A Molecular Orchestration of Plant Translation under Abiotic Stress.

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.

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA.

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.

Development of dual reporter vector system for estimating translational activity of regulatory elements.

BACKGROUND: 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. RESULTS: 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 ß-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. CONCLUSIONS: 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.

A high throughput assay of lichenase activity with Congo red dye in plants.

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 [Formula: see text]-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.