IC4R003-Microarray-2012-22722767
Contents
Project Title
Genome-Wide Analyses of Changes in Translation State Caused by Elevated Temperature in Oryza sativa
The Background of This Project
- Recently, genome-wide analyses of the translation state in response to some abiotic stresses in Arabidopsis thaliana have been reported (reviewed in Bailey-Serres et al. 2009). The translation state is a parameter reflecting the degree to which individual transcripts are translated into the corresponding proteins. Typically, the translation state is evaluated by comparing mRNA levels in polysomal (multiribosomal) and non- polysomal (free and/or monoribosomal) complexes in cell extracts fractionated through sucrose density gradients or by examining the correlation between total cellular and polysome-bound mRNA levels. The reported data showed that translation of A. thaliana mRNAs is differentially regulated: the majority of transcripts show varying degrees of translational repression, while a subset of transcripts escapes such repression and re- mains actively translated.
- Translational control under various abiotic stresses in the dicotyledonous model plant A. thaliana has been well documented. In the monocotyledonous plant, enhanced translation from anaerobic genes such as ADH in low oxygen conditions and genome-wide changes in the translational state during germination have been reported in maize (Bailey- Serres and Dawe 1996, Jiménez-López et al. 2011). In addition, global translational repression can be detected in response to heat and osmotic stress in rice (Matsuura et al. 2010b). These results indicated that translation states of individual mRNAs in monocotyledonous plant cells might be selectively regulated in response to abiotic stresses and that the underlying mechanisms might be similar among several plant species. However, some results have implied that there are differences in the precise molecular mechanisms of translational control in dicotyledonous and monocotyledonous plants. (i) The 5 0 -untranslated region (5 0 UTR) of the ADH mRNA works as a translational enhancer (Satoh et al. 2004, Sugio et al. 2008). Insertion of the 5 0 UTR of the tobacco ADH mRNA (NtADH) enhanced-glucuronidase (GUS) gene expression in A. thaliana and Nicotina tabacum cells. However, in the monocotyledonous model plant Oryza sativa, the 5 0 UTR of NtADH had little effect on GUS gene expression levels (Sugio et al. 2008). (ii) Under stress conditions, 5 0 UTRs are involved in mechanisms of escape from global translation repression in A. thaliana (Matsuura et al. 2008). The 5 0 UTR of the A. thaliana ADH mRNA (AtADH), which is important for escape from global translation repression in A. thaliana and N. tabacum cells, did not show the same function in O. sativa (our unpublished data). Thus, as a first step, it is important to characterize mRNAs that are translationally maintained or highly repressed in O. sativa and perform gene-level comparisons of translational behavior in O. sativa and A. thaliana.
- Here, we carried out a whole-genome microarray analysis to investigate changes in translation states in O. sativa in response to HS. Furthermore, using these microarray data sets, we searched for a group of genes encoding proteins with similar biological functions (functional categories) that showed a differential response relative to all other genes. This allowed characterization of translational control in response to abiotic stress, networks of co-regulated mRNAs and the biological significance of this process. Finally, these data were compared with data from A. thaliana exposed to HS.
Plant Materials & Treatment
- The O. sativa cv. Nipponbare suspension cell strain was cultured in R2S medium with constant agitation at 160 r.p.m. at 28 ℃ under continuous light. Stress-treated and control cells were prepared from the same batch of 3-day-old cultured cells. Cultured cells were incubated for 10 min in a water bath set at 41 ℃ for HS treatment or at 28 ℃ for control treatment. Two independent biological replicate samples were used for individual analyses.
Research Findings
- To monitor the translation state in O. sativa cultured cells, cell extracts were separated using sucrose density gradient centri- fugation (polysome fraction assay). Within 10 min of HS (shift from 28 to 41 ℃) polysome profiles (254 nm absorbance profile) were dramatically changed. The polysome (two or more ribosomes) peak was decreased with an increasing nonpolysome peak after 10 min of HS (Figure. 1). These changes indicate a decrease in polysome-bound mRNAs and a concomitant increase in monosome-bound or free mRNAs, suggesting global translational repression following exposure to HS. (Figure. 1) illustrates Changes in the translation state of O. sativa suspension-cultured cells exposed to 10 min of HS at 41 ℃. Absorbance profiles at 254 nm of sucrose density gradient-fractionated polysomes isolated from O. sativa suspension cultured cells exposed to 10 min of HS at 41 ℃(red) or untreated (blue). The direction of sedimentation is from right to left. Ethidium bromide staining of 28S and 18S rRNAs in sucrose gradient fractions. Centrifuged sucrose gradients were divided into eight fractions, and RNAs isolated from each fraction were subjected to denaturing formaldehyde–agarose gel electrophoresis.
- Our microarray data set was validated by quantitative real-time PCR (qRT-PCR) for 21 transcripts which were selected to represent a wide range of △PS values, PS values and changes of transcript levels in response to HS (log 10 ES) (description below). For calculation of △PS, transcripts were separated into eight fractions using sucrose density centrifugation, and the amount of transcripts in each fraction was evaluated. PS 28 ,PS 41 and △PS were determined from the qRT-PCR data and compared with the values from DNA microarray analysis (Fig. 3A, Table 1). All values showed high correlation (r = 0.86–0.96) between the two independent methods (Fig. 3B, Supplementary Fig. S2A, B). As a supplemental explanation, overall △PS values from qRT-PCR had a tendency to show smaller values than those from microarray. This was because we could not completely achieve fraction equivalence between the two methods when RNA was collected after sucrose density centrifugation.
Labs working on this Project
- Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan
- Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871 Japan
- Institute for Environmental Science and Technology, Saitama University, 255 Shimo-Ohkubo, Sakura-ku, Saitama, Saitama, 338-8570 Japan
Corresponding Author
Ko Kato (email: kou@bs.naist.jp )
