Introduction

The world population is expected to reach approximately 10 billion by 2050 (Tilman et al. 2011). Therefore, increasing crop productivity is critical for supporting the growing human population. Crop productivity is severely affected by abiotic stresses such as high salinity, drought, heat, and cold. Approximately 20% of irrigated agricultural land is damaged by high salinity (Pitman and Läuchli 2002). The increase in soil salinity in these areas is attributed to several factors such as irrigation with saline water, low precipitation, high surface evaporation, and weathering (Jamil et al. 2011). Rice (Oryza sativa) is more sensitive than other cereal crops, such as wheat (Triticum aestivum), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), to salinity stress. Rice yield decreases by 12% in more than 5% barley under 30 mM NaCl conditions (Chinnusamy et al. 2005). Salinity affects plant physiological and metabolic processes such as plant weight, height, yield, and root length (Liang et al. 2018). Plants exposed to salinity stress encounter osmotic stress first and then suffer from water imbalance and leaf senescence (Munns et al. 2006, 2008). In addition, ionic stress occurs in plants due to the accumulation of Na+, which interrupts protein synthesis, photosynthesis, and enzyme activity under salinity. Furthermore, osmotic stress and ionic toxicity can cause secondary stress by accumulating reactive oxygen species (ROS). Abiotic stresses such as salinity and drought induce ROS formation. Increased ROS levels cause the degradation of macromolecules, such as lipids, proteins, RNA, and DNA, leading to plant death (Mittler 2002). Therefore, plants have developed antioxidant defense systems using ROS-scavenging mechanisms under unfavorable conditions. To sustain redox homeostasis under stress, antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are used to relieve ROS (Hossain and Dietz 2016). In addition, osmotic stress due to salinity triggers the biosynthesis of the phytohormone abscisic acid (ABA), which regulates plant physiological processes in response to various abiotic stresses (Finkelstein et al. 2002; Xiong et al. 2002). High salinity promotes ABA accumulation by activating the expression of synthetic pathway genes in plants (Wei et al. 2020; Jia et al. 2002). ABA not only mediates salinity signals but also protects cells from salt stress by modulating the expression of stress-responsive genes (Danquah et al. 2014; Yoshida et al. 2014). For example, OsMADS25 overexpression enhances salt tolerance, partially depending on the ABA-mediated regulatory pathway in rice (Xu et al. 2018). Plants alter intracellular metabolic processes via changes in protein levels, such as by altering the synthesis and degradation of proteins or regulating transcription factors to avoid stress. The ubiquitin-26S proteasome system (UPS) is a core mechanism for controlling all basic cellular processes such as metabolism, protein degradation, and cell development and death (Ciechanover. 2006; Schwartz and Ciechanover 2009; Tai and Schuman. 2008; Varshavsky 2005). In this system, the conjugation cascade requires three classes of enzymes: E1 (a ubiquitin-activating enzyme in humans), E2 (a ubiquitin-conjugating enzyme in Arabidopsis thaliana), and E3 (a ubiquitin ligase enzyme) (Hochstrasser. 1996; Vierstra 2009). E3 ligase, which is a core enzyme of the UPS reaction, can be classified into two classes: REALLY INTERESTING NEW GENE (RING) and HOMOLOGOUS TO E6-AP COOH TERMINUS (HECT). This is based on the movement of activated ubiquitin molecules from E2 to substrate proteins (Moon et al. 2004). Approximately 425 RING E3 ligases have been identified in rice (Lim et al. 2013). The basic sequence of the RING domain, Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)-Cys-X2-Cys-X(4–48)-Cys-X2-Cys (where X is any amino acid) mediated by the binding of two zinc atoms, is well known (Goddard et al. 1991). RING E3 ligases function as important regulators in the response to various abiotic stresses, such as drought, salt, and extreme temperatures (Sadanandom et al. 2012; Stone 2019). A previous study has confirmed the transcriptional patterns of 47 RING finger proteins in rice under different abiotic stress conditions (Lim et al. 2013). Among these, OsSIRHC-2 (O. sativa salt-induced RING finger protein HC-2), a C3HC4 RING-type protein, is localized in the cytosol and shows E3-ubiquitin ligase activity in vitro. In addition, this gene is upregulated under salinity and drought stress (Lim et al. 2013); however, its molecular functions are yet to be fully elucidated. In this study, we further investigated the molecular functions and characterization of the OsSIRHC-2 gene using a biochemical assay for detecting Na+ accumulation. The expression of Na+ transporter genes, and phenotype analysis of OsSIRHC-2-overexpressing plants under salinity.

Materials and Methods

Rice Transgenic Plants

The coding sequence of OsSIRHC-2 (LOC_Os08g040130) was amplified from rice (Oryza sativa L. Spp. Japonica, cv. Dongjin byeo) cDNA using the Q5 polymerase 2X Master Mix (NEB, Massachusetts, United States) and appropriate primers. The PCR products were cloned into the pGA3425 binary plasmid vector tagged with the pUbi promoter. OsSIRHC-2-overexpressing plants were generated at the National Institute of Agriculture Sciences (Suwon, Korea). The cloned pUbi:OsSIRHC-2 recombinant plasmid was transferred into Agrobacterium tumefaciens strain EHA105 using the heat-shock method (Froger and Hall 2007). Rice plants were transformed using a previously described method (Toki et al. 2006). Dongjin-byeo rice seeds were sterilized using 2% sodium hypochlorite with Tween 20 (Sigma-Aldrich) and grown on 2N6D medium containing 0.4% phytagel at 30 °C for 5 days. Precultured calli were infected with EHA105 cells containing the pUbi:OsSIRHC-2 plasmid and cultured at 25 °C for 3 days in the dark. The callus was washed five times with sterilized water containing 500 mg L−1 carbenicillin and transferred to 2N6D medium containing 50 mg L−1 hygromycin and 400 mg L−1 carbenicillin for 1 month under light. After 1 month of incubation, resistant calli were transferred to a shoot induction medium containing 0.1 mg L−1 naphthaleneacetic acid (Duchefa Biochemie, Haarlem, Netherlands) and 2 mg L−1 kinetin (Duchefa Biochemie) at 32 °C for 3 weeks under light. Transformed plants were grown in a well-controlled greenhouse. Seeds of two independent T3 homozygous plants were selected for further study.

