Identification of salt tolerance‐associated presence–absence variations in the OsMADS56 gene through the integration of DEGs dataset and eQTL analysis

Ongoing soil salinization has emerged as a major adverse environmental stress that globally threatens crop growth and productivity (Munns et al., 2020). As one of the most important cereal crops, rice (Oryza sativa L.) is a glycophyte and hence often severely affected by salinity stress (Negrão et al., 2011). Developing salt tolerant rice cultivars by exploiting more genetic salt tolerant resources will greatly contribute to global food security. Presence–absence variations (PAVs), referring to the presence or absence of gene variability in diverse rice accessions, are an important source of genetic diversity and have been revealed to play key roles in the determination of plant evolution and agronomical traits (Della Coletta et al., 2021; Wang et al., 2023). However, it remains unclear how they modulate stress response, especially salt stress. Currently, with the availability of rice pan-genomic data, PAVs can be systematically identified and described, enabling in-depth exploration of their roles in rice genetic diversity and salt stress response. The expression of genes can be altered by nearby PAV due to their interruptions in gene or regulatory elements (Scott et al., 2021). Several eQTLs associated with salt tolerance have been identified using a set of SNPs generated from the super pan-genome of rice (Wei et al., 2024). Additionally, PAVs known as hidden variants have the ability to reveal new eQTLs that cannot be detected by SNPs (Shang et al., 2022). To discover PAVs affecting gene expression under normal and salt stress conditions, we identified PAV associated with gene expression levels (PAV-expression quantitative loci, PAV-eQTLs) among the Global MiniCore Rice Collection of 202 accessions that have been published previously (Shang et al., 2022; Wei et al., 2024). We defined the PAV located in the c. 2 kb as cis-eQTL and identified 2427 and 2898 cis-PAV-eGenes under normal and salt stress conditions, respectively. Based on occurrence in different conditions, 1692 belonged to static PAV-eGenes and 1206 eGenes pertained dynamic PAV-eGene only under salt stress environment (Fig. 1a, upper panel). Given the crucial role of transcription factors (TFs) involved in salt stress response, we focused on 22 members that overlapped with differentially expressed genes (DEGs) and dynamic PAV-eGenes under the salt stress condition and TF dataset, of which these data were sourced from PlantTFDB (Fig. 1a, lower panel; Supporting Information Table S1). Through examination of a Manhattan plot of PAV-eQTL c. 22 members, we found that 12 members have significant and unique association peaks (Fig. S1). The person correction of 22 genes between the Fragments per Kilobase of transcript per Million mapped reads (FPKM) and survival rate under salt stress condition was further analyzed, and the result indicated that 12 members showed a significant correlation between the expression levels and the survival rate (Figs 1b, S2). Next, we found that seven candidate genes can simultaneously meet both of the above conditions (Fig. S3). Finally, we further analyzed the significant positions where PAVs fall and combined with the gene function, focusing on OsMADS56 (also named as GL10) that regulated heading date and grain size (Figs 1c, S3; Table S1). Among these significant PAVs of OsMADS56, we found that one PAV (Chr10_20,863489) resulted in the complete absence of both the ATG and first exon (Fig. 1d). Based on sequence variations of this PAVs across 202 rice accessions, they were classified into two haplotypes (Hap1 and Hap2) of OsMADS56 (Fig. 1d). The Hap1 containing 1.0 Kb PAV was detected in six salt-tolerant cultivars (Fig. S4). Furthermore, these accessions containing Hap1 had much higher survival rate and lower dead leaf rate than those with Hap2 after NaCl treatment (Fig. 1e, left and middle panel). Additionally, the FPKM values of Hap1 were significantly higher than Hap2 (Fig. 1e, right panel), indicating that this PAV disrupted the expression of OsMADS56 to cause more sensitivity to salt tolerance. To further reveal the genetic effect of this natural variation in OsMADS56 on salt tolerance, we obtained the near isogenic lines (NILs) of OsMADS56, NIL-GL10 and NIL-gl10, corresponding to Hap1 and Hap2, respectively (Zhan et al., 2022), and found that the survival rate of NIL-GL10 was obviously higher than that of NIL-gl10 under salt stress (Fig. 1f). Collectively, these results support the notion that OsMADS56 is likely to be crucial for salt tolerance in rice. We found that OsMADS56 was significantly induced when exposed to 150 mM NaCl and reached the peak at 6 h (Fig. S5a). Subsequently, the