A Negative Regulator in Response to Salinity in Rice: Oryza sativa Salt-, ABA- and Drought-Induced RING Finger Protein 1 (OsSADR1)

A Negative Regulator in Response to Salinity in Rice: Oryza sativa Salt-, ABA- and... Abstract RING (Really Interesting New Gene) finger proteins play crucial roles in abiotic stress responses in plants. We report the RING finger E3 ligase gene, an Oryza sativa salt, ABA and drought stress-induced RING finger protein 1 gene (OsSADR1). We demonstrated that although OsSAR1 possesses E3 ligase activity, a single amino acid substitution (OsSADR1C168A) in the RING domain resulted in no E3 ligase activity, suggesting that the activity of most E3s is specified by the RING domain. Additional assays substantiated that OsSADR1 interacts with three substrates—no E3 ligase acti and OsPIRIN, and mediates their proteolysis via the 26S proteasome pathway. For OsSADR1, approximately 62% of the transient signals were in the cytosol and 38% in the nucleus. However, transiently expressed OsSADR1 was primarily expressed in the nucleus (70%) in 200 mM salt-treated rice protoplasts. The two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSADR1 in the cytosol and nucleus. Heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitive phenotypes for salt- and mannitol-responsive seed germination and seedling growth. With ABA, OsSADR1 overexpression in plants produced highly tolerant phenotypes, with morphological changes in root length and stomatal closure. The ABA-tolerant transgenic plants also showed hypersensitivity phenotypes under severe water deficit conditions. Taken together, OsSADR1 may act as a regulator in abiotic stress responses by modulating target protein levels. Introduction Abiotic stresses, such as drought, cold, high salinity, extreme temperatures and heavy metal accumulation, severely reduce the productivity of food crops worldwide by altering plant physiological and biochemical processes (Mickelbart 2015, Zhu 2016). Because of their sessile nature, plants have coped with these stresses through the evolution of defense mechanisms which allow them to adapt and survive under stressful environmental conditions. Recently, drought, salinity and extreme temperature have been major stresses that challenge adequate global food production. Rice (Oryza sativa) is known as a major staple food for more than half of the world’s population, and is also considered a model plant for cereal genomics; however, it can be severely injured by abiotic stresses (Lafitte et al. 2004). In particular, rice is highly sensitive to salt stress during the young seedling and reproductive stages (Reddy et al. 2017). Rice is also known as a semi-aquatic crop, and as such it is more susceptible to drought than other cereals. Drought stress affects every stage of rice growth; in particular, seedlings, vegetative stages and anthesis can be highly affected and stress can reduce grain yield (Basu et al. 2016). The salt stress tolerance mechanism may be categorized into three different aspects: osmotic stress tolerance, Na+ exclusion from the shoot and Na+ tissue tolerance (Munns and Tester 2008). The drought resistance mechanism of plants is also categorized into four types: drought tolerance, drought escape, drought avoidance and drought recovery. To evaluate the drought resistance of plants, different drought-related indicators such as leaf traits, root traits, water potential, ABA content and osmotic adjustment capabilities have been used (Fang and Xiong 2015). Both salt and drought create osmotic stress in plant cells. Because of this unique and overlapping hyperosmotic signal, ABA is accumulated and acts as a stress defensive mechanism under unfavorable conditions (Finkelstein et al. 2002, Zhu 2002). During osmotic stress, ABA plays an important role by reducing transpirational water loss through stomatal closure (Zhu 2002, Wang and Song 2008). It is closely related to reactive oxygen species, such as hydrogen peroxide (H2O2), leading to stomatal closure by increasing the concentration of ABA in guard cells (Zang et al. 2001). Ubiquitin (Ub) is a highly conserved, universally expressed and stable protein, and its ubiquitination acts as a post-transitional modification, which mediates growth and development of eukaryotic species by regulating transcriptional changes needed for abiotic stress adaption. In higher plants, Ub-mediated substrate degradation plays an important role in growth, hormonal signaling and abiotic stress responses; however, the process of ubiquitination is very complex where enzymes, namely E1 (Ub activating enzyme), E2 (Ub conjugating enzyme and E3 Ub ligases, are typically required (Viestra 2009). Among them, RING Ub E3 ligases play vital roles in the post-transitional modification of proteins via attachment of Ub (Deshaies and Joazeiro 2009). One of the E3 ligases is the Really Interesting New Gene (RING) finger protein, which consists of cysteine (Cys) and histidine (His) residues (in the order Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-Cys/His-X2-Cys-X4-48-Cys-X2-Cys, where X may be any amino acid) that bind two zinc atoms. The clear relationship between abiotic stress and ubiquitination remains unknown; however, many high salinity-induced rice proteins were regulated by ubiquitination, including salt-induced RING finger protein OsSIRP1, which negatively regulates germination and root growth in Arabidopsis (Hwang et al. 2016), microtubule-associated rice RING finger protein OsRMT1, which positively regulates salt stress (Lim et al. 2015), and OsRINGC2-1 and OsRING2-2, which enhance salt tolerance in Arabidopsis (Jung et al. 2012). Likewise, several drought-induced rice genes have been identified, which effect physiological and molecular changes via ubiquitination. For example, salt- and drought-induced OsSDIR1 regulates ABA signaling and induced drought stress tolerance in rice (Gao et al. 2011). Similarly, OsDIS1 acts negatively on drought stress tolerance by post-transitional regulation of OsNek6 (Ser/Thr protein kinase) in rice (Ning et al. 2011). In addition, chloroplast targeting rice E3 ligase OsCTR1 plays a positive role in drought tolerance in an ABA-responsive manner through post-transitional modification (Lim et al. 2015). Besides these, other genes have been identified which are involved in multiple abiotic stresses, such as the rice RING E3 ligase OsDSG1 (Delayed Seed Germination 1) with its mutant osdsg1 and gene silencing by RNA interference (RNAi) that improves germination under high salinity and drought, mediated through enhanced ABA-regulated responses (Park et al. 2010). Another is OsSRF1 (stress-related RING finger protein 1), which negatively regulates salt, cold and oxidative stress (Fang et al. 2015). Moreover, OsPUB15 (Plant U-box 15) prevents tissue damage and protects against osmotic stress during germination and growth of rice plants (Park et al. 2011). Previously, >100 NAC transcription factors, which consist of NAM (non-apical meristem), ATAF1-2 (Arabidopsis transcription activation factor) and CUC2 (cup-shaped cotyledon), have been identified as the largest important families of transcriptional regulators for various stress responses and development in Arabidopsis and rice (Nakashima et al. 2012, Nuruzzaman et al. 2013, Lv et al. 2016). For example, rice OsNAC2 is a negative regulator of high salinity- and drought-mediated stress by regulating LATE EMBRYOGENESIS ABUNDANT 3 (LEA3) and stress-activated protein kinase 1 (OsSAPK1) in rice (Shen et al. 2017). Stress-responsive rice SNAC genes, OsNAC2/6 and OsNAC10, can improve drought and salt tolerance when overexpressed (Nakashima et al. 2009, Jeong et al. 2010). Another Arabidopsis NAC gene, ANAC092, showed salt-promoted senescence and seed maturation under ANAC092-mediated gene regulatory networks (Balazadeh et al. 2010). Another homologous group of genes, OsNAC5/OsNAC009/OsNAC071 and OsNAC6, is induced by drought, high salinity and ABA stress, and then enhances stress tolerance by up-regulating the expression of stress-inducible rice genes (Takasaki et al. 2010). It is also known that the environmental stress-related NAC group, SNAC, can bind to the NACR (NAC recognition sequence; CACG core) (Thao et al. 2013, Nakashima et al. 2014). Arabidopsis SNAC genes, such as RD26 and ATAF1, and rice SNAC genes, such as SNAC1, OsNAC6/SNAC2 and OsNAC5, can improve drought and/or high salt stress tolerance when overexpressed (Tran et al. 2004, Hu et al. 2006, Takasaki et al. 2010, Nakashima et al. 2014). Herein, we suggest the molecular function of the O. sativa salt-, ABA- and drought-induced RING finger protein 1 (OsSADR1) gene, which is highly induced by salt, ABA and drought stress. Its subcellular localizations were the cytosol and nucleus in rice protoplasts, with different localization frequencies between normal and salt-stressed samples. The yeast two-hybrid (Y2H) system, bimolecular fluorescence (BiFC) assay and in vitro pull-down assay revealed that the OsSADR1 protein primarily interacts with nuclear proteins, such as stress-induced no apical meristem protein 2 (OsSNAC2), GRAS family transcription factor domain-containing protein 44 (OsGRAS-44) and one chloroplast-localized putative pirin-like protein (OsPIRIN). Furthermore, the heterogeneous overexpression of OsSADR1 in OsSADR1-overexpressing plants showed sensitivity to salt and drought, and hyposensitivity to ABA treatment. Results Expression pattern and sequence analysis of OsSADR1 In a previous study, expression levels of 44 rice RING finger protein (OsRFP) genes, which were randomly selected based on domain analysis, were examined under salt stress (Hwang et al. 2016). We further examined the expression patterns of 44 rice RING finger proteins in response to drought stress in 14-day-old root samples. In particular, transcription levels of one gene (LOC_Os11g07450, called OsSADR1) was highly induced from 1 h until 12 h (Supplementary Fig. S1). Therefore, this gene was selected for further study. In addition, transcript levels of OsSADR1 were identified via quantitative real-time PCR (qRT-PCR) during a different time course (0, 1, 3, 6, 12 and 24 h) (Fig. 1a). The gene was up-regulated from 1 h until 24 h (up approximately 25-fold) after the salt stress treatment. Under drought stress, the transcript levels of OsSADR1 were increased at 6 h and then decreased at 12 and 24 h. Interestingly, OsSADR1 was highly induced in root samples treated with 0.1 mM ABA 6 h. In addition, each of the genes known to be induced reliably by salt (OsNAC10 and OsSalt), drought and ABA (OsLEA3 and OsRAB16A) showed transcript increases according to the increasing time course of treatments, supporting the postulate that samples suffered from the treatment because of each of the stressors (Supplementary Fig. S2). Fig. 1 View largeDownload slide Expression pattern of the OsSADR1 gene and amino acid sequence analysis of its product. (a) qRT-PCR analysis of the OsSADR1 gene in salt- (200 mM), ABA- (1 μM) and drought-treated 2-week-old rice seedlings. As compared with controls (0 h), asterisks represent the statistically significant differences according to a two-tailed Student’s t-test; *P < 0.05, **P < 0.01. (b) Multiple alignment of OsSADR1 and other orthologs of RING-HC-type proteins, i.e. At1G47670 from Arabidopsis, GRMZM2G000014 from maize andBradi4G24480 from Brachypodium. The alignment was performed using Clustal2 software (http://ebi.ac.uk/clustalw/). NLS, nuclear localization signal. Fig. 1 View largeDownload slide Expression pattern of the OsSADR1 gene and amino acid sequence analysis of its product. (a) qRT-PCR analysis of the OsSADR1 gene in salt- (200 mM), ABA- (1 μM) and drought-treated 2-week-old rice seedlings. As compared with controls (0 h), asterisks represent the statistically significant differences according to a two-tailed Student’s t-test; *P < 0.05, **P < 0.01. (b) Multiple alignment of OsSADR1 and other orthologs of RING-HC-type proteins, i.e. At1G47670 from Arabidopsis, GRMZM2G000014 from maize andBradi4G24480 from Brachypodium. The alignment was performed using Clustal2 software (http://ebi.ac.uk/clustalw/). NLS, nuclear localization signal. OsSADR1 encodes a protein of 479 amino acids and SADR1 contains the RING-HC domain at the N- and C-termini with a nuclear localization signal (NLS). We retrieved some orthologs from Arabidopsis (At1G47570), Zea mays (GRMZ2G000014) and Brachypodium distachyon (Bradi4G24480) from The Institute for Genomic Research rice annotation database, and aligned their induced amino acids, finding a highly conserved RING-HC domain (Fig. 1b). However, the NLS sequence was only included at the N-terminal sequence of OsSADR1 and not in other orthologous genes. Collectively, the expression patterns of the OsSADR1 gene exhibited strong induction under salt, ABA and drought exposure in rice roots. Subcellular localization of OsSADR1 with an NLS The domain analysis showed that OsSADR1 contains NLS sequences at the C-terminus, suggesting that the protein might be localized to the nucleus. To examine this hypothesis, the constructed 35S:OsSADR1-enhnanced yellow fluorescent protein (EYFP) vector was transfected into rice protoplasts and transient expression of OsSADR1 showed two significantly different patterns of subcellular localization among protoplasts. We found that approximately 62% of the transient signal was detected in the cytosol and 38% in the nucleus (Fig. 2b, left; Supplementary Fig. S3). Subsequently, we determined whether the frequencies of the subcellular localizations could be changed under abiotic stress, such as salt and/or drought, which induced expression of the gene (Lim et al. 2015). Interestingly, in 200 mM salt-treated rice protoplasts, 30% of transiently expressed OsSADR1 was detected in the cytosol, whereas 70% was in the nucleus (Fig. 2b, right). The findings support the hypothesis of movement of the OsSADR1 protein between the cytosol and nucleus under salt stress. To confirm our hypothesis, transfected rice protoplasts were treated with 200 mM salt stress for 30 min and then expanded via a multiphoton confocal laser scanning microscope (model LSM 780 META; Carl Zeiss). As shown in Fig. 2a and b, the florescent signals of OsSADR1 proteins were detected in both the cytoplasm and nucleus. Interestingly, we found that the cytoplasm aggregations of the OsSADR1 protein had moved into the nucleus (Fig. 2c;Supplementary Movie S1). These findings might provide a clue to understanding the molecular functions of the OsSADR1 proteins in the nucleus under salt stress. Fig. 2 View largeDownload slide Subcellular localization of the OsSADR1 protein in rice protoplasts. (a) Confocal images of 35S:OsSADR1–EYFP protein in rice protoplasts. Nuclear localization patterns (top panel) and cytosol localization patterns (bottom panel of 35S:OsSADR1–EYFP. (b) Percentage of fluorescent signals of the OsSADR1–EYFP protein in each of the non-treated and 200 mM salt-treated rice protoplasts. Each of the three repeated normal and NaCl-treated protoplasts were detected for analysis (n = 50). (c) Time-course of confocal images of OsSADR1–EYFP protein. The images were detected after 15 min of 200 mM salt treatment. The white arrows indicated the movement of OsSADR1–EYFP fusion protein in rice protoplasts. Fig. 2 View largeDownload slide Subcellular localization of the OsSADR1 protein in rice protoplasts. (a) Confocal images of 35S:OsSADR1–EYFP protein in rice protoplasts. Nuclear localization patterns (top panel) and cytosol localization patterns (bottom panel of 35S:OsSADR1–EYFP. (b) Percentage of fluorescent signals of the OsSADR1–EYFP protein in each of the non-treated and 200 mM salt-treated rice protoplasts. Each of the three repeated normal and NaCl-treated protoplasts were detected for analysis (n = 50). (c) Time-course of confocal images of OsSADR1–EYFP protein. The images were detected after 15 min of 200 mM salt treatment. The white arrows indicated the movement of OsSADR1–EYFP fusion protein in rice protoplasts. E3 ligase activity of OsSADR1 with a RING-HC domain The alignment between OsSADR1 and its orthologs showed a well-conserved RING-HC domain (Fig. 1). To determine whether the OsSADR1 protein functions as an E3 Ub