Mutation of a gene with PWWP domain confers salt tolerance in rice

doi:10.21203/rs.3.rs-4872497/v1

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Mutation of a gene with PWWP domain confers salt tolerance in rice

https://doi.org/10.21203/rs.3.rs-4872497/v1

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Salinity is a major problem due to the continuous increase in the salinization of agricultural lands, particularly, paddy fields. Using a forward genetics approach, salt-insensitive TILLING (targeting-induced local lesions in genomes) line 3, sitl3, was selected from a core population induced by gamma-ray irradiation. Under salt stress, sitl3 had greater fresh weight and chlorophyll content, and lower H₂O₂ and Na⁺ contents than the wild-type. In the gene (LOC_Os07g46180) with two PWWP domains (OsPWWP4) of sitl3, a premature stop was caused by an SNP, and was named OsPWWP4p.Gly462* (a stop gain occurred from the 462th amino acid residue). The OsPWWP4 and substrate proteins (OsEULS2, OsEULS3, and OsEULD2) were identified using yeast two-hybrid, bimolecular fluorescence complementation, in vitro pull-down, and in vitro methyltransferase assays. Subcellular localization of OsPWWP4 and OsPWWP4p.Gly462*GFP-tagged proteins revealed they were both localized in the nucleus, while OsEULS2, OsEULS3, and OsEULD2 GFP-tagged proteins were found in the nucleus and cytosol of rice protoplasts. The expression levels of OsEULS2, OsEULS3, OsEULD2 under salt stress were higher in sitl3 than in wild-type plants. In contrast, OsPWWP4 expression was higher in the latter. Genes involved in the salt overly sensitive (SOS) pathway showed higher expression in the aerial tissues of silt3 than in the wild-type. Our data suggest that TILLING line sitl3 is a valuable genetic resource for understanding protein post-translational regulation-related salinity tolerance mechanisms such as methyltransferase activities, and for improving salt tolerance in rice through breeding.

Gamma-ray irradiation

Mutation

OsEUL family

OsPWWP4

premature stop

Salt stress

1. Salt-insensitive TILLING line () was selected by a forward genetics approach

2. is a new, useful genetic resource in salt-tolerance breeding programs

3. showed a guanine to thymine change in resulting in a premature stop

4. Mutations in affected partner-, TF-, and transport-gene expression

Rice is a major staple crop worldwide, particularly in Asia, where more than 4.7 billion people live. However, abiotic stress conditions are a major factor that negatively affects crop growth and productivity (Pathak et al., 2021). Among them, salinity is an emerging problem due to the high concentrations of salts in the soil and an increase in the salinization of agricultural lands (Shrivastava et al., 2015). Further, rice is a highly salt-sensitive plant, and its sensitivity largely depends on growth stage (Walia et al., 2005; Ganie et al., 2019). The seedling stage is the most sensitive, whereas tillering is the least sensitive stage (Chinnusamy et al., 2005). Nonetheless, the development of salt-tolerant rice varieties is necessary to achieve a sustainable yield under salinity conditions (Mickelbart et al., 2015; Krishnamurthy et al., 2016). Therefore, it is vital to study the entire genome composition and function of the corresponding genes to develop salt-tolerant rice varieties (Sahi et al., 2006).

The entire gene sequences of numerous major crops have been determined using whole-genome sequencing (WGS). Particularly, the rice genome sequence has been determined with over 99% accuracy by the International Rice Genome Sequencing Project (Goff et al., 2002; Matsumoto et al., 2008). Owing to whole-genome sequencing projects, the application of biotechnology has led to many advances in crop variety development. Conventional strategies for developing varieties using biotechnology include mutagenesis and transgenesis. Specifically, transgenesis can be applied in breeding to quickly and accurately introduce desirable genetic traits (Ahmar et al., 2020). However, the cultivation of genetically modified crops is still restricted by legal regulations in many countries (Dunwell., 2014; Blancke et al., 2015; Østerberg et al., 2017; Atanassova et al., 2018). Conversely, mutational breeding resulting from the modification of the original DNA of an organism via chemicals (e.g., sodium azide) or radiation (e.g., gamma rays) is not restricted by legal regulations (Ishikawa et al., 2012; Seo & Jang, 2023). Therefore, mutation breeding can improve plant traits within legal regulations and contribute to increasing the diversity of new varieties with specific traits (Harten, 1998; Khan and Goyal, 2009; Tripathi et al., 2023).

Post-translational regulation of target proteins in plants exposed to abiotic stress is a key physiological process (Krasensky and Jonak 2012; Stone., 2014). Plants alter intracellular metabolic processes, including protein synthesis, degradation, and transcription factor regulation, to overcome a range of abiotic stress conditions (Hirayama et al., 2010). Additionally, post-translational modification of proteins is a key mechanism that regulates various cellular signaling pathways in response to various abiotic stress in plants (Mazzucotelli et al., 2008). Protein modifications include phosphorylation, glycosylation, acetylation, and methylation (Kurotani et al., 2014). In particular, protein methylation has been recognized as an essential regulatory mechanism of abiotic stress responses in plants. This response can occur during and immediately after protein synthesis in ribosomes or later in mature protein molecules (Lee et al., 2005). Protein methylation is mediated by protein methyltransferases (PMTs), which are enzymes that transfer the methyl group of the cofactor S-adenosyl-L-methionine (SAM) to the arginine or lysine side chains of cytoplasmic and nuclear proteins (Schapira, 2016).

The PWWP domain functions as a DNA, histone, lysine, and protein methyltransferase (Qiu et al., 2002; Sankaran et al., 2016). Further, it consists of approximately 70 amino acids and is named the central core ‘Pro-Trp-Trp-Pro’ (Qin et al., 2014; Zhu et al., 2020). The PWWP domain is found in one or two copies of a DNA-binding protein that functions as a transcription factor that regulates developmental processes. Because of its position, the amino acid composition is close to that of the PWWP motif, and owing to the presence of patterns in other domains, it has been suggested that the PWWP domain is involved in protein-protein interactions (PPIs) (Stec et al., 2000). Indeed, PPI in plants are essential for studying the tolerance of plants to abiotic stress factors and can be demonstrated in protein response mechanisms to various abiotic stress factors (Zhang et al., 2022). For example, calcineurin B-like protein (CBL), which is related to calcium ions that regulate plant signaling, interacts with CBL-interacting protein kinases (CIPK) to respond to various abiotic stress conditions (Kanwar et al., 2014). The transcription factor OsbZIP20 interacts with OsSAPK10 to improve drought and salt tolerance in rice (Baoxiang et al., 2022).

