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The rice RING finger E3 ligase, OsHCI1, drives nuclear export of multiple
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substrate proteins and its heterogeneous overexpression enhances acquired
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thermotolerance
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Sung Don Lim, Hyun Yong Cho, Yong Chan Park, Deok Jae Ham, Ju Kyong Lee, and
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Cheol Seong Jang*
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Department of Applied Plant Sciences, Kangwon National University, Chuncheon 200-713, Korea
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* Corresponding author: Cheol Seong Jang
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E-mail: [email protected]
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Tel: +82-33-250-6416
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Fax: +82-33-244-6410
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ABSTRACT
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Thermotolerance is very important for plant survival when plants are subjected to lethally high
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temperature. However, thus far little is known about the functions of RING E3 ligase in
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response to heat shock in plants. We found that one rice gene encoding the RING finger protein
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was specifically induced by heat and cold stress treatments but not by salinity or dehydration
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and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results
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showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and
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extensively along the cytoskeleton at normal temperatures. In contrast, OsHCI1 may have
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accumulated in the vicinity of the nucleus under high temperatures. We found that OsHCI1
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physically interacted with nuclear substrate proteins including a basic helix-loop-helix
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transcription factor. Transient co-overexpression between OsHCI1 and each of three nuclear
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proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations.
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Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through
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acquired thermotolerance. We propose that OsHCI1 mediates nuclear-cytoplasmic trafficking of
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nuclear substrate proteins via monoubiquitination and drives an inactivation device for the
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nuclear proteins under heat shock.
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Keywords: abiotic stress, monoubiquitination, rice, RING E3 ligase, thermotolerance, nuclear-
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cytoplasmic trafficking
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Introduction
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Extreme temperature is a major agricultural problem limiting crop yields worldwide. A transient
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increase in temperature, usually 10–15°C above ambient, is generally considered heat shock or
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heat stress in living organisms, particularly in plants. Heat shock negatively affects plant growth,
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seed germination, photosynthesis, respiration, water relation, and membrane stability in plants
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(Wahid et al., 2007). At the cellular and molecular level, heat shock leads to adverse outcomes
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in plant cell functions, including alterations in cellular composition of membrane fluidity and
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permeability, enzyme activity, metabolism, production of active oxygen species, and gene
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expression (Alfonso et al., 2001; Kampinga et al., 1995; Larkindale et al., 2005; Larkindale and
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Huang, 2004; Larkindale and Knight, 2002). These alterations could cause reduced
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photosynthesis and carbon gain in plants, thereby leading to decreased growth and reproduction.
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For example, studies on the relationship between rice crop yields and temperature over the last
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two decades have demonstrated that grain yields decrease significantly by 10 % for each 1°C
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increase in the growing-season minimum temperature (Peng et al., 2004).
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Investigations into molecular mechanisms underlying thermoprotection have involved genetic
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and molecular approaches (Ahuja et al., 2010; Iba, 2002; Qin et al., 2011; Sung et al., 2003).
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Plants generally possess basal and acquired thermotolerance by two heat tolerance mechanisms
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(Vierling, 1991). Basal thermotolerance is defined as an inherent ability to survive high
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temperatures, whereas acquired thermotolerance is the ability to tolerate an otherwise lethally
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high temperature after being pre-exposed to a sub-lethal increased temperature, mimicking an
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“immunization” against high temperature. Once plants are exposed to high temperature, either
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basal, acquired, or both, thermotolerance mechanisms may be involved (Larkindale et al., 2005).
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One of the best known mechanisms regarding acquired thermotolerance is the induction of
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heat shock proteins (HSPs; Vierling, 1991). HSPs are molecular chaperone stress-response
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proteins that protect organisms against various stresses, particularly high temperature. HSPs
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preserve structural and functional protein integrity by binding to proteins that have become
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denatured or misfolded as a result of heat shock (Perez et al., 2009; Sarkar et al., 2009). Plant
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adaptations to high temperature are not only HSP-based mechanisms but also other components
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such as phospholipids, the dehydration-responsive element binding protein 2A (DREB2A), and
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S-nitroglutathione reductase (GSNOR) (Ahuja et al., 2010). For example, the heat stress
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transcription factor, HsfA3, which is transcriptionally induced during heat shock by DREB2A,
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regulates the expression of an HSP-encoding gene (Schramm et al., 2008). Furthermore,
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filamentous temperature sensitive H 11 protease and GSNOR activity contribute to plant
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adaption to high temperatures (Chen et al., 2006; Lee et al., 2008). Attachment of ubiquitin
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molecules (Ub, a small 76-amino acid protein) to target substrates for modification mediates a
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variety of cellular functions via the Ub/26S proteasome system in higher plants. In this pathway,
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the conjugation cascade subsequently requires three classes of enzymes, i.e., E1 (ubiquitin-
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activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase) (Vierstra,
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2009). Approximately 5% of the Arabidopsis proteome is postulated to be involved in the
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Ub/26S proteasome pathway, and about 1,300 genes are predicted to encode E3 ligase
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components in particular (Smalle and Vierstra, 2004). The E3 ligases specifically interact with
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target proteins to confer different fates by attachment of ubiquitin molecules. The substrate-
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ubiquitin structures determine the subcellular localization and different functions of many target
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proteins (Hicke and Dunn, 2003; Pickart, 2004; Roos-Mattjus and Sistonen, 2004). For example,
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attachment of single ubiquitin molecules to one or more lysines on target proteins, known as
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mono-ubiquitination or multimono-ubiquitination, activates a variety of their functions, e.g.,
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trafficking, subcellular localization, signal transduction, transcription regulation, and DNA
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repair (Deng et al., 2000; Hicke and Dunn, 2003; Kaiser et al., 2000; Wu et al., 2003). In
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contrast, polyubiquitinated substrate proteins destined for degradation are usually targeted by
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the 26S proteasome (Roos-Mattjus and Sistonen, 2004; Vierstra, 2009).
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The Ub/26S proteasome pathway is an important mechanism of tolerance against high
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temperature. For example, seedlings of Prosopis chilensis, which is a leguminous tree, are able
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to survive at 50°C after germination at 35°C (Medina and Cardemil, 1993). P. chilensis showed
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higher relative accumulation rates of free Ub, conjugated Ub, and HSP70 than cultivated
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Glycine max (soybean) under heat stress, suggesting that the ubiquitinated-proteolytic pathway
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is an important heat tolerance mechanism (Ortiz and Cardemil, 2001). In addition, small
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ubiquitin-like modifiers (SUMOs) that are ubiquitin-like polypeptides also attach to various
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target substrates and, thus, modify their cellular functions. In Arabidopsis, the findings that
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SUMO1/2 conjugates were highly accumulated by repeated heat shock, while HSP70-
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overexpressing plants showed fewer SUMO1/2 conjugates during heat shock, suggested that the
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accumulation of SUMO1/2 conjugates is relevant to thermotolerance (Kurepa et al., 2003).
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Plant single-subunit E3 ligases are generally classified into three groups based on the
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presence of the homologous E6-AP C-terminus, U-box, and RING domain (Smalle and Vierstra,
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2004). Of these, the RING domain of the really interesting new gene was the first to be
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identified as a novel cysteine-rich sequence (Freemont et al., 1991). The proteins harboring
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RING domain are believed to play E3 ligase for recognizing and ubiquitylation of substrate
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proteins. Subsequently, a number of RING E3s have been reported to play crucial roles in post-
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translational regulation of plant hormone signaling pathways e.g., abscisic acid (ABA) and
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environmental stresses. For example, the RING E3 ligase ABI3-interacting protein 2 is a
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negative regulator of ABA signaling by promoting degradation of ABSCISIC ACID-
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INSENSITIVE 3 (ABI3; Zhang et al., 2005). Another outstanding example is the KEEP ON
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GOING E3 ligase, which also regulates the protein level of ABI5, a basic domain/leucine zipper
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transcription factor, by 26S proteasomal degradation in an ABA-dependent manner (Stone et al.,
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2006). The Arabidopsis RING E3 ligases DREB2A-interacting protein 1 and 2 negatively
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modulate the expression of drought stress-response genes (Qin et al., 2008). Hot pepper RING
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membrane-anchor 1 homolog 1 (Rma1H1) functions as an E3 ligase plasma membrane
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aquaporin, PIP2;1, under water-deficient conditions (Lee et al., 2009). In Arabidopsis, the high
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expression of osmotically responsive gene 1 (HOS1) harboring a RING-like domain negatively
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regulates cold signal transduction (Lee et al., 2001a). Additionally, salt- and drought- induced
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ring finger 1 E3 ligase is believed to enhance salt stress-responsive ABA signaling (Zhang et al.,
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2007). However, RING E3 ligase and its substrate proteins on heat shock response via
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ubiquitination still remain unknown in plants.
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In this study, we identified the molecular functions of a rice RING domain E3 ligase, OsHCI1
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(Oryza sativa Heat and Cold Induced 1), which is highly induced under heat and cold stress
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conditions. Studies with a Golgi-localized OsHCI1-EYFP fusion protein showed that OsHCI1
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dynamically moved from the cytoplasm to the nucleus along cytoskeletal tracts under heat
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shock conditions. To shed light on the molecular function of this gene, we performed a yeast-
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two hybrid (Y2H) screen, a bimolecular fluorescence complementation (BiFC) assay, and an in
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vitro ubiquitination assay. The results demonstrated that OsHCI1 interacted with six substrate
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proteins and mediated subcellular trafficking of nuclear proteins to the cytoplasm via
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monoubiquitination. Furthermore, Arabidopsis overexpressing OsHCI1-EYFP exhibited a heat-
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tolerant phenotype, suggesting an important role of this protein in the regulation of heat5
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generated signals in plants.