ligase, we performed an in vitro ubiquitination assay. Immunoblot analysis with anti-Ub and anti-maltose-binding protein (MBP) showed that a high molecular mass of the poly-Ub chain of MBP-OsSADR1 while incubated with all the components (Fig. 3a, lane 4), whereas no chains were detected in the presence of empty MBP and in the absence of AtUBC10 or human E1 (Fig. 3a, lanes 1–3), respectively. It is believed that the RING domain was constructed by Cys and/or residues based on two zinc ions into an interwoven structure, and then acted as an E3 ligase (Freemont et al. 1991, Deshaies and Joazeiro 2009, Liu and Stone 2010). To confirm that OsSADR1 was assigned E3 ligase activity by the RING domain, a single amino acid substitution MBP–OsSADR1C168A, in which Cys168 was substituted with Ala168, was constructed (Supplementary Fig. S4A). In Fig. 3b, MBP–OsSADR1 shows high molecular mass ubiquitinated ladders, but no poly-Ub chain was observed with OsSADR1C168A using either antibody. Moreover, a time-course ubiquitination assay showed that MBP–OsSADR1 was detected in ubiquitinated ladders after 1 h and gradually reached its highest level at 4 h incubation (Supplementary Fig. S4B). These results showed that OsSADR1 harboring a RING-HC domain acted as an E3 Ub ligase. Fig. 3 View largeDownload slide In vitro ubiquitination assay of OsSADR1 E3 ligase. (a) The ubiquitination reaction contains E1 (human), AtE2 (Arabidopsis UBC10), maltose-binding protein-tagged OsSADR1 (MBP–OsSADR1), Ub and ATP. (b) The ubiquitination reaction of MBP, OsSADR1 and the single amino acid-changed mutant (MBP–OsSADR1C168A). Fig. 3 View largeDownload slide In vitro ubiquitination assay of OsSADR1 E3 ligase. (a) The ubiquitination reaction contains E1 (human), AtE2 (Arabidopsis UBC10), maltose-binding protein-tagged OsSADR1 (MBP–OsSADR1), Ub and ATP. (b) The ubiquitination reaction of MBP, OsSADR1 and the single amino acid-changed mutant (MBP–OsSADR1C168A). Interaction of OsSADR1 and each of the three substrate proteins in vitro and in vivo Many studies have revealed that RING finger proteins regulate their interaction proteins via ubiquitination (Pickart 2004, Lim et al. 2015, Park et al. 2015). To identify the substrate of OsSADR1, we performed a Y2H screening using a salt-treated rice root library. Three rice genes from these yeast colonies strongly interacted with OsSADR1, Os01g66120 (no apical meristem protein, OsSNAC2, Hu et al. 2008), Os11g47870 (GRAS family transcription factor domain-containing protein, OsGRAS-44, Zhao et al. 2016) and Os03g62790 (putative pirin-like protein, hereafter OsPIRIN), according to their α-galactosidase activity (Supplementary Fig. S5). To confirm their physical interactions, yeast GAL4 DNA-binding domain (BD)-tagged OsSADR1 and each of the interacting proteins, which tagged the yeast GAL4 activation domain, were co-transformed into the Y2H Gold strain and then separately spotted onto DDO and QDO/X/A media (Fig. 4a). Results showed that all the yeast colonies of the three interaction partners grew on DDO plates when co-expressed with both an empty DNA BD and BD-tagged OsSADR1. However, the three colonies of interacting proteins, which were co-expressed with an empty DNA BD, did not grow in the QDO/X/A medium, even when co-expressed with OsSADR1. In addition, the colonies of interacting partners with OsSADR1 demonstrated their α-galactosidase activity in the QDO/X/A medium. Fig. 4 View largeDownload slide Identification of the interaction substrate of OsSADR1 E3 ligase. (a) The full-length OsSADR1 and each of the interaction proteins, OsPIRIN (Os03g62790, putative pirin-like protein), OsSNAC2 (Os01g66120, no apical meristem protein) and OsGRAS-44 (Os11g47870, GRAS family transcription factor domain-containing protein), were cloned into pGBKT7 and pGADT7 vectors, respectively. Each of the positive substrates was co-transformed with OsSADR1 into the Y2H Gold yeast strain. Cells were dropped onto DDO and QDO/X/A medium. (b) Subcellular localization of interacting proteins. Each of the full-length genes was cloned into the 35S:EYFP vector and then transfected into rice protoplasts. (c) BiFC assay of the three substrate proteins with OsSADR1 E3 ligase. Each of the substrate proteins was cloned into pSPYNE(R), and OsSADR1 was cloned into pSPYCE(M). The cloned substrate proteins were co-transfected with OsSADR1 into rice protoplast. (d) In vitro pull-down assay of substrate proteins with OsSADR1. Each of the EYFP-tagged substrate proteins was separately infiltrated into N. benthamiana leaves. After 3 d, extracted substrate proteins were incubated with the His-Trx-tagged OsSADR1 protein for 1 h. Immune signals were detected with an anti-GFP and anti-His antibody. Fig. 4 View largeDownload slide Identification of the interaction substrate of OsSADR1 E3 ligase. (a) The full-length OsSADR1 and each of the interaction proteins, OsPIRIN (Os03g62790, putative pirin-like protein), OsSNAC2 (Os01g66120, no apical meristem protein) and OsGRAS-44 (Os11g47870, GRAS family transcription factor domain-containing protein), were cloned into pGBKT7 and pGADT7 vectors, respectively. Each of the positive substrates was co-transformed with OsSADR1 into the Y2H Gold yeast strain. Cells were dropped onto DDO and QDO/X/A medium. (b) Subcellular localization of interacting proteins. Each of the full-length genes was cloned into the 35S:EYFP vector and then transfected into rice protoplasts. (c) BiFC assay of the three substrate proteins with OsSADR1 E3 ligase. Each of the substrate proteins was cloned into pSPYNE(R), and OsSADR1 was cloned into pSPYCE(M). The cloned substrate proteins were co-transfected with OsSADR1 into rice protoplast. (d) In vitro pull-down assay of substrate proteins with OsSADR1. Each of the EYFP-tagged substrate proteins was separately infiltrated into N. benthamiana leaves. After 3 d, extracted substrate proteins were incubated with the His-Trx-tagged OsSADR1 protein for 1 h. Immune signals were detected with an anti-GFP and anti-His antibody. To identify the subcellular localization of interacting partners, 35S:mCherry-tagged recombinant proteins were constructed. Two proteins, OsSNAC2 and OsGRAS-44, were localized in the nucleus, whereas fluorescence signals of OsPIRIN protein were localized in the chloroplasts in rice protoplasts (Fig. 4b). To verify the physical interactions between OsSADR1 and each interacting protein, we performed the BiFC assay using 35S:HA-SPYCE(M) and 35S:cMyc-PYNE(R), which included the EYFP C-terminus and the EYFP N-terminus, respectively (Waadt et al. 2008), resulting in cytosol and nucleus target fluorescent signals that were detected in co-expression complexes [i.e. 35S:OsSADR1-SPYCE(M), 35S:OsSNAC2-SPYNE(R) and 35S:OsGRASS-44-SPYNE(R)]. However, the complex of OsSADR1/OsPIRIN–YFP was only detected in the cytosol. To examine further the dimeric complex of OsSADR1 and interacting partners, we first constructed bacterially expressed His-Trx-tagged OsSADR1 proteins. Then, OsSADR1-His-Trx protein and each of the EYFP-tagged interacting partners, OsSNAC2–EYFP, OsGRAS–44-EYFP and OsPIRIN–EYFP, were co-incubated with HisPur Cobalt resin. The bound proteins were eluted from the resin and finally an immunoblot analysis was performed using anti-His and anti-green fluorescent protein (GFP) antibodies (Fig. 4d). An in vitro pull-down assay showed that each of the interacting proteins was pulled down from the HisPur Cobalt resin by the His-Trx-tagged OsSADR1 protein. These results indicated that each partner protein could bind to OsSADR1-His-Trx, and that the OsSADR1 interaction with the three proteins, i.e. OsSNAC2, OsGRAS-44 and OsPIRIN, might regulate this cellular function in rice. OsSADR1 E3 ligase regulates protein levels of interacting proteins via the 26S proteasome system The Y2H, BiFC and in vitro pull-down assays showed that OsSADR1 interacted with the cytosol- and nuclear-localized proteins, OsSNAC2 and OsGRASS-44, respectively, and one chloroplast-localized protein, OsPIRIN. It is believed that the RING domain containing E3 ligase regulates their substrate protein via the 26S proteasome system (Smalle and Vierstra 2004, Vierstra 2009). Further experiments addressed the hypothesis that OsSADR1 ubiquitinates interacting proteins and is subjected to proteolytic degradation via the 26S proteasome system. To identify this, we first tested the in vitro ubiquitination of interacting partners with the OsSADR1 protein. The MBP-tagged OsSADR1 was incubated with each of the His-Trx-tagged interacting partners, i.e. OsSNAC2, OsGRASS-44 and OsPIRIN-His-Trx, and analyzed by immune blotting using anti-Trx antibodies (Fig. 5a). The results showed that each of the Ub-attached protein bands was found in the lanes with MBP–OsSADR1 combined with E1, E2 and Ub, whereas no Ub-attached band was observed in the lanes lacking E1, E2 or OsSADR1. Secondly, we attempted to measure the protein levels of interacting proteins in the presence or absence of OsSADR1 using an in vivo protein degradation assay. We separately expressed MBP–OsSADR1 and EYFP-tagged OsSNAC2, OsGRAS-44 and OsPIRIN, and then incubated them for various times, i.e. 0, 1 and 2 h, with or without MG132 (Fig. 5b). In the presence of OsSADR1, each of the partner protein levels was dramatically reduced at the 1–2 h reaction time (Fig. 5b, left panel). However, degradation of the interacting proteins did not occur in MG132-treated lanes. In addition, similar results showed that the protein levels of interacting proteins did not change when incubated with a single amino acid-substituted MBP–OsSADR1C168A (Fig. 5b, right panel). These results indicated that the protein–protein interactions of OsSADR1 and interacting proteins, OsSNAC2, OsGRAS-44 and OsPIRIN, leads to proteolysis via the 26S proteasome degradation pathway. Fig. 5 View largeDownload slide In vitro ubiquitination and in vivo degradation assay of substrate proteins by OsSADR1 E3 ligase. (a) In vitro ubiquitination assay of His-Trx-tagged substrate proteins with MBP–OsSADR1. Each interacting substrate protein was incubated with the MBP-tagged OsSADR1 protein, E1 (human) and AtUBC10 (Arabidopsis). Immune signals were detected with an anti-His antibody. (b) In vivo protein degradation assay of interacting proteins. Each of the cMyc-tagged recombinant substrate proteins, i.e. OsSNAC2-cMyc, OsGRAS-44-cMyc and OsPIRIN-cMyc, was constructed and separately infiltrated into N. benthamiana leaves. Three substrate proteins were extracted and then incubated at different time points (i.e. 0, 1, 2 and 3 h) with each MBP-tagged OsSADR1 or single amino acid-mutated OsSADR1C168A protein and analyzed by immunoblotting using an anti-cMyc antibody and anti-MBP antibody. MG132 was used as an inhibitor protein to block the 26S proteasome pathway. (c) The transcript levels of substrate genes. The 200 mM salt-treated rice samples were used for qRT-PCR. The transcript levels were standardized based on cDNA amplification with ActinII as an internal control. As compared with controls (0 h), asterisks represent statistically significant difference according to two-tailed Student’s t-test; *P < 0.05, **P < 0.01. Fig. 5 View largeDownload slide In vitro ubiquitination and in vivo degradation assay of substrate proteins by OsSADR1 E3 ligase. (a) In vitro ubiquitination assay of His-Trx-tagged substrate proteins with MBP–OsSADR1. Each interacting substrate protein was incubated with the MBP-tagged OsSADR1 protein, E1 (human) and AtUBC10 (Arabidopsis). Immune signals were detected with an anti-His antibody. (b) In vivo protein degradation assay of interacting proteins. Each of the cMyc-tagged recombinant substrate proteins, i.e. OsSNAC2-cMyc, OsGRAS-44-cMyc and OsPIRIN-cMyc, was constructed and separately infiltrated into N. benthamiana leaves. Three substrate proteins were extracted and then incubated at different time points (i.e. 0, 1, 2 and 3 h) with each MBP-tagged OsSADR1 or single amino acid-mutated OsSADR1C168A protein and analyzed by immunoblotting using an anti-cMyc antibody and anti-MBP antibody. MG132 was used as an inhibitor protein to block the 26S proteasome pathway. (c) The transcript levels of substrate genes. The 200 mM salt-treated rice samples were used for qRT-PCR. The transcript levels were standardized based on cDNA amplification with ActinII as an internal control. As compared with controls (0 h), asterisks represent statistically significant difference according to two-tailed Student’s t-test; *P < 0.05, **P < 0.01. Increased sensitivity response of OsSADR1-overexpressing Arabidopsis under salt and mannitol stress Our expression analysis showed that OsSADR1 was highly induced in salt-treated rice root samples (Fig. 1a). To study the function of OsSADR1 under salt stress, 35S:OsSADR1–EYFP-overexpressing Arabidopsis were constructed and then three independent transgenic lines (35S:OsSADR1-EYFP #1, #3 and #4) were selected, depending on the expression levels of OsSADR1 by RT-PCR (Supplementary Fig. S7). As shown in Fig. 6, phenotypic effects of OsSADR1-overexpressing lines exhibited a sensitivity response with 100–200 mM salt treatments (Fig. 6a). After 2 weeks of 100 mM salt treatment, EYFP-overexpressing control lines showed a greater root length than OsSADR1-overexpressing lines; 0.6 cm longer roots than those of the 35S-EYFP plants (Fig. 6b). Similarly, plants subjected to both 150 and 200 mM salt treatments also exhibited longer root lengths of 0.7 and 0.05 cm, respectively. For germination rates, each of the 35S:EYFP- and 35S:OsSADR1-overexpressing plants showed similar results; all seeds were germinated 2 d after seedlings (Supplementary Fig. S8A). However, 35S:EYFP-overexpressing lines had a 100% germination rate after 4 d of 100 mM salt treatment, whereas all seeds were germinated after 6–8 d under 150–200 mM salt-stressed conditions (Supplementary Fig. S8A). Fig. 6 View largeDownload slide Phenotypic effects of osmotic stresses on OsSADR1-overexpressing Arabidopsis. (a) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of salt during seedling growth. Three independent lines of Col-0/35S:OsSADR1 and wild-type plants were grown for 14 d on MS medium with 0, 100, 150 or 200 mM salt (scale bar = 2 cm). (b) Root length of wild-type and each of the three independent plants. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (c) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of mannitol during seedling growth. Plants were grown in 0, 100, 150 or 200 mM mannitol-containing medium (scale bar = 2 cm). (d) Root growth assay with various concentrations of mannitol. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Fig. 6 View largeDownload slide Phenotypic effects of osmotic stresses on OsSADR1-overexpressing Arabidopsis. (a) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of salt during seedling growth. Three independent lines of Col-0/35S:OsSADR1 and wild-type plants were grown for 14 d on MS medium with 0, 100, 150 or 200 mM salt (scale bar = 2 cm). (b) Root length of wild-type and each of the three independent plants. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (c) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of mannitol during seedling growth. Plants were grown in 0, 100, 150 or 200 mM mannitol-containing medium (scale bar = 2 cm). (d) Root growth assay with various concentrations of mannitol. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). It is believed that plants respond to different types of osmotic stress, such as NaCl (ionic) and mannitol (non-ionic), via physiological and biochemical changes (Gangopadhyay et al. 1997, Ghuge et al. 2010). To verify the ionic or non-ionic effect in Arabidopsis, 35S:OsSADR1-overexpressing plants were treated with various concentrations of mannitol, i.e. 0, 100, 150 and 200 mM (Fig. 6d). OsSADR1-overexpressing lines showed low elongation of root length for plants supplemented with various mannitol concentrations compared with 35S:EYFP-overexpressing control plants (Supplementary Fig. S8B). Interestingly, no difference was observed in both the 150 and 200 mM-treated 35S:EYFP and 35S:OsSADR1-overexpressing plants 2 weeks after germination; however, we found a shorter root length of overexpressed plants with 100 mM salt treatment. In addition, OsSADR1-overexpressing plants exhibited slower germination rates than did the 35S:EYFP control plants. In each 150 mM-treated 35S:EYFP plant, 100% germination occurred at 6 d, whereas each of the OsSADR1-overexpressing lines showed 100% germination rates at 8 d. Similarly, the germination rate of 35S:EYFP transgenic seedlings was significantly higher than that of OsSADR1-overexpressing seedlings with 200 mM mannitol treatment. These results might suggest that heterologous overexpression of OsSADR1 in Arabidopsis enhanced germination and root length, and acted as both an ionic and a non-ionic osmotic stressor. Hyposensitive response of OsSADR1-overexpressing Arabidopsis to ABA treatment To evaluate the phenotypic effect of OsSADR1 transgenic lines under ABA stress, each of the control and transgenic plants was planted on Murashige and Skoog (MS) medium containing different concentrations (0. 0.5 and 1 μM) of ABA. On control plates, plants did not exhibit any difference in germination rates, whereas OsSADR1-overexpressing plants exhibited faster germination rates than did control plants. All the overexpressed seeds were germinated by 2 d, but control seeds were fully germinated after 6 and 12 d under 0.5 and 1 μM ABA, respectively (Fig. 7a). Similarly, we measured root length of control and overexpressing plants after 14 d and found a longer root length for overexpressing plants than for control plants under both 0.5 and 1 μM (Fig. 7b, c). To examine whether the hyposensitive response to ABA treatment of OsSADR1-overexpressing plants affected stomatal movement, we performed an ABA-dependent stomatal closure assay using each transgenic line (#1, #3 and #4) (Fig. 7d). In the non-treated condition, the average width of stomatal apertures showed no significant difference between OsSADR1-overexpressing plants and control plants. However, 0.5 μM ABA-treated OsSADR1 transgenic plants exhibited larger stomatal apertures, #1 (0.3679 ± 0.0427), #3 (0.4105 ± 0.0865) and #4 (0.386 ± 0.0544), compared with those of the control plants (0.2778 ± 0.0873). In addition, the stomatal aperture of transgenic lines showed similar results with tolerance phenotypes, #1 (0.2399 ± 0.074), #3 (0.2941 ± 0.0726) and #4 (0.2201 ± 0.0503), which was more than the wild-type plant (0.1393 ± 0.0298) under 1 μM ABA treatment, suggesting that OsSADR1-overexpressing plants expressed hyposensitivity phenotypes in response to ABA stress. Fig. 7 View largeDownload slide ABA tolerance phenotype of OsSADR1-overexpressing Arabidopsis. (a) Comparisons of germination rates of OsSADR1-overexpressing and wild-type plant seeds after exposure to different concentrations of ABA for 14 d. The data are presented as means ± SD (n = 25). (b) Phenotypes of wild-type and transgenic plants in response to various concentrations of ABA during seedling growth. Plants were grown in 0.5 or 1 μM ABA-containing medium (scale bar = 2 cm). (c) Root length assay of the wild type and each of the independent transgenic lines. The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (D and E) Stomatal aperture of OsSADR1-overexpressing and wild-type plants in response to different concentrations of ABA (0, 0.5 and 1 μM). Stomatal aperture was quantified by counting 25 guard cells in each treatment (scale bar = 5 μm). The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Fig. 7 View largeDownload slide ABA tolerance phenotype of OsSADR1-overexpressing Arabidopsis. (a) Comparisons of germination rates of OsSADR1-overexpressing and wild-type plant seeds after exposure to different concentrations of ABA for 14 d. The data are presented as means ± SD (n = 25). (b) Phenotypes of wild-type and transgenic plants in response to various concentrations of ABA during seedling growth. Plants were grown in 0.5 or 1 μM ABA-containing medium (scale bar = 2 cm). (c) Root length assay of the wild type and each of the independent transgenic lines. The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (D and E) Stomatal aperture of OsSADR1-overexpressing and wild-type plants in response to different concentrations of ABA (0, 0.5 and 1 μM). Stomatal aperture was quantified by counting 25 guard cells in each treatment (scale bar = 5 μm). The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Overexpression of OsSADR1 confers drought tolerance in Arabidopsis It is believed that plants promote stomatal closure to conserve water by accumulating ABA in the guard cells under drought stress (Cominelli et al. 2010, Popko et al 2010, Wilkinson and Davies 2010, Lim et al 2015). In the present study, each of the three independent OsSADR1-overexpressing plants showed larger stomatal apertures than the control plants under ABA treatment (Fig. 7e). To identify the effect of OsSADR1 under water deficit conditions, 2-week-old transgenic and control plants were exposed to drought stress by withholding irrigation for 5 d. After the drought treatment, each of the plants was irrigated for 5 d and determined the survival rates (Fig. 8). In the plants grown under normal conditions, no significant differences were observed between the three independent and control plants. However, after 5 d of withholding irrigation, each of the transgenic plants showed lower tolerance phenotypes compared with the control plants. In addition, 5 d after rewatering, each of the independent transgenic plants showed lower survival rates, #1 (34.4 ± 3.85), #3 (22.5 ± 4.64) and #4 (16.9 ± 0.48), whereas 90% of the control plants survived. These data suggest that OsSADR1 E3 ligase acts as a negative regulator under drought conditions by the regulation of ABA-mediated stomatal closure. Fig. 8 View largeDownload slide Decreased water tolerance of OsSADR1-overexpressing Arabidopsis. Water loss rates of detached rosette leaves from 2-week-old control and OsSADR1 transgenic lines. The percentage and number of surviving plants per total number of tested plants are indicated under the photographs. The data are presented as the mean ± SD (n = 100). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Fig. 8 View largeDownload slide Decreased water tolerance of OsSADR1-overexpressing Arabidopsis. Water loss rates of detached rosette leaves from 2-week-old control and OsSADR1 transgenic lines. The percentage and number of surviving plants per total number of tested plants are indicated under the photographs. The data are presented as the mean ± SD (n = 100). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Discussion Several studies have provided important evidence regarding the molecular functions of RING E3 ligase under various environmental stresses, such as high salinity, temperature and water loss conditions in rice. For example, the rice RING E3 ligase Oryza sativa Chloroplast Targeting RING Finger Protein 1 (OsCTR1) is a positive regulator that is specifically induced during drought stress through the regulation of protein levels and inhibition of trafficking of chloroplast genes, OsCP12 and OsPR1 (Lim et al. 2014). Moreover, overexpression of the drought-induced OsRDC1, O. sativa RING domain-containing protein in transgenic rice plants resulted in improved tolerance to water deficits (Bae et al. 2011). Overexpression of rice OsRHP1, which has a putative RING-H2 domain, conferred enhanced drought and salt tolerance (Zeng et al. 2014). However, high salinity stress induced the O. sativa Salt-Induced RING Finger Protein 1 (OsSIRP1) gene, which exhibited negative regulation during seed germination and root growth (Hwang et al. 2016). Numerous studies have provided evidence that RING E3 ligases are involved, as either positive or negative regulators, in the modulation of plant defense responses to diverse abiotic stresses. In fact, the role of RING E3 ligases as either positive or negative regulators in response to different stresses mainly depends on the nature of the target proteins. Therefore, it is necessary to identify target proteins to gain a better understanding of the biological functions of these RING E3 ligases in response to abiotic stresses. However, understanding the detailed regulatory roles of RING E3 ligases at the molecular and biochemical levels is often hindered by the lack of information on such target proteins (Cho et al. 2017). In the present study, OsSADR1 was highly induced under salinity, ABA and water loss conditions in rice root samples (Fig. 1a). In addition, heterogeneous overexpression of OsSADR1 in Arabidopsis produced a sensitivity response to both high salinity and mannitol treatments (Fig. 6a, b). In plants, as sessile organisms, a key plant signaling hormone, ABA, is accumulated under osmotic stress, such as high salinity and drought (Hubbard et al. 2010, Yoshida et al. 2014). This accumulation causes expression of the ABA-responsive genes, which include ABA RESPONSE ELEMENT (ABRE), at their promoters, and regulates the osmotic stress response (Wilson et al. 2014, Kazan 2015). As shown in Fig. 7, ABA-insensitive phenotypes were observed in each of the OsSADR1-overexpressing lines, despite the control plants exhibiting a hypersensitivity response (Fig. 7a–e). These results suggest that OsSADR1 acts as a regulator in an ABA-dependent manner. Several RING finger proteins have been reported to regulate transcription factors. First, the Arabidopsis RING-type E3 ligase KEEP ON GOING (KEG) protein regulates the ABA-responsive transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5) under ABA stress. Moreover, KEG-overexpressing Arabidopsis plants exhibited an insensitivity response under high concentration ABA treatment via protein degradation of ABI5 (Liu and Stone 2010). Secondly, the HECT domain containing Arabidopsis UPL5 regulates leaf senescence through protein degradation of WRKY53, which acts in a regulatory network influencing transcription in Arabidopsis (Miao and Zentgraf 2010). Thirdly, overexpression of the tomato SEVEN IN ABSENTIA3 (SINA3) ubiquitinates the NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, promotes its degradation and represses the R protein-mediated hypersensitive response (HR) cell death in N. benthamiana (Miao et al. 2016). Fourthly, the Arabidopsis RGLG2, which includes RING and a so-called copine (or von Willebrand factor type A) domain, interacts with ETHYLENE RESPONSE FACTOR53 (AtERF53), which is a drought-induced transcription factor, and leads to protein degradation under drought stress (Cheng et al. 2012). In the present study, the subcellular localization of the OsSADR1 protein was detected in both the cytosol and nucleus in rice protoplasts (Fig. 2). Interestingly, approximately 62% of the cytosol-localized fluorescent signals were detected, whereas 38% of the nuclear signals were identified in non-treated rice protoplasts (Fig. 2b, left panel). In contrast, 30% of the OsSADR1 signals occurred in the cytosol, whereas 70% of the nuclear signals were detected in high salinity-treated rice protoplasts (Fig. 2b, right, c). These data suggest that the dynamic movement of OsSADR1 into the nucleus might regulate some nuclear proteins, such as transcription factors, via action similar to E3 ligase under high salinity conditions. Our results indicated a functional relationship among OsSADR1 and three interacting proteins, of which two, OsSNAC2 and OsGRAS-44, are nuclear-localized proteins, and one, OsPIRIN, is a chloroplast-localized protein (Fig. 4). Interestingly, fluorescence of OsSADR1 and the interacting proteins was observed in the cytosol (although signals of both interactions with OsSNAC2 and OsGRAS-44 were also observed to be nuclear) when transfected with OsSADR1 under BiFC analysis (Fig. 4c). In addition, interacting proteins were ubiquitinated by OsSADR1 E3 ligase, leading to reduced protein levels mediated by the 26S proteasome pathway (Fig. 5). Taken together, these data indicated that OsSADR1 could ubiquitinate interacting proteins in the cytosol. However, the OsSADR1 protein exhibited translocation from the cytosol to the nucleus when protoplasts were exposed to high salinity (Fig. 2). We hypothesized two possibilities regarding the interaction between OsSADR1 and interacting proteins in the cytosol. One attractive possibility is that OsSADR1 might interact with nuclear proteins, i.e. OsSNAC2 and OsGRAS-44, in the nucleus and then translocate from the nucleus to the cytosol. Lim et al. (2013) indicated that the Golgi apparatus-localized OsHCI1 relocates to the nucleus under heat stress, and then the targeted nuclear proteins, including OsbHLH065, send the transcription factors to the cytosol. An alternative hypothesis is that the OsSADR1 protein directly inhibits trafficking of two nuclear proteins in the cytosol. Similar findings were observed in a hot pepper (Capsicum annuum) E3 ligase, Rma1H1, which inhibited trafficking of the PIP2;1 aquaporin protein from the endoplasmic reticulum to the plasma membrane, leading to tolerance to dehydration under degradation of the PIP2;1 protein (Lee et al. 2009). However, much work remains before the hypothesis can be ruled out. Rice SNAC (STRESS-RESPONSIVE NAM, ATAF and CUC) genes were induced by various environmental conditions, and expression of stress-related genes includes the NAC recognition site (NACR) at their promoters. For example, rice SNAC1, encoding a NAC transcription factor, binds to the NACR of the OsERD1 promoter, which then leads to improved drought and salt resistance in vegetative stages (Hu et al. 2006). Similarly, SNAC2 also exhibits transcriptional activity and binds to NACR-like sequences (Hu et al. 2008), and OsSNAC2-overexpressing transgenic plants showed improved tolerance to cold and high salinity stress, whereas a sensitivity response was observed under ABA treatment. Despite tolerance responses of SNAC2-overexpressing plants, the results of the present study showed that heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitivity responses, i.e. low germination rates and short root length, under high salinity and mannitol stresses (Fig. 6; Supplementary Fig. S8). Interestingly, the OsSADR1-overexpressing plants showed insensitive responses, whereas OsSNAC2-overexpressing plants showed hypersensitivity responses under ABA treatment (Hu et al. 2008). In the present study, relative expression levels of OsSNAC2 were gradually induced under high salinity stress for 12 h. However, we also found that OsSNAC2 and OsSADR1 proteins interacted in the cytosol and levels of OsSNAC2 protein gradually decreased when it was incubated with the OsSADR1 protein (Figs. 4b, 5b). Hu et al. (2008) reported that transcriptional levels of the six NACR-containing genes of the Scarecrow-like transcription factors (OsGRAS-39, Os11g04400), O-methyltransferase family proteins (OsOMT, Os09g17560), sodium/hydrogen exchanger 3 family proteins (OsNHX1, Os07g47100), GDSL-like lipase/acylhydrolase family proteins (Os05g11910), UDP-glucose 4-epimerase (OsUGE, Os09g35800) and heavy metal- associated proteins (Os01g74490) were induced in OsSNAC2-overexpressing transgenic rice. We examined the expression levels of these six genes, resulting in high induction of all genes in the salinity-treated rice protoplasts, but low expression of them in OsSADR1-EYFP-transfected rice protoplasts (Supplementary Fig. S9). These data suggest that inhibition of the function of OsNAC2 transcription factors via protein degradation leads to a sensitivity response under high salinity, but an insensitive response under ABA treatment in OsSADR1-overexpressing plants by inhibition of the expression of NACR-containing genes. Several studies have shown that a few NAC transcription factors undergo intensive post-translational regulation via ubiquitination (Olsen et al. 2004, Puranik et al. 2012). For example, the Arabidopsis transcription activator NAC1 was ubiquitinated by SINAT5 E3 ligase, which is a homolog of the Drosophila protein SINA, under auxin signals in plant cells (Xie et al. 2002). The nuclear-localized RHA2a, containing the RING-H2 type domain, interacts with ABSCISIC ACID RESPONSIVE NAC (ANAC) and regulates its transcription activity (Greve et al. 2003). Supporting these data, our study provides a potential mechanism between OsSADR1 and OsSNAC2 proteins in rice. First, the OsSNAC2 gene was up-regulated under high salinity