Plant Growth Conditions and Phenotypic Assay

OsSIRHC-2-overexpressing and wild-type (WT) seeds were germinated at 30 °C in the dark for 2 days. The germinated seeds were transferred to plastic containers containing half-strength Kimura B nutrient solution (pH 5.6) and incubated in a growth chamber under 16 h/8 h light/dark conditions with 70% relative humidity. Ten-day-old seedlings were treated with 100 mM sodium chloride (NaCl). Phenotype characteristics, including shoot length, root length, shoot fresh weight, and fresh root weight, were measured in three biological replicates (n = 15). Plant leaf and root tissues were harvested, ground using liquid nitrogen, and quickly stored at − 70 °C for further studies.

ABA Sensitivity Assays

To determine the sensitivity of OsSIRHC-2-overexpressing plants to ABA at the germination stage, we grew seeds of OsSIRHC-2-overexpressing and WT plants in half-strength MS medium containing 0, 1, and 3 μM ABA. Seeds were grown under 16 h/8 h light/dark conditions in a growth chamber. The germination rate was determined after 5 days. When seeds were sprouted the measurement was carried out assuming that they had germinated.

RNA Extraction and Gene Expression Analysis

Total RNA was extracted using TRIzol® (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. Total RNA (1 µg) was used for cDNA synthesis using the PrimeScript™ 1st-Strand cDNA Synthesis Kit (Takara Bio, Ohtsu, Japan). Quantitative PCR (qPCR) was conducted using the CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), SYBR® Green TOP Real qPCR 2X PreMIX (Enzynomics™, Daejeon, Korea), 25 ng of cDNA, and appropriate gene-specific primers. Primers were designed using Beacon Designer™ (PRIMER Biosoft, Palo Alto, CA, USA) and synthesized by Macrogen (Seoul, Korea). The amplification conditions were as follows: pre-denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 10 s, annealing at the appropriate annealing temperature for the primers, and extension at 70 °C for 30 s. OseEF-1a (LOC_Os03g08010) was used as the internal control. Relative expression levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001).

In Vitro Ubiquitination Assay of OsSIRHC-2

To determine the E3 ligase activity of OsSIRHC-2, the full-length OsSIRHC-2 gene was amplified using Pfu Turbo DNA polymerase (Strategene, LA Jolla, CA, USA), and the PCR products were cloned into the pMAL-c5X-MBP tagged vector (New England BioLabs, Ipswich, MA, USA). To generate a point mutation, an amino acid substitution (OsSIRHC-2C269A) in the RING finger domain was cloned into the pMAL-c5X-MBP tagged vector. Recombinant MBP-OsSIRHC2 and the mutated proteins were expressed in Escherichia coli strain BL21 (DE3) and purified by affinity chromatography using an amylose resin column (New England BioLabs). E2 (Arabidopsis UBC10) was cloned into a pET-28a (+)-6 X-His-tagged vector (Novagen, Gibbstown, NJ, USA) and purified using the Ni–NTA Purification System (Invitrogen Life Technologies). For the in vitro ubiquitination assay, purified MBP-OsSIRHC-2 and mutated MBP-OsSIRHC-2C269A (250 ng) each was mixed with 50 ng human E1 (Sigma-Aldrich), 250 ng purified E2 (AtUBC10, At5g53300), bovine ubiquitin (Sigma-Aldrich), and reaction buffer (pH 7.5, 50 mM Tris–HCl; 10 mM MgCl2; 0.05 mM ZnCl2; 0.2 mM DTT; 1 mM ATP; 10 mM phosphocreatine; and 0.1 unit of creatine kinase), and the reaction was incubated at 30 °C for 3 h. For immunoblotting, samples were mixed with 5 × sample loading buffer and heated at 95 °C for 5 min. Samples were loaded onto a 7% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and the protein bands were transferred onto nitrocellulose membranes. Ubiquitinated OsSIRHC-2 proteins were detected by protein gel blotting using primary ubiquitin antibodies (Sigma-Aldrich) and secondary anti-rabbit IgG goat antibodies. The cells were photographed using a ChemiDoc™XRS imaging system (Bio-Rad).

Rice Protoplast Extraction and Subcellular Localization

Rice seeds were germinated and grown in half Kimura B solution in a growth chamber at 30 °C/25 °C (16 h/8 h light/dark) with 70% relative humidity for 14 d. Protoplasts were isolated from 2-week-old WT leaves. Leaves from approximately 30 seedlings were chopped with a razor blade and incubated in 30 mL of filtered enzyme solution containing 1% cellulase R-10 (Yakult Honsa Co. Ltd, Tokyo, Japan), 0.25% macerozyme R-10 (Yakult Honsa), 500 mM mannitol (Sigma-Aldrich), 1 mM CaCl2 (Sigma-Aldrich), 0.1% BSA (Sigma-Aldrich), and 10 mM MES (Duchefa Biochemie) at pH 5.6, for 5 h in the dark with gentle rocking (40 rpm) at 25 °C. The mixture was then filtered using 40-μm nylon mesh filters (SPL, Gyeonggi-do, Korea). The protoplasts were collected by centrifugation at 300×g for 4 min. Settled protoplast pellets were resuspended in 30 mL of W5 solution containing 154 mM NaCl (Duchefa Biochemie), 125 mM CaCl2 (Sigma-Aldrich), 5 mM KCl (Sigma-Aldrich), 5 mM glucose (Duchefa Biochemie), and 1.5 mM MES (Duchefa Biochemie), and then centrifuged at 300 × g for 4 min. After first washing with W5 solution, the pelleted protoplasts were resuspended by MMG solution [0.4 M mannitol (Duchefa Biochemie), 15 mM MgCl2 (Sigma-Aldrich), and 4.7 mM MES (Duchefa Biochemie), pH 5.6]. OsSIRHC-2 was cloned into the 35S:EYFP vector and the recombinant vector (10 μg) was transfected into protoplasts with the MMG solution and mixed carefully. The mixture was added to a prepared PEG solution [40% PEG 4000 (Sigma-Aldrich), 100 mM Ca (NO3)2, and 400 mM mannitol] and gently mixed. The prepared samples were then incubated at 25 °C for 30 min in the dark. After incubation, the protoplast pellets were collected by centrifugation at 300×g for 4 min. The pelleted protoplasts were gently resuspended in 1 mL W5 solution and incubated for 16 h overnight at room temperature in the dark. Protoplasts were observed using a confocal laser-scanning microscope at the Central Laboratory of Kangwon National University (Chuncheon, Korea). Photographs were acquired using a ZNE (ZEISS, Oberkochen, Germany).