Materials and Methods
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Plant materials and heat shock treatments
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Seeds of rice (Oryza sativa L. cv. Donganbyeo) were grown on mesh supported in plastic
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containers with 1/2 Murashige and Skoog (MS) nutrient solution in a growth chamber (16/8-h
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light/dark photoperiod at 25°C with 70 % relative humidity). Two-week-old seedlings were
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exposed to high-salinity (250 mM NaCl), dehydration, cold (4°C), and heat (45°C) stress. The
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high-salinity and dehydration stress treatments were performed as described by Lim et al.
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(2010). Two-week-old seedlings were transferred to fresh MS nutrient solution with each of
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ABA (0.1 mM), JA (0.1 mM), and SA (1 mM). For ethylene treatments, seedlings were moved
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into air-tight plastic containers with fresh MS solution for the ethylene treatment. Ethylene gas
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(50 μL L-1) was injected into the plastic boxes using a syringe (Wuriyanghan et al., 2009). Leaf
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tissues were sampled at 0, 1, 6, 12, 24, and 48 h after the stress treatment. Healthy samples
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without stress treatment were harvested as controls at the same times. All leaf samples were
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ground using liquid nitrogen and immediately stored at -80°C until total RNA extraction.
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Dry seeds of A. thaliana ecotype Columbia were grown, and two constructs of 35S:EYFP
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(EV) and 35S:OsHCI1-EYFP were transformed via Agrobacterium tumefaciens (GV3101) using
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the floral dip method (Zhang et al., 2006). The assessment of segregation of kanamycin
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resistance in T3 transformants was conducted < 1 month after harvest for the assay. Three
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independent OsHCI1-overexpressing lines and a control plant (35S:EYFP) were tested
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according to Lakindale et al (2005) to observe the heat shock effect. Transgenic Arabidopsis
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plants were grown on MS agar plates for 7 days then dipped into water baths at either 38°C or
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45°C, as appropriate. Basal thermotolerance treatments were performed by heating the plants in
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sealed plates at 45°C for 1 h. Acquired thermotolerance treatments were conducted by heating
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the plants initially to 38°C for 90 min, and then they were moved to a growth chamber (24°C)
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for 120 min before finally heating to 45°C for 3 h. Both heat shock treatments were performed
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in the dark. Heat-treated plants were recovered in a growth chamber at 24°C for 5 days in the
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light.
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To evaluate the expression patterns of six interacting protein genes with OsHCI1, rice plants
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were grown on MS agar plates for 14 days. Then basal or acquired heat treatments were
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performed as described above. Leaves were sampled at different time points using liquid
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nitrogen and immediately stored at -80°C until total RNA extraction.
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Rice protoplast isolation and transfection
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Protoplasts were isolated from 2-week-old seedlings (Kim et al., 2012). Seeds of rice were
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grown on 1/2 MS nutrient solution in a growth chamber (16/8-h light/dark photoperiod at 25°C
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with 70% relative humidity). Young leaves and sheaths were chopped and dipped in enzyme
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solution [0.5 M manitol, 1.5 % cellulose RS (Yakult Honsa Co., Ltd, Tokyo, Japan), 0.75 %
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mecerozyme R10 (Yakult Honsa), 1 mM CaCl2, and 0.1 % BSA] with carbenicillin (100 mg l-1).
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This mixture was incubated on a shaking incubator for 16 h at room temperature then filtered
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through Miracloth. Protoplasts were pelleted by centrifugation for 4 min at 300 × g were
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resuspended in an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5
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mM glucose, and 1.5 mM MES, adjusted to pH 5.7) and incubated in ice for 5 h. Protoplasts
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were centrifuged and resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl2, and 4.7
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mM MES, adjusted to pH 5.7). Plasmid DNA (10 or 20 µg) was added to the protoplast solution
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and transfected with 40 % polyethylene glycol (PEG) solution [40 % PEG 4000, 0.4 M mannitol,
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and 100 mM Ca (NO3)2] for 20 min at room temperature. W5 solution was added stepwise to
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dilute the PEG solution and discarded. Transfected protoplasts were incubated overnight at
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room temperature and then observed under confocal microscopy.
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Gene expression study
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Total RNA was extracted using TRIzol® regent, according to the manufacturer’s protocol
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(Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis, from 500 ng total RNA was
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conducted using a cDNA Synthesis kit (Takara-Bio, Ohtsu, Japan). Semi-quantitative reverse
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transcription-polymerase chain reaction (RT-PCR) was performed as described previously (Lim
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et al., 2010). Gene-specific primers were designed using Primer-BLAST (NCBI,
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http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Reliable genes such as OsSalT for salt stress
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(Claes et al., 1990), OsbZIP23 for dehydration (Xiang et al., 2008), LIP19 for cold (Shimizu et
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al., 2005), and OsHsp90-1 for heat (Hu et al., 2009) were used as positive controls to verify the
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stress treatments, respectively. Quantitative real-time PCR was performed with a Rotor-Gene Q
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(Qiagene, USA) by monitoring the SYBR Green fluorescence signal during DNA synthesis. The
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real-time PCR results were calculated by using the Delta-Delta CT Method (Livak and
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Schmittgen, 2001). Os18S-rRNA (Os09g00999) was used as an internal control. Primers with
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restriction enzyme sites used in this study are listed in Supplementary Table S1.
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Yeast two-hybrid (Y2H) screening and Y2H assays
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A full-length coding sequence of OsHCI1 was amplified and cloned in-frame with the GAL4
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DNA binding domain of the GBKT7-BD vector to generate the GAL4 DNA-BD fusion
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construct. A rice cDNA library was generated from 14-day-old seedlings treated with salt stress
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(250 mM NaCl). Then, yeast transformation and library screening were conducted in
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accordance with the recommended procedures (Make Your Own “Mate & Plate
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System; Matchmaker
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System 2, Clontech, Palo Alto, CA, USA). The full-length OsHCI1coding sequence was fused
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to the yeast GAL DNA-binding domain and used as a bait protein for screening. A rice cDNA
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library from salt-treated seedlings was fused to the yeast GAL4 activation domain (AD) as a
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prey protein. A total of 280 yeast transformants were selected on a synthetic defined (SD)
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medium lacking Leu and Trp supplemented with 40 μg/ml X-α-Gal and 70 ng/ml aureobasidin
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A (AbA) (DDO/X/A) and re-patched on SD medium lacking Ade, His, Leu, and Trp with 40
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μg/ml X-α-Gal and 70 ng/ml AbA (QDO/X/A).
TM
TM
” Library
Gold Yeast Two-Hybrid System; YeastmakerTM Yeast Transformation
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Six full-length interaction partners were amplified by RT-PCR using primers listed in the
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Supplementary Table S1 to confirm a positive interaction with OsHCI1. Each PCR product was
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digested with the appropriate restriction enzyme and introduced into the pGADT7 vector. These
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constructs with pGBKT7-OsHCI1 were co-transformed into the Y2H Gold yeast strain.
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Transformed yeast cells were separately grown onto SD/-Leu/-Trp and SD/-Ade/-His/-Lue/-
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Trp/X-α-Gal/Aba with 70 ng/ml (AbA) for 5 days at 30°C. All experiments were repeated three
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times.
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Subcellular localization
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Two fluorescence protein constructs were prepared for the subcellular localization assay. For the
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35S:EYFP and 35S:DsRed2 constructs, the coding sequence of the EYFP and DsRed2 were
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amplified using a high-fidelity Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA)
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from pEYFP-C1 and pDsRed2-C1 (Clontech) as templates, respectively, with primers harboring
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multiple cloning sites (Supplementary Table S1). The PCR products then were cloned into the
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pBIN35S binary vector under the control of the CaMV 35S promoter. The coding region of the
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full-length cDNA of OsHCI1 was amplified from rice cDNA with appropriate primer pairs and
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then inserted into the pBIN35S-EYFP vector between the XbaI and KpnI sites for the
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subcellular localization study. A single amino acid substitution (OsHCI1C172A) in the RING
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domain of OsHCI1 was generated using the QuikChange® Site-Directed Mutagenesis kit
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(Stratagene) with the OsHCI1C172A-F and OsHCI1C172A-R primer pair. Additionally, full-
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length cDNAs of the six OsHCI1 interacting partners were cloned into the pBIN35S-DsRed2
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vector with appropriate enzyme sites, respectively. The plasmid containing an organelle marker
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for the Golgi apparatus (Nelson et al., 2007) was kindly provided by the Arabidopsis Biological
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Resource Center.
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BiFC assay
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The full-length OsHCI1 cDNAs and the six interacting partners were amplified by PCR using
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appropriate primers to generate BiFC constructs. PCR products were digested and then ligated
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into 35S-HA-SPYCE(M) and 35S-c-myc-SPYNE(R)173 vectors, respectively (Waadt et al.,
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2008). Primers and restriction enzymes used for cloning are presented in Supplementary Table
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S1.
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Agrobacterium tumefaciens strain GV3101, harboring each construct, was inoculated for 16 h
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at 28°C for transient expression. These cells were harvested and re-suspended in infiltration
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buffer (10 mM MES, 10 mM MgCl2, 0.2 mM acetosyringone, pH 5.6) to a final concentration
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at an optical density at 600 nm = 0.5. Equal volumes of different combinations of
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Agrobacterimum strains were mixed and coinfiltrated into 5-week-old Nicotiana benthamiana
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leaves with a syringe. Infiltrated plants were placed at 25°C for 3 d to detect YFP fluorescence.
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In vitro ubiquitination assay
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Full-length OsHCI1 cDNA was amplified by PCR with primer pairs (Supplementary Table S1).
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The amplicon was digested with NotI and BamHI and then ligated into a digested pMAL-c5X
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vector (New England BioLabs, Ipswich, MA, USA) with the same enzymes. Recombinant MBP
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fusion protein and non-recombinant MBP (negative control) were expressed in E. coli strain
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BL21 (DE3) pLysS (Promega, Madison, WI, USA), purified by affinity chromatography using
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amylose resin (New England BioLabs), and used for the in vitro self-ubiquitination assay. The
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full-length cDNAs of AtUBC10 and AtUBC11 were amplified and then introduced into the
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pET-28a (+) vector (Novagen, Gibbstown, NJ, USA) with a 6x His-tag. The fusion 6x His-
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tagged AtUBC10 and AtUBC11 were expressed in E. coli strain BL21 (DE3) pLysS and
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purified using the Ni-NTA Purification System (Invitrogen).
