Hideyuki Matsuura

Genome-Wide Analyses of Early Translational Responses to Elevated Temperature and High Salinity in Arabidopsis thaliana

Abiotic stress influences the translation of mRNAs in plants. To gain a global view of the early translational response to abiotic stress, we investigated genome-wide changes in mRNA translation in Arabidopsis thaliana suspension cell cultures exposed to brief periods of two types of stress: elevated temperature (37 degrees C) and high salinity (200 mM NaCl). Microarray analyses revealed that polysome association of most transcripts, which were monitored by using polysomal- and non-polysomal-associated RNA pools, was variably depressed by both stresses within 10 min. We also inspected coordination of changes in translational profiles with transcriptional profiles, and found no simple correlations between the changes in these two processes under both stresses. In addition, we uncovered that the 10 min heat- and salt-inducible changes in polysome association of individual transcripts affected specific biological functions differently; some functional classes were recalcitrant to the overall depression, while others were hypersensitive to it. Heat and salt stresses imposed similar, but not identical, changes in polysome association of individual transcripts, and the functional categories with differential responses from all other genes (i.e. recalcitrant or hypersensitive functional categories) displayed some overlap between the two stresses, suggesting similar underlying mechanisms. Our results highlight the importance of dynamic changes in mRNA translation, which include selective translation and extensive repression of a subset of transcripts, in plant abiotic stress responses.

The 5'-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells.

The 5-untranslated region (5-UTR) functions as a translational enhancer in monocotyledonous plant cells is necessary to express a foreign gene efficiently. Here, we show that the 5-UTR of the rice alcohol dehydrogenase gene contributes to efficient translation in not only dicotyledonous plant cells, but also in monocotyledonous rice cells.

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.

Effect of the sequence context of the AUG initiation codon on the rate of translation in dicotyledonous and monocotyledonous plant cells.

The sequence context around the AUG initiation codon strongly contributes to the translation initiation step in mammalian and plant cells. Here, we investigated the effect of the three nucleotides immediately upstream of the initiating AUG (positions -3 to -1) on the translation efficiency of a reporter gene, beta-glucuronidase, in dicotyledonous and monocotyledonous plant cells.

Preferential translation mediated by Hsp81-3 5′-UTR during heat shock involves ribosome entry at the 5′-end rather than an internal site in Arabidopsis suspension cells

Translational inhibition of most mRNAs and preferential translation of mRNAs coding heat shock proteins (Hsps) occur in most cells under heat shock stress. For most Hsp mRNAs, preferential translation in heat-shocked cells is conferred by their 5-untranslated regions (5-UTRs). However, the preferential translation directed by 5-UTRs during heat shock remains mostly unknown in plants. Here, we found that the mRNA of Hsp81-3, which is an Arabidopsis Hsp90 family gene, continued to be associated with polysomes in heat-shocked Arabidopsis suspension-cultured cells. The Hsp81-3 5-UTR was found to contribute to the efficient translation of capped reporter mRNAs in heat-shocked Arabidopsis protoplasts using a transient expression assay. Further characterization of the Hsp81-3 5-UTR revealed that the anterior half of the 5-UTR is important for the efficient translation in heat-shocked protoplasts. Moreover, the Hsp81-3 5-UTR was highly capable of enhancing translation from uncapped reporter mRNAs relative to the 5-UTR of a housekeeping gene in both normal and heat-shocked protoplasts. These Hsp81-3 5-UTR-directed translations both in capped and uncapped reporter mRNAs were substantially reduced by the insertion of an upstream AUG at the 5-end of the 5-UTR, indicating that ribosomes are recruited to the 5-end of the Hsp81-3 5-UTR regardless of temperature and the presence or absence of the cap structure. These results suggest that the preferential translation of Hsp81-3 mRNA in heat-shocked Arabidopsis cells involves a ribosome scanning from the 5-end of the 5-UTR rather than ribosome entry to the internal site.

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.

Molecular Cloning and Partial Characterization of a Peroxidase Gene Expressed in the Roots of Portulaca oleracea cv., One Potentially Useful in the Remediation of Phenolic Pollutants

Portulaca (Portulaca oleracea cv.) efficiently removes phenolic pollutants from hydroponic solution. In plant roots, peroxidase (PRX) is thought to be involved in the removal of phenolic pollutants by the cross-linking them to cell wall polysaccharides or proteins at the expense of reduction of hydrogen peroxide (H2O2). In this study, we found that portulaca roots secreted an acidic PRX isozyme that had relatively high H2O2 affinity. We isolated five PRX genes, and the recombinant PRX proteins produced in cultured tobacco cells were partially characterized. Among these genes, PoPRX2 probably encoded the acidic PRX isozyme. PoPRX2 had an extra N-terminal region which has not been reported for other PRX proteins. We found that PoPRX2 oxidized phenolic pollutants, including bisphenol A, octylphenol, nonylphenol, and 17β-estradiol. In addition, we found that the Cys261 residue of PoPRX2 played an important role in the determination of affinity for H2O2 and stability toward H2O2.

Development of surface-engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene-excimer fluorescence

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface‐engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level‐dependent synthesis of PC2 from glutathiones by AtPCS1‐displaying yeast cells, followed by simple discriminative detection of PC2 via sensing of excimer fluorescence of thiol‐labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose‐dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross‐reaction, the proposed simple biosensor holds promise as a method useful for cost‐effective screening of Cd contamination in environmental and food samples. The AtPCS1‐displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS.

A Short Period of Mannitol Stress but Not LiCl Stress Led to Global Translational Repression in Plants

In plant cells, high salinity stress induces rapid inhibition of general protein synthesis. In this study, we found that treatment with mannitol, but not lithium stress, led to rapid global translational repression, suggesting that a rapid response at the level of translation might be induced by the osmotic but not the ionic components of salinity stress.

Paper-based colorimetric biosensor for antibiotics inhibiting bacterial protein synthesis.

Due to the presence of antibiotics in environmental water and their potential influence on the occurrence of antibiotic-resistant bacteria, development of a detection method suitable for the screening of environmental water for antibiotics is required. In this study, we developed a simple colorimetric paper-based biosensor based on a novel principle for the detection of antibiotics inhibiting bacterial protein synthesis, including aminoglycosides, tetracycline, chloramphenicol, and macrolides. This biosensor is based on the detection of a color change induced by β-galactosidase, which is synthesized on freeze-dried paper discs containing an inxa0vitro transcription/translation system. When a water sample without antibiotics is applied to the paper discs, β-galactosidase can be synthesized, and it hydrolyzes a colorimetric substrate, resulting in a color change from yellow to purple. By contrast, in the presence of antibiotics, the color change can be hampered due to an inhibition of β-galactosidase synthesis. We investigated the effect of the incubation temperature and pH of water samples and confirmed that the paper discs showed the color change to purple in the ranges of 15-37°C and pH 6-10. We observed concentration-dependent color variations of the paper discs by the naked eye and further estimated detection limits to be 0.5, 2.1, 0.8, and 6.1xa0μg/mL for paromomycin, tetracycline, chloramphenicol, and erythromycin, respectively, using digitized pictures. The paper-based biosensor proved to detect 0.5xa0μg/mL paromomycin, spiked in real environmental water samples, by the naked eye.