Visualization of Na+ Accumulation

Na+-ion-specific fluorescent dye CoroNa Green solution (Invitrogen Life Technologies) was used to visualize Na+ accumulation in the roots. Ten-day-old seedlings of OsSIRHC-2-transgenic and WT plants were grown under 0 or 100 mM NaCl conditions for 24 h. The root tips were cut into approximately 1.5-cm pieces and washed with distilled water. Root tips were then stained with 25 μM CoroNa Green staining solution dissolved in dimethyl sulfoxide and incubated for 3 h at room temperature. After washing the tissue with distilled water, the prepared samples were placed on a glass slide, and Na+ accumulation was detected using green fluorescence signal SR-CLSM (LSM880, Carl Zeiss). Photographs were acquired using ZNE (ZEISS).

Measurement of Chlorophyll Content

The chlorophyll content of 10-day-old rice leaves was measured. Fresh rice leaf tissue (50 mg) was ground in liquid nitrogen and 80% acetone (1.5 mL) was added to it. The mixed samples were then incubated in the dark at 4 °C for 30 min. The incubated samples were collected by centrifugation at 13,000×g and 4 °C for 15 min, and the supernatants were transferred to fresh microtubes. The absorbance of the prepared samples was measured at 663 nm and 645 nm using a microplate reader (SPECTROstar Nano, BMG Labtech), and the chlorophyll content was calculated as previously described (Ni et al. 2009).

Measurement of H2O2, Proline, and Soluble Sugar Content

The H2O2 content was measured as previously described (Gay and Gebicki 2000). Rice root (200 mg) and leaf (100 mg) samples were mixed with 0.1% trichloroacetic acid (Sigma-Aldrich), incubated at 4 °C for 30 min, and centrifuged at 12,000×g at 4 °C for 30 min. The supernatant was transferred to a new microcentrifuge tube and reacted with solution A (2.5 M H2SO4 and 25 mM ammonium ferrous II sulfate) and solution B (100 mM sorbitol and 125 μM xylenol orange). The absorbance of the reacted samples was measured at 560 nm by using a microplate reader (SPECTROstar Nano; BMG LabTech). The proline content of the rice root and leaf tissues was measured as previously described (Carillo and Gibon 2011). Leaf and root tissues (100 mg) were mixed with 1 mL of 70% ethanol and incubated at room temperature for 20 min. Thereafter, 50 μL of the mixture was mixed with 100 μL of the reaction solution [20% ethanol and 1% ninhydrin (w/v) in 60% acetic acid (v/v)] and heated at 95 °C for 20 min in a water bath. The prepared samples were immediately cooled to room temperature and the absorbance was measured at 520 nm using a microplate reader (SPECTROstar Nano). The soluble sugar content was measured as previously described (Song et al. 2011). Rice leaf and root tissues (100 mg) were mixed with 1 mL 80% ethanol and incubated at 80 °C for 30 min. Thereafter, the volume was adjusted to 10 mL using 80% ethanol. To determine the soluble sugar content, 1 mL of the extract was mixed with 5 mL anthrone reagent dissolved in 0.2% H2SO4. The mixed samples were then incubated at 95 °C for 15 min. The sample was then cooled to room temperature, and the absorbance was measured at 620 nm using a microplate reader (SPECTROstar Nano).

Measurement of Ionic Concentrations

Ten-day-old seedlings of WT and OsSIRHC-2-overexpressing plants were grown in a growth chamber (16 h/8 h, light/dark cycle) with 70% relative humidity. Both the plants were treated with 0 or 100 mM NaCl for 24 h. Na+ and K+ concentrations in the leaves and roots were measured using inductively coupled plasma-optical emission spectroscopy (Optima 7300 dv, PerkinElmer) at the Central Laboratory of Kangwon National University (Chuncheon, Korea). Experiments were performed using three biological replicates (n = 15).

Yeast-Two Hybrid (Y2H) Screening

To screen for the substrate proteins of OsSIRHC-2, The Matchmaker® Gold Yeast Two-Hybrid System was used as previously described (Chapagain et al. 2018). OsSIRHC-2-BD was transformed into a yeast strain and mated with a NaCl-treated rice library in yeast strains. To identify protein–protein interactions, the full-length genes of OsHKT1;4 (LOC_Os04g51830, sodium transporter), OsHKT1;5 (LOC_Os01g20160, sodium transporter), OsAKT1 (LOC_Os04g45990, potassium transporter), and OsHAK7 (LOC_Os07g47350, potassium transporter) were transformed into a pGADT7-AD vector and co-transformed with OsSIRHC-2-BD into the Y2H Gold yeast strain. Co-transformed yeast cells were incubated on a double dropout (DDO) medium lacking leucine and tryptophan for 3–5 days at 30 °C. Each of the colonies was then grown in liquid DDO medium to OD600 = 1.0, and dotted onto DDO/AbA and lacking leucine, tryptophan, adenine, and histidine medium QDO/X-α-gal/AbA for 3 days at 30 °C.