Lectins selectively recognize and reversibly bind specific glycans or free sugars present in glycoproteins and glycolipids (Peumans et al., 1995). As carbohydrate-binding proteins, lectins have been identified in important food crops such as tomatoes, potatoes, and wheat, as well as in viruses, fungi, animals, and other plants; moreover, there is evidence that important groups of lectins are involved in plant defense (Peumans et al., 1995; Damme et al., 1998a and 1998b). Additionally, previous studies have shown that plants synthesize lectins in response to specific stressors and changing environmental conditions, and that they are upregulated under stressful conditions. These lectins are referred to as ‘inducible’ lectins, and have been detected in the nucleus and cytoplasm (Damme et al., 2003; Damme et al., 2004; Damme et al., 2008). Euonymus lectin (EUL) is an inducible lectin derived from the fleshy arils of Euonymus europaeus and possesses lectin activity (Fouquaert et al., 2008; Lannoo and Damme., 2010). Particularly, rice EULs are classified into five types based on their structure: OsEULS2, OsEULS3, OsEULD1a, OsEULD1b, and OsEULD2 (Fouquaert et al., 2009; Fouquaert and Damme, 2012). Furthermore, EUL transcripts respond to various abiotic stressors, e.g., NaCl, Mannitol, and ABA (Atalah et al., 2014). In Arabidopsis thaliana they are involved in the response to osmotic stress and are upregulated by treatment with ABA, NaCl, and methyl jasmonate (Van Hove et al., 2014). In rice, the OsEULS2, OsEULS3, OsEULD1a, OsEULD1b, and OsEULD2 transcripts are differentially expressed under drought, salt, and ABA treatments (Atalah et al., 2014; Lambin et al., 2020) and, although, their expression pattern has been reported, no studies have been conducted on the gene family involved in plant tolerance mechanisms to abiotic stress.

In a previous study, a TILLING (targeting-induced local lesions in genomes) population was created using gamma ray irradiation (Cho et al., 2010). The genotypes were fixed by advancing to the M₁₀ generation, and lines were selected for abiotic stress tolerance (Choi et al., 2022; Lim et al., 2022; Seo et al., 2022; Seo and Jang., 2023). In this study, a salt-tolerant line was screened from the M₁₀ mutant population and selected for further study. The selected mutant was named salt-insensitive TILLING 3 (sitl3) based on previous studies (Lim et al., 2022, Seo et al., 2022), and single nucleotide polymorphisms (SNPs) were confirmed in the genomic DNA and CDS of sitl3. The mutated gene possessed a PWWP4 domain and its substrate genes (OsEULS2, OsEULS3, and OsEULD2) were identified. We investigated the molecular mechanisms underlying salt tolerance in sitl3 mutants.

2.1. Plant materials, growth conditions, and salt-insensitive mutant line screening

A gamma-irradiated M₁₀ core rice mutant population was constructed using the single-seed descent method (Cho et al., 2010). A total of 150 M₁₀ rice mutants and wild type (WT) rice (O. sativa cv. ‘Donganbyeo’), were germinated in a petri dish with moistened filter paper at 28°C for 2 d. Germinated seeds were transferred to a transparent plastic box and cultured in hydroponics using 1/2 Kimura solution for 7 d (Chen et al., 2006) under a 16/8h light/dark regime at 30/28°C, respectively, and 70% humidity. To select salt-insensitive lines, WT and mutant plants were treated with 150 mM NaCl for 7 d, after which, the shoot and root lengths, and fresh weight of each individual plant were measured in three biological replicates (n = 30) per line.

2.2. Phenotype analysis of sitl3 mutant

For chlorophyll analysis, 300 mg of leaves of WT and sitl3 (three biological, n = 30) were ground and placed in a 15 mL tube, added with 5 mL of 80% acetone, incubated in the dark for 30 min, before centrifuging at 12,000 g and 4°C for 15 min. Then, 200 µL of the supernatant were transferred to a 96-well microplate for each of the three biological replicates. A SPECTROstar Nano (BMG Labtech, Ortenberg, Germany) was used to measure total chlorophyll content at 645 and 663 nm. Chlorophyll a and b contents were calculated after Ni et al. (2009).

Oxygen peroxide (H₂O₂) content was measured in roots and leaves of WT and sitl3 plants as described by Gay and Janusz (2000). A total of 300 mg of root and leaf tissues of each WT and sitl3 plant (three biological, n = 30) were incubated in 0.1% TCA solution (Trichloroacetic acid; Sigma Aldrich, St. Louis, MO, USA) at 4°C for 30 min, and then centrifuged at 12,000 g for 30 min. After centrifugation, the supernatant was transferred to a microtube and added with solution A (25 mM ammonium ferrous (II) sulfate in 2.5 M H₂SO₄) and solution B (100 mM sorbitol, 125 µM xylenol orange). The concentration of H₂O₂ was calculated using a standard curve (y = 0.0039x + 0.1148) previously prepared. The results were obtained using a SPECTROstar Nano (BMG Labtech).

2.3. Ionomic analysis

To conduct ionomic analysis, one-week-old WT and silt3 plants (three biological, n = 30) were treated with 150 mM NaCl for 7 days in a growth chamber under a 16/8h light/dark regime at 30/28°C, respectively, and 70% humidity. After treatment, root and leaf samples were harvested, oven-dried at 80°C for 16 h, and subjected to ion (Na, K, P, and Ca) analysis using inductively coupled plasma-optical emission spectroscopy (ICP-OES; Optima 700 dv; Perkin Elmer, Waltham, MA, USA) at the Central Laboratory of Kangwon National University (Chuncheon, Korea).

2.4. Whole-genome re-sequencing

For whole-genome resequencing, genomic DNA was extracted from the leaves of two-week-old WT and sitl3 plants using the CTAB method (Saghai-Maroof et al., 1984). DNA libraries were constructed using the Illumina TruSeq Nano DNA Library Kit (Illumina, San Diego, CA, USA). After the raw sequence data were compressed into FASTQ format, low-quality reads were removed using the NGS QC Toolkit (Patel and Mukesh, 2012). High-quality reads were aligned using the Oryza sativa cv. The Nipponbare genome reference (Os-Nipponbare-Reference-IRGSP-1.0) obtained from the RAP-DB database (https://rapdb.dna.affrc.go.jp/) by BWA (Burrows-Wheeler Alignment) software (Li and Richard., 2009). The aligned files were then formed into sequence-alignment map files and converted into binary-alignment map (BAM) files (Li et al., 2009). Data filtering was performed to remove duplicate reads using Picard tools (https://broadinsitute.github.io/picard/).

2.5. Confirmation of mutation caused by SNPs

To confirm the presence of SNPs in OsPWWP4, genomic DNA was extracted using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For OsPWWP4, primers were prepared for the forward and reverse regions (Table S1) and amplified using Q5 High-Fidelity DNA polymerase (NEB, Ipswich, MA, USA).