stress, which led to the induction of downstream genes with an NACR (Fig. 9a, left). In our relative expression analysis, both OsSNAC2 and OsSADR1 showed gradual up-regulation under high salinity stress (Figs. 1a, 5c). However, OsSNAC2 exhibited decreased expression in the 24 h samples, despite continuing to induce expression of the OsSADR1 gene. This might suggest that after activation of downstream genes of OsSNAC2, the OsSNAC2 protein was regulated by OsSADR1 E3 ligase through translocation to the cytosol. Secondly, the OsSNAC2-overexpressing transgenic plants showed a tolerance response (Hu et al., 2008), whereas OsSADR1-overexpressing plants showed hypersensitivity under high salinity stress (Fig. 6). In addition, the OsNAC2 transgenic plants showed sensitivity under ABA treatment (Hu et al. 2008), whereas OsSADR1 transgenic plants showed an insensitive response (Fig. 7). Moreover, hypersensitivity phenotypes of OsSADR1-transgenic plants were observed under water deficit conditions compared with the control plants (Fig. 8). Interestingly, salt-related downstream genes with an NACR were not induced in OsSADR1-transfected rice protoplasts under high salt treatments, whereas they were highly induced in salt-treated non-transfected protoplasts (Supplementary Fig. S9). These results suggest that overexpression of OsSADR1 leads to degradation and subsequently OsSNAC2 transcription factor and, thus, could not activate its downstream genes with an NACR (Fig. 9b, right). Fig. 9 View largeDownload slide A model representing the regulation of the SNAC2 transcription factor between wild-type (WT) and OsSADR1-overexpressing plants during abiotic stresses. The upstream transcription factors may bind to the promoter of regulated OsSNAC2 genes and influence their transcription. In wild-type plants, translated OsSNAC2 protein moved to the nucleus and acted as a transcription factor of NACR-containing stress-induced target genes. After transcription of these genes, the OsSNAC2 transcription factor binds to OsSADR1 E3 ligase and is degraded by the 26S proteasome system in the cytosol. In OsSADR1 E3 ligase-overexpressing plants, translated OsSNAC2 protein directly binds to OsSADR1 before acting as a transcription factor, and then is degraded by the 26S proteasome system. Fig. 9 View largeDownload slide A model representing the regulation of the SNAC2 transcription factor between wild-type (WT) and OsSADR1-overexpressing plants during abiotic stresses. The upstream transcription factors may bind to the promoter of regulated OsSNAC2 genes and influence their transcription. In wild-type plants, translated OsSNAC2 protein moved to the nucleus and acted as a transcription factor of NACR-containing stress-induced target genes. After transcription of these genes, the OsSNAC2 transcription factor binds to OsSADR1 E3 ligase and is degraded by the 26S proteasome system in the cytosol. In OsSADR1 E3 ligase-overexpressing plants, translated OsSNAC2 protein directly binds to OsSADR1 before acting as a transcription factor, and then is degraded by the 26S proteasome system. We found hypersensitive phenotypes of OsSADR1-overexpressing plants even though the gene was highly induced by salt and drought stress (Figs. 1a, 6, 8). Moreover, OsSADR1-overexpressing plants were hyposensitive to ABA treatment, despite the up-regulated expression pattern of OsSADR1 under the treatment (Fig. 1a, 7). These observations raised some questions. (i) Why did transgenic Arabidopsis show hypersensitivity phenotypes to salinity and drought stress, despite the induction of OsSADR1 in plants under these stresses? (2) Why did the transgenic plants exhibit insensitivity to ABA even though OsSADR1 was highly induced under ABA treatment? Previously, a host of similar findings have been reported. For example, OsSIRP1 was highly induced under salinity stress, but its transgenic plants exhibited a hypersensitivity phenotype under high salinity stress (Hwang et al. 2016). In addition, another rice RING finger protein, OsDIS1 (O. sativa drought-induced SINA protein 1), showed up-regulation by drought treatment, but the OsDIS1-overexpressing plants exhibited a sensitivity response under drought stress (Ning et al. 2011). Similarly, the pepper gene, CaREL1 (Capsicum annuum RING E3 Ligase 1), was induced by ABA treatment and its transgenic plants had larger stomatal closures and higher germination rates and root lengths than did the control plants (Lim et al 2017). Our results confirmed that OsSADR1 interacts with the OsSNAC2 transcription factor, which induces the NACR-containing downstream genes under salt stress, which then led to regulation of protein levels (Figs. 4, 5a, b). The hypothesis that fine regulation of protein accumulation maintains cell balance might be able to explain inconsistencies in that SNAC2 induced under salinity is regulated by OsSADR1 induced by the stress in wild-type plants (Fig. 9, right). The down-regulation of the substrate genes (e.g. OsSNAC2) at 24 h after the salt treatment, despite high induction of OsSARD1 at 24 h, might support our hypothesis. However, constitutive overexpression of OsSADR1 in the transgenic plants might subsequently block the OsSNAC2 function to induce the downstream genes via degradation, resulting in the hypersensitive responses to salinity and drought, and insensitivity to ABA (Fig. 9, left). Our findings on the movement of OsSADR1 to the nucleus by salinity, degradation of transcription factors, especially that of OsSNAC2 via the Ub–26S proteasome system, and down-regulation of the downstream genes OsSNAC2 in OsSARD1-transfected protoplasts might provide important clues to molecular functions of the OsSADR1 gene under abiotic stress. In conclusion, OsSADR1 might act as a negative regulator in response to abiotic stresses, especially salinity, primarily depending on the hypersensitivity response of OsSADR1-overexpressing plants under abiotic stresses; however, further studies are required to elucidate this characteristic. Materials and Methods Plant materials and growth conditions Rice seeds (O. sativa L. ‘Donganbyeo’) were grown with commercial soil in a growth chamber (16/8 h light/dark photoperiod at 25/23°C with 70% relative humidity) for 2 weeks. The seedlings were treated with 200 mM salt and their roots were harvested at 1, 3, 6, 12 or 24 h. For drought stress, 2-week-old seedlings were positioned in the space between two pieces of tissue paper in the growth chamber, and then harvested at the same times as those in the salt treatment. For ABA treatment, 2-week-old rice seedlings were treated at different concentrations, 0.5 and 1 μM. Samples were ground using liquid nitrogen and immediately stored at –80°C until use. Gene expression analysis To identify the expression patterns of genes, total RNA was extracted using the TRIzol® reagent according to the manufacture’s protocol (Invitrogen). First-strand cDNA synthesis from total RNA (3 μg) was conducted using a cDNA Synthesis Kit (TAKARA-BIO INC.). For qRT-PCR, cDNA was used after being mixed with a TOPreal™ qPCR 2X Premix with SYBR green (Enzynomics) and PCR was performed using the CFX96 real-time PCR Detection System (BioRad Laboratories). OsActinII was used as an internal control. A list of all primers used in this study is provided in Supplementary Table S1. Confocal microscopy and imaging To identify the subcellular localization of O. sativa genes, OsSADR1, OsSNAC2, OsGRAS-44 and OsPIRIN cDNAs were cloned into EYFP vectors using salt-treated rice root samples. In addition, the full-length clone of OsSADR1 and each of the interacting genes were inserted into 35S:HA-SPYCE(M) and 35S:c-Myc-SPYNE(R) vectors for BiFC assay. Each of the constructed plasmids was transfected into the protoplast using a 40% polyethylene glycol (PEG) solution [40% PEG, 400 mM mannitol, 100 mM Ca(NO3)2] for 30 min at room temperature. Finally, the W5 solution was added to dilute the PEG solution and then discarded. The transfected protoplasts were resuspended with W5 and incubated overnight at room temperature. The transfected protoplasts were observed using a confocal microscope after 16 h of incubation. Fluorescent images were obtained using a multiphoton confocal laser scanning microscope (model LSM 510 META; Carl Zeiss) at the Korea Basic Science Institute Chuncheon Center, Korea. In vitro ubiquitination assay To construct the recombinant proteins, the full-length OsSADR1 gene was amplified and cloned into a pMAL-c5x vector (New England BioLabs). A single amino acid substitution (OsSADR1C168A) in the RING finger domain of OsSADR1 was generated using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene). Each recombinant MBP fusion protein was expressed by Escherichia coli strain BL21 (DE3) pLysS (Promega) and then purified by affinity chromatography using an amylose resin (New England BioLabs). For the in vitro self-ubiquitination assay, purified MBP–OsSADR1 was mixed with human E1 (Sigma-Aldrich), 6× His-tagged AtUBC10 and bovine Ub (Sigma-Aldrich), and then incubated in a ubiquitination reaction buffer [1 M Tris–HCl, pH 7.5; 40 mM ATP; 100 mM MgCl2; 40 mM dithiothreitol (DTT)]. After a 3 h incubation period at 30°C, the reaction was stopped by adding 6× SDS sample buffer, analyzed via 12% SDS–PAGE and then transferred to a nitrocellulose membrane. Immunoblot analyses were conducted using an anti-Ub antibody (Sigma-Aldrich) with a secondary goat anti-rabbit IgG peroxidase antibody (Sigma-Aldrich). Antibody detection was conducted using the chemiluminescent substrate SuperSignal® West Pico (ThermoScientific) for horseradish peroxidase (HRP). Photographs were obtained using ChemiDoc™ XRS+ (Bio-Rad). To confirm that OsSADR1 mediated ubiquitination of the three interacting proteins, OsSNAC2, OsGRAS44 and OsPIRIN-His-Trx, fusion proteins were affinity-purified and incubated together with purified MBP–OsSADR1 in the ubiquitination mixture for 3 h. The mixture was then subjected to SDS–PAGE and immunoblot analysis. Screening the interacting proteins via yeast two-hybrid system To identify the substrates of the OsSADR1 protein, yeast transformation and library screening were conducted in accordance with the recommended commercial procedures (Make Your Own ‘Mate & Plate’ Library system; Matchmaker Gold Yeast Two-Hybrid system; Yeastmaker Yeast Transformation System 2). A rice cDNA library in pGADT7-AD, which included the GAL4 activation domain, was generated from salt stress-treated rice root samples. Then, the full-length coding sequence of OsSADR1 was amplified and cloned into the pGBKT7-BD vector, which included the GAL4 DNA-binding domain as a bait strain. The yeast library screening was performed as previously described by Park et al. (2015). For screening, the BD-OsSADR1-transformed yeast strain was mated with salt-treated rice root library yeast strains. Then, positive interactions were selected on double dropout media lacking leucine and tryptophan (DDO/X/A) and supplemented with 40 μg ml−1 X-α-gal and 42 ng ml−1 aureobasidin A. After first screening, blue colonies were patched out onto higher stringency QDO/X/A, which lacked adenine, histidine, leucine and tryptophan. A total of 54 clones were identified as positive interacting colonies of OsSADR1, sequenced and verified by The Institute for Genomic Research (TRGR) rice annotation database. For each of the three genes, OsSNAC2, OsGRAS-44 and OsPIRIN, the full-length coding region was cloned into a pGADT7-AD vector and co-transformed into the Y2H Gold strain with the pGBKT7-OsSADR1 vector. Each of the co-transformed yeast cells was grown on DDO medium for 5 d at 30°C. Then, each of the clones was cultured in liquid DDO medium adjusted to OD600 = 1.0 and spotted onto DDO and QDO/X/A media. In vitro pull-down assay To observe the protein–protein interaction between OsSADR1 and partner proteins, i.e. OsSNAC2, OsGRAS-44 and OsPIRIN, MBP tagged OsSADR1 and His-Trx tagged each of the partner proteins, which were then expressed in E. coli strain BL21. After sonication of each protein expressed in BL21, bacterial lysate containing MBP–OsSADR1 was bound with amylose resin (New England BioLabs) in Poly-Prep Chromatography Columns (New England BioLabs). After incubation, amylose resin–MBP–OsSADR1 was mixed with or without lysates containing His-Trx-tagged partner proteins and rotated at 4°C for 2 h. Columns were washed five times using column buffer (20 mM Tris–HCl, 20 mM NaCl, 1 mM EDTA and 1 mM DTT). The isolated proteins were then mixed with the addition of 6× SDS sample buffer followed by 5 min of boiling at 95°C. Then, they were separated using 10% SDS–PAGE, transferred to a nitrocellulose membrane, and immune signals were detected using an anti-MBP antibody and anti-Trx antibody. Protein degradation assay To perform the degradation assay, the full-length cDNA of partner genes was cloned into a 35S:8cMyc vector. Each of the constructed vectors was transformed to Agrobacterium strain GV3101 and infiltrated in Nicotiana benthamiana leaves. After 3 d, leaves were homogenized and incubated with a Plant Total Protein Extraction Kit (Sigma-Aldrich), and each of the expressed proteins, including the total N. benthamiana proteins, was mixed with the MBP-tagged OsSADR1 protein or MBP-tagged OsSADR1C168A at a volume ratio of 1 : 1 at 4°C. The 26S proteasome inhibitor, MG132, was added to the corresponding samples to a final concentration of 50 μM. The reaction was stopped by the addition of sample buffer at different time points, i.e. 1–3 h. Immunoblotting was performed using anti-MBP and anti-cMyc antibodies. Arabidopsis transformation and treatment For the development of OsSADR1-overexpressing plants, the constructed 35S:OsSADR1-EYFP vector was transformed into Agrobacterium strain GV3101 and carried (ecotype Columbia, Col-0) according to the floral dip method (Clough 2005), with some modifications. Each of the T3 seeds of the transgenic plants (lines #1, #3 and #4) were selected for germination on half-strength MS agar plates containing 50 mg l−1 kanamycin. For identification of the phenotypic differences between control plants (35S:EYFP) and OsSADR1-overexpressing Arabidopsis lines under salt, mannitol and ABA stress, transgenic plants were treated with different concentrations: salt and mannitol (0, 100, 150 and 200 mM) or ABA (0, 0.5 and 1 μM). For measurement of the stomatal aperture, each of the 2-week-old ABA-treated transgenic plant leaves was examined under a Leica microscope (DM500). Supplementary Data Supplementary data are available at PCP online. Funding This research was supported by the Ministry of Education, Science, and Technology [Basic Science Research Program through the National Research Foundation of Korea (NRF) (2016R1A2B4015626)]. Disclosures The authors have no conflicts of interest to declare. References Bae H.S., Kim S.K., Cho S.K., Kang B.G., Kim W.T. ( 2011) Overexpression of OsRDCP1, a rice RING domain-containing E3 ubiquitin ligase, increased tolerance to drought stress in rice (Oryza sativa L.). Plant Sci.  6: 775– 782. Google Scholar CrossRef Search ADS   Balazadeh S., Siddiqui H., Allu A.D., Matallana-Ramirez L.P., Caldana C., Mehrnia M. ( 2010) A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence. Plant J.  62: 250– 264. Google Scholar CrossRef Search ADS PubMed  Basu S., Ramegowda V., Kumar A., Pereira A. ( 2016) Plant adaptation to drought stress. F1000Res . 5: 1554. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations BD binding domain BiFC bimolecular fluorescence GRAS-44 GRAS family transcription factor domain-containing protein 44 DTT dithiothreitol EYFP enhanced yellow fluorescent protein MBP maltose-binding protein MS Murashige and Skoog NAC NAM-ATAF1,2-CUC2 NACR NAC recognition site NLS nuclear localization signal PEG polyethylene glycol PIRIN putative pirin-like protein qRT-PCR quantitative reaal-time PCR RING Really Interesting New Gene SADR1 salt-, ABA- and drought-induced RING finger protein 1 SNAC stress-induced no apical meristem protein 2 Ub ubiquitin Y2H yeast two-hybrid © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

A Negative Regulator in Response to Salinity in Rice: Oryza sativa Salt-, ABA- and Drought-Induced RING Finger Protein 1 (OsSADR1)