Statistical Analysis

All data were evaluated using a two-tailed Student’s t-test (Millar. 2001). The significance of the data was determined at three different P-value values (95%, P < 0.05; 99%, P < 0.01; and 99.9%, P < 0.001). The resulting data were analyzed using Microsoft Excel 2016 (Microsoft).

Results

Expression Patterns of OsSIRHC-2 Under Abiotic Stress Conditions

In a previous study, OsSIRHC-2 was found to be highly expressed under saline and drought conditions (Lim et al. 2013). We evaluated the expression patterns of OsSIRHC-2 in rice leaves and roots in response to treatment with 100 mM NaCl, 20% PEG, 42 °C heat, 4 °C cold, and 100 µM ABA at various time points (0, 0.5, 1, 6, 12, and 24 h) (Figs. 1, S1). The expression of OsSIRHC-2 increased after 1 to 24 h in the leaves (4.8-fold) and roots (3.7-fold) under salinity stress (100 mM NaCl) (Fig. 1A, B). Under 20% PEG, OsSIRHC-2 expression increased up to 2.0-fold in leaves and 2.4-fold in roots at 24 h (Fig. S1A, B). In response to heat stress (42 °C), OsSIRHC-2 expression increased by 1.9-fold and 1.9-fold in leaves and roots, respectively, at 24 h (Fig. S1C, D). Under cold treatment (4 °C), OsSIRHC-2 expression increased 1.8-fold and 1.7-fold in the leaves and roots, respectively, at 24 h (Fig. S1E, F). Under 100 µM ABA treatment, the transcript level of OsSIRHC-2 increased up to 5.2-fold and 14.4-fold in leaves and roots, respectively, at 24 h (Fig. S1G, H).

Fig. 1
figure 1

HYPERLINK "sps:id::fig1||locator::gr1||MediaObject::0" Expression patterns of OsSIRHC-2 in the leaves and roots of WT plants (Oryza sativa cv. Dongjin-byeo) under salinity and PEG (20%) treatment. q-PCR analysis of OsSIRHC-2 expression in 10-day-old seedlings was examined after 0-, 0.5-, 1-, 6-, 12-, 24-h treatment with 100 mM NaCl A and B expression of OsSIRHC-2 in leaves and roots after 100 mM NaCl treatment, respectively. All q-PCRs were conducted with three biological replicates at each time point (ten plants were measured for each replicate). Gene expression levels were normalized to those of OseEF1-a. Student's t-test: *P < 0.05 and **P < 0.01

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OsSIRHC-2 is a C3HC4-Type RING E3 Ubiquitin Ligase

To identify the function of OsSIRHC-2, we performed a domain analysis and an in vivo ubiquitination assay. Domain analysis of OsSIRHC-2, which encodes 401 amino acids, was conducted using the National Center for Biotechnology Information Conserved Domain Database. We compared the C3HC4-type RING domains of OsSIRHC-2 and its orthologs in S. bicolor (Sb07g002700), Brachypodium distachyon (Bradi3g14420), and Arabidopsis thaliana (AT1G79380) and found that the RING domains were highly conserved (Fig. 2A). OsSIRHC-2 harbors a C3HC4-type RING domain (352–393 residues). In a previous in vitro ubiquitination assay, OsSIRHC-2 displayed E3-ubiquitin ligase activity (Lim et al. 2013). To confirm the E3-ubiquitin ligase activity of OsSIRHC-2 and its mutant, OsSIRHC-2C370A, in which the third cysteine residue of the RING structure was replaced with alanine, we performed an in vitro ubiquitination assay using an empty vector as a negative control. The results showed that the negative control and mutant OsSIRHC-2C370A did not show any antibody response to the polyubiquitin chain (Fig. 2B). In contrast, OsSIRHC-2 exhibited a clear polyubiquitin chain. These results demonstrated the E3-ubiquitin ligase activity of the RING domain of OsSIRHC-2.

Fig. 2
figure 2

Molecular characterization of OsSIRHC-2. A Domain analysis and multiple amino acids alignment of OsSIRHC-2 and its orthologues in Sorghum (Sb07g002700), Brachypodium (Bradi3g14420), and Arabidopsis (AT1G79380). B In vitro ubiquitination assay of OsSIRHC-2 and OsSIRHC-2C269A as a variant with a point mutation resulting in amino acid change. C Subcellular localization of 35S:OsSIRHC-2-YFP. The 35S:EYFP-fused OsSIRHC-2 was observed in rice protoplasts under normal conditions. The vector 35S:YFP was used as empty vector control. Scale bar = 5 μm

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OsSIRHC-2 is Localized in the Cytosol

In a previous study, OsSIRHC-2 was localized in the cytosol of heterogeneous Nicotiana tabacum (Lim et al. 2013). To identify the subcellular localization of OsSIRHC-2 in rice, we used the 35S:OsSIRHC-2-YFP recombinant vector and 35S:EYFP, localized in the cytosol, as the positive control (Fig. 2C). Similar signals were observed for the positive control and OsSIRHC-2. These results clearly showed that OsSIRHC-2 is localized in the cytosol.