To obtain the full-length CDS sequences of OsPWWP4, and OsPWWP4p.Gly462*, total RNA was extracted from one-week-old WT and sitl3 plants using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For cDNA synthesis, a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) was used with extracted RNA as a template. The synthesized cDNA was amplified using Q5 High-Fidelity DNA polymerase (NEB) to obtain the full sequence, and then cloned into a directional D-topo vector (Invitrogen, Waltham, USA).

2.6. Yeast-two-hybrid assays and yeast co-transformation

A yeast two-hybrid assay was used according to manufacturer instructions to identify partner proteins that interacted with OsPWWP4 (Clontech, Mountain View, CA, USA). A rice cDNA library containing the GAL4 activation domain vector, pGADT7-AD, was constructed; the samples included two-week-old seedlings treated with salt for 0, 1, 3, 6, 12, and 24 h. Partner genes were identified by sequencing the blue colonies shown in QDO/X/A, and the partner gene family was listed by referring to a previous report (Fouquaert and Damme, 2012). To confirm the interaction of the EUL protein family and OsPWWP4 or OsPWWP4p.Gly462*, the yeast strain was co-transformed with BD-(OsPWWP4 and OsPWWP4p.Gly462*) vectors and AD-OsEUL family gene (OsEULS2, OsEULS3, OsEULD1a, OsEULD1b, and OsEULD2) vectors, individually, and then they were grown on SD/-Leu/-Trp (DDO) and SD/- Ade/-His/-Leu/-Trp (QDO) plates at 30°C for 3 d.

2.7. Subcellular localization and bimolecular fluorescence complementation assays

To confirm the subcellular localization of OPWWP4 and OsPWWP4p.Gly462*, the full length genes were cloned into a binary vector (pGWB406; Nakagawa et al., 2007), respectively. Ten micrograms of 35S::OsPWWP4-sGFP, 35S::OsPWWP4p.Gly462*-sGFP, 35S::OsEULS2-sGFP, 35S::OsEULS3-sGFP, 35S::OsEULD1a, 35S::OsEULD1b, 35S::OsEULD2, and 35S::sGFP were transfected into rice protoplasts using a 40% PEG 4000 solution as described previously (Lim et al., 2014).

To confirm the interactions and subcellular localization of the EUL Family genes in OsPWWP4 and the mutant, a bimolecular fluorescence complementation (BiFC) assay was conducted. OsPWWP4, OsPWWP4p.Gly462*, OsEULS2, OsEULS3, and OsEULD2 were cloned into the BiFC vectors OsPWWP4-YFPn, OsPWWP4p.Gly462*-YFPn, OsEULS2-YFPc, OsEULS3-YPFc, and OsEULD2-YFPc, respectively. Ten micrograms each of OsPWWP4-YFPn, OsPWWP4p.Gly462*-YFPn, OsEULS2-YFPc, OsEULS3-YPFc, OsEULD2-YFPc, and YFPn-YFPc (negative control) were transfected into rice protoplasts using a 40% PEG 4000 solution as described previously (Lim et al., 2014). Each protoplast containing the recombinant sGFP vector was incubated overnight in the dark and observed under a super-sensitive high-resolution confocal laser scanning microscope (ZEISS, Oberkochen, Germany). Subcellular localization was imaged using ZEN software (ZEISS).

2.8. Protein expression and in vitro methyltransferase activity assay

To confirm the expression of OsPWWP4, the mutant protein, and partner proteins OsPWWP4 and OsPWWP4p.Gly462*-full-length genes were restricted to NheI (Thermo Fisher Scientific, Waltham, USA) and XhoI enzyme (Thermo Fisher Scientific) cloned into the 6X His-tagged pET-28a (+) vector (Novagen, Gibbstown, NJ, USA), and then, OsEULS2, OsEULS3, and OsEULD2 full-length genes were restricted to Sal I and EcoR I enzymes (Thermo Fisher Scientific) cloned into the maltose binding pMAL-c5x vector (NEB, Ipswich, MA, USA). The recombinant protein vectors were transformed into E. coli strain BL21 (DE3) PLysS (Promega, Madison, WI, USA). The transformed BL21 strains were cultured at 37°C for 90 min after adding 0.3 mM Isopropyl β-D-1-thiogalactopyranoside (ITPG). Recombinant pET-28a (+) OsPWWP4 and OsPWWP4p.Gly462* proteins were purified using a Ni–NTA Purification System (Invitrogen Life Technologies). Recombinant pMAL-c5x OsEULS2, OsEULS3, and OsEULD2 proteins were isolated using amylose resin (NEB). Recombinant proteins were separated using 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. Immunoblotting was performed using recombinant pET-28a (+) (OsPWWP4 and OsPWWP4p.Gly462*), polyhistidine (Sigma-Aldrich), and recombinant pMAL-c5x (OsEULS2, OsEULS3, and OsEULD2) proteins using anti-MBP (Sigma-Aldrich). respectively. Both recombinant proteins were used as secondary anti-MBP mouse antibodies (Sigma-Aldrich). The reaction with labeled antibodies was detected using Westar Nova 2.0 Pico Substrate (Cyanagen, Bologna, Italy). Protein bands were visualized using a ChemiDocTM XRS + imaging system (Bio-Rad, Hercules, CA, USA).

In vitro methyltransferase assay was performed using a Methyltransferase Activity (MT) Assay Kit (Colorimetric) (BioVision, Milpitas, CA, USA). Briefly, the purified recombinant proteins OsPWWP4, OsPWWP4p.Gly462*, OsEULS2, OsEULS3, and OsEULD2 were incubated at 37°C in MT Assay buffer. The sample reaction mix was composed of enzyme mix I, II, III, OxiRedTM probe, and SAM cofactor (50 mM), and adjusted to 50 µL of reaction mix with a combination of OsPWWP4+(OEULS2, OsEULS3, or OsEULD2), and OsPWWP4p.Gly462*+(OEULS2, OsEULS3, or OsEULD2), respectively. Each sample was added to 50 µL of MT Assay Buffer in a 96-well clear plate for a total reaction-mix volume of 100 µL. Absorbance at 570 nm was measured every 30 s for 45 min at 37°C using a SPECTROstar Nano (BMG Labtech) in kinetic mode.