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The in vitro self-ubiquitination assay was conducted as described previously by Hardtke et al.
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(2002) with some modifications. Purified MBP-OsHCI1 (250 ng) was mixed with 50 ng yeast
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E1 (Boston Biochemicals, Cambridge, MA, USA), 250 ng purified Arabidopsis E2 (AtUBC10
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and AtUBC11), and 10 μg bovine ubiquitin (Sigma-Aldrich, St. Louis, MO, USA) incubated in
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ubiquitination reaction buffer [50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.05 mM ZnCl2, 1 mM
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ATP, 0.2 mM DTT, 10 mM phosphocreatine, and 0.1 unit of creatine kinase (Sigma-Aldrich)].
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After 3 h incubation at 30°C, the reaction was halted at different time points by adding 2× SDS
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sample buffer followed by 5 min of boiling at 95°C. Ten microliters of each reaction was
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analyzed via 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
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then transferred to a nitrocellulose membrane. Immunoblot analyses were conducted using anti-
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ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with a secondary goat
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anti-rabbit IgG peroxidase antibody (Sigma-Aldrich). Detection was conducted using the
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chemiluminescent substrate SuperSignal® West Pico (Thermo Scientific, Waltham, MA, USA)
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for HRP and imaged on X-ray film (Kodak, Rochester, NY, USA). To confirm that OsHCI1
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mediated ubiquitination of the six interacting proteins, OsPGLU1, OsbHLH065, OsGRP1, and
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OsPOX1-His-Trx fusion proteins were affinity-purified, and 200 ng of purified proteins was
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incubated together with purified MBP-OsHCI1 in the ubiquitination mixture for 3 h. The
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mixture was then subjected to 10 % SDS-PAGE and immunoblot analysis.
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Confocal microscopy and imaging
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Transformed tobacco leaves were cut 3–5 days after infection for microscopic analyses.
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Fluorescent images were obtained using a Multiphoton confocal laser scanning microscope
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(model LSM 510 META NLO and LSM 780 NLO, Carl Zeiss, Oberkochen, Germany) at the
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Korea Basic Science Institute, Chuncheon Center. Excitation/emission wavelengths were
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514/535 590 nm for EYFP and BiFC constructs and 543/565 615 nm for the DsRed2 and
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mCherry construct. All images were acquired using either a C-Apochromat (40×/1.2 water
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immersion) objective. To prevent cross-talk between EYFP and mCherry signals, the spectral
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images were acquired using the lambda mode. Scanned images were captured as single optical
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sections or as a Z-series of optical sections. Image processing was carried out using an LSM 5
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Image Browser (Zeiss) and Adobe Photoshop 9.0 software (Mountain View, CA, USA).
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RESULTS
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OsHCI1 is upregulated by heat and cold
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We previously defined expression diversity of members of the rice RING finger protein genes
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based on their expression profiles via in silico analysis (Lim et al., 2010). Subsequently, in an
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effort to isolate RING finger protein gene(s) that play a critical role in extreme temperature 48
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RING finger protein genes were randomly selected and examined for their expression patterns
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via semi-quantitative RT-PCR (data not shown). Interestingly, one gene (Os10g30850) was
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highly induced at 1–48 h after heat treatment (45°C), whereas OsHsp90-1 used for validation of
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the stress treatment was similarly induced by the stress (Hu et al., 2009) (Fig. 1A).
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Consequently, the gene was named Oryza sativa heat and cold inducible gene 1 (OsHCI1). We
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further examined expression patterns of the gene against other abiotic stresses such as cold
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(4°C), salinity, and dehydration (Fig. 1A). The gene was upregulated at 12–48 h by cold stress,
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whereas the LIP19 (Shimizu et al., 2005) gene was induced at 1–48 h. However, both the
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salinity and dehydration stresses exhibited no induction of the gene through 48 h after the
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treatments. We employed two reliable stress-inducible genes as a quality control, OsSalT (Claes
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et al., 1990) and OsbZIP23 (Xiang et al., 2008), for salinity and dehydration, respectively. High
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induction of the OsSalT and OsbZIP23 genes served as evidence that the plants have been
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subjected severe stresses, supporting no response of the OsHCI1 gene to either stress.
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Consequently, the gene was named Oryza sativa heat and cold inducible gene 1 (OsHCI1). We
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further confirmed the transcript levels of the OsHCI1 gene via quantitative real-time PCR,
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which revealed high expression patterns under heat and cold stresses but not under salt and
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drought stresses (Supplementary Fig. S1).
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When plants are subjected to heat shock, phytohormones including ABA, salicylic acid (SA),
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and ethylene act as key signals (Larkindale and Knight, 2002). Therefore, we further examined
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phytohormonal regulation during OsHCI1 gene expression (Fig. 1B). Under 0.1 mM ABA
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treatment, OsHCI1 was induced at 3 h, the highest transcript level occurred at 12 h, and then
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gradually decreased to 48 h, whereas OsSalT exhibited an increase at 3 h and then steady
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expression until 48 h. In the case of jasmonic acid (JA), the OsPBZ1 gene, which is inducible by
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hormone treatment (Lee et al., 2001b), showed a slight induction at 3 h and then a subsequent
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increase up to 48 h, whereas OsHCI1 exhibited a somewhat slight induction at 3–24 h. In
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addition, OsHCI1 gene expression increased at 3 h, reached its highest transcript level at 12 h,
12
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and then showed no induction until 48 h. However, OsPR1b (Agrawal et al., 2000) was induced
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at 6 h and gradually and slightly increased until 24 h. Additionally, the transcription level of
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OsHCI1 under 50 μL L-1 ethylene treatment increased at 6 h then reached its highest level at 12
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h. Collectively, the OsHCI1 expression patterns under phytohormonal treatments were induced
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gradually at 12 h then decreased its transcript levels until 24 h. These results indicate that the
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OsHCI1 gene rapidly responds to hormone treatments.
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Dynamics of OsHCI1-EYFP subcellular localization
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It is generally believed that subcellular localization of a protein of interest is crucial to
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understand its cellular function. To examine subcellular localization of the OsHCI1 protein, we
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constructed one binary vector harboring the enhanced yellow fluorescence protein (EYFP)
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under the control of a CaMV 35S promoter. Transient expression of 35S:EYFP was diffuse in
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both the cytosol and the nucleus in tobacco epidermal cells (Fig. 2A, upper panel). We further
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generated the 35S:OsHCI1-EYFP construct, which is transiently expressed in tobacco leaves.
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OsHCI1 fluorescence displayed a punctuate pattern; the fluorescence appeared to localize in the
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dispersed organization of Golgi stacks in most (about 93%) tobacco cells (Fig. 2A, lower panel).
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In contrast, about 7 % of the transformed tobacco cells showed an additional reticulate
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fluorescence with a punctate pattern, which seemed to target endoplasmic reticulum (ER)
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network patterns (Supplementary Fig. S2A, C). To confirm whether the destination of the
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OsHCI1 protein alone was the Golgi apparatus, we employed the G-rk-mCherry organelle
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marker localized to the Golgi body. Both constructs, OsHCI1-EYFP and G-rk-mCherry, were
328
transiently co-expressed with p19 in tobacco cells (Nelson et al., 2007). The OsHCI1-EYFP
329
signal was closely overlapped by that of G-rk-mCherry (Fig. 2D), indicating that the final
330
destination of OsHCI1 was the Golgi complex. The ER localization may represent newly
331
synthesized OsHCI1-EYFP protein that has not yet been transported to the Golgi stack.
332
Furthermore, the punctate patterns of OsHCI1-EYFP fluorescence were also displayed around
333
the nuclear envelope (Fig. 2A in lower panel, D). In addition, we found dynamic movement in
334
which the OsHCI1-EYFP fluorescent signals moved rapidly and extensively along the
335
cytoskeleton of leaf epidermis cells (Supplementary Fig. S2B; Supplementary Movie S1).The
336
finding that the subcellular localization of fluorescently tagged fusion proteins is changed by
337
environmental stress (Lee et al., 2001a; von Arnim and Deng, 1994) allowed us to ask whether
338
the Golgi localization of the OsHCI1 protein could be altered by heat shock. Thus, we
339
transiently expressed 35S:EYFP and 35S:OsHCI1-EYFP in tobacco leaves, and then incubated
13
340
them for 1 h at 38°C or 45°C. Interestingly, strong OsHCI1-EYFP signals were found in the
341
nucleus (Fig. 2B, C, lower panel). We were concerned that heterogeneous expression of
342
OsHCI1 caused protein mislocalization and functional diversity. Subsequently, the constructs
343
were expressed in rice protoplasts, which were then incubated for 15 min at 38°C or 45°C,
344
resulting in a similar expression pattern compared to that of tobacco (Fig. 2E). These results
345
support the previous finding, i. e., there is no significant difference in protein localization
346
between tobacco and rice cells. Under moderate heat treatment (38°C), approximately 55.0% of
347
cells exhibited a nuclear localized pattern of the OsHCI1-EYFP protein and approximately
348
35.0% of cells displayed this pattern in both the Golgi and nucleus. However, approximately
349
10% of cells still showed only the Golgi localized pattern of rice protoplasts (Fig. 2F). Similarly,
350
approximately 66.6 % and 21.6 % of cells showed nuclear localization and both the Golgi and
351
nucleus pattern, respectively, under severe heat treatment (45°C). By contrast, approximately
352
11.6 % of cells only displayed a Golgi localized pattern at 45°C.
