The complexities of translational strategies make this stage of implementing genetic information one of the most challenging to comprehend and, simultaneously, perhaps the most engaging. It is evident that this diverse range of strategies results not only from a long evolutionary history, but is also of paramount importance for refining gene expression and metabolic modulation. This notion is particularly accurate for organisms that predominantly exhibit biochemical and physiological reactions with a lack of behavioural ones. Plants are a group of organisms that exhibit such features. Addressing unfavourable environmental conditions plays a pivotal role in plant physiology. This is particularly evident with the changing conditions of global warming and the irrevocable loss or depletion of natural ecosystems. In conceptual terms, the plant response to abiotic stress comprises a set of elaborate and intricate strategies. This is influenced by a range of abiotic factors that cause stressful conditions, and molecular genetic mechanisms that fine-tune metabolic pathways allowing the plant organism to overcome non-standard and non-optimal conditions. This review aims to focus on the current state of the art in the field of translational regulation in plants under abiotic stress conditions. Different regulatory elements and patterns are being assessed chronologically. We deem it important to focus on significant high-performance techniques for studying the genetic information dynamics during the translation phase.

Regulation at the level of translation plays an essential role in modulating gene expression. Given the importance of knowledge about translationally active mRNAs as an indicator of translation efficiency, it is crucial for researchers to have at their disposal organised sources of functional information on translation - plant translatomic maps similar to transcription maps. The development of a general experimental and computational methodology for constructing translatome maps is essential to address this challenge. In this manuscript, we discuss what researchers can do to design plant translatome maps, namely, we review: (i) experimental methods to obtain translationally active mRNA fractions, considering their advantages and limitations; (ii) additional methodological approaches to obtain complete pools of translationally active mRNAs; (iii) sequencing methods to qualitatively and quantitatively assess different mRNA fractions; and (iv) multivariate analyses of sequencing results by bioinformatics methods, including metrics for assessing mRNA translational activity.

Stress is one of the important factors affecting the growth and development of plants. In recent years, a large body of data on transcriptional changes in response to stress in plants has been obtained. Despite the fact that the important role of the regulation of translation in response to stress has been proven in plants, knowledge in this area remains extremely scarce. In this review, we focused on mRNA translation in the cytoplasm of higher plants. Most of the current research on the effects of translation has been done in yeast and mammalian systems. Translation factors and mechanisms are mostly conserved among eukaryotes; however, some differences are known to exist among plants. A comprehensive understanding of the complex apparatus of translation and its regulation in plants is required. In this work, we have tried to update modern ideas about the regulation of translation under conditions of abiotic stress.

For the needs of modern biotechnology, a quantitative approach to the control of regulatory elements at all stages of gene expression has long become indispensable. Such a control regime is impossible without a quantitative analysis of the role of each regulatory element or pattern used. Therefore, it seems important to modify and develop the accuracy, reproducibility, and availability of methods for quantifying the contribution of each regulatory code to the implementation of genetic information. A 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 β-glucuronidase) under the control of different promoters. Herewith we also propose a new method for quantification of the effect of tested regulatory elements on expression, which relies on assessment of the enzyme activities of reporter proteins taking into account the transcription of their genes. In our view, this approach makes it possible to precisely determine the amounts of reporter proteins and their transcripts at all stages of expression. The efficiency of the proposed system has been validated by the analysis of the roles of known translation enhancers at the stages of transcription and translation.

BACKGROUND:RNA-seq has become a standard technology to quantify mRNA. The measured values usually vary by several orders of magnitude, and while the detection of differences at high values is statistically well grounded, the significance of the differences for rare mRNAs can be weakened by the presence of biological and technical noise.RESULTS:We have developed a method for cleaning RNA-seq data, which improves the detection of differentially expressed genes and specifically genes with low to moderate transcription. Using a data modeling approach, parameters of randomly distributed mRNA counts are identified and reads, most probably originating from technical noise, are removed. We demonstrate that the removal of this random component leads to the significant increase in the number of detected differentially expressed genes, more significant pvalues and no bias towards low-count genes.CONCLUSION:Application of RNAdeNoise to our RNA-seq data on polysome profiling and several published RNA-seq datasets reveals its suitability for different organisms and sequencing technologies such as Illumina and BGI, shows improved detection of differentially expressed genes, and excludes the subjective setting of thresholds for minimal RNA counts. The program, RNA-seq data, resulted gene lists and examples of use are in the supplementary data and at https://github.com/Deyneko/RNAdeNoise .

Late blight is one of the most economically important diseases affecting potato and causing a significant loss in yield. The development of transgenic potato plants with enhanced resistance to infection by Phytophthora infestans may represent a possible approach to solving this issue. A comparative study of the leaf response in control potato plants (S.tuberosum L. cultivar Skoroplodnyi), control transgenic plants expressing the reporter gene of thermostable lichenase (transgenic licBM3 line) and transgenic plants expressing cyanobacterial hybrid genes ∆9-acyl-lipid desaturase (transgenic desC lines) and ∆12-acyl-lipid desaturase (transgenic desA lines) to infection with P. infestans has been performed. The expression of desaturase genes in potato plants enhanced their tolerance to potato late blight agents as compared with the control. The lipid peroxidation level raised in the leaves of the control and transgenic desA plants on third day after inoculation with P. infestans zoospores and remained the same in the transgenic desC plants. The number of total phenolic compounds was increased as early as on the second day after infection in all studied variants and continued to remain the same, except for transgenic desC plants. Accumulation of flavonoids, the main components of the potato leaf phenolic complex, raised on the second day in all studied variants, remained unchanged on the third day in the control plants and decreased in most transgenic plants expressing desaturase genes. The results obtained in our study demonstrate that the expression of genes of Δ9- and Δ12-acyl-lipid desaturases in potato plants enhanced their resistance to P. infestans as compared with the control non-transgenic plants due to concomitant accumulation of phenolic compounds, including flavonoids, in the leaves. All these changes were more pronounced in transgenic desC plants, which indicates that the Δ9-acyllipid desaturase gene appears to be a potential inducer of the production of biological antioxidants in plant cells.