2.9. In vitro pull-down assay

To confirm partner proteins and OsPWWP4 or the mutant protein, an in vitro pull-down assay was performed using recombinant His-tagged OsPWWP4 and OsPWWP4p.Gly462*- and MBP-tagged OsEULS2, OsEULS3, and OsEULD2 proteins using a Pull-Down Assay Kit (Thermo Fisher Scientific). The eluted proteins were denatured by boiling in 5X SDS sample buffer. Thereafter, the eluted proteins were separated using 7% SDS-PAGE and transferred onto a nitrocellulose membrane. The reaction with appropriate labeled antibodies (primary; poly His and MBP, secondary; mouse) was detected using Westar Nova 2.0 Pico Substrate (Cyanagen). Protein bands were visualized using a ChemiDocTM XRS + imaging system (Bio-Rad).

2.10. Gene expression analysis

Wild-type and sitl3 tissue samples were subjected to various treatments for gene expression analysis. Particularly, shoot and root samples were harvested from seven-day-old seedlings at 0, 1, 3, 6, 12, and 24 h after treatment with 150 mM NaCl and leaf blades, leaf sheaths, and roots samples were harvested from seven-day-old seedlings at 7 d after treatment with 150 mM NaCl. Additionally, shoot and root samples were harvested from seven-day-old seedlings at 0, 1, 3, 6, 12, and 24 h after treatment with 100 µM ABA. Total RNA was extracted from NaCl- and ABA-treated tissue samples using the RNeasy Plant Mini Kit (Qiagen), followed by treatment with DNase I (Qiagen), according to manufacturer instructions. The extracted RNA was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).

Expression levels of OsPWWP4, OsEUL family members, transcription factors (TFs), and transporter genes in sitl3 were compared to those in the WT. TFs genes including, OsWRKY8, OsWRKY23, OsWRKY45, OsWRKY72, OsWRKY89, OsNAC4, OsNAC5, OsNAC10, OsNAC58, OsbZIP23, and OsbZIP72 overlapped in the promoter regions of three genes (OsEULS2, OsEULS3, and OsEULD2) and were randomly selected from the website; PlantPAN 4.0 (http://plantpan.itps.ncku.edu.tw/plantpan4/index.html) (Fig. S2, Table S6-S8). Transporter genes including, OsSOS1, OsSOS2 (OsCIPK24), OsSOS3 (OsCBL4), OsCIPK15, OsNSCC2, OsTPC1, OsMSR2, OsNHX1, and OsCAX1 were selected as previously described (Reddy et al., 2017; Lim et al. 2022).

Real-time qPCR was performed using the CFX96 Connect Real-Time PCR System (Bio-Rad Laboratories), and the SYBR Green TOP Real qRT-PCR PreMIX (Enzynomics). The expression levels of abovementioned genes were calculated using the 2-ΔΔCT method (Livak and Thomas., 2001), and the housekeeping eEF-1a (Jain et al., 2006) gene was used as an internal control. The specific primer sequences used for qPCR are listed in Table S2.

3.1. Phenotypic evaluation of salt tolerance in sitl3

A total of 150 TILLING rice lines were screened to select a salt-insensitive rice mutant, (Cho et al., 2010; Lim et al., 2022; Seo et al., 2022). Among them, one salt tolerance line was additionally selected and then named salt-insensitive TILLING line 3 (sitl3), according to nomenclature of previous studies (Lim et al., 2022, Seo et al., 2022).

Wild-type and sitl3 seedlings were grown in a plastic box for hydroponic cultivation (Kimura B nutrient solution) for 21 days after germination (DAG). No significant differences were observed for either shoot or root length, or fresh weight between WT and sitl3 seedlings at 2, 5, 7, 14, or 21 DAG (Fig. S1A-C). When the agronomic traits were investigated at harvest in untreated seedlings, there were no significant differences among the eight categories; neither was there any difference for yield between the two types of plant under normal conditions (Fig. S2E).

First, salt insensitivity was evaluated at the seedling stage. Seven-day-old WT and sitl3 plants were treated with 150 mM NaCl for 7 days (Fig. 1A), and shoot and root length and fresh weight, chlorophyll and H₂O₂ contents were measured. When measuring shoot and root length we confirmed that the shoot length of sitl3 was greater than that of the WT in the 150 mM NaCl treatment group but there was no significant difference in root length between the two (Fig. 1B). Similarly, shoot weight of sitl3 seedlings was greater than that of WT seedlings under 150 mM NaCl, but there was no significant difference between them in root weight (Fig. 1C). Chlorophyll content was measured using chlorophyll a, chlorophyll b, and total chlorophyll (a + b). We found no significant differences between WT and sitl3 plants before NaCl treatment. In contrast, under 150 mM NaCl, chlorophyll content, including chlorophyll a and chlorophyll b, was higher in sitl3 plants than in WT plants (Fig. 1D). In turn, no significant difference in H₂O₂ accumulation was found in the leaf blades, leaf sheaths, and roots between untreated WT and sitl3 seedlings under 150 mM NaCl. In contrast, the same three tissues of silt3 accumulated less H₂O₂ than those of WT seedlings (Fig. 1E). Additionally, salt insensitivity of the WT and sitl3 plants during the entire growth period in the soil was evaluated (Fig S2). There were no significant differences in shoot height, tiller number, or chlorophyll content between untreated WT and sitl3 plants during vegetative growth (Fig. S2A-C). However, when grown under 0.3 dS/m NaCl treatment, sitl3 plants showed a significant difference in chlorophyll content (Fig. S2C) but not in shoot height or tiller number (Fig. 2B). In turn, differences in culm length, panicle length, number of panicles per hill, number of spikelets per panicle, total number of spikelets, maturity rate, total grain weight and 1,000 grain weight were measured between WT and sitl3 under 0.3 dS/m NaCl at the reproductive and harvest stages. We found that the 1,000 grain weight and total grain weight of sitl3 were significantly greater than those of the WT (Fig. S2D, F).

Germination rates were measured for 5 d under NaCl and ABA treatment conditions. In the NaCl treatment, sitl3 seeds exhibited significantly higher germination rates than the WT under 100 to 200 mM NaCl (Fig. S3A). Interestingly, the seed germination rate of sitl3 seeds was significantly lower at 5 d after treatment with 3 µM ABA and at 2 d after treatment with 5 µM ABA; whereas no significant difference was found for germination rate between the two when treated at 2 µM ABA (Fig. S3B).

Ionomic analysis was performed to investigate the accumulation of Na⁺ and K⁺ under salt stress. The Na⁺ contents in WT and sitl3 were not significantly different between shoots and roots before treatment. However, after 150 mM NaCl treatment, the accumulated Na⁺ in sitl3 was 0.65-fold in shoots and 0.84-fold in roots relative to WT (Fig. 1F). However, there was no difference in K⁺ content between control and treatment conditions (Fig. 1G).