353
Expression pattern and subcellular localization of proteins interacting with OsHCI1
354
A Y2H screen was performed to identify proteins that interact with OsHCI1. Twenty-four
355
positive clones were selected, sequenced, and their α-galactosidase activity was measured
356
(Supplementary Fig. S3). To confirm these positive interactions with OsHCI1, full-length
357
coding sequences of the top six genes, which exhibited strong α-galactosidase activity, were
358
cloned into GAL4 AD, respectively. Full-length OsHCI1 and each interacting protein were co-
359
transformed into the Y2H Gold strain, and grown on QDO/X/A medium (Supplementary Fig.
360
S4). The six interacting protein genes were 20S proteasome subunit α7 (named OsPSA7,
361
Os01g59600), periplasmic beta-glucosidase (OsBGLU1, Os03g53800), ethylene-responsive
362
protein (OsbHLH065, Os04g41570, Li et al., 2006), glycine-rich cell wall structural protein
363
(OsGRP1, Os05g02770), peroxidase (OsPOX1, Os07g48020) and 14-3-3 protein (Os14-3-3,
364
Os11g34450).
365
We also examined the expression patterns of the interacting partner genes with OsHCI1 under
366
two different heat stresses via semi-quantitative RT-PCR with rice seedlings treated by basal or
367
acquired heat shock treatments (Fig. 3) The results showed that the OsHCI1 transcript was
368
highly induced by basal heat treatment (45°C for 24 h) and its transcripts level was
369
downregulated when seedlings were recovered at 24°C for 2 h. For acquired heat shock
370
treatment, OsHCI1 was slightly induced by mild heat treatment at 38°C for 90 min and down
14
371
regulated at 24°C for 2 h. The OsHCI1 transcript was highly accumulated by re-heat shock
372
treatment at 45°C for 24 h and downregulated by 24°C for 2 h.
373
We next evaluated the expression patterns of the six interacting partner genes under both heat
374
shock conditions (Fig. 3). OsPSA7 and Os14-3-3 transcript levels were stable under both heat
375
stress conditions. In contrast, OsPGLU1, OsbHLH065, OsGRP1, and OsPOX1 showed
376
strikingly decreased transcript levels at 45°C during the basal and acquired heat treatments. In
377
addition, both genes, OsPGLU1 and OsbHLH065, displayed a slight decrease at 38°C.
378
Expression patterns of OsPGLU1, OsbHLH065, and OsGRP1 were likely to have a reverse
379
correlation with that of OsHCI1. Three genes, OsPGLU1, OsbHLH065, and OsGRP1, were
380
downregulated at 45°C during basal and acquired heat treatments; their transcript levels were
381
upregulated following the recovery period at 24°C. These results suggest that heat shock results
382
in high expression of the OsHCI1 transcript or protein, which can affect the transcript levels of
383
its interacting genes.
384
We questioned the subcellular localization of each OsHCI1 interaction partner that showed
385
dynamic subcellular localization. The six interaction partners were tagged with DsRed2, and
386
each construct was transiently expressed together with p19 in tobacco leaves. A series of
387
DsRed2 signal z-stack images were captured and merged after 5 days of agro-infiltration. As
388
shown in Fig. 4, DsRed2 fluorescent signals of the OsPGLU1, OsbHLH065, and OsGRP1
389
proteins were only associated with the nucleus, whereas OsPSA7-DsRed2 was found in both the
390
cytoplasm and the nucleus. In contrast, OsPOX1-DsRed2 was observed in a punctuate/dot form
391
pattern (Fig. 4E), and Os14-3-3-DsRed2 was localized to the cytoplasm and cytoskeleton (Fig.
392
5F).
393
394
Subcellular localization of the complex of OsHCI1 and each interacting protein
395
We employed BiFC technology to visualize the interactions between OsHCI1 and each of the
396
interaction partners in living cells (Waadt and Kudla, 2008). Full-length coding sequences of
397
OsHCI1 and each of the six interacting protein genes were cloned into the 35S-HA-SPYCE(M)
398
and 35S-c-myc-SPYNE(R)173 vectors, respectively. After 5 days of agro-infiltration, we
399
observed yellow fluorescent protein (YFP) signals of all BiFC complex formations in tobacco
400
cells. All of the YFP signals except that of OsPSA7 appeared to associate with the cytoplasm
401
and nucleus (Fig. 5); however, the OsPGLU1-, OsbHLH065-, and OsGRP1-DsRed alone
402
protein signals were detected only in the nucleus (Fig. 4B-D). In contrast, the OsHCI1 BiFC
403
complex with OsPSA7 was localized to the cytoplasm with a punctuate complex (Fig. 5A).
15
404
405
OsHCI1 functions as an E3 ligase and mediates ubiquitination of interacting proteins
406
The OsHCI1 gene encoded a 246-amino acid protein with a predicted molecular mass of 28.8
407
kDa and harbored a single RING-HC domain in its C-terminal region (Supplementary Fig. S5).
408
It is generally believed that many proteins harboring the RING-HC domain function are Ub E3
409
ligases (Stone et al., 2005). We performed an in vitro ubiquitination assay to test whether the
410
OsHCI1 protein has E3 Ub ligase activity. A purified MBP-OsHCI1 fusion protein was mixed
411
with ubiquitin, ATP, yeast E1 activating enzyme, and Arabidopsis E2 conjugating enzymes
412
(AtUBC10 and AtUBC11) and then incubated at 30°C for 3 h. An immunoblot analysis with
413
anti-Ub showed that ubiquitinated proteins were detected in the presence of all of these
414
components (Fig. 6A). Furthermore, we observed clearer ubiquitinated proteins in the presence
415
of the AtUBC10 enzyme but not AtUBC11 (Fig. 6A, lanes 6 and 7). In time-course experiments,
416
MBP-OsHCI1 began to cause high molecular mass ubiquitinated ladders after 30 min that
417
gradually reached their highest level after at 2 h incubation (Fig. 6B). However, no ubiquitinated
418
ladders were found at 0 h. These results suggest that the OsHCI1 protein possesses E3 ligase
419
activity in the presence of E1 and E2 enzymes.
420
We fused the six interaction proteins with His and Trx tags to determine whether OsHCI1
421
mediated ubiquitination of the six interacting proteins. The recombinant fusion proteins were
422
expressed in the BL21 (DE3) pLysS E. coli strain. However, His and Trx tagged OsPSA7 and
423
Os14-3-3 fusion proteins were not expressed well in this E. coli system. Therefore, we
424
conducted an in vitro ubiquitination assay with OsPGLU1, OsbHLH065, OsGRP1, and
425
OsPOX1 as substrates. In the presence of E1, E2, and MBP-OsHCI1 as E3 ligases, an additional
426
higher molecular weight band was detected by anti-Trx immunoblot analysis (Fig. 6C-F).
427
Interestingly, nuclear-localized OsPGLU1, OsbHLH065, and OsGRP1 proteins had one
428
additional ubiquitin monomer, whereas the OsPOX1 protein had poly-ubiquitinated chains on
429
the original fusion protein bands. Collectively, the OsHCI1 protein was a functional E3 ligase
430
and, mediated multiple substrate mono- and polyubiquitination.
431
OsHCI1 translocates nuclear substrate proteins into the cytoplasm
432
The findings that the Golgi-localized OsHCI1 protein relocated to the nucleus along the
433
cytoskeleton under heat shock and that it mediated monoubiquitination of each of the three
434
nuclear-localized substrates in an in vivo ubiquitination assay led to the hypothesis that OsHCI1
435
E3 translocates its substrate proteins for heat-stress regulation. To test this hypothesis, we first
16
436
investigated whether nuclear-localized OsPGLU1, OsbHLH065, and OsGRP1 could be
437
relocated by themselves under a heat shock condition in tobacco leaves. Nuclear localization of
438
the OsPGLU1-, OsbHLH065-, and OsGRP1-DsRed2 signals was not significantly different
439
between normal and heat shock conditions (Fig. 4; Supplementary Fig. S6). Next, a single
440
amino acid substitution (OsHCI1C172A) in the RING domain of OsHCI1 was generated to obtain
441
a non-functional RING E3 ligase. MBP-OsHCI1C172A did not show self-ubiquitination activity
442
in vitro (Supplementary Fig. S7). In addition, subcellular localization of OsHCI1 C172A –EYFP
443
was highly similar to that of wild-type OsHCI1-EYFP in tobacco leaves under normal and heat
444
shock conditions (Supplementary Fig. S8). We transiently co-expressed combinations of each of
445
the OsHCI1-EYFP, OsHCI1C172A-EYFP, and empty-EYFP constructs with OsbHLH065-DsRed2
446
in tobacco leaves. The fluorescence signal of OsbHLH065-DsRed2 was detected in the
447
cytoplasm and in the nucleus when co-expressed with OsHCI1-EFYP under heat shock and
448
normal conditions (Fig. 7; Supplementary Fig. S9). In contrast, no alterations in their subcellular
449
localizations were observed when the OsHCI1C172A-EYFP or empty-EYFP construct was co-
450
expressed (Fig. 7B, C). Furthermore, co-expression of OsPGLU1- and OsGRP1-DsRed2 with
451
OsHCI1-EYFP showed the same patterns of fluorescence signals as OsbHLH065-DsRed2 in the
452
cytoplasm and in the nucleus (Supplementary Fig. S10).
453
454
We questioned whether regulation of dynamic translocation under heat shock misleads
455
through heterogeneous expression. In an effort to verify the mechanism in rice cells the nuclear-
456
localized OsbHLH065-DsRed2 was transformed in rice protoplasts. The fluorescent signal of
457
OsbHLH065-DsRed2 was associated with the nucleus under normal conditions (Fig. 8A).
458
However, the signal displayed both the nucleus and cytoplasm as punctuate formations under
459
heat shock (Fig. 8B). Subsequently, OsbHLH065-DsRed2 and OsHCI1-EYFP were
460
cotransformed into rice protoplasts and then by heat shock. The OsHCI1-EYFP fluorescence
461
clearly moved from the cytoplasm to the nucleus, whereas the OsbHLH065-DsRed2 signal was
462
displayed in both the nucleus and cytoplasm (Fig. 8C).
