**2. Experimental Approaches to Determine Differentially-Translated mRNAs in Plants**

Initially, proteomics methods were used to identify the correlation between the observed fluctuations in the expression of a transcript and the actual level of peptides in plants [3]. However, the proteomics approaches have certain limitations in the case of a spatiotemporal study of a large pool of translated mRNAs and are mainly applied for assessing translation of the known peptides and proteins. Moreover, the methods of proteomics are laborious and expensive, while preparation of the specimens, quantification of proteome, and subsequent peptide sequencing require specialized technical experience [4]. Advances in high-throughput technologies, such as microarrays and deep sequencing, have made it possible to develop the new experimental approaches to studying mRNA

translation efficiency on a global scale. Three basic experimental approaches are currently used for these purposes: (a) polysome profiling; (b) translating ribosome affinity purification (TRAP); and (c) ribosome profiling or Ribo-Seq. These approaches are based on (i) the production of the mRNA pool with the ribosomes arrested on them; (ii) separation of actively-translated mRNAs (polysomal mRNAs and mRNAs bound to several ribosomes), moderately translated mRNAs (monosomal mRNAs and mRNAs bound to one ribosome), and untranslated mRNAs (steady-state mRNAs that are not bound to ribosomes); and (iii) subsequent quantitative assessment of an individual transcript or an mRNA population represented in polysomal complexes relative to the total amount of the transcript in the assayed plant specimens. Note that polysomes are several ribosomes performing translation from one mRNA and this process is regulated for individual mRNAs.