K, Na, P, and Ca contents were measured in leaf blades, leaf sheaths, and roots of seven-day-old WT and sitl3 plants for 7 d in untreated and 150 mM NaCl-treated plants (Fig. S4). K contents in the three tissues did not significantly differ between WT and sitl3 plants under control or saline conditions (Fig. S4A). In contrast, Na contents were higher in the three tissues of silt3 than in those of the WT (Fig. S4B). In contrast, although there was no difference in P between WT and sitl3 before treatment, it was significantly lower in sitl3 than in WT roots and leaf bladesࣧbut not in leaf sheathsࣧunder salt stress (Fig. S4C). In turn, Ca contents did not differ in untreated nor in salt-treated groups between WT and sitl3 plants, except for a lower content in sitl3 leaf blades than that in WT plants under salt stress (Fig. S4D).

3.2. Whole genome re-sequencing and selection of candidate gene corresponding to salinity tolerance

In a previous study, whole-genome resequencing was performed to identify mutations in 150 core TILLING lines, including sitl3 (Hwang et al., 2020). Genetic mutations were detected in sitl3 to compare genetic polymorphisms with those in the WT genome. In all, 167,646,640 of the 196,179,185 WT reads (84.46%) and 37,158,152 of 43,603,544 sitl3 reads (85.22%) were mapped to the reference genome ( i.e., Oryza sativa cv. Nipponbare) (Table. S3). Genetic variants of 82 SNPs and 53 indels were detected in the sitl3 genome after manual evaluation of each variant individually (Fig. 2A). Further, SNPs and indels were distributed over 12 chromosomes and homozygous variants were mapped to 12 chromosomes (Fig. 2B). Mutations were classified according to their location on the genome, and the degree of their impact on the genome was predicted and evaluated. Among 82 SNPs and 53 indels, 17 SNPs (20.73%) and 7 indels (13.20%) were observed in genic regions (Table S4-5). Additionally, in the genic region, 10 of the 17 SNPs (58.82%) and one of the 7 indels (14.28%) occurred in the CDS region. SNPs had eight missense variants, one synonymous variant, and one stop gain (Fig. 2C), whereas indels had one frameshift variant (Table. S5). Both mutations had a high impact on genetic variation, with SNP and indels. One mutated gene with the SNP encoded a PWWP domain-containing protein (LOC_Os07g46180), and the other with indel was annotated retrotransposons (Fig. 2C and Table. S5). Therefore, the gene encoding the PWWP domain-containing protein was named Oryza sativa PWWP4 (OsPWWP4), in accordance to the nomenclature of a previous report (Guo et al., 2022), and was selected as a candidate for the salt tolerance phenotype of sitl3 for further study.

3.3. Molecular characterization of OsPWWP4 and OsPWWP4p.Gly462*

Further studies were performed to confirm the SNP of OsPWWP4 in sitl3. First, the reference genomic DNA and CDS sequences were retrieved from an internet website (phytozome 13, https://phytozome-next.jgi.doe.gov/). OsPWWP4 is located on chromosome 7 and consists of six exons and five introns with 11,146 nucleotides long of genomic DNA and 4,344 nucleotides long of CDS (Fig. 3A). Sanger sequencing confirmed that the WT gene had the same sequences as the reference gene from gDNA and CDS. In contrast, guanine in the mutant gene of gDNA and CDS at positions 4464th and 1384th was changed to thymine, resulting in premature termination (Fig. 3B-C). Therefore, the mutant gene with only one PWWP domain was named OsPWWP4p.Gly462* according to its nomenclature, although the WT gene consisted of two PWWP domains (Fig. 3B).

3.4. Interaction of OsPWWP4 or OsPWWP4p.Gly462* with OsEUL families

To identify partner proteins (s) of OsPWWP4, we performed yeast two-hybrid screening using a salt-treated rice cDNA library. As a result of this screening, we found 10 blue colonies on DDO/X/A media, followed by patching on QDO/X/A, and finally selected one colony (Fig. S5A). The colony was BLASTed, and five candidate genes belonging to the EUL family (OsEULS2, OsEULS3, OsEULD1a, OsEULD1b, and OsEULD2) were highly matched (Fig. S5B, C). The EUL family consists of five genes in the rice genome and is divided according to its structure, including the genuine EUL domain and inter-domain linker (Fouquaert et al., 2012) (Fig. 4A). In addition, the phylogenetic tree based on the amino acid sequences revealed three subgroups: OsEULD1a-OsEULD2, OsEULS2-OsEULS3, and OsEULD1b (Fig. 4B). Each gene of the EUL family was cloned into the pGADT7-AD vector, and a co-transformation experiment was performed with OsPWWP4 or OsPWWP4p.Gly462*. Five proteins co-transformations of OsPWWP4 with each of the three proteins (OsEULS2, OsEULS3, and OsEULD2) grew well in QDO/X/A medium. In contrast, no co-transformation of OsPWWP4p.Gly462* with each of the five proteins was observed (Fig. 4C). The positive control (P53 + T) but not the negative control (Lam + T) grew well (Fig. 4C).

3.5. Subcellular localization and bimolecular fluorescence complementation assays

To investigate the subcellular localization of OsPWWP4, OsPWWP4p.Gly462* and their partner proteins (OsEULS2, OsEULS3, and OsEULD2), these genes were expressed in rice protoplasts. The positive control, 35S:: sGFP, was highly expressed in the cytosol. In turn, 35S::sGFP-OsPWWP4 and 35S::sGFP-OsPWWP4p.Gly462*, and OsMeCP-DsRed2, were highly expressed in the nucleus (Kim et al., 2019). The partner genes 35S::sGFP-OsEULS2, 35S::sGFP-OsEULS3, and 35S::sGFP-OsEULD2 were expressed in the nucleus and cytosol (Fig. 5A). Rice protoplasts were co-infected with OsPWWP4, OsEULS2, OsEULS3, and OsEULD2), and with OsPWWP4p.Gly462*, EULS2, EULS3, and EULD2 for the BiFC assay. When 35S::YFPn + 35S::YFPc co-infection was used as the negative control, no signal was obtained. Interestingly, all six combinations of OsPWWP4 or OsPWWP4p.Gly462*, and each of the three partner proteins, showed strong yellow fluorescence signals in the nucleus (Fig. 5B).

3.5. In vitro pull-down assay and methyltransferase activity of OsPWWP4 and the mutant protein

To verify interactions of OsPWWP4 or OsPWWP4p.Gly462* with each of the partners (OsEULS2, OsEULS3, and OsEULD2), we constructed each of the His-tagged OsPWWP4 or OsPWWP4p.Gly462* protein followed by constructing each of the MBP-tagged OsEULS2, OsEULS3, or OsEULD2 protein. In vitro pull-down assays showed strong interactions between OsPWWP4 and OsEULS2, and between OsEULS3, and OsEULD2, resulting in the detection of MBP-tagged OsEULS2, OsEULS3, and OsEULD2 proteins pulled down by His-tagged OsPWWP4 using HisPur™ Cobalt resin (Fig. 6A). Interestingly, another in vitro pull-down assay of OsPWWP4p.Gly462* and each of the three partner proteins confirmed all the interactions between the mutant protein and each protein (Fig. 6B).