463
OsHCI1-overexpressing Arabidopsis enhances heat shock tolerance
464
The distinct induction of OsHCI1 expression by temperature extremes and the dynamics of its
465
subcellular translocation under heat treatment conditions suggest a crucial role of the gene in
466
thermotolerance. To test this possibility, several independent Arabidopsis transgenic lines (T3)
17
467
were developed with strong OsHCI1 gene expression and compared to plants without the gene
468
(35S:EYFP), which served as controls (Fig. 9A). Plants were tested for basal heat treatment by
469
heating directly to 45°C for 1 h, which resulted in no recovery (0 %) in all tested control lines,
470
whereas transgenic lines showed approximately 4-7 % of survival rates at 5 days after treatment
471
(Fig. 9B). For acquired heat treatment, the plants were subjected to heating to 38°C for 90 min
472
and subsequently cooled for 2 h at room temperature (24°C). After pretreatment, plants were
473
subjected to heating to 45°C for 3 h and then allowed to recover for 5 days at 24°C (Fig. 9B).
474
The OsHCI1-overexpressing lines showed strikingly high survival rates of approximately 55–
475
65 %; however, most control plants did not recover (Fig. 9C).
476
477
Discussion
478
Our findings regarding dynamic movement of OsHCI1 under heat shock, translocations of
479
target proteins co-expressed with OsHCI1, and acquired thermotolerance via heterogeneous
480
overexpression might provide some clues regarding a new molecular mechanism for the heat
481
stress-regulated RING E3 ligase. RING E3 ligases have been recently reported as major players
482
in plant responses to environmental stresses. For example, HOS1 RING E3 ligase is a negative
483
regulator of plant cold responses by mediating degradation of ICE1, which binds the CBF
484
promoter and induces its transcription (Dong et al., 2006), and Rma1H1 RING E3 ligase
485
functions in the downregulation of plasma membrane aquaporin levels as a response to drought
486
stress (Lee et al., 2009). However, the role of RING E3 ligases in the adaptation to heat shock in
487
plants has remained largely unknown.
488
The finding that OsHCI1 gene expression patterns were specifically and somewhat rapidly
489
increased by heat and cold stresses but not by salt and drought stresses indicates that the gene is
490
associated closely with thermal stress in rice (Fig. 1A; Supplementary Fig. S1). Subcellular
491
localization of OsHCI1 was mainly associated with the Golgi apparatus and these punctuate
492
signals rapidly moved to the nucleus under heat shock (Fig. 2). Wild-type OsHCI1-EYFP
493
expression effectively moved its nuclear target substrate proteins to the cytoplasm (Figs. 7, 8;
494
Supplementary Fig. S10) and attachment of the ubiquitin molecule on the nuclear substrates by
495
OsHCI1 fusion protein via in vitro ubiquitination assay might support this translocation of
496
nuclear substrate proteins to the cytoplasm (Fig. 6). In addition, heterogeneous overexpression
497
of OsHCI1 in Arabidopsis resulted in rising survival rates through acquired heat treatment (Fig.
18
498
9). These results suggest that the OsHCI1 E3 ligase might function in the heat shock response in
499
plants.
500
A hypothesis regarding E3 ligase translocation for functional activation might be postulated
501
by several findings. For example, the COP1 RING E3 ligase is localized to the nucleus in the
502
dark but translocates to the cytoplasm under light signals (Deng et al., 2000; von Arnim and
503
Deng, 1994). Similarly, the Arabidopsis HOS1 protein exhibits nucleo-cytoplasmic partitioning
504
in response to cold stimuli (Lee et al., 2001a). Recently, two alternative splicing forms of
505
Arabidopsis XBAT35 RING E3 ligase have been reported that display dual targeting of this E3
506
ligase to the nuclear and cytoplasmic compartment, suggesting a novel player in ethylene-
507
mediated regulation of the apical hook curvature (Carvalho et al., 2012). The OsHCI1 protein,
508
whose localization is confined to the Golgi stack under control conditions, accumulated in the
509
nucleus in response to heat shock (Fig. 2). In addition, OsHCI1 protein interacts with substrate
510
proteins localized in both the nucleus and the cytoplasm and relocates nuclear substrate proteins
511
to the cytoplasm (Figs. 4, 5, 7). These findings suggest that the nucleo-cytoplasmic partitioning
512
of E3 ligases is an extensive regulatory mechanism to control cellular responses to
513
environmental stimuli. However, the OsHCI1 protein can also interact with its substrate proteins
514
and relocates them to the cytoplasm under both normal and heat shock conditions (Fig. 5;
515
Supplementary Fig. S9). It is possible that overexpression of OsHCI1 might lead to interaction
516
with its nuclear proteins under normal conditions. However, further studies are necessary to test
517
this possibility.
518
Plants and other organisms have the intrinsic ability to acquire thermotolerance for survival
519
under lethally high temperatures. It is generally known that the ability accelerate transcription
520
and translation of HSPs and decrease normal protein synthesis (Barnabas et al., 2008; Vierling,
521
1991). Thus, translational modifications of transcription factors might be necessary to decrease
522
synthesis of normal proteins under heat shock. A number of studies regarding the transcriptional
523
regulation of targets via post-translational modification of transcript factors, such as ABI3, ABI5,
524
DREB2A, and ICE1 by E3 ligases and 26 proteasomes have been reported (Dong et al., 2006;
525
Qin et al., 2008; Stone et al., 2006; Zhang et al., 2005). Similarly, we provide evidence
526
supporting that OsHCI1 interacts with multiple substrates including the OsbHLH065
527
transcription factor with a basic helix-loop-helix transcription factor, which is highly
528
downregulated by heat shock treatment (Fig. 3). Interestingly, we found that the transcription
529
levels of three nuclear-targeted partner genes displayed a significant decrease following
530
overexpression of OsHCI1 in rice protoplasts (Supplementary Fig. S11). These results led to the
19
531
hypothesis that OsHCI1 plays a crucial role in the thermotolerance mechanism via post-
532
translational modifications. Significant future work on target protein degradation by OsHCI1 via
533
the 26S proteasome is warranted.
534
A large body of evidence demonstrates the role of E3 ligase in the differential control of
535
mono- vs. polyubiquitination of target proteins. For example, ubiquitination by Mdm2, an
536
oncogenic E3 ligase, causes two alternative p53 fates depending on Mdm2 levels. When Mdm2
537
levels are high, Mdm2 drives p53 degradation via polyubiquitination, whereas low levels
538
promote p53 nuclear exclusion via monoubiquitination (Li et al., 2003). Human Nedd4-1, an E3
539
ligase, catalyzes mono-ubiquitination of hDCNL1, which drives its nuclear export (Wu et al.,
540
2011). We observed that OsHCI1 drives two different ubiquitination types depending on the
541
target proteins (Fig. 6). In addition, co-expression of each of three nuclear-localized proteins
542
and wild-type OsHCI1 promoted nuclear export of target proteins to the cytoplasm, while non-
543
functional OsHCI1C172A did not affect (Fig. 7). Collectively, our findings suggest that OsHCI1
544
may mediate a nuclear-cytoplasmic translocation of nuclear target substrates via
545
monoubiquitination, demonstrating an inactivation device of nuclear proteins in this
546
compartment under heat shock (Li et al., 2003). An alternative hypothesis is that translocation of
547
the target proteins drives another cellular program to mediate thermotolerance mechanisms in
548
plant cells (Mihara et al., 2003). However, much work is needed to rule out this hypothesis.
549
Why OsHCI1 drives different ubiquitination processes depending on the target protein
550
localization is a mystery. A simple hypothesis may be that different fates of the target proteins
551
exist under heat shock.
552
The finding that heterogenous OsHCI1 overexpression in Arabidopsis enhances heat shock
553
tolerance suggests that this gene is involved in acquried thermotolerance (Fig. 9). An
554
outstanding report suggested that the protection mechanism against heat-induced oxidative
555
damage involves phytohormones such as ABA, SA, and ethylene in Arabidopsis (Larkindale
556
and Knight, 2002). As shown Fig. 1B, phytohormone treatment, i.e. ABA, which causes a rapid
557
increase in OsHCI1 transcripts, suggests that the gene is related to the ABA-dependent pathway
558
involved in temperature stress responses (Yamaguchi-Shinozaki and Shinozaki, 2006).
559
Furthermore, induction of OsHCI1 by SA and ethylene treatments might be consistent with the
560
previously reported relationship among SA, ethylene, and thermotolerance (Dat et al., 1998;
561
Wang and Li, 2006). We tested whether the OsHCI1 gene is related to the ABA-dependent
562
pathway involved in acquired thermotolerance. However, constitutive expression of OsHCI1 did
563
not confer sensitivity or insensitivity to ABA during seed germination, cotyledon greening, or
20
564
root growth (Supplementary Fig. S12), suggesting that the OsHCI1 E3 Ub ligase is involved in
565
the ubiquitination of unidentified proteins, which might function in the heat response in
566
transgenic Arabidopsis plants in a ABA-independent manner.
567
In this study, we demonstrated the specific expression patterns of the OsHCI1 transcript and
568
dynamic movement of OsHCI1-EYFP under normal and heat shock conditions. In addition,
569
OsHCI1 functions as an E3 ligase that mediated ubiquitination of substrate proteins in vitro.
570
OsHCI1-overexpressing Arabidopsis showed higher tolerance than control plants under heat
571
shock conditions. These results demonstrate that accumulation of the OsHCI1 RING E3 ligase
572
by heat shock mediates nuclear-cytoplasmic trafficking of nuclear substrate proteins via
573
monoubiquitination to improve heat tolerance as an inactivation mechanism. Our results are an
574
excellent example of the post-translational regulation of the heat tolerance mechanism via the
575
Ub/26S proteasome system in plant cells.
576
Supplementary Data
577
Supplementary Table S1. Primer list
578
Supplementary Fig. S1. Quantitative real-time PCR analysis of OsHCI1 gene in 2-weeks-old
579
rice plants subjected to heat (45°C), cold (4°C), NaCl (250 mM), and dehydration. 18S-rRNA
580
was used as an internal control for normalization. Values are presented as means ± standard
581
deviations.