Characteristic of OsSIRHC-2-Overexpressing Plants to Salinity Stress

To evaluate the insensitivity of OsSIRHC-2-overexpressing plants to salt stress, the phenotypes of the rice transgenic lines and the WT were analyzed. First, q-PCR analysis of five independent overexpressing transgenic rice lines was performed and the results were compared with those of the WT plants (Fig. S2). The results were significantly higher (approximately 3- to 160-fold) in the transgenic lines than in WT. Therefore, we chose both the #1–2 (150-fold) and #7–2 (70-fold) overexpressing lines for further studies. To evaluate how OsSIRHC-2-overexpression affects plants, we compared the phenotypes of OsSIRHC-2-overexpressing and WT plants. Phenotypic analyses, such as the lengths and fresh weights of shoots and roots, and the number of roots, were evaluated after growth in Kimura B solution for 10 days (Fig. 3A). Transgenic plants exhibited shorter lengths and lower shoot weights than WT plants under control conditions (0 mM NaCl) (Fig. 3B, C). There was no significant difference in root length between the transgenic and WT plants, but root weights were higher in transgenic plants than in WT plants under control conditions (Fig. 3D, E). Under 100 mM NaCl, higher shoot and root weights were observed in transgenic than in WT plants (Fig. 3C, E). Although shorter shoot lengths were observed in transgenic than in WT plants, no significant difference in root length was observed between the two (Fig. 3B, D). Under 0 and 100 mM NaCl, a greater number of roots occurred in the transgenic than in the WT plants (Fig. S3). Next, we evaluated the phenotypes under abiotic stress, 20% PEG, and heat (42 °C) and cold (4 °C) conditions. However, under the three abiotic stresses, there were no significant differences in the phenotypes between the transgenic and WT plants (data not shown). These results indicate that the overexpression of OsSIRHC-2 enhanced insensitivity to 100 mM NaCl but retarded shoot growth under normal conditions.

Fig. 3
figure 3

Phenotypes of OsSIRHC-2-overexpressing plants. A Representative images of two independent OsSIRHC-2-overexpressing lines (OE-1 and OE-7) and wild-type (WT) plants. 2-week-old OsSIRHC-2-overexpressing and WT plants treated with 0 or 100 mM NaCl using three biological replicates (fifteen plants were measured for each replicate). Pictures were taken after 1 week of treatment with NaCl. Scale bar = 5 cm. B, C Length and weight of shoots were measured in transgenic and WT plants under 0 and 100 mM NaCl treatment, respectively. D, E Length and weight of roots were measured in transgenic and WT plants under 0 and 100 mM NaCl treatment, respectively. Student’s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001; ns, nonsignificant

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It is believed that salinity stress negatively affects the chloroplast membrane and chlorophyll content, thereby reducing photosynthetic efficiency (Ali et al. 2004). Chlorophyll content was measured in the leaves of the transgenic and WT plants grown in 0 and 100 mM NaCl. Chlorophyll content was significantly higher in transgenic than in WT plants under both conditions (Fig. 4A). We also measured H2O2, soluble sugar, and proline contents in the leaves and roots of transgenic and WT plants grown in 0 and 100 mM NaCl. High salinity induces ROS such as H2O2 in plants (Pang and Wang 2008). Therefore, plants have developed various molecular mechanisms to avoid ROS stress and prevent their accumulation in inner cells. In addition, soluble sugars and proline accumulation play important roles in various abiotic stresses (Hayat et al. 2012; Nemati et al. 2011). There were no significant differences in H2O2, soluble sugar, and proline contents in the leaves or roots with 0 mM NaCl (Fig. 4B–G). However, transgenic plants exhibited significantly lower H2O2 content than WT plants in both leaves and roots under salinity (Fig. 4B, C), suggesting that WT plants were more damaged than transgenic plants at 100 mM NaCl. Soluble sugar and proline contents were higher in transgenic plants than in WT plants at 100 mM NaCl (Fig. 4D–G). Therefore, a greater accumulation of soluble sugars and proline could make transgenic plants more insensitive to salinity stress than WT plants.

Fig. 4
figure 4

Contents of A chlorophyll, B, C H2O2, D, E proline, and F, G soluble sugars in OsSIRHC-2-overexpressing and WT plants under 0 and 100 mM NaCl treatment. Chlorophyll content was measured in only leaves but the other contents in both leaves and roots. Experiments were performed with three biological replicates (ten plants were measured for each replicate). Student’s t-test: *P < 0.05, **P < 0.01, and ***P < 0.001; ns, nonsignificant

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ABA Sensitivity of OsSIRHC-2-Overexpressing Plants

To test the ABA sensitivity of OsSIRHC-2-overexpressing plants, we conducted germination assays and q-PCR for ABA pathway-related genes. For the germination assay, transgenic and WT seeds were grown on a half-strength MS agar medium containing 0, 1, or 3 µM ABA. In the absence of ABA, there were no significant differences in germination rates between the transgenic lines and WT plants (Fig. 5A). However, with 1 and 3 µM ABA, the germination rates of transgenic lines were lower than those of WT seeds from 2 to 5 days and 3 to 5 days, respectively (Fig. 5B, C). These results support the hypothesis that transgenic lines exhibit higher ABA sensitivity than WT plants.

Fig. 5
figure 5

Abscisic acid sensitivity assays. Seeds of OsSIRHC-2-overexpressing and WT plants were grown on each half of MS agar mediums containing A 0, B 1, or C 3 µM ABA. Experiments were performed with three biological replicates (Fifteen plants were measured for each replicate). Blue triangle for WT, orange square for OE-1, and green circle for OE-7. D, day. Student’s t-test: **P < 0.01 and ***P < 0.001

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Subsequently, we evaluated the expression patterns of ABA signaling pathway-related genes, namely OsLEA3 (a group 3 late embryogenesis abundant protein; Moons et al. 1997), OsP5Cs (a pyrroline-5-carboxylate synthetase, Igarashi et al. 1997), OsProT (a proline transporter, Igarashi et al. 2000), and NAC5 (a stress indicator gene, Sperotto et al. 2009), in the leaves and roots of transgenic and WT plants (Fig. 6). There were no significant differences in the expression levels between transgenic and WT plants under non-treatment conditions. In contrast, at 100 mM NaCl, transgenic lines showed significantly higher expression levels than WT plants. These results suggest that the molecular function of OsSIRHC-2 is related to the ABA signaling pathway.