The PWWP domain was expected to possess methyltransferase activity (Qiu et al., 2002; Sankaran et al., 2016). To confirm the presence of OsPWWP4 and OsPWWP4p.Gly462* with methyltransferase activity towards OsEULS2, OsEULS3, and OsEULD2, the in vitro methyltransferase assay was performed. The expected sizes of the OsPWWP4 and OsPWWP4p.Gly462* recombinants were 163 and 55 kDa including 5 kDa of the pET-28a (+) vector using a prediction program (https://www.bioinformatics.org/), respectively. Additionally, the three partner proteins OsEULS2, OsEULS3, and OsEULD2 were expected to be 66, 76, and 88 kDa, respectively, including the 43 kDa of the pMAL vector. Expressed sizes of purified OsPWWP4 and OsPWWP4p.Gly462*, and OsEULS2, OsEULS3, and OsEULD2 proteins were confirmed using western blotting analysis, finding all same sizes, as expected (Fig. S6A).

An in vitro methyltransferase assay was performed using the purified protein (Fig. S6B-C). The protein combinations were OsPWWP4 and each of OsEULS2, OsEULS3, OsEULD2, and OsPWWP4p.Gly462* and EULS2, EULS3, and EULD2, including positive and negative controls. Unfortunately, despite the use of several vectors and expression systems, the high-molecular-weight protein OsPWWP4 (163 kDa), including the 5 kDa protein of pET-28a(+), showed severe fragmentation during protein purification. The other proteins, including the mutant protein, were expressed and purified without fragmentation (Fig. S6A). Optical density (OD) was measured using a spectrometer at 30 s intervals over 45 min after incubation. The OD value gradually increased until 45 min and, finally, OD values of OsPWWP4 and each of OsEULS2, OsEULS3, and OsEULD2 values were 0.288, 0.261, and 0.295, and values of OsPWWP4p.Gly462* and each of EULS2, EULS3, and EULD2 were 0.279, 0.297, and 0.292, respectively, resulting in no significant difference between OsPWWP4 and the mutant protein (Fig. S6B-C). The OD value of the negative control was 0.156, while that of the positive control was 0.222 (Fig. S6B-C).

3.7. Relative expression level of OsPWWP4, and OsEUL genes

Next, we examined the relative expression levels of OsPWWP4 as well as the mutant gene and partner genes in WT and sitl3 plants under salt stress and ABA treatment. Then, expression levels of the genes were measured at 0, 1, 3, 6, 12, and 24 h under 150 mM NaCl (Fig. S7A) or 100 µM ABA treatment (Fig. S7B) in WT and sitl3. Under saline conditions, the expression levels of OsPWWP4 and its mutant genes increased in the shoots during 6 h and then decreased after 12 h in both WT and sitl3 plants. Interestingly, the expression levels were significantly lower in shoots of sitl3 than in those of WT plants at 6–24 h after treatment, although there was no difference between non-saline and early salt treatments. In the roots, the expression levels of the sitl3, OsPWWP4, mutant gene were significantly lower than those of the WT from 6 to 24 h after salt treatment, under which, the relative expression of the EUL family genes, such as OsEULS2, OsEULS3, and OsEULD2, was significantly higher in the shoots and roots of sitl3 than in those of WT plants, at least one or more sampling time points, particularly at 6 to 12 h after treatment.

Additionally, under the ABA treatment, the relative expression of OsPWWP4 gradually increased after 24 h. However, the OsPWWP4 mutant gene of sitl3 showed a lower level of expression than OsPWWP4 in the WT from 12 to 24 h after salt treatment. In contrast, partner genes showed higher expression in sitl3 than in WT from 3 to 24 h (OsEULS2 and OsEULS3) or from 1 to 24 h (OsEULD2) after salt treatment.

3.8. Relative expression level of transcriptional factors related to EUL family genes

To further verify the molecular regulation of OsPWWP4 and EUL family gene expression during the plant response to salinity, the expression of transcription factors related to partner genes was measured in both WT and sitl3 plants under salt stress. First, upstream sequences (2 kb long) of the three partner genes were scanned with a website program, PlantPAN4.0 (Chow et al., 2019) to detect transcription binding sites and the corresponding transcription factors. We retrieved overlapping transcription factors that were predicted to exist in the promoters of all three partner genes (Fig. S8, Table S6-S8). Among them, five WRKYs, four NACs, and two bZIPs transcription factors related to salt stress were randomly selected based on previous reports, and their expression levels were evaluated in leaf blades, leaf sheaths, and roots of both one-week-old plants under non-treatment and 150 mM NaCl for 7 d (Fig. 7). Five WRKY transcription factors, OsWRKY8, OsWRKY23, OsWRKY45, OsWRK72, and OsWRKY89 were selected, and their expression was significantly increased in leaf blades and roots or in all three all tissues of sitl3 relative to those of WT plants under salinity. However, there were no significant differences in the expression of the genes tested in any of the three tissues between the WT and sitl3 untreated controls, except for OsWRKY8 in the leaf blades and OsWRKY45 in the roots of sitl3 plants.

Expression analysis of four NAC genes, OsNAC4, OsNAC5, OsNAC10, and OsNAC58, revealed significant differences in expression levels in at least one or two tissues between WT and sitl3 plants, resulting in higher levels of OsNAC4, OsNAC5, and OsNAC58 in sitl3 than in WT plants under salt stress; however, the expression of OsNAC10 was higher in the roots of WT plants. No significant difference was found between the tissues of both plant types under control conditions, except for OsNAC4 in the leaf blades of sitl3. Among the two bZIP transcription factors, the expression of OsbZIP23 was higher in roots and leaf blades but not in leaf sheaths under salt stress, while no significant changes were observed in any tissue under control conditions. Lastly, there was no difference in expression of OsbZIP72 between WT and sitl3 plants in any of the three tissues studied under control or salinity conditions.

The expression of OsPWWP4 (or the mutant gene) and its three partner genes were also examined in the same tissues. Expression of OsPWWP4 was higher in the three tissues of WT than those in the mutant gene in sitl3 under salinity conditions, whereas no difference was observed in any of the three tissues under control conditions. Interestingly, the expression of the three partner genes was significantly higher in sitl3 than in WT plants under salt stress; however, no difference was found between the two plant types under control conditions, except in the roots of OsEULS2.

