Transcriptional Downregulation of Rice rpL32 Gene under Abiotic Stress Is Associated with Removal of Transcription Factors within the Promoter Region Pradipto Mukhopadhyay, Malireddy K. Reddy, Sneh Lata Singla-Pareek, Sudhir K. Sopory* Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, Delhi, India Abstract Background: The regulation of ribosomal proteins in plants under stress conditions has not been well studied. Although a few reports have shown stress-specific post-transcriptional and translational mechanisms involved in downregulation of ribosomal proteins yet stress-responsive transcriptional regulation of ribosomal proteins is largely unknown in plants. Methodology/Principal Findings: In the present work, transcriptional regulation of genes encoding rice 60S ribosomal protein L32 (rpL32) in response to salt stress has been studied. Northern and RT-PCR analyses showed a significant downregulation of rpL32 transcripts under abiotic stress conditions in rice. Of the four rpL32 genes in rice genome, the gene on chromosome 8 (rpL32_8.1) showed a higher degree of stress-responsive downregulation in salt sensitive rice variety than in tolerant one and its expression reverted to its original level upon withdrawal of stress. The nuclear run-on and promoter:reporter assays revealed that the downregulation of this gene is transcriptional and originates within the promoter region. Using in vivo footprinting and electrophoretic mobility shift assay (EMSA), cis-elements in the promoter of rpL32_8.1 showing reduced binding to proteins in shoots of salt stressed rice seedlings were identified. Conclusions: The present work is one of the few reports on study of stress downregulated genes. The data revealed that rpL32 gene is transcriptionally downregulated under abiotic stress in rice and that this transcriptional downregulation is associated with the removal of transcription factors from specific promoter elements. Citation: Mukhopadhyay P, Reddy MK, Singla-Pareek SL, Sopory SK (2011) Transcriptional Downregulation of Rice rpL32 Gene under Abiotic Stress Is Associated with Removal of Transcription Factors within the Promoter Region. PLoS ONE 6(11): e28058. doi:10.1371/journal.pone.0028058 Editor: Steven R. Ellis, University of Louisville, United States of America Received December 26, 2010; Accepted October 31, 2011; Published November 23, 2011 Copyright: 2011 Mukhopadhyay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by grants from the Department of Biotechnology (grant number BT/PR2965/AGR/155-III/2002), Government of India and internal grants from International Centre for Genetic Engineering and Biotechnology. PM is thankful to University Grant Commission (UGC), Government of India for providing research fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: sopory@icgeb.res.in Introduction Plants encounter a number of abiotic stresses which limit agricultural production. Classical breeding programs, as also the initial attempts with genetic engineering to raise stress tolerant plants have met with limited success [1]. Through recent advances in genomics and proteomics, some knowledge about stress perception, stress signaling and transcription regulators has been gained from model organisms such as Arabidopsis, Synechocystis and Clamydomonas [2–4]. Yet a more detailed understanding of stress response and adaptation is desirable, especially for monocot crop plants like rice [5]. An in depth knowledge about the regulation of stress-responsive genes will help in developing strategies to increase the stress tolerance level of socially and economically important crops. It is well known that growth is severely affected in plants under stress conditions. Ribosomal proteins are essential for proper growth and development of any organism. Not much was known about regulation of expression of genes encoding ribosomal proteins in plants until recently. Many ribosomal proteins have been reported to be up- or downregulated under various stress conditions [6–10]. Recently, chloroplast encoded rpL33 was shown to be necessary for plant survival under cold stress in Arabidopsis [11]. Pathways of translational downregulation of ribosomal proteins as observed in mammals and transcriptional regulation of ribosomal proteins, somewhat similar to yeast, have been described in plants earlier [12–17]. Post-transcriptional regulation for ribosomal proteins has also been suggested [7,18– 21]. Although altered expression of ribosomal proteins under stress has been shown to involve the post-transcriptional and transla- tional regulatory mechanisms, stress-specific transcriptional regu- lation of ribosomal proteins is still to be reported in plants. In this work, we have studied the transcriptional regulation of genes encoding 60S ribosomal protein rpL32 in rice under salt stress. Homologues of rpL32 have been shown to bind RNA in archaea and maize, possess transcriptional transactivation activity, mediate telomere silencing in yeast, and promote proper growth and development in Drosophila [22–25]. From the analysis of differential cDNA libraries of rice (previous work in our lab; unpublished) and barley [26], the downregulation of this gene under salt stress was postulated. The present work has shown that rpL32 gene is downregulated at the transcriptional level under abiotic stress and that this downregulation is linked to the removal of proteins from cis-elements in the promoter region. Our work presents a study of transcriptional downregulation of genes which PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e28058