Fig. 6
figure 6

Analysis of relative expression patterns of ABA-dependent pathway-related genes in leaves and roots of OsSIRHC-2-overexpressing and WT plants under 0 and 100 mM NaCl. The samples were harvested at 10 d after 0 and 100 mM NaCl treatment. A, E OsLEA3, B, F OsP5Cs, C, G OsProT, D, H OsNAC5. q-PCR was performed with three technical and biological replicates (ten plants were harvested for each replicate). Gene expression levels were normalized to those of OseEF-1a. Student's t-test: **P < 0.01, and ***P < 0.001; ns, nonsignificant

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OsSIRHC-2-Overexpressing Plants Exhibited Low Na+ Accumulation

Under salinity stress, Na+ causes cellular damage in plants. Additionally, a high accumulation of Na+ induces K+ deficiency, leading to an ionic imbalance in rice roots. Under saline conditions, K+ deficiency in plant cells causes a decrease in plant development because K+ ions play an important role in the cell membrane and enzyme activity (Rahman et al. 2017). CoroNa Green binds Na+ ions only in confined cells (Meier et al. 2006). The fluorescence intensity of CoroNa Green increases in response to increasing Na+ accumulation (Nath et al. 2016). To determine whether OsSIRHC-2-overexpressing plants absorbed less Na+ ions than WT plants, both transgenic and WT plants were treated with 0 or 100 mM NaCl for 1 d, and their root samples were visualized by CoroNa Green staining using a confocal microscope (Fig. 7A, B). No significant difference was observed between transgenic and WT plants treated with 0 mM NaCl (Fig. 7A). At 100 mM NaCl, a lower fluorescence intensity was detected in transgenic plants than in WT plants (Fig. 7B, C). In addition, transgenic plants showed lower Na+ concentrations in their roots under 0 and 100 mM NaCl conditions. K+ concentrations showed significant differences between transgenic and WT plants at 0 and 100 mM NaCl (Fig. 7E). Under normal conditions, transgenic plants had lower K+ concentrations than WT plants; however, a greater accumulation of K+ was observed in transgenic plants than in WT plants. Subsequently, we evaluated the Na+/K+ ratio of both plants at 0 and 100 mM NaCl (Fig. 7F), which resulted in a lower Na+/K+ ratio in transgenic plants than in WT plants. These results suggest that transgenic plants have lower Na+ ion absorption rates under salinity stress than WT plants.

Fig. 7
figure 7

Staining of CoroNa Green in root tips and measurement of Na+ and K+ concentration in roots of OsSIRHC-2-overexpressing and WT plants treated with 0 and 100 mM NaCl for 24 h. A, B Ten-day-old seedlings were treated with 0 or 100 mM NaCl for 24 h, respectively. Experiments were performed with three biological replicates (Six plants were measured for each replicate). Green signals of root tips were taken after 24 h. Scale bar = 100 μm. C Root tip fluorescence intensity was measured in OsSIRHC-2-overexpressing and WT plants after 0 and 100 mM NaCl treatment. D Na+ concentration, E K+ concentration, and F Na+/K+ ratio in roots of OsSIRHC-2-overexpressing and WT plants. Experiments were performed with three biological replicates (Ten plants were measured for each replicate). Student's t-test: *P < 0.05, **P < 0.01, and ***P < 0.001; ns, nonsignificant

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Expression of Na+ or K+ Transporter Genes in OsSIRHC-2-Overexpressing Plants

To understand the mechanism of OsSIRHC-2-overexpressing plants in salinity insensitivity, we analyzed the expression of Na+ or K+ transporter genes in the leaves and roots at 0 and 100 mM NaCl (Fig. 8). Under normal conditions, sodium transporter genes, such as OsHKT1;4 and OsHKT2;1, showed lower expression levels in the leaves of transgenic plants than in WT plants (Fig. 8A, C). In contrast, in roots, lower OsHKT1;4 expression but higher OsHKT2;1 expression was observed in transgenic plants than in WT plants (Fig. 8K, M). After treatment with 100 mM NaCl, the expression levels of OsHKT1;4 and OsHKT2;1 in the leaves and roots of transgenic plants were lower than those of WT plants (Fig. 8A, C, K, M). In contrast, the expression of OsHKT1;5, which mediates Na+ exclusion in the phloem to prevent Na+ transfer to young blades (Kobayashi et al. 2017), was not significantly different in the leaf of transgenic and WT plants under normal conditions but was lower in the roots of transgenic than in WT plants (Fig. 8B, L). However, at 100 mM NaCl, the expression levels in the leaves and roots of the transgenic lines were significantly higher than those in WT plants (Fig. 8B, L). The vacuolar Na+/H+ antiporter gene OsNHX1 showed no significant difference in expression in the leaves of transgenic and WT plants under normal conditions (Fig. 8D). However, under 100 mM NaCl treatment, the expression level was significantly lower in the leaves of transgenic plants than in WT plants (Fig. 8D). In contrast, in the roots, OsNHX1 expression in transgenic plants was higher than that in WT plants under 0 and 100 mM NaCl (Fig. 8N). OsSOS1, a Na+/H+ encoding antiporter gene that excludes intracellular Na+ to the extracellular space (Parida and Das 2005), showed no significant difference in expression in leaves of transgenic lines and WT plants under normal conditions; however, under 100 mM NaCl, OsSOS1 expression in transgenic plants was slightly higher than that in WT plants (Fig. 8E). However, in roots under 0 and 100 mM NaCl, OsSOS1 expression showed a slight increase in transgenic plants than in WT plants (Fig. 8O).