A broad and amazingly intricate network of mechanisms underlying the decoding of a plant genome into the proteome forces the researcher to design new strategies to enhance both the accumulation of recombinant proteins and their purification from plants and to improve the available relevant strategies. In this paper, we propose new approaches to optimize a codon composition of target genes (case study of interferon-αA) and to search for regulatory sequences (case study of 5′UTR), and we demonstrated their effectiveness in increasing the synthesis of recombinant proteins in plant systems. In addition, we convincingly show that the approach utilizing stabilization of the protein product according to the N-end rule or a new protein-stabilizing partner (thermostable lichenase) is sufficiently effective and results in a significant increase in the protein yield manufactured in a plant system. Moreover, it is validly demonstrated that thermostable lichenase as a protein-stabilizing partner not only has no negative effect on the target protein activity (interferon-αA) integrated in its sequence, but rather enhances the accumulation of the target protein product in plant cells. In addition, the retention of lichenase enzyme activity and interferon biological activity after the incubation of plant protein lysates at 65 °C and precipitation of nontarget proteins with ethanol is applicable to a rapid and inexpensive purification of fusion proteins, thereby confirming the utility of thermostable lichenase as a protein-stabilizing partner for plant systems.

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

Abstract Background Since the beginning of the use of reporter proteins for expression analysis, a variety of approaches have been developed and proposed; both qualitative and quantitative. The lack of simple methods for direct observation of gene expression in living organisms makes it necessary to continue to propose new methods. In this work, we consider a method for the quantitative analysis of the expression of thermostable lichenase from Clostridium thermocellum used as a sensitive reporter protein. Results In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusions The specific interaction between the dye Congo red and $$\beta$$ β -d-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

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

The transcript Euonymus europaeus was collected de novo. A comparative analysis of transcriptomes from the stage of the globular embryo and the mature fruit made it possible to identify key DGAT genes at the contrasting stages of the development of E. europaeus fruits.

The polysome profiling method was used to separate mRNAs depending on their loading by ribosomes into polysomal and monosomal fractions. Pools separation of such mRNAs and analysis of transcripts (mRNAs) which are associated with each mRNA pool due to RNA sequencing allowed to get an idea of the translational efficiency of individual mRNAs. Moreover, subsequent in silico analysis make possible searching of regulatory contexts in the 5'-UTR of plant A. thaliana, which may be potentially important for efficient translation of mRNA.

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.

A large data array on plant gene expression accumulated thanks to comparative omic studies directs the efforts of researchers to the specific or fine effects of the target gene functions and, as a consequence, elaboration of relatively simple and concurrently effective approaches allowing for the insight into the physiological role of gene products. Numerous studies have convincingly demonstrated the efficacy of transient expression strategy for characterization of the plant gene functions. The review goals are (i) to consider the advantages and limitations of different plant systems and methods of transient expression used to find out the role of gene products; (ii) to summarize the current data on the use of the transient expression approaches for the insight into fine mechanisms underlying the gene function; and (iii) to outline the accomplishments in efficient transient expression of plant genes. In general, the review discusses the main and critical steps in each of the methods of transient gene expression in plants; areas of their application; main results obtained using plant objects; their contribution to our knowledge about the fine mechanisms of the plant gene functions underlying plant growth and development; and clarification of the mechanisms regulating complex metabolic pathways.

Motivation Polysome profiling is novel, and yet has proved to be an effective approach to detect mRNAs with differential ribosomal load and explore the regulatory mechanisms driving efficient translation. Genes encoding regulatory proteins, having a great influence of the organism, usually reveal moderate to low transcriptional levels, compared, for example, to genes of house-keeping machinery. This complicates the reliable detection of such genes in the presence of technical and/or biological noise.Results In this work we investigate how cleaning of polysome profiling data on Arabidopsis thaliana influences the ability to detect genes with low level of total mRNA, but with a highly differential ribosomal load, i.e. genes translationally active. Suggested data modelling approach to identify a background level of mRNA counts individually for each dataset, shows higher power in detection of low transcribed genes, compared to the use of thresholds for the minimal required mRNA counts or the use of raw data. The significant increase in detected number of regulation–related genes was demonstrated. The described approach is applicable to a wide variety of RNA-seq data. All identified and classified mRNAs with high and low translation status are made available in supplementary material.

The control of translation in the course of gene expression regulation plays a crucial role in plants’ cellular events and, particularly, in responses to environmental factors. The paradox of the great variance between levels of mRNAs and their protein products in eukaryotic cells, including plants, requires thorough investigation of the regulatory mechanisms of translation. A wide and amazingly complex network of mechanisms decoding the plant genome into proteome challenges researchers to design new methods for genome-wide analysis of translational control, develop computational algorithms detecting regulatory mRNA contexts, and to establish rules underlying differential translation. The aims of this review are to (i) describe the experimental approaches for investigation of differential translation in plants on a genome-wide scale; (ii) summarize the current data on computational algorithms for detection of specific structure–function features and key determinants in plant mRNAs and their correlation with translation efficiency; (iii) highlight the methods for experimental verification of existed and theoretically predicted features within plant mRNAs important for their differential translation; and finally (iv) to discuss the perspectives of discovering the specific structural features of plant mRNA that mediate differential translation control by the combination of computational and experimental approaches.