The Molecular Chaperone Binding Protein BiP Prevents Leaf Dehydration-Induced Cellular Homeostasis Disruption Humberto H. Carvalho 1,3 , Ota ´ vio J. B. Brustolini 1,2 , Maiana R. Pimenta 3 , Giselle C. Mendes 1,3 , Bianca C. Gouveia 1,2 , Priscila A. Silva 1,2 , Jose ´ Cleydson F. Silva 3 , Clenilso S. Mota 1 , Juliana R. L. Soares-Ramos 2 , Elizabeth P. B. Fontes 1,2 * 1 National Institute of Science and Technology in Plant-Pest Interactions, Universidade Federal de Vic ¸osa, Vic ¸osa, MG, Brazil, 2 Departamento de Bioquı ´mica e Biologia Molecular/Bioagro, Universidade Federal de Vic ¸osa, Vic ¸osa, MG, Brazil, 3 Departamento de Biologia Vegetal, Universidade Federal de Vic ¸osa, Vic ¸osa, MG, Brazil Abstract BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought- induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (y w ) values. In the WT leaves, a y w reduction of 21.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP- overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf y w = 21.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf y w = 21.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at y w = 21.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at y w = 21.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (y w = 21.0, 21.7 and 22.0 MPa) and 35S::BiP-4 leaves at y w = 21.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (y w = 21.6 MPa) to respond to drought than that of WT (y w = 21.0). Therefore, BiP overexpression maintains cellular homeostasis under water stress conditions and thus ameliorates endogenous osmotic stress. Citation: Carvalho HH, Brustolini OJB, Pimenta MR, Mendes GC, Gouveia BC, et al. (2014) The Molecular Chaperone Binding Protein BiP Prevents Leaf Dehydration-Induced Cellular Homeostasis Disruption. PLoS ONE 9(1): e86661. doi:10.1371/journal.pone.0086661 Editor: Lam-Son Phan Tran, RIKEN Center for Sustainable Resource Science, Japan Received September 6, 2013; Accepted December 12, 2013; Published January 29, 2014 Copyright: ß 2014 Carvalho 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 research was funded through the following grants from Brazilian Government Agencies: CNPq grants 573600/2008-2 and 470287/2011-0 (to EPBF); FAPEMIG grant CBB-APQ-00070-09; and FINEP grant 01.09.0625.00 (to EPBF). GCM and PAAR were supported by CNPq graduate fellowships, and HHC was supported by a FAPEMIG graduate 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: bbfontes@ufv.br Introduction The endoplasmic reticulum (ER) is a major biosynthetic organelle in all eukaryotic cells and it is the center secretory proteins synthesis, which occurs in ER membrane-associated polysomes, and protein processing, which occurs in the luminal space. The ER quality control (ER-QC) system mediates and monitors secretory proteins processing and folding, identifies and presents misfolded proteins to the ER-associated degradation (ERAD) machinery and thereby ensures that only properly folded proteins proceed to their final destinations in the secretory pathway [1]. To perform this task, ER-QC relies on molecular chaperone activities that not only assist in proper folding but also monitor the unfolded status of the secretory proteins. One such ER-resident molecular chaperone is the binding protein (BiP), which has been demonstrated to have multiple functions. BiP mediates the folding and maturation of secretory proteins, the targeting of misfolded proteins for degradation, the translocation of secretory proteins into the ER lumen and the regulation of the unfolded protein response (UPR), a signaling cascade that allows the ER lumen to communicate with the nucleus and cytoplasm of ER-stressed cells [2–4]. As in other eukaryotes, plant BiP is induced by any conditions that disrupt ER homeostasis and cause unfolded protein accumu- lation in the lumen of the organelle, a condition known as ER stress [5,6]. To cope with the effects of ER stress, the cytoprotective UPR is activated and ER-resident molecular chaperone-, ERAD- and secretory route component-encoding genes are induced to increase the ER protein folding and processing capacity under stress conditions [2], [7], [8]. However, under persistent stress conditions resulting in an insufficient PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e86661