A Plant Biologist’s Toolbox to Study Translation

Abstract

Across a broad range of species and biological questions, more and more studies are incorporating translation data to better assess how gene regulation occurs at the level of protein synthesis. The inclusion of translation data improves upon, and has been shown to be more accurate than, transcriptional studies alone. However, there are many different techniques available to measure translation and it can be difficult, especially for young or aspiring scientists, to determine which methods are best applied in specific situations. We have assembled this review in order to enhance the understanding and promote the utilization of translational methods in plant biology. We cover a broad range of methods to measure changes in global translation (e.g., radiolabeling, polysome profiling, or puromycylation), translation of single genes (e.g., fluorescent reporter constructs, toeprinting, or ribosome density mapping), sequencing-based methods to uncover the entire translatome (e.g., Ribo-seq or translating ribosome affinity purification), and mass spectrometry-based methods to identify changes in the proteome (e.g., stable isotope labeling by amino acids in cell culture or bioorthogonal noncanonical amino acid tagging). The benefits and limitations of each method are discussed with a particular note of how applications from other model systems might be extended for use in plants. In order to make this burgeoning field more accessible to students and newer scientists, our review includes an extensive glossary to define key terms.

Lost in Translation: Physiological Roles of Stored mRNAs in Seed Germination

“…Various RBPs have been identified in seeds by proteome analyses [35,[93][94][95], and some of these RBPs may interact with specific sequence motifs in stored mRNAs, thereby regulating their localization, stability, or translation. Recently, many research tools have been developed to examine the functions of translation control and RBPs [96,97]. These techniques will further advance the study of stored mRNAs.…”

Photoprotective Acclimation of the Arabidopsis thaliana Leaf Proteome to Fluctuating Light

“…To clarify the relative contributions of protein synthesis and degradation to the observed changes in the abundance of different proteins, future studies should aim to determine the rates of protein translation, e.g., by ribosome profiling (Juntawong et al, 2014;Mazzoni-Putman and Stepanova, 2018), and degradation, e.g., using metabolic stable isotope labeling (Li et al, 2017), during acclimation to FL conditions.…”

Directions for research and training in plant omics: Big Questions and Big Data

“…These include identification of causative mutations from forward genetic screens (Cuperus et al., ), determination of copy number variants (reviewed in Zmienko, Samelak, Kozlowski, & Figlerowicz ()), and elucidation of chromosomal architecture (Grob, Schmid, Luedtke, Wicker, & Grossniklaus, ), methylation status (Cokus et al., ; Lister et al., ), cis ‐regulatory regions and motifs (Lu, Hofmeister, Vollmers, DuBois, & Schmitz, ; O'Malley et al., ; Zhang, Zhang, Wu, & Jiang, ), and sites of transcription factor‐DNA interaction (Kaufmann et al., ). With regard to the transcriptome, nucleic acid sequencing is being used to determine translation efficiency (reviewed in Mazzoni‐Putman & Stepanova, ), the RNA interactome of RNA‐binding proteins (Meyer et al., ; Zhang et al., ), and dynamics of RNA structure and stability (Ding et al., ; Li et al., ; Su et al., ). Low cost sequencing of transcriptomes along with reaction miniaturization is also powering large‐scale data collection of single‐cell transcriptomes (Birnbaum, ).…”