3.9. Relative expression level of salt stress-related genes

In addition, the expression levels of genes related to the plant responses to salinity were evaluated using the same samples (Fig. 8). There was no significant difference in the expression levels of three OsSOS1-3 genes (Xiang et al., 2007) between WT and sitl3 plants before treatment, except in the roots of OsSOS3. However, the expression levels after treatment increased and were higher in sitl3 than in the WT plant tissues, except for the roots of OsSOS2 and OsSOS3 under salinity. The expression pattern of the OsCIPK15 gene, a protein kinase that interacts with OsSOS3 (Kanwar et al., 2014) was similar to that of OsSOS2.

As for OsTPC1, which imports calcium into the cytosol (Kurusu et al., 2012), it showed higher expression in the leaf sheaths and roots of sitl3 than in those of the WT, before treatment; however, no difference was found between the two plant types under salt stress. The expression of OsMSR2, which responds to Ca²⁺ signaling and the activity OsNHX1 Na⁺ transport (Xu et al., 2013), was higher in the three tissues of sitl3 than in those of the WT, whereas no significant difference was detected under control conditions, except in the roots. Expression of OsNHX1 (Chen et al., 2007) showed no significant differences in the leaf blades and sheaths before treatment; however, higher expression was observed in both tissues in sitl3 than in the WT. OsCAX1, which exchanges Ca²⁺ and H⁺ in the tonoplast (Kamiya 2006), showed no difference between both plants in expression, in any of the three tissues studied, except for leaf blades under saline conditions.

Salt stress affects vast agricultural areas worldwide, reducing crop yield and quality (El Sabagh et al., 2020). Ongoing research focuses on improving salt tolerance, and it is important to study the composition of the entire genome and the functions of the corresponding genes to gain a thorough understanding of salinity tolerance at the molecular level, such as to enable the development of salinity-tolerant rice varieties (Sahi et al., 2006; Ganie et al., 2019; Qin et al., 2020). Therefore, a crop genomics-assisted breeding platform composed of a core TILLING line population with a fixed generation and their entire genome information and molecular and physio-biochemical characterization analysis would empower the selection and development of abiotic stress-tolerant varieties without legal regulation (Cho et al., 2010, Choi et al., 2022, Lim et al., 2022, Seo et al., 2022, Seo and Jang, 2023). For example, the salt-insensitive TILLING line 1 (sitl1) was mutated with a frameshift in the OsCZMT1 protein, whereby, its ability to transport Na⁺ and Mg²⁺ was reduced, thus resulting in salt tolerance (Lim et al., 2022). In turn, in the salt-insensitive TILLING line 2 (sitl2), a frameshift mutation in the Os4BGlu18 protein, promoted the accumulation of lignin and reduced the absorption rate of Na⁺, thus enhancing salt tolerance (Seo et al., 2022). Phenotypic evaluation under the constructed platform showed that salt-insensitive TILLING line 3 (sitl3) exhibited greater fresh weight, root length, and chlorophyll content, and lower H₂O₂ and Na⁺ concentrations than the WT under salt stress; however, there were no significant reductions in agronomic traits of sitl3, plants, including growth and yield in the field.

Reactive oxygen species (ROS) can cause oxidative damage to plants (Lin et al., 2000; Zhu., 2001), and H₂O₂ accumulation reflects the degree of damage caused by salinity in plants. In this study, we observed that sitl3 plants were less damaged under salt stress than the WT, thus showing enhanced salinity tolerance.

In plants growing under salt stress, Na⁺ absorption and accumulation of other ions through the regulation of ion transporters have been reported to respond to salt stress and maintain the K⁺/Na⁺ ratio (Shabala et al., 2008; Hauser et ai., 2010; Shabala and Igor, 2014). Herein, the concentration of Na⁺ increased, whereas that of K⁺ decreased after salinity treatment in both WT and sitl3 plants, compared to the concentrations observed before treatment. The concentration of Na⁺ showed significantly lower accumulation in both shoots (0.65-fold) and roots (0.83-fold) in sitl3 than in the WT, but K⁺ contents showed no significant difference. The low Na⁺ accumulation in salt-tolerant lines is consistent with previous reports (Lim et al., 2022, Seo et al., 2022), providing important clues regarding the salt tolerance mechanism at play in sitl3. Specifically, Ca²⁺ is a stress-signaling ion. Further, SOS2 protein kinase and its associated Ca²⁺ sensor, SOS3, regulate SOS1 and the H⁺/Na⁺ antiporter that controls long-distance Na⁺ transport from roots and shoots, and CAX1, an H⁺/Ca²⁺ antiporter localized in the vacuolar membrane, thus providing a mechanistic link between Ca²⁺ and Na⁺ homeostasis in plants (Shi et al., 2000, Shi et al., 2002; Cheng et al., 2004). The SOS2-SOS3 complex reportedly regulates salt stress tolerance via the phosphorylation of OsSOS1 (Qiu et al., 2002; Quintero et al., 2002; Kim et al., 2013). As a result, ICP-OES of P showed significantly reduced accumulation in roots and leaf blades of sitl3 plants under salt stress, whereas Ca accumulated to a significantly lesser extent only in leaf blades of sitl3 plants than in those of the WT. Although adequate P fertilization can increase salt tolerance in crops, excessive P accumulation can negatively affect salt tolerance (Bargaz et al., 2016). For example, high Na⁺ and P concentrations negatively affect maize growth, whereas P deficiency improves salt tolerance in maize, which is associated with the accumulation of osmolytes, a reduction in Na⁺ accumulation, and selective absorption of K⁺ over Na⁺ (Tang et al., 2019). In another study, Na⁺ accumulation increased because of the increased concentration of P in soybeans (Phang et al., 2009). Although much work remains to be done, this finding suggests that the salt tolerance of sitl3 might change low ion accumulation, including that of P, by the action of multiple complex ion-transport related genes. The findings regarding the higher expression of OsSOS1-3 and OsCIPK15, a protein kinase that interacts with OsSOS3 (Kanwar et al., 2014) in two or three tissues of sitl3 than in WT under salinity might indicate greater activation of the SOS pathway in sitl3 plants despite the absence of any significant difference between WT and sitl3 plants before NaCl treatment. In addition, considering the decrease in Ca accumulation in the leaf blades of sitl3, it is worth investigating the relationship between salinity tolerance and the SOS pathway, including Ca²⁺ signaling and activation of SOS1 and OsCAX1 to export Na⁺ to the external environment or sequestration to the vacuole at sitl3 (Köster, et al., 2019).