582
Supplementary Fig. S2. Subcellular localization of the OsHCI1-EYFP fusion protein. The
583
Golgi stacks of OsHCI1-EYFP are highly mobile and are associated with the endoplasmic
584
reticulum (ER) network. A, The OsHCI1-EYFP fluorescent signal was detected on both sides of
585
the Golgi and reticulate ER network in some cells. The OsHCI1-EYFP construct was transiently
586
expressed with p19 in tobacco leaves for 5 days, and images were captured by confocal
587
microscopy. Arrow heads indicate the position of the nucleus. B, Time-lapse confocal images of
588
OsHCI1-EYFP signals. Arrow heads indicate movement of the OsHCI1-EYFP punctate spot
589
signals. C, Quantification of OsHCI1-EYFP localization patterns. Transformed tobacco cells
590
were counted base on its localization patterns: the Golgi only pattern and the Golgi plus ER
591
patterns as shown Supplementary Fig. S2A.
592
Supplementary Fig. S3. Positive clones from yeast two-hybrid screening. ‘+’ indicates cell
593
growth on QDO/X/A (synthetic defined medium lacking Ade, His, Leu, and Trp with 40 μg/ml
21
594
X-α-Gal and 70 ng/ml aureobasidin) or density of α-GAL activity (‘+’ weak, ‘++’strong, ‘+++’
595
very strong).
596
Supplementary Fig. S4. Identification of OsHCI1 interaction with six proteins. The full-length
597
OsHCI1 was cloned into pGBKT7, and full-length OsPSA7 (Os01g59600, 20S proteasome
598
subunit
599
(Os04g41570, basic/Helix-Loop-Helix transcription factor), OsGRP1 (Os05g02770, glycine-
600
rich cell wall structural protein), OsPOX1 (Os07g48020, peroxidase), and Os14-3-3
601
(Os11g34450, 14-3-3 protein) were cloned into pGADT7. The combination of indicated
602
constructs was co-transformed into the Y2H Gold yeast strain. Yeast cells were dropped onto
603
DDO and QDO/X/A medium, and grown for 5 d separately to test protein-protein interactions.
604
Supplemented BD-murine p53 with AD-SV40 large T-antigen were used as positive controls
605
(PC). BD-lamin and AD-SV40 large T-antigen combinations were used as negative controls
606
(NC).
607
Supplementary Fig. S5. Sequence analysis of OsHCI1. A, Multiple alignments of OsHCI1
608
homologs from different plant species. The derived amino acid sequence of OsHCI1
609
(Os10g30850) is compared with those of RING proteins from rice (Os10g35670, Os07g46700,
610
and
611
(XP_00.552240.1), Arabidopsis (AT5g01520), and sorghum (XP_002463337.1). Protein
612
homology
613
(http://www.ncbi.nlm.nih.gov/BLAST/). The multiple sequence alignment was processed with
614
ClustalW2 software (http://www.ebi.ac.uk/clustalw/), and results were edited in the GeneDoc
615
program (http://www.nrbsc.org/gfx/genedoc/). The C-terminal RING-HC motifs of OsHCI1 and
616
seven homologs are indicated by a green solid line. B, Sequence comparison of the RING-HC
617
motif of OsHCI1 and other RING proteins. The conserved mtal-ligand residues of cysteine or
618
histidine forming RING-HC motif at the C-terminal end are marked with asterisks. C,
619
Phylogenetic trees were generated using the neighbor joining method in MEGA software
620
version 5.05 (http://megasoftware.net/). Boostrap values were supported from 1,000 replicates.
621
Supplementary Fig. S6. Subcellular localization of nuclear localized OsPGLU-, OsbHLH065-,
622
and OsGRP1-DsRed2 fusion proteins under heat shock. The full-length OsPGLU1 (A),
623
OsbHLH065 (B), and OsGRP1 (C) fusion proteins were transiently expressed with p19 in
624
Nicotiana leaves. Tobacco leaves were incubated at 45°C for 1 h after 5 days of agro-infiltration.
625
Images were captured and merged by z-series optical sections.
α7),
OsPGLU1
Os07g17400),
maize
searches
(Os03g53800,
(Gene
were
Bank
periplasmic
accession
performed
beta-glucosidase),
no.
with
OsbHLH065,
NP_001137047.1),
the
BLASTP
soybean
program
22
626
Supplementary Fig. S7. The ubiquitination reaction contains E1, E2 (Arabidopsis UBC10),
627
MBP-OsCTR1, Ub, and ATP. PolyUb chains were appeared by immunoblotting with a Ub-
628
specific antibody. Wild-type MBP-OsHCI1 and single amino acid changed mutant (MBP-
629
OsHCI1C172A) were incubated in the presence of E1, atUBC10, ATP, and Ub. The changed
630
amino acid residue in the RING finger domain is indicated. Asterisk indicates unspecific band.
631
Supplementary Fig. S8. Subcellular localization of the OsHCI1C172A-EYFP fusion protein.
632
The 35S: OsHCI1C172A-EYFP construct was transiently expressed in tobacco leaves and
633
incubated at 25°C, 38°C, or 45°C for 1 h or 15 min, respectively. Images were captured and
634
merged by single or z-series optical sections. Arrow heads indicate the position of the nucleus.
635
Dotted line outlines the cell shape and nuclear staining was performed with Hoechst 33258.
636
Supplementary Fig. S9. OsHCI1 protein mediates nuclear-cytoplasmic trafficking in tobacco
637
leaves. Full-length OsbHLH065-DsRed2 fusion protein was transiently co-expressed with wild-
638
type OsHCI1-EYFP protein in tobacco leaves at 25°C. Images were captured and merged by z-
639
series optical sections. Nuclear staining was performed with Hoechst 33258. Arrow heads
640
indicate the position of nucleus.
641
Supplementary Fig. S10. OsHCI1 protein mediates nuclear-cytoplasmic trafficking in tobacco
642
leaves. Full-length OsPGLU1- (A) and OsGRP1-DsRed2 fusion proteins (B) were transiently
643
co-expressed with wild-type OsHCI1-EYFP, OsHCI1C172A-EYFP protein, or empty-EYFP in
644
tobacco leaves. Tobacco leaves were incubated at 45°C for 1 h after 5 days of agro-infiltration.
645
Images were captured and merged by z-series optical sections. Nuclear staining was performed
646
with Hoechst 33258. Arrow heads indicate the position of nucleus.
647
648
Supplementary Fig. S11. Expression patterns of interacting protein genes in overexpression
649
OsHCI1-EYFP in rice protoplast. Rice protoplasts were prepared from 2-weeks-old plants
650
transformed with 35S:EYFP (A) and 35S:OsHCI1-EYFP (B). C, RT-PCR analysis of nuclear-
651
localized OsPGLU1, OsbHLH065, and OsGRP1 transcripts in OsHCI1-overexpressed rice
652
protoplasts. Os18S-rRNA was used as an internal control.
653
Supplementary Fig. S12. Phenotype of 35S:EYFP and 35S:OsHCI1-EYFP plants in response
654
to different concentrations of ABA during seed germination and seedling growth. A, For
655
germination rate, three independent lines of Col-0/35S:OsHCI1-EYFP and 35S:EYFP as control
656
plants were grown for 10 d on MS medium without ABA and increasing ABA concentration
23
657
(0.7, 1, and 1.5 μM). Standard error bars are indicated (n = 30). C and D, Root growth assay in
658
plants exposed to different ABA concentrations for 7 d. Seedlings were germinated on MS
659
medium prior to transfer to ABA-supplemented or control plates. Standard error bars are
660
indicated (n = 90).
661
662
Supplementary Movie S1. Dynamic movement of the Golgi-localized OsHCI1-EYFP fusion
663
protein along the actin cytoskeleton. Fluorescence images corresponding to Supplementary Fig.
664
S2B were used to create a time-lapse movie of the expressed 35S:OsHCI1-EYFP construct.
665
Acknowledgements
666
We would like to express special thanks to Dr. Beom-Gi Kim, Rural Development
667
Administration, Suwon, Korea and Prof. Sung Chul Lee, and Dr. Chae Woo Lim, Chung Ang
668
Univ. Korea, for valuable comments and technical support. This work was supported by the
669
Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture,
670
Forestry, and Fisheries (number 308020-05-4-SB050), the National Research Foundation of
671
Korea (NRF) funded by the Ministry of Education, Science, and Technology (NRF-2010-
672
0007088), and 2011 Research Grant From Kangwon National University (number 120110129)
673
to CSJ.
674
Abbreviations
675
RING
676
677
really interesting new gene
ABA
678
679
abscisic acid
JA
680
681
jasmonic acid
SA
682
683
salicylic acid
BiFC
684
685
686
687
bimolecular fluorescence complementation
PEG
polyethylene glycol
DDO/X/A
24
688
synthetic defined (SD) medium lacking Leu and Trp supplemented with 40 μg/ml X-α-
689
Gal and 70 ng/ml aureobasidin A (AbA)
690
691
QDO/X/A
SD medium lacking Ade, His, Leu, and Trp with 40 μg/ml X-α-Gal and 70 ng/ml AbA
692
25
693
References
694
Agrawal GK, Rakwal R, Jwa NS. 2000. Rice (Oryza sativa L.) OsPR1b gene is
695
phytohormonally regulated in close interaction with light signals. Biochem Biophys Res
696
Commun 278, 290-298.
697
Ahuja I, de Vos RCH, Bones AM, Hall RD. 2010. Plant molecular stress responses face
698
climate change. Trends in Plant Science 15, 664-674.
699
Alfonso M, Yruela I, Almarcegui S, Torrado E, Perez MA, Picorel R. 2001. Unusual
700
tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.)
701
Merr. cell cultures deficient in fatty acid desaturation. Planta 212, 573-582.