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy009
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Abstract

Abstract RING (Really Interesting New Gene) finger proteins play crucial roles in abiotic stress responses in plants. We report the RING finger E3 ligase gene, an Oryza sativa salt, ABA and drought stress-induced RING finger protein 1 gene (OsSADR1). We demonstrated that although OsSAR1 possesses E3 ligase activity, a single amino acid substitution (OsSADR1C168A) in the RING domain resulted in no E3 ligase activity, suggesting that the activity of most E3s is specified by the RING domain. Additional assays substantiated that OsSADR1 interacts with three substrates—no E3 ligase acti and OsPIRIN, and mediates their proteolysis via the 26S proteasome pathway. For OsSADR1, approximately 62% of the transient signals were in the cytosol and 38% in the nucleus. However, transiently expressed OsSADR1 was primarily expressed in the nucleus (70%) in 200 mM salt-treated rice protoplasts. The two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSADR1 in the cytosol and nucleus. Heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitive phenotypes for salt- and mannitol-responsive seed germination and seedling growth. With ABA, OsSADR1 overexpression in plants produced highly tolerant phenotypes, with morphological changes in root length and stomatal closure. The ABA-tolerant transgenic plants also showed hypersensitivity phenotypes under severe water deficit conditions. Taken together, OsSADR1 may act as a regulator in abiotic stress responses by modulating target protein levels. Introduction Abiotic stresses, such as drought, cold, high salinity, extreme temperatures and heavy metal accumulation, severely reduce the productivity of food crops worldwide by altering plant physiological and biochemical processes (Mickelbart 2015, Zhu 2016). Because of their sessile nature, plants have coped with these stresses through the evolution of defense mechanisms which allow them to adapt and survive under stressful environmental conditions. Recently, drought, salinity and extreme temperature have been major stresses that challenge adequate global food production. Rice (Oryza sativa) is known as a major staple food for more than half of the world’s population, and is also considered a model plant for cereal genomics; however, it can be severely injured by abiotic stresses (Lafitte et al. 2004). In particular, rice is highly sensitive to salt stress during the young seedling and reproductive stages (Reddy et al. 2017). Rice is also known as a semi-aquatic crop, and as such it is more susceptible to drought than other cereals. Drought stress affects every stage of rice growth; in particular, seedlings, vegetative stages and anthesis can be highly affected and stress can reduce grain yield (Basu et al. 2016). The salt stress tolerance mechanism may be categorized into three different aspects: osmotic stress tolerance, Na+ exclusion from the shoot and Na+ tissue tolerance (Munns and Tester 2008). The drought resistance mechanism of plants is also categorized into four types: drought tolerance, drought escape, drought avoidance and drought recovery. To evaluate the drought resistance of plants, different drought-related indicators such as leaf traits, root traits, water potential, ABA content and osmotic adjustment capabilities have been used (Fang and Xiong 2015). Both salt and drought create osmotic stress in plant cells. Because of this unique and overlapping hyperosmotic signal, ABA is accumulated and acts as a stress defensive mechanism under unfavorable conditions (Finkelstein et al. 2002, Zhu 2002). During osmotic stress, ABA plays an important role by reducing transpirational water loss through stomatal closure (Zhu 2002, Wang and Song 2008). It is closely related to reactive oxygen species, such as hydrogen peroxide (H2O2), leading to stomatal closure by increasing the concentration of ABA in guard cells (Zang et al. 2001). Ubiquitin (Ub) is a highly conserved, universally expressed and stable protein, and its ubiquitination acts as a post-transitional modification, which mediates growth and development of eukaryotic species by regulating transcriptional changes needed for abiotic stress adaption. In higher plants, Ub-mediated substrate degradation plays an important role in growth, hormonal signaling and abiotic stress responses; however, the process of ubiquitination is very complex where enzymes, namely E1 (Ub activating enzyme), E2 (Ub conjugating enzyme and E3 Ub ligases, are typically required (Viestra 2009). Among them, RING Ub E3 ligases play vital roles in the post-transitional modification of proteins via attachment of Ub (Deshaies and Joazeiro 2009). One of the E3 ligases is the Really Interesting New Gene (RING) finger protein, which consists of cysteine (Cys) and histidine (His) residues (in the order Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-Cys/His-X2-Cys-X4-48-Cys-X2-Cys, where X may be any amino acid) that bind two zinc atoms. The clear relationship between abiotic stress and ubiquitination remains unknown; however, many high salinity-induced rice proteins were regulated by ubiquitination, including salt-induced RING finger protein OsSIRP1, which negatively regulates germination and root growth in Arabidopsis (Hwang et al. 2016), microtubule-associated rice RING finger protein OsRMT1, which positively regulates salt stress (Lim et al. 2015), and OsRINGC2-1 and OsRING2-2, which enhance salt tolerance in Arabidopsis (Jung et al. 2012). Likewise, several drought-induced rice genes have been identified, which effect physiological and molecular changes via ubiquitination. For example, salt- and drought-induced OsSDIR1 regulates ABA signaling and induced drought stress tolerance in rice (Gao et al. 2011). Similarly, OsDIS1 acts negatively on drought stress tolerance by post-transitional regulation of OsNek6 (Ser/Thr protein kinase) in rice (Ning et al. 2011). In addition, chloroplast targeting rice E3 ligase OsCTR1 plays a positive role in drought tolerance in an ABA-responsive manner through post-transitional modification (Lim et al. 2015). Besides these, other genes have been identified which are involved in multiple abiotic stresses, such as the rice RING E3 ligase OsDSG1 (Delayed Seed Germination 1) with its mutant osdsg1 and gene silencing by RNA interference (RNAi) that improves germination under high salinity and drought, mediated through enhanced ABA-regulated responses (Park et al. 2010). Another is OsSRF1 (stress-related RING finger protein 1), which negatively regulates salt, cold and oxidative stress (Fang et al. 2015). Moreover, OsPUB15 (Plant U-box 15) prevents tissue damage and protects against osmotic stress during germination and growth of rice plants (Park et al. 2011). Previously, >100 NAC transcription factors, which consist of NAM (non-apical meristem), ATAF1-2 (Arabidopsis transcription activation factor) and CUC2 (cup-shaped cotyledon), have been identified as the largest important families of transcriptional regulators for various stress responses and development in Arabidopsis and rice (Nakashima et al. 2012, Nuruzzaman et al. 2013, Lv et al. 2016). For example, rice OsNAC2 is a negative regulator of high salinity- and drought-mediated stress by regulating LATE EMBRYOGENESIS ABUNDANT 3 (LEA3) and stress-activated protein kinase 1 (OsSAPK1) in rice (Shen et al. 2017). Stress-responsive rice SNAC genes, OsNAC2/6 and OsNAC10, can improve drought and salt tolerance when overexpressed (Nakashima et al. 2009, Jeong et al. 2010). Another Arabidopsis NAC gene, ANAC092, showed salt-promoted senescence and seed maturation under ANAC092-mediated gene regulatory networks (Balazadeh et al. 2010). Another homologous group of genes, OsNAC5/OsNAC009/OsNAC071 and OsNAC6, is induced by drought, high salinity and ABA stress, and then enhances stress tolerance by up-regulating the expression of stress-inducible rice genes (Takasaki et al. 2010). It is also known that the environmental stress-related NAC group, SNAC, can bind to the NACR (NAC recognition sequence; CACG core) (Thao et al. 2013, Nakashima et al. 2014). Arabidopsis SNAC genes, such as RD26 and ATAF1, and rice SNAC genes, such as SNAC1, OsNAC6/SNAC2 and OsNAC5, can improve drought and/or high salt stress tolerance when overexpressed (Tran et al. 2004, Hu et al. 2006, Takasaki et al. 2010, Nakashima et al. 2014). Herein, we suggest the molecular function of the O. sativa salt-, ABA- and drought-induced RING finger protein 1 (OsSADR1) gene, which is highly induced by salt, ABA and drought stress. Its subcellular localizations were the cytosol and nucleus in rice protoplasts, with different localization frequencies between normal and salt-stressed samples. The yeast two-hybrid (Y2H) system, bimolecular fluorescence (BiFC) assay and in vitro pull-down assay revealed that the OsSADR1 protein primarily interacts with nuclear proteins, such as stress-induced no apical meristem protein 2 (OsSNAC2), GRAS family transcription factor domain-containing protein 44 (OsGRAS-44) and one chloroplast-localized putative pirin-like protein (OsPIRIN). Furthermore, the heterogeneous overexpression of OsSADR1 in OsSADR1-overexpressing plants showed sensitivity to salt and drought, and hyposensitivity to ABA treatment. Results Expression pattern and sequence analysis of OsSADR1 In a previous study, expression levels of 44 rice RING finger protein (OsRFP) genes, which were randomly selected based on domain analysis, were examined under salt stress (Hwang et al. 2016). We further examined the expression patterns of 44 rice RING finger proteins in response to drought stress in 14-day-old root samples. In particular, transcription levels of one gene (LOC_Os11g07450, called OsSADR1) was highly induced from 1 h until 12 h (Supplementary Fig. S1). Therefore, this gene was selected for further study. In addition, transcript levels of OsSADR1 were identified via quantitative real-time PCR (qRT-PCR) during a different time course (0, 1, 3, 6, 12 and 24 h) (Fig. 1a). The gene was up-regulated from 1 h until 24 h (up approximately 25-fold) after the salt stress treatment. Under drought stress, the transcript levels of OsSADR1 were increased at 6 h and then decreased at 12 and 24 h. Interestingly, OsSADR1 was highly induced in root samples treated with 0.1 mM ABA 6 h. In addition, each of the genes known to be induced reliably by salt (OsNAC10 and OsSalt), drought and ABA (OsLEA3 and OsRAB16A) showed transcript increases according to the increasing time course of treatments, supporting the postulate that samples suffered from the treatment because of each of the stressors (Supplementary Fig. S2). Fig. 1 View largeDownload slide Expression pattern of the OsSADR1 gene and amino acid sequence analysis of its product. (a) qRT-PCR analysis of the OsSADR1 gene in salt- (200 mM), ABA- (1 μM) and drought-treated 2-week-old rice seedlings. As compared with controls (0 h), asterisks represent the statistically significant differences according to a two-tailed Student’s t-test; *P < 0.05, **P < 0.01. (b) Multiple alignment of OsSADR1 and other orthologs of RING-HC-type proteins, i.e. At1G47670 from Arabidopsis, GRMZM2G000014 from maize andBradi4G24480 from Brachypodium. The alignment was performed using Clustal2 software (http://ebi.ac.uk/clustalw/). NLS, nuclear localization signal. Fig. 1 View largeDownload slide Expression pattern of the OsSADR1 gene and amino acid sequence analysis of its product. (a) qRT-PCR analysis of the OsSADR1 gene in salt- (200 mM), ABA- (1 μM) and drought-treated 2-week-old rice seedlings. As compared with controls (0 h), asterisks represent the statistically significant differences according to a two-tailed Student’s t-test; *P < 0.05, **P < 0.01. (b) Multiple alignment of OsSADR1 and other orthologs of RING-HC-type proteins, i.e. At1G47670 from Arabidopsis, GRMZM2G000014 from maize andBradi4G24480 from Brachypodium. The alignment was performed using Clustal2 software (http://ebi.ac.uk/clustalw/). NLS, nuclear localization signal. OsSADR1 encodes a protein of 479 amino acids and SADR1 contains the RING-HC domain at the N- and C-termini with a nuclear localization signal (NLS). We retrieved some orthologs from Arabidopsis (At1G47570), Zea mays (GRMZ2G000014) and Brachypodium distachyon (Bradi4G24480) from The Institute for Genomic Research rice annotation database, and aligned their induced amino acids, finding a highly conserved RING-HC domain (Fig. 1b). However, the NLS sequence was only included at the N-terminal sequence of OsSADR1 and not in other orthologous genes. Collectively, the expression patterns of the OsSADR1 gene exhibited strong induction under salt, ABA and drought exposure in rice roots. Subcellular localization of OsSADR1 with an NLS The domain analysis showed that OsSADR1 contains NLS sequences at the C-terminus, suggesting that the protein might be localized to the nucleus. To examine this hypothesis, the constructed 35S:OsSADR1-enhnanced yellow fluorescent protein (EYFP) vector was transfected into rice protoplasts and transient expression of OsSADR1 showed two significantly different patterns of subcellular localization among protoplasts. We found that approximately 62% of the transient signal was detected in the cytosol and 38% in the nucleus (Fig. 2b, left; Supplementary Fig. S3). Subsequently, we determined whether the frequencies of the subcellular localizations could be changed under abiotic stress, such as salt and/or drought, which induced expression of the gene (Lim et al. 2015). Interestingly, in 200 mM salt-treated rice protoplasts, 30% of transiently expressed OsSADR1 was detected in the cytosol, whereas 70% was in the nucleus (Fig. 2b, right). The findings support the hypothesis of movement of the OsSADR1 protein between the cytosol and nucleus under salt stress. To confirm our hypothesis, transfected rice protoplasts were treated with 200 mM salt stress for 30 min and then expanded via a multiphoton confocal laser scanning microscope (model LSM 780 META; Carl Zeiss). As shown in Fig. 2a and b, the florescent signals of OsSADR1 proteins were detected in both the cytoplasm and nucleus. Interestingly, we found that the cytoplasm aggregations of the OsSADR1 protein had moved into the nucleus (Fig. 2c;Supplementary Movie S1). These findings might provide a clue to understanding the molecular functions of the OsSADR1 proteins in the nucleus under salt stress. Fig. 2 View largeDownload slide Subcellular localization of the OsSADR1 protein in rice protoplasts. (a) Confocal images of 35S:OsSADR1–EYFP protein in rice protoplasts. Nuclear localization patterns (top panel) and cytosol localization patterns (bottom panel of 35S:OsSADR1–EYFP. (b) Percentage of fluorescent signals of the OsSADR1–EYFP protein in each of the non-treated and 200 mM salt-treated rice protoplasts. Each of the three repeated normal and NaCl-treated protoplasts were detected for analysis (n = 50). (c) Time-course of confocal images of OsSADR1–EYFP protein. The images were detected after 15 min of 200 mM salt treatment. The white arrows indicated the movement of OsSADR1–EYFP fusion protein in rice protoplasts. Fig. 2 View largeDownload slide Subcellular localization of the OsSADR1 protein in rice protoplasts. (a) Confocal images of 35S:OsSADR1–EYFP protein in rice protoplasts. Nuclear localization patterns (top panel) and cytosol localization patterns (bottom panel of 35S:OsSADR1–EYFP. (b) Percentage of fluorescent signals of the OsSADR1–EYFP protein in each of the non-treated and 200 mM salt-treated rice protoplasts. Each of the three repeated normal and NaCl-treated protoplasts were detected for analysis (n = 50). (c) Time-course of confocal images of OsSADR1–EYFP protein. The images were detected after 15 min of 200 mM salt treatment. The white arrows indicated the movement of OsSADR1–EYFP fusion protein in rice protoplasts. E3 ligase activity of OsSADR1 with a RING-HC domain The alignment between OsSADR1 and its orthologs showed a well-conserved RING-HC domain (Fig. 1). To determine whether the OsSADR1 protein functions as an E3 Ub ligase, we performed an in vitro ubiquitination assay. Immunoblot analysis with anti-Ub and anti-maltose-binding protein (MBP) showed that a high molecular mass of the poly-Ub chain of MBP-OsSADR1 while incubated with all the components (Fig. 3a, lane 4), whereas no chains were detected in the presence of empty MBP and in the absence of AtUBC10 or human E1 (Fig. 3a, lanes 1–3), respectively. It is believed that the RING domain was constructed by Cys and/or residues based on two zinc ions into an interwoven structure, and then acted as an E3 ligase (Freemont et al. 1991, Deshaies and Joazeiro 2009, Liu and Stone 2010). To confirm that OsSADR1 was assigned E3 ligase activity by the RING domain, a single amino acid substitution MBP–OsSADR1C168A, in which Cys168 was substituted with Ala168, was constructed (Supplementary Fig. S4A). In Fig. 3b, MBP–OsSADR1 shows high molecular mass ubiquitinated ladders, but no poly-Ub chain was observed with OsSADR1C168A using either antibody. Moreover, a time-course ubiquitination assay showed that MBP–OsSADR1 was detected in ubiquitinated ladders after 1 h and gradually reached its highest level at 4 h incubation (Supplementary Fig. S4B). These results showed that OsSADR1 harboring a RING-HC domain acted as an E3 Ub ligase. Fig. 3 View largeDownload slide In vitro ubiquitination assay of OsSADR1 E3 ligase. (a) The ubiquitination reaction contains E1 (human), AtE2 (Arabidopsis UBC10), maltose-binding protein-tagged OsSADR1 (MBP–OsSADR1), Ub and ATP. (b) The ubiquitination reaction of MBP, OsSADR1 and the single amino acid-changed mutant (MBP–OsSADR1C168A). Fig. 3 View largeDownload slide In vitro ubiquitination assay of OsSADR1 E3 ligase. (a) The ubiquitination reaction contains E1 (human), AtE2 (Arabidopsis UBC10), maltose-binding protein-tagged OsSADR1 (MBP–OsSADR1), Ub and ATP. (b) The ubiquitination reaction of MBP, OsSADR1 and the single amino acid-changed mutant (MBP–OsSADR1C168A). Interaction of OsSADR1 and each of the three substrate proteins in vitro and in vivo Many studies have revealed that RING finger proteins regulate their interaction proteins via ubiquitination (Pickart 2004, Lim et al. 2015, Park et al. 2015). To identify the substrate of OsSADR1, we performed a Y2H screening using a salt-treated rice root library. Three rice genes from these yeast colonies strongly interacted with OsSADR1, Os01g66120 (no apical meristem protein, OsSNAC2, Hu et al. 2008), Os11g47870 (GRAS family transcription factor domain-containing protein, OsGRAS-44, Zhao et al. 2016) and Os03g62790 (putative pirin-like protein, hereafter OsPIRIN), according to their α-galactosidase activity (Supplementary Fig. S5). To confirm their physical interactions, yeast GAL4 DNA-binding domain (BD)-tagged OsSADR1 and each of the interacting proteins, which tagged the yeast GAL4 activation domain, were co-transformed into the Y2H Gold strain and then separately spotted onto DDO and QDO/X/A media (Fig. 4a). Results showed that all the yeast colonies of the three interaction partners grew on DDO plates when co-expressed with both an empty DNA BD and BD-tagged OsSADR1. However, the three colonies of interacting proteins, which were co-expressed with an empty DNA BD, did not grow in the QDO/X/A medium, even when co-expressed with OsSADR1. In addition, the colonies of interacting partners with OsSADR1 demonstrated their α-galactosidase activity in the QDO/X/A medium. Fig. 4 View largeDownload slide Identification of the interaction substrate of OsSADR1 E3 ligase. (a) The full-length OsSADR1 and each of the interaction proteins, OsPIRIN (Os03g62790, putative pirin-like protein), OsSNAC2 (Os01g66120, no apical meristem protein) and OsGRAS-44 (Os11g47870, GRAS family transcription factor domain-containing protein), were cloned into pGBKT7 and pGADT7 vectors, respectively. Each of the positive substrates was co-transformed with OsSADR1 into the Y2H Gold yeast strain. Cells were dropped onto DDO and QDO/X/A medium. (b) Subcellular localization of interacting proteins. Each of the full-length genes was cloned into the 35S:EYFP vector and then transfected into rice protoplasts. (c) BiFC assay of the three substrate proteins with OsSADR1 E3 ligase. Each of the substrate proteins was cloned into pSPYNE(R), and OsSADR1 was cloned into pSPYCE(M). The cloned substrate proteins were co-transfected with OsSADR1 into rice protoplast. (d) In vitro pull-down assay of substrate proteins with OsSADR1. Each of the EYFP-tagged substrate proteins was separately infiltrated into N. benthamiana leaves. After 3 d, extracted substrate proteins were incubated with the His-Trx-tagged OsSADR1 protein for 1 h. Immune signals were detected with an anti-GFP and anti-His antibody. Fig. 4 View largeDownload slide Identification of the interaction substrate of OsSADR1 E3 ligase. (a) The full-length OsSADR1 and each of the interaction proteins, OsPIRIN (Os03g62790, putative pirin-like protein), OsSNAC2 (Os01g66120, no apical meristem protein) and OsGRAS-44 (Os11g47870, GRAS family transcription factor domain-containing protein), were cloned into pGBKT7 and pGADT7 vectors, respectively. Each of the positive substrates was co-transformed with OsSADR1 into the Y2H Gold yeast strain. Cells were dropped onto DDO and QDO/X/A medium. (b) Subcellular localization of interacting proteins. Each of the full-length genes was cloned into the 35S:EYFP vector and then transfected into rice protoplasts. (c) BiFC assay of the three substrate proteins with OsSADR1 E3 ligase. Each of the substrate proteins was cloned into pSPYNE(R), and OsSADR1 was cloned into pSPYCE(M). The cloned substrate proteins were co-transfected with OsSADR1 into rice protoplast. (d) In vitro pull-down assay of substrate proteins with OsSADR1. Each of the EYFP-tagged substrate proteins was separately infiltrated into N. benthamiana leaves. After 3 d, extracted substrate proteins were incubated with the His-Trx-tagged OsSADR1 protein for 1 h. Immune signals were detected with an anti-GFP and anti-His antibody. To identify the subcellular localization of interacting partners, 35S:mCherry-tagged recombinant proteins were constructed. Two proteins, OsSNAC2 and OsGRAS-44, were localized in the nucleus, whereas fluorescence signals of OsPIRIN protein were localized in the chloroplasts in rice protoplasts (Fig. 4b). To verify the physical interactions between OsSADR1 and each interacting protein, we performed the BiFC assay using 35S:HA-SPYCE(M) and 35S:cMyc-PYNE(R), which included the EYFP C-terminus and the EYFP N-terminus, respectively (Waadt et al. 2008), resulting in cytosol and nucleus target fluorescent signals that were detected in co-expression complexes [i.e. 35S:OsSADR1-SPYCE(M), 35S:OsSNAC2-SPYNE(R) and 35S:OsGRASS-44-SPYNE(R)]. However, the complex of OsSADR1/OsPIRIN–YFP was only detected in the cytosol. To examine further the dimeric complex of OsSADR1 and interacting partners, we first constructed bacterially expressed His-Trx-tagged OsSADR1 proteins. Then, OsSADR1-His-Trx protein and each of the EYFP-tagged interacting partners, OsSNAC2–EYFP, OsGRAS–44-EYFP and OsPIRIN–EYFP, were co-incubated with HisPur Cobalt resin. The bound proteins were eluted from the resin and finally an immunoblot analysis was performed using anti-His and anti-green fluorescent protein (GFP) antibodies (Fig. 4d). An in vitro pull-down assay showed that each of the interacting proteins was pulled down from the HisPur Cobalt resin by the His-Trx-tagged OsSADR1 protein. These results indicated that each partner protein could bind to OsSADR1-His-Trx, and that the OsSADR1 interaction with the three proteins, i.e. OsSNAC2, OsGRAS-44 and OsPIRIN, might regulate this cellular function in rice. OsSADR1 E3 ligase regulates protein levels of interacting proteins via the 26S proteasome system The Y2H, BiFC and in vitro pull-down assays showed that OsSADR1 interacted with the cytosol- and nuclear-localized proteins, OsSNAC2 and OsGRASS-44, respectively, and one chloroplast-localized protein, OsPIRIN. It is believed that the RING domain containing E3 ligase regulates their substrate protein via the 26S proteasome system (Smalle and Vierstra 2004, Vierstra 2009). Further experiments addressed the hypothesis that OsSADR1 ubiquitinates interacting proteins and is subjected to proteolytic degradation via the 26S proteasome system. To identify this, we first tested the in vitro ubiquitination of interacting partners with the OsSADR1 protein. The MBP-tagged OsSADR1 was incubated with each of the His-Trx-tagged interacting partners, i.e. OsSNAC2, OsGRASS-44 and OsPIRIN-His-Trx, and analyzed by immune blotting using anti-Trx antibodies (Fig. 5a). The results showed that each of the Ub-attached protein bands was found in the lanes with MBP–OsSADR1 combined with E1, E2 and Ub, whereas no Ub-attached band was observed in the lanes lacking E1, E2 or OsSADR1. Secondly, we attempted to measure the protein levels of interacting proteins in the presence or absence of OsSADR1 using an in vivo protein degradation assay. We separately expressed MBP–OsSADR1 and EYFP-tagged OsSNAC2, OsGRAS-44 and OsPIRIN, and then incubated them for various times, i.e. 0, 1 and 2 h, with or without MG132 (Fig. 5b). In the presence of OsSADR1, each of the partner protein levels was dramatically reduced at the 1–2 h reaction time (Fig. 5b, left panel). However, degradation of the interacting proteins did not occur in MG132-treated lanes. In addition, similar results showed that the protein levels of interacting proteins did not change when incubated with a single amino acid-substituted MBP–OsSADR1C168A (Fig. 5b, right panel). These results indicated that the protein–protein interactions of OsSADR1 and interacting proteins, OsSNAC2, OsGRAS-44 and OsPIRIN, leads to proteolysis via the 26S proteasome degradation pathway. Fig. 5 View largeDownload slide In vitro ubiquitination and in vivo degradation assay of substrate proteins by OsSADR1 E3 ligase. (a) In vitro ubiquitination assay of His-Trx-tagged substrate proteins with MBP–OsSADR1. Each interacting substrate protein was incubated with the MBP-tagged OsSADR1 protein, E1 (human) and AtUBC10 (Arabidopsis). Immune signals were detected with an anti-His antibody. (b) In vivo protein degradation assay of interacting proteins. Each of the cMyc-tagged recombinant substrate proteins, i.e. OsSNAC2-cMyc, OsGRAS-44-cMyc and OsPIRIN-cMyc, was constructed and separately infiltrated into N. benthamiana leaves. Three substrate proteins were extracted and then incubated at different time points (i.e. 0, 1, 2 and 3 h) with each MBP-tagged OsSADR1 or single amino acid-mutated OsSADR1C168A protein and analyzed by immunoblotting using an anti-cMyc antibody and anti-MBP antibody. MG132 was used as an inhibitor protein to block the 26S proteasome pathway. (c) The transcript levels of substrate genes. The 200 mM salt-treated rice samples were used for qRT-PCR. The transcript levels were standardized based on cDNA amplification with ActinII as an internal control. As compared with controls (0 h), asterisks represent statistically significant difference according to two-tailed Student’s t-test; *P < 0.05, **P < 0.01. Fig. 5 View largeDownload slide In vitro ubiquitination and in vivo degradation assay of substrate proteins by OsSADR1 E3 ligase. (a) In vitro ubiquitination assay of His-Trx-tagged substrate proteins with MBP–OsSADR1. Each interacting substrate protein was incubated with the MBP-tagged OsSADR1 protein, E1 (human) and AtUBC10 (Arabidopsis). Immune signals were detected with an anti-His antibody. (b) In vivo protein degradation assay of interacting proteins. Each of the cMyc-tagged recombinant substrate proteins, i.e. OsSNAC2-cMyc, OsGRAS-44-cMyc and OsPIRIN-cMyc, was constructed and separately infiltrated into N. benthamiana leaves. Three substrate proteins were extracted and then incubated at different time points (i.e. 0, 1, 2 and 3 h) with each MBP-tagged OsSADR1 or single amino acid-mutated OsSADR1C168A protein and analyzed by immunoblotting using an anti-cMyc antibody and anti-MBP antibody. MG132 was used as an inhibitor protein to block the 26S proteasome pathway. (c) The transcript levels of substrate genes. The 200 mM salt-treated rice samples were used for qRT-PCR. The transcript levels were standardized based on cDNA amplification with ActinII as an internal control. As compared with controls (0 h), asterisks represent statistically significant difference according to two-tailed Student’s t-test; *P < 0.05, **P < 0.01. Increased sensitivity response of OsSADR1-overexpressing Arabidopsis under salt and mannitol stress Our expression analysis showed that OsSADR1 was highly induced in salt-treated rice root samples (Fig. 1a). To study the function of OsSADR1 under salt stress, 35S:OsSADR1–EYFP-overexpressing Arabidopsis were constructed and then three independent transgenic lines (35S:OsSADR1-EYFP #1, #3 and #4) were selected, depending on the expression levels of OsSADR1 by RT-PCR (Supplementary Fig. S7). As shown in Fig. 6, phenotypic effects of OsSADR1-overexpressing lines exhibited a sensitivity response with 100–200 mM salt treatments (Fig. 6a). After 2 weeks of 100 mM salt treatment, EYFP-overexpressing control lines showed a greater root length than OsSADR1-overexpressing lines; 0.6 cm longer roots than those of the 35S-EYFP plants (Fig. 6b). Similarly, plants subjected to both 150 and 200 mM salt treatments also exhibited longer root lengths of 0.7 and 0.05 cm, respectively. For germination rates, each of the 35S:EYFP- and 35S:OsSADR1-overexpressing plants showed similar results; all seeds were germinated 2 d after seedlings (Supplementary Fig. S8A). However, 35S:EYFP-overexpressing lines had a 100% germination rate after 4 d of 100 mM salt treatment, whereas all seeds were germinated after 6–8 d under 150–200 mM salt-stressed conditions (Supplementary Fig. S8A). Fig. 6 View largeDownload slide Phenotypic effects of osmotic stresses on OsSADR1-overexpressing Arabidopsis. (a) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of salt during seedling growth. Three independent lines of Col-0/35S:OsSADR1 and wild-type plants were grown for 14 d on MS medium with 0, 100, 150 or 200 mM salt (scale bar = 2 cm). (b) Root length of wild-type and each of the three independent plants. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (c) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of mannitol during seedling growth. Plants were grown in 0, 100, 150 or 200 mM mannitol-containing medium (scale bar = 2 cm). (d) Root growth assay with various concentrations of mannitol. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Fig. 6 View largeDownload slide Phenotypic effects of osmotic stresses on OsSADR1-overexpressing Arabidopsis. (a) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of salt during seedling growth. Three independent lines of Col-0/35S:OsSADR1 and wild-type plants were grown for 14 d on MS medium with 0, 100, 150 or 200 mM salt (scale bar = 2 cm). (b) Root length of wild-type and each of the three independent plants. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (c) Phenotypes of wild-type and 35S:OsSADR1-overexpressing plants in response to various concentrations of mannitol during seedling growth. Plants were grown in 0, 100, 150 or 200 mM mannitol-containing medium (scale bar = 2 cm). (d) Root growth assay with various concentrations of mannitol. The data are presented as the mean ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). It is believed that plants respond to different types of osmotic stress, such as NaCl (ionic) and mannitol (non-ionic), via physiological and biochemical changes (Gangopadhyay et al. 1997, Ghuge et al. 2010). To verify the ionic or non-ionic effect in Arabidopsis, 35S:OsSADR1-overexpressing plants were treated with various concentrations of mannitol, i.e. 0, 100, 150 and 200 mM (Fig. 6d). OsSADR1-overexpressing lines showed low elongation of root length for plants supplemented with various mannitol concentrations compared with 35S:EYFP-overexpressing control plants (Supplementary Fig. S8B). Interestingly, no difference was observed in both the 150 and 200 mM-treated 35S:EYFP and 35S:OsSADR1-overexpressing plants 2 weeks after germination; however, we found a shorter root length of overexpressed plants with 100 mM salt treatment. In addition, OsSADR1-overexpressing plants exhibited slower germination rates than did the 35S:EYFP control plants. In each 150 mM-treated 35S:EYFP plant, 100% germination occurred at 6 d, whereas each of the OsSADR1-overexpressing lines showed 100% germination rates at 8 d. Similarly, the germination rate of 35S:EYFP transgenic seedlings was significantly higher than that of OsSADR1-overexpressing seedlings with 200 mM mannitol treatment. These results might suggest that heterologous overexpression of OsSADR1 in Arabidopsis enhanced germination and root length, and acted as both an ionic and a non-ionic osmotic stressor. Hyposensitive response of OsSADR1-overexpressing Arabidopsis to ABA treatment To evaluate the phenotypic effect of OsSADR1 transgenic lines under ABA stress, each of the control and transgenic plants was planted on Murashige and Skoog (MS) medium containing different concentrations (0. 