The plant hormone ABA acts as a major endogenous messenger in response to abiotic stress (Zhu, 2002). The mechanisms that respond to salt stress consist of ABA-dependent and ABA-independent pathways, which can be verified by germination rate tests (Huang et al., 2021). For example, the OsbHLH035 mutant has an ABA-dependent pathway, resulting in a decrease in the germination rate compared to the WT in response to ABA, and a higher germination rate upon NaCl treatment (Chen et al., 2018). Consistently, the germination assay confirmed that sitl3 had a high germination rate under NaCl treatment and a low germination rate under ABA treatment. Indeed, qRT-PCR analysis showed similar results; specifically, the relative expression levels of OsPWWP4 and partner genes such as OsEULS2, OsEULS3, and OsEULD2 in sitl3 were increased upon ABA treatment relative to the WT. These results support the hypothesis that the salt tolerance mechanism in sitl3 is related to the ABA-dependent pathway, although the issue warrants further research.

Among post-translational protein modifications, methylation plays a vital role in defense mechanisms in response to abiotic stress, as well as during normal plant development, by methyltransferase-mediated transfer of methyl groups (Teerawanichpan et al., 2009; Sun et al., 2022). The PWWP domain functions as a DNA, histone, lysine, and protein methyltransferase (Qiu et al., 2002; Sankaran et al., 2016). For example, the H3K4 methyltransferase OsSDG721 with a PWWP domain reportedly enhances saline–alkaline stress tolerance in rice (Liu et al., 2021). However, there is a lack of conclusive studies on the role of PWWP in the response to abiotic stress, especially salinity. Therefore, an OsPWWP4 gene mutation with a premature stop codon would be a high priority for understanding salt tolerance related to methyltransferase activity in sitl3. As a result of Y2H, we detected the interaction of OsPWWP4 with OsEUL family members such as OsEULS2, OsEULS3, or OsEULD2, but not OsPWWP4p.Gly462*, and with the partner proteins. In contrast, BiFC analysis and pull-down assays revealed clear interactions between PWWP4 and OsPWWP4p.Gly462* of the EUL family. In addition, a methyltransferase activity assay showed similar activities between OsPWWP4 and OsPWWP4p.Gly462* with partner proteins such as OsEULS2, OsEULS3, and OsEULD2. These results suggest that a premature stop in OsPWWP4p.Gly462* might not affect (at least not completely) its interaction with partner proteins and methyltransferase activity. It has been suggested that the N-terminal PWWP domain plays an innate role in the interaction and methyltransferase activity, although the second domain is absent in OsPWWP4p.Gly462*. We recognized that the severe fragmentation of the high molecular-weight protein OsPWWP4 during protein expression and purification should be resolved to rule out differences in activity between OsPWWP4 and the mutant protein. Nevertheless, it is an attractive hypothesis that the lesser extent of methyltransferase activity of the mutant to partner proteins than in the WT, as suggested in a previous report (Seo and Jang, 2023) enhances salt tolerance in sitl3. However, other effect(s) of the gene mutation should be ruled out to understand the enhancement of salinity tolerance in sitl3.

Observation of gene expression changes in sitl3 compared with the WT is critical to rule out the mechanism of salt tolerance in sitl3 (Zhang et al., 2022). The expression levels of OsPWWP4 and the mutant gene were higher in WT than in sitl3 under salt stress. Conversely, the expression levels of members of OsEUL family genes showed higher expression in stil3 than in the WT under salt stress. These results suggest an antagonistic relationship between OsPWWP4 and each of its partner genes via methylation in response to salinity. Accordingly, 10 of the 12 tested putative transcription factors that regulate EUL family genes were expressed to a greater extent in at least one tissue or more in sitl3 than in the WT under salinity conditions. In contrast, nine of the 12 genes showed non-significant differences in expression level between the two plant types in all three tissues studied under control conditions. These results suggest that the upregulation of EUL family genes in sitl3 plants is activated by transcription factors in response to salinity.

One of the mechanisms of salinity tolerance in plants is the restriction of Na⁺ accumulation in the cytosol via its sequestration into the vacuole, as well as the increase of export, thus reducing the possibility of Na⁺ toxicity effects (Tester et al., 2003; Munns et al., 2008). The observation of higher expression of OsNHK1, which transports Na⁺ into the vacuole and is activated by OsMSR2 (Xu et al., 2013), as well as OsCAX1 in two tissues of sitl3 to a greater extent than in WT under salinity conditions suggests that less accumulation in the cytosol in sitl3 enhances salt tolerance, although the issue needs conclusive elucidation.

In this study, we characterized a rice sitl3 mutant showing a salt-tolerant phenotype. The phenotypes and characterization of OsPWW4 including interaction of partner genes and expression of transcription factors and transporters, as well as related genes in sitl3 provide valuable insights into the molecular mechanisms to improve salinity tolerance induced by mutations in OsPWWP4, although much work remains needed to thoroughly clarify the molecular events involved. Therefore, sitl3 might be a valuable genetic resource for understanding the salinity tolerance mechanisms related to post-translational regulation, such as the activity of methyltransferases, to improve rice salt tolerance through breeding.

Author contributions

CSJ conceived the study. HUS conducted the experiments and analyzed the data. HUS and CSJ drafted the manuscript. All the authors have read and approved the final manuscript.

Acknowledgements

This work was supported by a grant from the Republic of Korea and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education(2021R1A6A1A03044242).

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Mutation of a gene with PWWP domain confers salt tolerance in rice

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Abstract

Figures

Highlights

1. Introduction

2. Materials and Methods

2.1. Plant materials, growth conditions, and salt-insensitive mutant line screening

2.2. Phenotype analysis of sitl3 mutant

2.3. Ionomic analysis

2.4. Whole-genome re-sequencing

2.5. Confirmation of mutation caused by SNPs

2.6. Yeast-two-hybrid assays and yeast co-transformation

2.7. Subcellular localization and bimolecular fluorescence complementation assays

2.8. Protein expression and in vitro methyltransferase activity assay

2.9. In vitro pull-down assay

2.10. Gene expression analysis

3. Results

3.1. Phenotypic evaluation of salt tolerance in sitl3

3.2. Whole genome re-sequencing and selection of candidate gene corresponding to salinity tolerance

3.3. Molecular characterization of OsPWWP4 and OsPWWP4p.Gly462*

3.4. Interaction of OsPWWP4 or OsPWWP4p.Gly462* with OsEUL families

3.5. Subcellular localization and bimolecular fluorescence complementation assays

3.5. In vitro pull-down assay and methyltransferase activity of OsPWWP4 and the mutant protein

3.7. Relative expression level of OsPWWP4, and OsEUL genes

3.8. Relative expression level of transcriptional factors related to EUL family genes

3.9. Relative expression level of salt stress-related genes

4. Discussion

Declarations

Author contributions

Acknowledgements

References

Supplementary Files

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