702
Barnabas B, Jager K, Feher A. 2008. The effect of drought and heat stress on reproductive
703
processes in cereals. Plant Cell Environ 31, 11-38.
704
Carvalho SD, Saraiva R, Maia TM, Abreu IA, Duque P. 2012. XBAT35, a novel Arabidopsis
705
RING E3 ligase exhibiting dual targeting of its splice isoforms, is involved in ethylene-mediated
706
regulation of apical hook curvature. Mol Plant 5, 1295-1309.
707
Chen J, Burke JJ, Velten J, Xin Z. 2006. FtsH11 protease plays a critical role in Arabidopsis
708
thermotolerance. Plant J 48, 73-84.
709
Claes B, Dekeyser R, Villarroel R, Van den Bulcke M, Bauw G, Van Montagu M, Caplan A.
710
1990. Characterization of a rice gene showing organ-specific expression in response to salt
711
stress and drought. Plant Cell 2, 19-27.
712
Dat JF, Foyer CH, Scott IM. 1998. Changes in salicylic acid and antioxidants during induced
713
thermotolerance in mustard seedlings. Plant Physiol 118, 1455-1461.
714
Deng L, Wang C, Spencer E, Yang L, Braun A, You J, Slaughter C, Pickart C, Chen ZJ.
715
2000. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-
716
conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351-361.
717
Dong CH, Agarwal M, Zhang Y, Xie Q, Zhu JK. 2006. The negative regulator of plant cold
718
responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1.
719
Proc Natl Acad Sci U S A 103, 8281-8286.
720
Freemont PS, Hanson IM, Trowsdale J. 1991. A novel cysteine-rich sequence motif. Cell 64,
721
483-484.
722
Hardtke CS, Okamoto H, Stoop-Myer C, Deng XW. 2002. Biochemical evidence for
723
ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8). Plant J 30, 385-
724
394.
725
Hicke L, Dunn R. 2003. Regulation of membrane protein transport by ubiquitin and ubiquitin26
726
binding proteins. Annu Rev Cell Dev Biol 19, 141-172.
727
Hu WH, Hu GC, Han B. 2009. Genome-wide survey and expression profiling of heat shock
728
proteins and heat shock factors revealed overlapped and stress specific response under abiotic
729
stresses in rice. Plant Science 176, 583-590.
730
Iba K. 2002. Acclimative response to temperature stress in higher plants: approaches of gene
731
engineering for temperature tolerance. Annu Rev Plant Biol 53, 225-245.
732
Kaiser P, Flick K, Wittenberg C, Reed SI. 2000. Regulation of transcription by ubiquitination
733
without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4.
734
Cell 102, 303-314.
735
Kampinga HH, Brunsting JF, Stege GJ, Burgman PW, Konings AW. 1995. Thermal protein
736
denaturation and protein aggregation in cells made thermotolerant by various chemicals: role of
737
heat shock proteins. Exp Cell Res 219, 536-546.
738
Kim H, Hwang H, Hong JW, Lee YN, Ahn IP, Yoon IS, Yoo SD, Lee S, Lee SC, Kim BG.
739
2012. A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the
740
ABA signal transduction pathway in seed germination and early seedling growth. J Exp Bot 63,
741
1013-1024.
742
Kurepa J, Walker JM, Smalle J, Gosink MM, Davis SJ, Durham TL, Sung DY, Vierstra
743
RD. 2003. The small ubiquitin-like modifier (SUMO) protein modification system in
744
Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress. J Biol Chem
745
278, 6862-6872.
746
Larkindale J, Hall JD, Knight MR, Vierling E. 2005. Heat stress phenotypes of Arabidopsis
747
mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant
748
Physiol 138, 882-897.
749
Larkindale J, Huang B. 2004. Thermotolerance and antioxidant systems in Agrostis
750
stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and
751
ethylene. J Plant Physiol 161, 405-413.
752
Larkindale J, Knight MR. 2002. Protection against heat stress-induced oxidative damage in
753
Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol 128,
754
682-695.
755
Lee H, Xiong L, Gong Z, Ishitani M, Stevenson B, Zhu JK. 2001a. The Arabidopsis HOS1
756
gene negatively regulates cold signal transduction and encodes a RING finger protein that
757
displays cold-regulated nucleo--cytoplasmic partitioning. Genes Dev 15, 912-924.
758
Lee HK, Cho SK, Son O, Xu ZY, Hwang I, Kim WT. 2009. Drought Stress-Induced Rma1H1,
759
a RING Membrane-Anchor E3 Ubiquitin Ligase Homolog, Regulates Aquaporin Levels via
27
760
Ubiquitination in Transgenic Arabidopsis Plants. Plant Cell 21, 622-641.
761
Lee MW, Qi M, Yang Y. 2001b. A novel jasmonic acid-inducible rice myb gene associates with
762
fungal infection and host cell death. Mol Plant Microbe Interact 14, 527-535.
763
Lee U, Wie C, Fernandez BO, Feelisch M, Vierling E. 2008. Modulation of nitrosative stress
764
by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis.
765
Plant Cell 20, 786-802.
766
Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W. 2003. Mono- versus
767
polyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972-1975.
768
Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z, Guo J, Liang W, Chen L, Yin J, Ma H,
769
Wang J, Zhang D. 2006. Genome-wide analysis of basic/helix-loop-helix transcription factor
770
family in rice and Arabidopsis. Plant Physiol 141, 1167-1184.
771
Lim SD, Yim WC, Moon JC, Kim DS, Lee BM, Jang CS. 2010. A gene family encoding
772
RING finger proteins in rice: their expansion, expression diversity, and co-expressed genes.
773
Plant Molecular Biology 72, 369-380.
774
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time
775
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408.
776
Medina C, Cardemil L. 1993. Prosopis chilensis is a plant highly tolerant to heat shock. Plant,
777
Cell & Environment 16, 305-310.
778
Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, Moll UM. 2003. p53
779
has a direct apoptogenic role at the mitochondria. Mol Cell 11, 577-590.
780
Nelson BK, Cai X, Nebenfuhr A. 2007. A multicolored set of in vivo organelle markers for co-
781
localization studies in Arabidopsis and other plants. Plant Journal 51, 1126-1136.
782
Ortiz C, Cardemil L. 2001. Heat-shock responses in two leguminous plants: a comparative
783
study. J Exp Bot 52, 1711-1719.
784
Peng SB, Huang JL, Sheehy JE, Laza RC, Visperas RM, Zhong XH, Centeno GS, Khush
785
GS, Cassman KG. 2004. Rice yields decline with higher night temperature from global
786
warming. Proceedings of the National Academy of Sciences of the United States of America 101,
787
9971-9975.
788
Perez DE, Hoyer JS, Johnson AI, Moody ZR, Lopez J, Kaplinsky NJ. 2009. BOBBER1 Is a
789
Noncanonical Arabidopsis Small Heat Shock Protein Required for Both Development and
790
Thermotolerance. Plant Physiology 151, 241-252.
791
Pickart CM. 2004. Back to the future with ubiquitin. Cell 116, 181-190.
792
Qin F, Sakuma Y, Tran LSP, Maruyama K, Kidokoro S, Fujita Y, Fujita M, Umezawa T,
793
Sawano Y, Miyazono KI, Tanokura M, Shinozaki K, Yamaguchi-Shinozaki K. 2008.
28
794
Arabidopsis DREB2A-interacting proteins function as RING E3 ligases and negatively regulate
795
plant drought stress-responsive gene expression. Plant Cell 20, 1693-1707.
796
Qin F, Shinozaki K, Yamaguchi-Shinozaki K. 2011. Achievements and challenges in
797
understanding plant abiotic stress responses and tolerance. Plant Cell Physiol 52, 1569-1582.
798
Roos-Mattjus P, Sistonen L. 2004. The ubiquitin-proteasome pathway. Ann Med 36, 285-295.
799
Sarkar NK, Kim YK, Grover A. 2009. Rice sHsp genes: genomic organization and expression
800
profiling under stress and development. BMC Genomics 10, 393.
801
Schramm F, Larkindale J, Kiehlmann E, Ganguli A, Englich G, Vierling E, von Koskull-
802
Doring P. 2008. A cascade of transcription factor DREB2A and heat stress transcription factor
803
HsfA3 regulates the heat stress response of Arabidopsis. Plant J 53, 264-274.
804
Shimizu H, Sato K, Berberich T, Miyazaki A, Ozaki R, Imai R, Kusano T. 2005. LIP19, a
805
basic region leucine zipper protein, is a Fos-like molecular switch in the cold signaling of rice
806
plants. Plant Cell Physiol 46, 1623-1634.
807
Smalle J, Vierstra RD. 2004. The ubiquitin 26S proteasome proteolytic pathway. Annual
808
Review of Plant Biology 55, 555-590.
809
Stone SL, Hauksdottir H, Troy A, Herschleb J, Kraft E, Callis J. 2005. Functional analysis
810
of the RING-type ubiquitin ligase family of Arabidopsis. Plant Physiol 137, 13-30.
811
Stone SL, Williams LA, Farmer LM, Vierstra RD, Callis J. 2006. KEEP ON GOING, a
812
RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid
813
signaling. Plant Cell 18, 3415-3428.
814
Sung DY, Kaplan F, Lee KJ, Guy CL. 2003. Acquired tolerance to temperature extremes.
815
Trends in Plant Science 8, 179-187.
816
Vierling E. 1991. The roles of heat shock proteins in plants. Annu Rev Plant Physiology Plant
817
Mol. Biol. 42, 579-620.
818
Vierstra RD. 2009. The ubiquitin-26S proteasome system at the nexus of plant biology. Nature
819
Reviews Molecular Cell Biology 10, 385-397.
820
von Arnim AG, Deng XW. 1994. Light inactivation of Arabidopsis photomorphogenic
821
repressor COP1 involves a cell-specific regulation of its nucleocytoplasmic partitioning. Cell 79,
822
1035-1045.
