Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia-up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions. ribosome profiling | uORF | alternative splicing | long intergenic noncoding RNA | translational efficiency
Soil flooding creates composite and complex stress in plants known as either submergence or waterlogging stress depending on the depth of the water table. In nature, these stresses are important factors dictating the species composition of the ecosystem. On agricultural land, they cause economic damage associated with longterm social consequences. The understanding of the plant molecular responses to these two stresses has benefited from research studying individual components of the stress, in particular low-oxygen stress. To a lesser extent, other associated stresses and plant responses have been incorporated into the molecular framework, such as ion and ROS signaling, pathogen susceptibility, and organ-specific expression and development. In this review, we aim to highlight known or suspected components of submergence/waterlogging stress that have not yet been thoroughly studied at the molecular level in this context, such as miRNA and retrotransposon expression, the influence of light/dark cycles, protein isoforms, root architecture, sugar sensing and signaling, post-stress molecular events, heavy-metal and salinity stress, and mRNA dynamics (splicing, sequestering, and ribosome loading). Finally, we explore biotechnological strategies that have applied this molecular knowledge to develop cultivars resistant to flooding or to offer alternative uses of flooding-prone soils, like bioethanol and biomass production.
Polyribosomes (polysomes) form as multiple ribosomes engage in translation on a single mRNA. This process is regulated for individual mRNAs by both development and the environment. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts by high-speed differential centrifugation. These ribonucleoprotein complexes can be further purified by centrifugation through sucrose density gradients. By fractionation of the gradient the amount of an individual mRNA in a sub-population of polysomes can be quantitatively determined. Here, we describe methods for the isolation and quantification of polysome complexes from plant tissues. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level. A modification of the conventional polysome isolation procedure is described for transgenic Arabidopsis thaliana that express an epitope-tagged version of ribosomal protein L18 (RPL18) that facilitates capture of ribosomes from crude cell extracts by a one-step immunoprecipitation method.
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