0.5 and 1 μM) of ABA. On control plates, plants did not exhibit any difference in germination rates, whereas OsSADR1-overexpressing plants exhibited faster germination rates than did control plants. All the overexpressed seeds were germinated by 2 d, but control seeds were fully germinated after 6 and 12 d under 0.5 and 1 μM ABA, respectively (Fig. 7a). Similarly, we measured root length of control and overexpressing plants after 14 d and found a longer root length for overexpressing plants than for control plants under both 0.5 and 1 μM (Fig. 7b, c). To examine whether the hyposensitive response to ABA treatment of OsSADR1-overexpressing plants affected stomatal movement, we performed an ABA-dependent stomatal closure assay using each transgenic line (#1, #3 and #4) (Fig. 7d). In the non-treated condition, the average width of stomatal apertures showed no significant difference between OsSADR1-overexpressing plants and control plants. However, 0.5 μM ABA-treated OsSADR1 transgenic plants exhibited larger stomatal apertures, #1 (0.3679 ± 0.0427), #3 (0.4105 ± 0.0865) and #4 (0.386 ± 0.0544), compared with those of the control plants (0.2778 ± 0.0873). In addition, the stomatal aperture of transgenic lines showed similar results with tolerance phenotypes, #1 (0.2399 ± 0.074), #3 (0.2941 ± 0.0726) and #4 (0.2201 ± 0.0503), which was more than the wild-type plant (0.1393 ± 0.0298) under 1 μM ABA treatment, suggesting that OsSADR1-overexpressing plants expressed hyposensitivity phenotypes in response to ABA stress. Fig. 7 View largeDownload slide ABA tolerance phenotype of OsSADR1-overexpressing Arabidopsis. (a) Comparisons of germination rates of OsSADR1-overexpressing and wild-type plant seeds after exposure to different concentrations of ABA for 14 d. The data are presented as means ± SD (n = 25). (b) Phenotypes of wild-type and transgenic plants in response to various concentrations of ABA during seedling growth. Plants were grown in 0.5 or 1 μM ABA-containing medium (scale bar = 2 cm). (c) Root length assay of the wild type and each of the independent transgenic lines. The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (D and E) Stomatal aperture of OsSADR1-overexpressing and wild-type plants in response to different concentrations of ABA (0, 0.5 and 1 μM). Stomatal aperture was quantified by counting 25 guard cells in each treatment (scale bar = 5 μm). The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Fig. 7 View largeDownload slide ABA tolerance phenotype of OsSADR1-overexpressing Arabidopsis. (a) Comparisons of germination rates of OsSADR1-overexpressing and wild-type plant seeds after exposure to different concentrations of ABA for 14 d. The data are presented as means ± SD (n = 25). (b) Phenotypes of wild-type and transgenic plants in response to various concentrations of ABA during seedling growth. Plants were grown in 0.5 or 1 μM ABA-containing medium (scale bar = 2 cm). (c) Root length assay of the wild type and each of the independent transgenic lines. The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). (D and E) Stomatal aperture of OsSADR1-overexpressing and wild-type plants in response to different concentrations of ABA (0, 0.5 and 1 μM). Stomatal aperture was quantified by counting 25 guard cells in each treatment (scale bar = 5 μm). The data are presented as means ± SD (n = 25). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Overexpression of OsSADR1 confers drought tolerance in Arabidopsis It is believed that plants promote stomatal closure to conserve water by accumulating ABA in the guard cells under drought stress (Cominelli et al. 2010, Popko et al 2010, Wilkinson and Davies 2010, Lim et al 2015). In the present study, each of the three independent OsSADR1-overexpressing plants showed larger stomatal apertures than the control plants under ABA treatment (Fig. 7e). To identify the effect of OsSADR1 under water deficit conditions, 2-week-old transgenic and control plants were exposed to drought stress by withholding irrigation for 5 d. After the drought treatment, each of the plants was irrigated for 5 d and determined the survival rates (Fig. 8). In the plants grown under normal conditions, no significant differences were observed between the three independent and control plants. However, after 5 d of withholding irrigation, each of the transgenic plants showed lower tolerance phenotypes compared with the control plants. In addition, 5 d after rewatering, each of the independent transgenic plants showed lower survival rates, #1 (34.4 ± 3.85), #3 (22.5 ± 4.64) and #4 (16.9 ± 0.48), whereas 90% of the control plants survived. These data suggest that OsSADR1 E3 ligase acts as a negative regulator under drought conditions by the regulation of ABA-mediated stomatal closure. Fig. 8 View largeDownload slide Decreased water tolerance of OsSADR1-overexpressing Arabidopsis. Water loss rates of detached rosette leaves from 2-week-old control and OsSADR1 transgenic lines. The percentage and number of surviving plants per total number of tested plants are indicated under the photographs. The data are presented as the mean ± SD (n = 100). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Fig. 8 View largeDownload slide Decreased water tolerance of OsSADR1-overexpressing Arabidopsis. Water loss rates of detached rosette leaves from 2-week-old control and OsSADR1 transgenic lines. The percentage and number of surviving plants per total number of tested plants are indicated under the photographs. The data are presented as the mean ± SD (n = 100). Asterisks represent a significant difference in the mean value in OsSADR1-overexpressing plants compared with the control (**P < 0.01, *P < 0.05, t-test). Discussion Several studies have provided important evidence regarding the molecular functions of RING E3 ligase under various environmental stresses, such as high salinity, temperature and water loss conditions in rice. For example, the rice RING E3 ligase Oryza sativa Chloroplast Targeting RING Finger Protein 1 (OsCTR1) is a positive regulator that is specifically induced during drought stress through the regulation of protein levels and inhibition of trafficking of chloroplast genes, OsCP12 and OsPR1 (Lim et al. 2014). Moreover, overexpression of the drought-induced OsRDC1, O. sativa RING domain-containing protein in transgenic rice plants resulted in improved tolerance to water deficits (Bae et al. 2011). Overexpression of rice OsRHP1, which has a putative RING-H2 domain, conferred enhanced drought and salt tolerance (Zeng et al. 2014). However, high salinity stress induced the O. sativa Salt-Induced RING Finger Protein 1 (OsSIRP1) gene, which exhibited negative regulation during seed germination and root growth (Hwang et al. 2016). Numerous studies have provided evidence that RING E3 ligases are involved, as either positive or negative regulators, in the modulation of plant defense responses to diverse abiotic stresses. In fact, the role of RING E3 ligases as either positive or negative regulators in response to different stresses mainly depends on the nature of the target proteins. Therefore, it is necessary to identify target proteins to gain a better understanding of the biological functions of these RING E3 ligases in response to abiotic stresses. However, understanding the detailed regulatory roles of RING E3 ligases at the molecular and biochemical levels is often hindered by the lack of information on such target proteins (Cho et al. 2017). In the present study, OsSADR1 was highly induced under salinity, ABA and water loss conditions in rice root samples (Fig. 1a). In addition, heterogeneous overexpression of OsSADR1 in Arabidopsis produced a sensitivity response to both high salinity and mannitol treatments (Fig. 6a, b). In plants, as sessile organisms, a key plant signaling hormone, ABA, is accumulated under osmotic stress, such as high salinity and drought (Hubbard et al. 2010, Yoshida et al. 2014). This accumulation causes expression of the ABA-responsive genes, which include ABA RESPONSE ELEMENT (ABRE), at their promoters, and regulates the osmotic stress response (Wilson et al. 2014, Kazan 2015). As shown in Fig. 7, ABA-insensitive phenotypes were observed in each of the OsSADR1-overexpressing lines, despite the control plants exhibiting a hypersensitivity response (Fig. 7a–e). These results suggest that OsSADR1 acts as a regulator in an ABA-dependent manner. Several RING finger proteins have been reported to regulate transcription factors. First, the Arabidopsis RING-type E3 ligase KEEP ON GOING (KEG) protein regulates the ABA-responsive transcription factor ABSCISIC ACID-INSENSITIVE5 (ABI5) under ABA stress. Moreover, KEG-overexpressing Arabidopsis plants exhibited an insensitivity response under high concentration ABA treatment via protein degradation of ABI5 (Liu and Stone 2010). Secondly, the HECT domain containing Arabidopsis UPL5 regulates leaf senescence through protein degradation of WRKY53, which acts in a regulatory network influencing transcription in Arabidopsis (Miao and Zentgraf 2010). Thirdly, overexpression of the tomato SEVEN IN ABSENTIA3 (SINA3) ubiquitinates the NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, promotes its degradation and represses the R protein-mediated hypersensitive response (HR) cell death in N. benthamiana (Miao et al. 2016). Fourthly, the Arabidopsis RGLG2, which includes RING and a so-called copine (or von Willebrand factor type A) domain, interacts with ETHYLENE RESPONSE FACTOR53 (AtERF53), which is a drought-induced transcription factor, and leads to protein degradation under drought stress (Cheng et al. 2012). In the present study, the subcellular localization of the OsSADR1 protein was detected in both the cytosol and nucleus in rice protoplasts (Fig. 2). Interestingly, approximately 62% of the cytosol-localized fluorescent signals were detected, whereas 38% of the nuclear signals were identified in non-treated rice protoplasts (Fig. 2b, left panel). In contrast, 30% of the OsSADR1 signals occurred in the cytosol, whereas 70% of the nuclear signals were detected in high salinity-treated rice protoplasts (Fig. 2b, right, c). These data suggest that the dynamic movement of OsSADR1 into the nucleus might regulate some nuclear proteins, such as transcription factors, via action similar to E3 ligase under high salinity conditions. Our results indicated a functional relationship among OsSADR1 and three interacting proteins, of which two, OsSNAC2 and OsGRAS-44, are nuclear-localized proteins, and one, OsPIRIN, is a chloroplast-localized protein (Fig. 4). Interestingly, fluorescence of OsSADR1 and the interacting proteins was observed in the cytosol (although signals of both interactions with OsSNAC2 and OsGRAS-44 were also observed to be nuclear) when transfected with OsSADR1 under BiFC analysis (Fig. 4c). In addition, interacting proteins were ubiquitinated by OsSADR1 E3 ligase, leading to reduced protein levels mediated by the 26S proteasome pathway (Fig. 5). Taken together, these data indicated that OsSADR1 could ubiquitinate interacting proteins in the cytosol. However, the OsSADR1 protein exhibited translocation from the cytosol to the nucleus when protoplasts were exposed to high salinity (Fig. 2). We hypothesized two possibilities regarding the interaction between OsSADR1 and interacting proteins in the cytosol. One attractive possibility is that OsSADR1 might interact with nuclear proteins, i.e. OsSNAC2 and OsGRAS-44, in the nucleus and then translocate from the nucleus to the cytosol. Lim et al. (2013) indicated that the Golgi apparatus-localized OsHCI1 relocates to the nucleus under heat stress, and then the targeted nuclear proteins, including OsbHLH065, send the transcription factors to the cytosol. An alternative hypothesis is that the OsSADR1 protein directly inhibits trafficking of two nuclear proteins in the cytosol. Similar findings were observed in a hot pepper (Capsicum annuum) E3 ligase, Rma1H1, which inhibited trafficking of the PIP2;1 aquaporin protein from the endoplasmic reticulum to the plasma membrane, leading to tolerance to dehydration under degradation of the PIP2;1 protein (Lee et al. 2009). However, much work remains before the hypothesis can be ruled out. Rice SNAC (STRESS-RESPONSIVE NAM, ATAF and CUC) genes were induced by various environmental conditions, and expression of stress-related genes includes the NAC recognition site (NACR) at their promoters. For example, rice SNAC1, encoding a NAC transcription factor, binds to the NACR of the OsERD1 promoter, which then leads to improved drought and salt resistance in vegetative stages (Hu et al. 2006). Similarly, SNAC2 also exhibits transcriptional activity and binds to NACR-like sequences (Hu et al. 2008), and OsSNAC2-overexpressing transgenic plants showed improved tolerance to cold and high salinity stress, whereas a sensitivity response was observed under ABA treatment. Despite tolerance responses of SNAC2-overexpressing plants, the results of the present study showed that heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitivity responses, i.e. low germination rates and short root length, under high salinity and mannitol stresses (Fig. 6; Supplementary Fig. S8). Interestingly, the OsSADR1-overexpressing plants showed insensitive responses, whereas OsSNAC2-overexpressing plants showed hypersensitivity responses under ABA treatment (Hu et al. 2008). In the present study, relative expression levels of OsSNAC2 were gradually induced under high salinity stress for 12 h. However, we also found that OsSNAC2 and OsSADR1 proteins interacted in the cytosol and levels of OsSNAC2 protein gradually decreased when it was incubated with the OsSADR1 protein (Figs. 4b, 5b). Hu et al. (2008) reported that transcriptional levels of the six NACR-containing genes of the Scarecrow-like transcription factors (OsGRAS-39, Os11g04400), O-methyltransferase family proteins (OsOMT, Os09g17560), sodium/hydrogen exchanger 3 family proteins (OsNHX1, Os07g47100), GDSL-like lipase/acylhydrolase family proteins (Os05g11910), UDP-glucose 4-epimerase (OsUGE, Os09g35800) and heavy metal- associated proteins (Os01g74490) were induced in OsSNAC2-overexpressing transgenic rice. We examined the expression levels of these six genes, resulting in high induction of all genes in the salinity-treated rice protoplasts, but low expression of them in OsSADR1-EYFP-transfected rice protoplasts (Supplementary Fig. S9). These data suggest that inhibition of the function of OsNAC2 transcription factors via protein degradation leads to a sensitivity response under high salinity, but an insensitive response under ABA treatment in OsSADR1-overexpressing plants by inhibition of the expression of NACR-containing genes. Several studies have shown that a few NAC transcription factors undergo intensive post-translational regulation via ubiquitination (Olsen et al. 2004, Puranik et al. 2012). For example, the Arabidopsis transcription activator NAC1 was ubiquitinated by SINAT5 E3 ligase, which is a homolog of the Drosophila protein SINA, under auxin signals in plant cells (Xie et al. 2002). The nuclear-localized RHA2a, containing the RING-H2 type domain, interacts with ABSCISIC ACID RESPONSIVE NAC (ANAC) and regulates its transcription activity (Greve et al. 2003). Supporting these data, our study provides a potential mechanism between