823
Waadt R, Kudla J. 2008. In Planta Visualization of Protein Interactions Using Bimolecular
824
Fluorescence Complementation (BiFC). CSH Protoc 2008, pdb prot4995.
825
Waadt R, Schmidt LK, Lohse M, Hashimoto K, Bock R, Kudla J. 2008. Multicolor
826
bimolecular fluorescence complementation reveals simultaneous formation of alternative
827
CBL/CIPK complexes in planta. Plant Journal 56, 505-516.
29
828
Wahid A, Gelani S, Ashraf M, Foolad MR. 2007. Heat tolerance in plants: An overview.
829
Environmental and Experimental Botany 61, 199-223.
830
Wang LJ, Li SH. 2006. Salicylic acid-induced heat or cold tolerance in relation to Ca(2+)
831
homeostasis and antioxidant systems in young grape plants. Plant Science 170, 685-694.
832
Wu G, Xu G, Schulman BA, Jeffrey PD, Harper JW, Pavletich NP. 2003. Structure of a beta-
833
TrCP1-Skp1-beta-catenin complex: destruction motif binding and lysine specificity of the
834
SCF(beta-TrCP1) ubiquitin ligase. Mol Cell 11, 1445-1456.
835
Wu K, Yan H, Fang L, Wang X, Pfleger C, Jiang X, Huang L, Pan ZQ. 2011. Mono-
836
ubiquitination drives nuclear export of the human DCN1-like protein hDCNL1. Journal of
837
Biological Chemistry 286, 34060-34070.
838
Wuriyanghan H, Zhang B, Cao WH, Ma B, Lei G, Liu YF, Wei W, Wu HJ, Chen LJ, Chen
839
HW, Cao YR, He SJ, Zhang WK, Wang XJ, Chen SY, Zhang JS. 2009. The ethylene
840
receptor ETR2 delays floral transition and affects starch accumulation in rice. Plant Cell 21,
841
1473-1494.
842
Xiang Y, Tang N, Du H, Ye H, Xiong L. 2008. Characterization of OsbZIP23 as a key player
843
of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and
844
salinity and drought tolerance in rice. Plant Physiol 148, 1938-1952.
845
Yamaguchi-Shinozaki K, Shinozaki K. 2006. Transcriptional regulatory networks in cellular
846
responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57, 781-803.
847
Zhang X, Garreton V, Chua NH. 2005. The AIP2 E3 ligase acts as a novel negative regulator
848
of ABA signaling by promoting ABI3 degradation. Genes Dev 19, 1532-1543.
849
Zhang XR, Henriques R, Lin SS, Niu QW, Chua NH. 2006. Agrobacterium-mediated
850
transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols 1, 641-646.
851
Zhang YY, Yang CW, Li Y, Zheng NY, Chen H, Zhao QZ, Gao T, Guo HS, Xie Q. 2007.
852
SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid
853
signaling in Arabidopsis. Plant Cell 19, 1912-1929.
854
855
856
30
857
Figure legends
858
Fig. 1. Expression level of OsHCl1 gene in rice plants subjected to four abiotic stresses and four
859
hormonal treatments. Rice seedlings were subjected to abiotic stresses and plant hormone
860
treatments. A, heat (45°C), cold (4°C), NaCl (250 mM), and dehydration (dehydration on two
861
pieces of tissues paper). OsHsp90-1, LIP19, OsSalT, and OsbZIP23 were used as reliable stress-
862
inducible genes for each abiotic stress treatment, respectively. B, 0.1 mM abscisic acid (ABA),
863
0.1 mM jasmonic acid (JA), 1 mM salicylic acid (SA), 50 μL L-1 ethylene (Ethyl). OsSalT,
864
OsPBZ1, OsPR1b, and OsERF3, were used as reliable genes for each hormonal treatment,
865
respectively. C and T indicate untreated control and stress-treated samples, respectively. The
866
experiments were performed with three biological replicates.
867
Fig. 2. Subcellular localization of the OsHCI1-EYFP fusion protein. Each construct was
868
transiently expressed in tobacco leaves and rice protoplasts. Images were captured and merged
869
by single or z-series optical sections. Arrow heads indicate the position of the nucleus.
870
Transiently expressed 35S:EYFP and 35S:OsHCI1-EYFP fusion protein were expressed in
871
tobacco leaves and incubated 25°C (A), 38°C (B) or 45°C (C) for 1 h. Nuclear staining was
872
performed with Hoechst 33258. D, Co-localization of OsHCI1-EYFP and the G-rk-mCherry-
873
Golgi marker in tobacco leaves. E, Transiently expressed 35S:EYFP and 35S:OsHCI1-EYFP
874
fusion protein were expressed in rice protoplasts and incubated 25°C, 38°C or 45°C for 15 min.
875
The green and red colors represent EYFP and chlorophyll autofluorescent signals, respectively.
876
Arrows indicate punctuate spots of OsHCI1-EYFP fluorescence. Dotted line outlines the cell
877
shape. F, Quantification of OsHCI1-EGFP localization patterns under different temperatures.
878
Protoplasts were counted based on their localization patterns: the Golgi only pattern, the nucleus
879
(NC) only pattern, and the Golgi plus NC patterns. Golgi-localized sixty rice protoplasts were
880
counted at different temperature.
881
Fig. 3. Expression patterns of the response of interacting protein genes with OsHCI1 under heat
882
treatment. Two-weeks-old rice seedlings were exposed to basal (A) or acquired heat stress (B)
883
and then placed to normal temperature for 2 h. Each leaf sample was harvested at different time
884
points.
885
Fig. 4. Subcellular localization of six interacting proteins. The full-length OsPSA7 (A),
886
OsPGLU1 (B), OsbHLH065 (C), OsGRP1 (D), OsPOX1 (E), Os14-3-3 (F), and empty EYFP
887
(G) were tagged with DsRed2 and transiently expressed with p19 in Nicotiana leaves. Images
31
888
were captured and merged by z-series optical sections after 5 days of agro-infiltration.
889
Fig. 5. BiFC assay for six substrate proteins confirms the interaction with OsHCI1 in living
890
cells. Full-length OsPSA7 (A), OsPGLU1 (B), OsbHLH065 (C), OsGRP1 (D), OsPOX1 (E),
891
and Os14-3-3 (F) were cloned into pSPYNE(R) and OsHCI1 was cloned into pSPYCE(M).
892
Combinations of each construct and SPYNE(R):empty (G, negative control) with
893
OsHCI1:SPYCE(M) were transiently expressed with p19 in Nicotiana leaves. Images were
894
captured and merged by z-series optical sections after 5 days of agro-infiltration.
895
Fig. 6. OsHCI1 functions as an E3 ubiquitin ligase and mediates OsPGLU1, OsbHLH065,
896
OsGRP1, and OsPOX1 protein ubiquitination in vitro. A, E3 ligase activity of OsHCI1 in vitro.
897
Maltose binding protein tagged OsHCI1 fusion protein was assayed for E3 ligase activity in the
898
presence of yeast E1, Arabidopsis E2s (AtUBC10 and AtUBC11), and Ub. B, MBP-OsHCI1
899
was incubated for the indicated time periods in the presence of yeast E1, E2 (AtUBC10), ATP,
900
and Ub. Ubiquitinated proteins were detected by immunoblot analysis using an anti-Ub
901
antibody. OsHCI1 mediates the ubiquitination of OsGLU1 (C), OsbHLH065 (D), OsGRP1 (E),
902
and OsPOX1 (F) proteins. The full-length OsGLU1, OsbHLH065, OsGRP1, and OsPOX1 genes
903
were cloned into His and Trx tags pET-32a (+) vector (Novagen) and these purified fusion
904
proteins were used as the substrate for the assay. Anti-Trx was used in the immunoblot analysis
905
for detecting His-Trx-tagged substrate proteins.
906
Fig. 7. OsHCI1 protein mediates nuclear-cytoplasmic trafficking in tobacco leaves. Full-length
907
OsbHLH065-DsRed2 fusion proteins were transiently co-expressed with wild-type OsHCI1-
908
EYFP protein (A), OsHCI1C172A-EYFP protein (B), or empty-EYFP construct (C) in tobacco
909
leaves. Tobacco leaves were incubated at 45°C for 1 h after 5 days of agro-infiltration. Images
910
were captured and merged by z-series optical sections. Nuclear staining was performed with
911
Hoechst 33258. Arrow heads indicate the position of nucleus.
912
Fig. 8. OsHCI1 protein mediates nuclear-cytoplasmic trafficking of the OsbHLH065
913
transcription factor in rice protoplasts. Full-length OsbHLH065-DsRed2 was transfected into
914
rice protoplasts (A). Transformed protoplasts were incubated at 45°C for 15 min (B). Full-length
915
OsbHLH065-DsRed2 was transfected with OsHCI1-EYFP into the rice protoplast and the
916
transformed protoplasts were incubated at 45°C for 15 min (C). Nuclear staining was performed
917
with Hoechst 33258. Arrows indicate the nuclear exported OsbHLH065.
32
918
Fig. 9. Thermotolerance phenotype of 35S:OsHCI1-EYFP. Seven-day-old seedlings of
919
35S:EYFP(EV) and 35S:OsHCI1-EYFP T3 transgenic plants (three independent lines) were
920
grown on agar plates in the light for 7d and heated to 38°C for 90 min, cooled 24°C for 2 h, then
921
heated to 45°C for 3 h (acquired thermotolerance) or heated to 45°C for 60 min (basal
922
thermotolerance). A, RT-PCR analysis of seven independent Col-0/35S:OsHCI1 T3 transgenic
923
plants, control wild-type (WT), and empty vector (EV). B, The phenotypes of the control (EV)
924
and three independent OsHCI1-overexpressed plants were treated at various high temperatures.
925
Images were captured 5 days after heat shock treatment. C, Percentage of surviving plants
926
relative to the control (EV) on the same plate was determined 5 d after heat shock. Error bars
927
represent ± standard deviation (n = 30) from the average value over all experiments. The
928
experiments were performed with four biological replicates.
929
33