High-throughput molecular and bioinformatic characterization of translational regulation in plants

Dehydration is a major abiotic stress that leads to severe losses in crop productivity. Plant tolerance of dehydration involves programmed changes in gene expression that are necessary for physiological and morphological adaptations. Transcript abundance is frequently used to monitor alterations in gene expression in response to dehydration stress. However, transcript abundance may not accurately reflect gene expression due to extensive regulation at post-transcriptional and post-translational levels. Here, the significance of translational regulation in the response to dehydration stress was evaluated in leaves of Nicotiana tobacum and Arabidopsis thaliana . Fractionation of polysome complexes and monosome/mRNP complexes was used to evaluate the effect of dehydration stress on levels of protein synthesis. A strong correlation between polysome level and leaf relative water content was observed in apical leaves and basal leaves of tobacco, as well as evidence that polysome levels are developmentally regulated. Stress significantly reduced polysome levels with concomitant increase in monosomes, indicating that the stress primarily impairs the initiation of translation. RNA blot analysis of mRNP and polysomal complexes revealed that dehydration-inducible lipid transfer protein and osmotin mRNA maintained ribosome association, whereas rubisco small subunit protein and eukaryotic initiation factor 4A mRNA showed reduced ribosome association. To further evaluate dehydration-stress induced translational regulation, DNA microarray analysis were performed using Affymetrix Arabidopsis GeneChips to quantitatively evaluate alterations in translational status (proportion on mRNA in polysomes: ribosome loading). The analysis revealed a tremendous variation in the proportion of individual mRNA in polysomes under non-stress and dehydration stress conditions. Strikingly, mRNAs with a two-fold or greater increase in mRNA abundance showed little reduction in ribosome loading, whereas the majority of cellular mRNAs (71%) showed significantly reduced ribosome loading, agreeing with the observations for the four tobacco mRNAs. In addition, mRNAs encoding transcription factors and signaling molecules primarily had low levels of ribosome loading as opposed to mRNAs encoding proteins involved in energy and metabolism. Remarkably, cytosolic ribosomal protein mRNA significantly reduced ribosome loading in response to dehydration stress with little change in mRNA abundance. The majority of mRNAs with strong reduction in mRNA abundance showed significant reduction in ribosome loading, indicating the connection between translation and mRNA stability for a subset of mRNAs. Bioinformatic characterization of mRNA nucleotide composition, sequence and structural features identified several determinants in the 5′ - and 3′-untranslated regions (UTRs) of mRNA that contribute significantly to the variation in ribosome loading under both nonstress and stress conditions. The potential for formation of secondary structures in the 5′-UTR had a more pronounced effect on ribosome loading under dehydration stress than non-stress. Also, nucleotide composition near to the initiation codon contributed to stress-regulated translation. The results indicate that differential ribosome loading observed in the two conditions is a consequence of the multifaceted qualities of the RNA sequence determinants that control the level of translation for individual mRNAs. This work provides a foundation for future study of mRNA sequence elements as well as characterization of genes that are translationally regulated in response to dehydration stress.