OsSADR1 and OsSNAC2 proteins in rice. First, the OsSNAC2 gene was up-regulated under high salinity stress, which led to the induction of downstream genes with an NACR (Fig. 9a, left). In our relative expression analysis, both OsSNAC2 and OsSADR1 showed gradual up-regulation under high salinity stress (Figs. 1a, 5c). However, OsSNAC2 exhibited decreased expression in the 24 h samples, despite continuing to induce expression of the OsSADR1 gene. This might suggest that after activation of downstream genes of OsSNAC2, the OsSNAC2 protein was regulated by OsSADR1 E3 ligase through translocation to the cytosol. Secondly, the OsSNAC2-overexpressing transgenic plants showed a tolerance response (Hu et al., 2008), whereas OsSADR1-overexpressing plants showed hypersensitivity under high salinity stress (Fig. 6). In addition, the OsNAC2 transgenic plants showed sensitivity under ABA treatment (Hu et al. 2008), whereas OsSADR1 transgenic plants showed an insensitive response (Fig. 7). Moreover, hypersensitivity phenotypes of OsSADR1-transgenic plants were observed under water deficit conditions compared with the control plants (Fig. 8). Interestingly, salt-related downstream genes with an NACR were not induced in OsSADR1-transfected rice protoplasts under high salt treatments, whereas they were highly induced in salt-treated non-transfected protoplasts (Supplementary Fig. S9). These results suggest that overexpression of OsSADR1 leads to degradation and subsequently OsSNAC2 transcription factor and, thus, could not activate its downstream genes with an NACR (Fig. 9b, right). Fig. 9 View largeDownload slide A model representing the regulation of the SNAC2 transcription factor between wild-type (WT) and OsSADR1-overexpressing plants during abiotic stresses. The upstream transcription factors may bind to the promoter of regulated OsSNAC2 genes and influence their transcription. In wild-type plants, translated OsSNAC2 protein moved to the nucleus and acted as a transcription factor of NACR-containing stress-induced target genes. After transcription of these genes, the OsSNAC2 transcription factor binds to OsSADR1 E3 ligase and is degraded by the 26S proteasome system in the cytosol. In OsSADR1 E3 ligase-overexpressing plants, translated OsSNAC2 protein directly binds to OsSADR1 before acting as a transcription factor, and then is degraded by the 26S proteasome system. Fig. 9 View largeDownload slide A model representing the regulation of the SNAC2 transcription factor between wild-type (WT) and OsSADR1-overexpressing plants during abiotic stresses. The upstream transcription factors may bind to the promoter of regulated OsSNAC2 genes and influence their transcription. In wild-type plants, translated OsSNAC2 protein moved to the nucleus and acted as a transcription factor of NACR-containing stress-induced target genes. After transcription of these genes, the OsSNAC2 transcription factor binds to OsSADR1 E3 ligase and is degraded by the 26S proteasome system in the cytosol. In OsSADR1 E3 ligase-overexpressing plants, translated OsSNAC2 protein directly binds to OsSADR1 before acting as a transcription factor, and then is degraded by the 26S proteasome system. We found hypersensitive phenotypes of OsSADR1-overexpressing plants even though the gene was highly induced by salt and drought stress (Figs. 1a, 6, 8). Moreover, OsSADR1-overexpressing plants were hyposensitive to ABA treatment, despite the up-regulated expression pattern of OsSADR1 under the treatment (Fig. 1a, 7). These observations raised some questions. (i) Why did transgenic Arabidopsis show hypersensitivity phenotypes to salinity and drought stress, despite the induction of OsSADR1 in plants under these stresses? (2) Why did the transgenic plants exhibit insensitivity to ABA even though OsSADR1 was highly induced under ABA treatment? Previously, a host of similar findings have been reported. For example, OsSIRP1 was highly induced under salinity stress, but its transgenic plants exhibited a hypersensitivity phenotype under high salinity stress (Hwang et al. 2016). In addition, another rice RING finger protein, OsDIS1 (O. sativa drought-induced SINA protein 1), showed up-regulation by drought treatment, but the OsDIS1-overexpressing plants exhibited a sensitivity response under drought stress (Ning et al. 2011). Similarly, the pepper gene, CaREL1 (Capsicum annuum RING E3 Ligase 1), was induced by ABA treatment and its transgenic plants had larger stomatal closures and higher germination rates and root lengths than did the control plants (Lim et al 2017). Our results confirmed that OsSADR1 interacts with the OsSNAC2 transcription factor, which induces the NACR-containing downstream genes under salt stress, which then led to regulation of protein levels (Figs. 4, 5a, b). The hypothesis that fine regulation of protein accumulation maintains cell balance might be able to explain inconsistencies in that SNAC2 induced under salinity is regulated by OsSADR1 induced by the stress in wild-type plants (Fig. 9, right). The down-regulation of the substrate genes (e.g. OsSNAC2) at 24 h after the salt treatment, despite high induction of OsSARD1 at 24 h, might support our hypothesis. However, constitutive overexpression of OsSADR1 in the transgenic plants might subsequently block the OsSNAC2 function to induce the downstream genes via degradation, resulting in the hypersensitive responses to salinity and drought, and insensitivity to ABA (Fig. 9, left). Our findings on the movement of OsSADR1 to the nucleus by salinity, degradation of transcription factors, especially that of OsSNAC2 via the Ub–26S proteasome system, and down-regulation of the downstream genes OsSNAC2 in OsSARD1-transfected protoplasts might provide important clues to molecular functions of the OsSADR1 gene under abiotic stress. In conclusion, OsSADR1 might act as a negative regulator in response to abiotic stresses, especially salinity, primarily depending on the hypersensitivity response of OsSADR1-overexpressing plants under abiotic stresses; however, further studies are required to elucidate this characteristic. Materials and Methods Plant materials and growth conditions Rice seeds (O. sativa L. ‘Donganbyeo’) were grown with commercial soil in a growth chamber (16/8 h light/dark photoperiod at 25/23°C with 70% relative humidity) for 2 weeks. The seedlings were treated with 200 mM salt and their roots were harvested at 1, 3, 6, 12 or 24 h. For drought stress, 2-week-old seedlings were positioned in the space between two pieces of tissue paper in the growth chamber, and then harvested at the same times as those in the salt treatment. For ABA treatment, 2-week-old rice seedlings were treated at different concentrations, 0.5 and 1 μM. Samples were ground using liquid nitrogen and immediately stored at –80°C until use. Gene expression analysis To identify the expression patterns of genes, total RNA was extracted using the TRIzol® reagent according to the manufacture’s protocol (Invitrogen). First-strand cDNA synthesis from total RNA (3 μg) was conducted using a cDNA Synthesis Kit (TAKARA-BIO INC.). For qRT-PCR, cDNA was used after being mixed with a TOPreal™ qPCR 2X Premix with SYBR green (Enzynomics) and PCR was performed using the CFX96 real-time PCR Detection System (BioRad Laboratories). OsActinII was used as an internal control. A list of all primers used in this study is provided in Supplementary Table S1. Confocal microscopy and imaging To identify the subcellular localization of O. sativa genes, OsSADR1, OsSNAC2, OsGRAS-44 and OsPIRIN cDNAs were cloned into EYFP vectors using salt-treated rice root samples. In addition, the full-length clone of OsSADR1 and each of the interacting genes were inserted into 35S:HA-SPYCE(M) and 35S:c-Myc-SPYNE(R) vectors for BiFC assay. Each of the constructed plasmids was transfected into the protoplast using a 40% polyethylene glycol (PEG) solution [40% PEG, 400 mM mannitol, 100 mM Ca(NO3)2] for 30 min at room temperature. Finally, the W5 solution was added to dilute the PEG solution and then discarded. The transfected protoplasts were resuspended with W5 and incubated overnight at room temperature. The transfected protoplasts were observed using a confocal microscope after 16 h of incubation. Fluorescent images were obtained using a multiphoton confocal laser scanning microscope (model LSM 510 META; Carl Zeiss) at the Korea Basic Science Institute Chuncheon Center, Korea. In vitro ubiquitination assay To construct the recombinant proteins, the full-length OsSADR1 gene was amplified and cloned into a pMAL-c5x vector (New England BioLabs). A single amino acid substitution (OsSADR1C168A) in the RING finger domain of OsSADR1 was generated using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene). Each recombinant MBP fusion protein was expressed by Escherichia coli strain BL21 (DE3) pLysS (Promega) and then purified by affinity chromatography using an amylose resin (New England BioLabs). For the in vitro self-ubiquitination assay, purified MBP–OsSADR1 was mixed with human E1 (Sigma-Aldrich), 6× His-tagged AtUBC10 and bovine Ub (Sigma-Aldrich), and then incubated in a ubiquitination reaction buffer [1 M Tris–HCl, pH 7.5; 40 mM ATP; 100 mM MgCl2; 40 mM dithiothreitol (DTT)]. After a 3 h incubation period at 30°C, the reaction was stopped by adding 6× SDS sample buffer, analyzed via 12% SDS–PAGE and then transferred to a nitrocellulose membrane. Immunoblot analyses were conducted using an anti-Ub antibody (Sigma-Aldrich) with a secondary goat anti-rabbit IgG peroxidase antibody (Sigma-Aldrich). Antibody detection was conducted using the chemiluminescent substrate SuperSignal® West Pico (ThermoScientific) for horseradish peroxidase (HRP). Photographs were obtained using ChemiDoc™ XRS+ (Bio-Rad). To confirm that OsSADR1 mediated ubiquitination of the three interacting proteins, OsSNAC2, OsGRAS44 and OsPIRIN-His-Trx, fusion proteins were affinity-purified and incubated together with purified MBP–OsSADR1 in the ubiquitination mixture for 3 h. The mixture was then subjected to SDS–PAGE and immunoblot analysis. Screening the interacting proteins via yeast two-hybrid system To identify the substrates of the OsSADR1 protein, yeast transformation and library screening were conducted in accordance with the recommended commercial procedures (Make Your Own ‘Mate & Plate’ Library system; Matchmaker Gold Yeast Two-Hybrid system; Yeastmaker Yeast Transformation System 2). A rice cDNA library in pGADT7-AD, which included the GAL4 activation domain, was generated from salt stress-treated rice root samples. Then, the full-length coding sequence of OsSADR1 was amplified and cloned into the pGBKT7-BD vector, which included the GAL4 DNA-binding domain as a bait strain. The yeast library screening was performed as previously described by Park et al. (2015). For screening, the BD-OsSADR1-transformed yeast strain was mated with salt-treated rice root library yeast strains. Then, positive interactions were selected on double dropout media lacking leucine and tryptophan (DDO/X/A) and supplemented with 40 μg ml−1 X-α-gal and 42 ng ml−1 aureobasidin A. After first screening, blue colonies were patched out onto higher stringency QDO/X/A, which lacked adenine, histidine, leucine and tryptophan. A total of 54 clones were identified as positive interacting colonies of OsSADR1, sequenced and verified by The Institute for Genomic Research (TRGR) rice annotation database. For each of the three genes, OsSNAC2, OsGRAS-44 and OsPIRIN, the full-length coding region was cloned into a pGADT7-AD vector and co-transformed into the Y2H Gold strain with the pGBKT7-OsSADR1 vector. Each of the co-transformed yeast cells was grown on DDO medium for 5 d at 30°C. Then, each of the clones was cultured in liquid DDO medium adjusted to OD600 = 1.0 and spotted onto DDO and QDO/X/A media. In vitro pull-down assay To observe the protein–protein interaction between OsSADR1 and partner proteins, i.e. OsSNAC2, OsGRAS-44 and OsPIRIN, MBP tagged OsSADR1 and His-Trx tagged each of the partner proteins, which were then expressed in E. coli strain BL21. After sonication of each protein expressed in BL21, bacterial lysate containing MBP–OsSADR1 was bound with amylose resin (New England BioLabs) in Poly-Prep Chromatography Columns (New England BioLabs). After incubation, amylose resin–MBP–OsSADR1 was mixed with or without lysates containing His-Trx-tagged partner proteins and rotated at 4°C for 2 h. Columns were washed five times using column buffer (20 mM Tris–HCl, 20 mM NaCl, 1 mM EDTA and 1 mM DTT). The isolated proteins were then mixed with the addition of 6× SDS sample buffer followed by 5 min of boiling at 95°C. Then, they were separated using 10% SDS–PAGE, transferred to a nitrocellulose membrane, and immune signals were detected using an anti-MBP antibody and anti-Trx antibody. Protein degradation assay To perform the degradation assay, the full-length cDNA of partner genes was cloned into a 35S:8cMyc vector. Each of the constructed vectors was transformed to Agrobacterium strain GV3101 and infiltrated in Nicotiana benthamiana leaves. After 3 d, leaves were homogenized and incubated with a Plant Total Protein Extraction Kit (Sigma-Aldrich), and each of the expressed proteins, including the total N. benthamiana proteins, was mixed with the MBP-tagged OsSADR1 protein or MBP-tagged OsSADR1C168A at a volume ratio of 1 : 1 at 4°C. The 26S proteasome inhibitor, MG132, was added to the corresponding samples to a final concentration of 50 μM. The reaction was stopped by the addition of sample buffer at different time points, i.e. 1–3 h. Immunoblotting was performed using anti-MBP and anti-cMyc antibodies. Arabidopsis transformation and treatment For the development of OsSADR1-overexpressing plants, the constructed 35S:OsSADR1-EYFP vector was transformed into Agrobacterium strain GV3101 and carried (ecotype Columbia, Col-0) according to the floral dip method (Clough 2005), with some modifications. Each of the T3 seeds of the transgenic plants (lines #1, #3 and #4) were selected for germination on half-strength MS agar plates containing 50 mg l−1 kanamycin. For identification of the phenotypic differences between control plants (35S:EYFP) and OsSADR1-overexpressing Arabidopsis lines under salt, mannitol and ABA stress, transgenic plants were treated with different concentrations: salt and mannitol (0, 100, 150 and 200 mM) or ABA (0, 0.5 and 1 μM). For measurement of the stomatal aperture, each of the 2-week-old ABA-treated transgenic plant leaves was examined under a Leica microscope (DM500). Supplementary Data Supplementary data are available at PCP online. Funding This research was supported by the Ministry of Education, Science, and Technology [Basic Science Research Program through the National Research Foundation of Korea (NRF) (2016R1A2B4015626)]. Disclosures The authors have no conflicts of interest to declare. References Bae H.S., Kim S.K., Cho S.K., Kang B.G., Kim W.T. ( 2011) Overexpression of OsRDCP1, a rice RING domain-containing E3 ubiquitin ligase, increased tolerance to drought stress in rice (Oryza sativa L.). Plant Sci.  6: 775– 782. Google Scholar CrossRef Search ADS   Balazadeh S., Siddiqui H., Allu A.D., Matallana-Ramirez L.P., Caldana C., Mehrnia M. ( 2010) A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence. Plant J.  62: 250– 264. Google Scholar CrossRef Search ADS PubMed  Basu S., Ramegowda V., Kumar A., Pereira A. ( 2016) Plant adaptation to drought stress. F1000Res . 5: 1554. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations BD binding domain BiFC bimolecular fluorescence GRAS-44 GRAS family transcription factor domain-containing protein 44 DTT dithiothreitol EYFP enhanced yellow fluorescent protein MBP maltose-binding protein MS Murashige and Skoog NAC NAM-ATAF1,2-CUC2 NACR NAC recognition site NLS nuclear localization signal PEG polyethylene glycol PIRIN putative pirin-like protein qRT-PCR quantitative reaal-time PCR RING Really Interesting New Gene SADR1 salt-, ABA- and drought-induced RING finger protein 1 SNAC stress-induced no apical meristem protein 2 Ub ubiquitin Y2H yeast two-hybrid © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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