Fig. 8
figure 8

Relative expression patterns of Na+ and K+ transporter genes in leaves and roots of transgenic and WT plants. Ten-day-old seedlings were treated with 0 or 100 mM NaCl for 24 h. Expression patterns of A OsHKT1;4, B OsHKT1;5, C OsHKT2;1, D OsNHX1, E OsSOS1, F OsAKT1, G OsHAK2, H OsHAK7, I OsGORK, and J OsSKOR in leaves, and K OsHKT1;4, L OsHKT1;5, M OsHKT2;1, N OsNHX1, O OsSOS1, P OsAKT1, Q OsHAK2, R OsHAK7, S OsGORK, and T OsSKOR in roots, respectively. q-PCR was performed with three technical and biological replicates at each time point (ten plants were measured for each replicate). Gene expression levels were normalized to that of OseEF-1a. Student's t-test: *P < 0.05, **P < 0.01, and ***P < 0.001; ns, nonsignificant

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Subsequently, the expression of K+ transporter genes, such as OsAKT1, OsHAK2, and OsHAK7, was evaluated under normal and saline conditions in the leaves and roots. The expression of OsAKT1 was lower in the leaves and roots of transgenic plants than in those of WT plants under both conditions (Fig. 8F, P). A slight increase in OsHAK2 expression was observed in the leaves of transgenic plants under normal conditions but not in the roots (Fig. 8G, Q). Under 100 mM NaCl, OsHAK2 expression exhibited a slight increase in the leaves and roots of transgenic than in WT plants (Fig. 8H, R). Notably, another K+ transporter gene, OsHAK7, exhibited contrasting results in leaves and roots; it showed a significant increase in the leaves of transgenic plants than in WT plants under normal and 100 mM NaCl conditions (Fig. 8H). In contrast, in the roots, OsHAK7 expression was lower in transgenic plants than in WT plants under normal and 100 mM NaCl conditions (Fig. 8R). The potassium efflux channel gene OsGORK (guard cell outward rectifying K+ Channel, Huang et al. 2020) exhibited higher expression in the leaves and roots of transgenic lines than in WT plants under normal conditions, whereas low expression in both tissues of transgenic lines was found under 100 mM NaCl than those of WT plants (Fig. 8I, S). Expression of another potassium channel gene, OsSKOR (stelar K + outward rectifier, Huang et al. 2020) was not significantly different in the leaves and roots of transgenic lines and WT plants under normal conditions; however, expression under 100 mM NaCl was higher in both tissues of transgenic lines than in WT plants (Fig. 8J, T).

Discussion

The negative effects of salinity stress on growth, productivity, and metabolism are well documented. Understanding the molecular mechanisms involved in the response to salinity stress is essential for improving salinity tolerance in rice. The ubiquitin–proteasome system (UPS) has been studied for several decades (Deshaies and Joazeiro 2009). In the UPS, E3 ligases play an important role in regulating target proteins via the 26S proteasome pathway. For example, OsSIRH2-14, an E3 ligase RING finger protein induced by salinity stress, regulates OsHKT2;1 via the 26S proteasome system under saline conditions, and OsSIRH2-14-overexpressing-plants have shown enhanced salinity tolerance compared to WT by maintaining low Na+ concentrations (Park et al. 2019). Another RING E3 ligase, OsRFPv6, plays a role in maintaining low Na+ accumulation in transgenic plants (Kim et al. 2021). The molecular mechanisms that enhance salinity tolerance in rice via RING E3 ligases have been well characterized but needed more research. Therefore, the molecular mechanisms related to other RING E3 ligases, such as OsSIRHC-2, are worth studying to improve the salinity tolerance of rice. In this study, we evaluated gene expression levels under abiotic stresses, including salinity. However, there was no significant difference in the phenotypes of the transgenic and WT plants under abiotic stress, except for salinity stress (Figs. 1, S1). Additionally, we studied the subcellular localization of OsSIRHC-2 in rice protoplasts and performed an in vitro OsSIRHC-2 ubiquitination assay. These findings suggest that the overexpression of OsSIRHC-2 might be specific to salinity rather than tolerance to multiple stressors. The phytohormone ABA is an important indicator of abiotic stress responses and negatively affects seed germination (Nambara and Marion-Poll 2005; Zhu 2002). The high expression level of OsSIRHC-2 under ABA treatment (Fig. S1G, H), low germination rates (Fig. 5A–C), and the different expression patterns of ABA-dependent pathway-related genes (Fig. 6A–H) in transgenic and WT plants suggest that the overexpression of OsSIRHC-2 may be associated with the ABA-dependent pathway in salinity tolerance. However, further research is required to verify this. These results also suggest that understanding the molecular functions of OsSIRHC-2 is crucial for enhancing salinity tolerance in rice.

Expression analyses of Na+ and K+ transporter genes in OsSIRHC-2-overexpressing plants compared to those of WT plants provided important clues that ruled out the role of OsSIRHC-2 in enhancing the salinity tolerance of plants. In plants, maintaining a high cytosolic K+/Na+ ratio has been reported to be a key determinant of salt tolerance (Gupta and Huang 2014; Shabala and Cuin 2008). Plants exposed to salinity have evolved to use inorganic ions (mainly Na+ and K+) to maintain cell turgor pressure (Flowers and Colmer 2008; Shabala and Mackay 2011). In this study, OsSIRHC-2-overexpressing plants exhibited improved salt tolerance by maintaining low Na+ and high K+ accumulation and a high K+/Na+ ratio in the roots under high salinity. In addition, there was a significant difference in the expression levels of Na+ and K+ transporters between the transgenic and WT plants under both normal and saline conditions (Fig. 8). The HKT family includes Na+/K+-and Na+-specific transporter genes that regulate Na+ efflux and influx under high salinity (Assaha et al. 2017). OsHKT1;4, which functions as a Na+ transporter, regulates the accumulation of Na+ in leaf sheaths under high salinity (Suzuki et al. 2016), and OsHKT1;4-overexpressing plants show an increased sensitivity to salt stress compared to WT plants (Oda et al. 2018). In our study, the low expression level of OsHKT1;4 in both leaves and roots under normal and saline conditions contributed to enhancing salt insensitivity in OsSIRHC-2-overexpressing plants. Other Na+ transporters, OsHKT1;5, which mediate Na+ exclusion from young leaf blades to the xylem and phloem parenchyma cells to prevent Na+ accumulation and oshkt1;5 plants showed a remarkable disturbance in Na+ homeostasis (Kobayashi et al. 2017). In this study, high expression levels of OsHKT1;5 in OsSIRHC-2-overexpressing plants may contribute to the low accumulation of Na+ in roots under high salinity conditions compared to WT plants. OsHKT2;1 plays a major role in Na+ influx into K+-starved roots and its expression decreases under salt stress (Horie et al. 2007; Zhang et al. 2018). In this study, OsSIRHC-2-overexpressing plants showed lower expression of OsHKT2;1 under saline conditions than WT plants in both the leaves and roots. These results suggest that the overexpression of OsSIRHC-2 positively mediates salt insensitivity by regulating the expression pattern of Na+ transporter genes in rice. Na+/H+ antiporters, tonoplast-localized NHX1, and plasma membrane-localized SOS1 contribute to enhancing salt tolerance by controlling the Na+ content in the cytoplasm under salt stress (Zhang et al. 2017). For example, overexpression of SOS1 in Arabidopsis plants showed a healthier phenotype than WT plants under salt stress. (Yang et al. 2009). High OsSOS1 expression was observed in roots and leaves under saline conditions in OsSIRHC-2-overexpressing plants. The results indicated low Na+ accumulation in the roots. Another Na+/H+ exchanger, OsNHX1, catalyzes the antiport of Na+ and H+ across membranes and plays an important role in salt tolerance by sequestrating Na+ into the vacuole (Fukuda et al. 1999). OsNHX1-overexpressing lines showed reduced Na+ content compared to WT plants (Liu et al. 2010). We identified that OsSIRHC-2-overexpressing plants showed contrasting expression patterns of OsNHX1 in both roots and leaves under high salinity compared to WT plants. These results suggest that the movement of Na+ to aerial plants can be restricted because of the sequestration of Na ions in the root.

