Efficient transgene expression by alleviation of translational repression in plant cells.

Global translational repression under abiotic stress influences translation of both endogenous and transgene mRNAs. Even in plant cell culture, hypoxia and nutrient deficient stress arise during the growth process. In this study, we first demonstrated the existence of global translational repression in Arabidopsis T87 cultured cells over a time course following inoculation. Next, we performed genome-wide analysis, which revealed that the translational states of endogenous mRNAs differed significantly between growth and stationary phase cells. This analysis showed that translation from most mRNAs was repressed upon stationary phase. Otherwise, a part of mRNA including alcohol dehydrogenase (ADH) gene was recalcitrant to the repression. Furthermore, by polysome analysis and followed quantitative reverse transcription PCR analysis of transformants having 5'untranslated regions (UTRs) of ADH or translationally repressed At3g47610 mRNA fused to reporter gene, we demonstrated that polysomal associations of reporter mRNAs were in accordance with those the mRNAs from which their 5'UTR derived, suggesting that the 5'UTR is an important determinant of the translational state of mRNAs in stationary phase cells. Finally, we demonstrated the effectiveness of 5'UTR of ADH mRNA in transformants derived from the BY-2 tobacco cell line. These results suggested that 5'UTR of ADH mRNA would be a useful element for efficient transgene expression upon stationary phase.

A computational and experimental approach reveals that the 5'-proximal region of the 5'-UTR has a Cis-regulatory signature responsible for heat stress-regulated mRNA translation in Arabidopsis.

Translation of specific plant mRNAs is differentially regulated under certain abiotic stress conditions such as heat, oxygen deprivation and dehydration. The majority of transcripts exhibit varying degrees of translational repression, whereas a subset of transcripts escape such repression and remain actively translated. The underlying mechanisms that mediate this control, and in particular the identities of the regulatory RNA elements involved, remain poorly understood. Using a combined computational and experimental approach, we identified a novel cis-regulatory element in the 5'-untranslated region (5'-UTR) that affects differential translation in response to heat stress (HS) in Arabidopsis thaliana. First, we selected a set of genes with distinct translational responses to HS, based on our previously reported genome-wide data regarding changes in polysome loading induced by HS in A. thaliana cultured cells. We evaluated the 5'-UTRs of these messages for their ability to mediate expression, when fused to reporter mRNAs, in protoplasts under HS. The data from the reporter assay and the nucleotide sequences of the 5'-UTRs tested were used to define regulatory elements in the 5'-UTRs, with the help of a partial least square (PLS) regression model. The computational analysis using PLS and subsequent experimental characterization of a series of 5'-UTR mutants provided evidence that the 5'-proximal sequence of the 5'-UTR is a primary and position-dependent determinant of 5'-UTR-mediated differential translation in response to HS. Finally, we discuss the possible mechanism underlying HS regulation of differential mRNA translation.

Genome-wide analyses of changes in translation state caused by elevated temperature in Oryza sativa.

It has been reported that the translational status of mRNAs responds dramatically to abiotic stresses. While many useful results have demonstrated translational control in dicotyledonous model plants, little is known about changes in the translation state in response to abiotic stresses in monocotyledonous plants. To understand global changes in translation of mRNAs, we performed genome-wide analyses using Oryza sativa treated with heat stress (HS). These analyses showed that most mRNAs were translationally repressed, while the translation of some mRNAs was maintained. In addition to other regulatory steps in gene expression, including transcription and processing, it is thought that translational regulation is a critical step in adaptation to new conditions because of the functional tendencies of proteins that are either translationally maintained or highly repressed upon HS. When we compared the functional tendencies of translationally regulated proteins in rice with those in Arabidopsis thaliana cells exposed to HS, some showed similar regulation, arguing for both common and different features of translational regulation in the two plants.