context, etc..; (2) to identify and determine the statistically significant representation of potential regulatory contexts at mRNA with different translational efficiency. A user can make versatile in silico analysis of full-length or truncated transcripts as well as their coding or noncoding regions. Every step of the analysis is accompanied by a graphical interpretation of the results.

BackgroundMolecular biology has always shown some similarities with computer science. So, considering transient expression, one can see an analogy with a DDoS attack on a computing system. Like the DDoS attack, transient expression can carry a payload. In particular, analysis of the structure of cell mechanisms and signal amplification in the study of very subtle mechanisms of regulation.ResultsA new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coli

Transient heterologous gene expression in two model plant species, Nicotiana benthamiana and N. excelsior, has been used to study the localization of the heterologous Δ9 acyl-lipid desaturase (Δ9 desaturase) of Synechococcus vulcanus in different cell compartments and its functional activity in the cases of the cytosol, chloroplast, and endoplasmic reticulum (ER) localization. The functional activity and substrate specificity of the heterologous desaturase under the conditions of transient expression have been confirmed by comparison of fatty acid (FA) profiles. The Δ9 desaturase, responsible for the synthesis of oleic and palmitoleic acids, has also been shown to strongly promote the accumulation of polyunsaturated FAs. The results convincingly demonstrate that the Δ9 desaturase of the thermophilic cyanobacterium transiently expressed in two Nicotiana species considerably alters lipid metabolism in their leaves towards a higher FA unsaturation. The functional activity of Δ9 desaturase depends on both the model plant species, N. benthamiana or N. excelsior, and the cellular localization of the enzyme. The method of transient expression of heterologous genes in plants is highly effective, inexpensive, and not time-consuming, which makes it attractive for estimating the functional activity and/or substrate specificity of heterologous desaturases.

A bireporter vector for the study of translational cis-regulatory elements has been created and tested.

We create web database JetGene that allows to estimate the variation of length, nucleotide composition, codon usage frequency and to study nucleotides surrounding of the start codon. JetGene allows user to compare two samples of mRNA.

The paradox of misfit between the levels of mRNAs and their protein products in the eukaryotic cells, including plant cells, encountered by researchers, direct their efforts towards the study into fine mechanisms of translation. Translation is an intricate biological process with numerous players, including mRNAs, tRNAs, ribosomes, and manifold protein factors. Certainly, each of them is important for efficient translation. However, the mRNAs itself contain numerous regulatory elements, such as 5'UTR, the context around the AUG start codon, and codon composition; each element separately or in combination can determine the fate of an individual mRNA in translational process. The previous reviews mainly focus on individual key stages in translation or the aspects of its control. Our goal here is (i) to summarize the recent data on the specific structure–function features of plant mRNAs and their correlation with translational efficiency; (ii) to brief new experimental and theoretical approaches to gaining the insight into the complex network of translation; and (iii) to assess the relevance of this knowledge to both the plant functional genomics and biotechnological application.

Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.

Based on the results of our own research and literature data, we will present the main theoretical and experimental approaches to studying the translation efficiency of plant mRNA and highlight their contribution to functional plant genomics and biotechnology.

We have proposed and tested a method for characterization of the signal sequences and determinations of target protein localization in a plant cell. This method, called the AgI-PrI, implies extraction of protoplasts from plant tissues after agroinfiltration. The suggested approach combines the advantages of two widely used methods for transient gene expression in plants-agroinfiltration and transfection of isolated protoplasts. The AgI-PrI technic can be applied to other plant species.

The review describes current approaches to the optimization of pharmacokinetic properties of pharmaceutical proteins in order to achieve the maximum therapeutic effect. Examples of such technologies, including PEGylation, PEG mimetics, glycosylation, protein–protein fusion, and their advantages and limitations are discussed.

A recombinant DNA in which the interferon αA (IFN–αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN–αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN–αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.