In plants, members of the K+ transporter gene family play an important role in maintaining intercellular K+ homeostasis, which is related to plant adjustment to salinity (Shen et al. 2015). Over the past decades, several families of K+ transporters have been identified, such as shaker-like and KUP/HAK/KT transporters (Maser et al. 2001; Very and Sentenac 2003). In rice, the HAK family, which is widely known as K+ transporters, plays an important role in regulating the K+ uptake response to salinity (Li et al. 2018). OsAKT1, which is known as an inward rectifying K+ channel in rice roots, is highly downregulated under saline conditions (Fuchs et al. 2005). In this study, we observed low OsAKT1 expression levels in both tissues of transgenic plants under normal and saline conditions compared with WT plants. In addition, OsAKT1 has been reported to be K+ permeable and participates in Na+ uptake (Golldack et al. 2003, Okada et al. 2008). Therefore, the low expression of OsAKT1 under normal and high-salinity conditions might correspond to low Na+ uptake and accumulation in transgenic plants. A previous study indicated that OsHAK2 mediates Na+ and K+ transport, and its K+ transport activity is inhibited by high Na+ concentrations (Horie et al. 2011). OsHAK2 expression in transgenic plants showed a slight increase under saline conditions in transgenic plants, which is consistent with higher K+ accumulation in transgenic plants than in WT plants. High expression levels of OsHAK7, which is known as a K+ transporter, have been reported in the excessive accumulation of Na+ in salt-tolerant rice varieties (Mekawy et al. 2015). The contrasting expression pattern of OsHAK7 between the leaves and roots in transgenic plants was similar to that in OsRFPv6-overexpressing plants, which showed salt insensitivity (Kim et al. 2021). Shaker-like K+ channels, such as OsGORK and OsSKOR, mainly regulate K+ homeostasis in plants (Hedrich 2012). OsGORK, an outward-rectifying potassium efflux channel, is central to stress-induced K+ loss from the cytosol (Adem et al. 2020). We found high or low expression patterns of OsGORK under normal or saline conditions in both roots and leaves, consistent with the low or high K+ content in the roots and leaves of transgenic plants. In addition, OsSKOR plays a role in root stellar tissues and loads K+ into the xylem (Kim et al. 2015). We observed increased expression of OsSKOR in both the leaves and roots of transgenic plants under high salinity. These results demonstrate a contrast in K+ content under normal and salt stress conditions. Another hypothesis for improved K+ retention might be the decrease in ROS-activated nonselective cation channels due to the low H2O2 content in transgenic lines under salinity (Demidchik et al. 2010).

The findings related to the low expression of Na+ or K+ transporter genes in transgenic plants under controlled conditions suggest that OsSIRHC-2 regulates their low expression via the UPS. We used Y2H to select the target proteins of OsSIRHC-2; however, despite several selections, positive clones were not found. The interactions of the four transporters with low expression in transgenic plants (OsHKT1;4, OsAKT1, OsHKT1;5, and OsHKT7) were examined using OsSIRHC-2, and no clear protein–protein interactions were observed (Fig. S4). These results might be supported by the deficiency of direct OsSIRHC-2 regulation of the examined transporters. Thus, their low expression might be mediated by transcription factor(s), which might be regulated by OsSIRHC-2; however, further research is required.

The examined physiological indicators, such as chlorophyll, H2O2, proline, and soluble sugar content, support the tolerance phenotypes of transgenic plants under salinity stress. Under normal conditions, the roots had a higher weight and the shoots had a lower weight in transgenic plants than in WT plants. These results suggest a negative effect of OsSIRHC-2 overexpression in transgenic plants, at least on shoot growth during the vegetative stage, but further research is required.

This study identified the expression levels, subcellular localization, and E3 ligase activity of OsSIRHC-2. The molecular functions of OsSIRHC-2 were elucidated by evaluating physiological indicators, transporter gene expression, and phenotyping of transgenic plants. These results demonstrate that the overexpression of OsSIRHC-2 enhanced salinity tolerance through the low absorption of Na+ via changes in the expression of Na+ or K+ transporter genes (Fig. S5). However, the identification of interacting proteins requires further research. Understanding the salt tolerance mechanisms of OsSIRHC-2 may provide a crucial strategy for adapting to salinity stress in plants.