pOsMADS56::GUS transgenic seedlings were used to further verify the response to salt stress. In accordance with the quantitative real-time polymerase chain reaction assays, the expression of OsMADS56 was enhanced after exposed to NaCl treatment for 1 h, peaked at 6 h, and then decreased (Fig. S5b). To confirm the function of OsMADS56 in salt stress, the loss-of-function osmads56 mutants mediated by CRISPR/Cas9 system and OsMADS56 overexpressing transgenic plants driven by the Ubiquitin promoter were obtained. We selected two T1 homozygous frame-shift osmads56 mutants (Cas9-72 and Cas9-73) and two independent overexpression lines with elevated OsMADS56 expression (OE-2 and OE-6) in XS134 background, as well as one T1 homozygous frame-shift mutant (Cas9-6) in ZH11 to further investigate their responses to 150 mM NaCl (Fig. S6). Compared with the wild-type (WT), OsMADS56 overexpressing lines exhibited several specific advantages under NaCl treatment, including higher seed germination rates during the germination stage (Figs 1g, S7), lesser inhibition effects on shoot length, root length, and fresh weight in the post-germination stage (Fig. S8), as well as higher survival rates at the four-leaf stage (Fig. 1h), suggesting that overexpression of OsMADS56 caused hyposensitivity to salt stress in rice. Nevertheless, osmads56 mutants from XS134 and ZH11 both exhibited much more severe salt stress-sensitive characteristics than their corresponding WT (Figs 1g,h, S7–S9). Furthermore, our RNA-seq data showed that the transcriptions of positively regulated genes for salt tolerance, such as SKC1 and OsSIK2, were drastically enhanced in overexpression lines under salt stress, compared with WT, which were notably reduced in the osmads56 mutants (Fig. S10). Together, these data indicated that OsMADS56 plays a positive regulatory role in rice salt tolerance. Emerging evidence suggests that salt stress-induced osmotic stress causes increased reactive oxygen species (ROS) generation, which in turn creates oxidative stress that results in physiological damage to plant cells (Castro et al., 2021). To detect the ROS accumulation, we compared 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining in osmads56 variants, OsMADS56 overexpression lines, and WT leaves. Without NaCl treatment, there was no noticeable difference between above plants. However, after being exposed to 150 mM NaCl treatment, the DAB and NBT staining results showed that more ROS accumulated in the osmads56 mutants, but less in the OsMADS56 overexpression lines, than in their corresponding WT (Fig. 1i). Since the ROS accumulation is related to the activity of ROS-scavenging enzymes, we then measured the antioxidant enzyme activities, such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). As expected, all of the mentioned enzyme activities were increased under osmotic stress treatment; however, the enzyme activities of osmads56 mutants were clearly lower than those of WT, whereas the enzyme activities were evidently higher in the OsMADS56 OE plants than in the WT (Fig. 1i). Consequently, we speculated that the OsMADS56 gene could improve the salt stress tolerance through enhancing the activity of certain ROS-scavenging enzymes in rice. To investigate the genetic interactions between OsMADS56 and other salt tolerance genes, all 202 accessions from the pan-genomic population were classified by functional nucleotide polymorphisms (FNPs) of GS3, SKC1, STG5, and MKK10.2. Coincidentally, in the presence of OsMADS56, significant differences were observed in salt tolerance between the groups containing GS3/SKC1 and gs3/skc1, implying that OsMADS56 had an additive effect on salt tolerance for these genes (Fig. 1j). A similar additive effect of OsMADS56 also occurred on the STG5 and MKK10.2 genes in terms of salt tolerance (Fig. S11). These results indicate that different combinations of OsMADS56 and these genes' FNPs have significant effects on salt tolerance, suggesting their potential synergistic regulation of salt tolerance. In conclusion, we innovatively determined the effects of PAVs on salt stress response in rice and successfully identified a novel salt tolerance gene OsMADS56, suggesting that super pan-genome and transcriptomics technologies rendered an effective way to identify functional PAV-eQTLs in salinity tolerance. OsMADS56 overexpression lines reduced the accumulation of ROS by up-regulating ROS-scavenging enzyme activities, thereby enhancing the salt stress tolerance in rice. Our findings also provide insights into an alternative multi-gene aggregation strategy for salt tolerance, implying that OsMADS56 might be a promising genetic resource for developing salt tolerance rice cultivars. These discoveries are expected to contribute to rice production in the face of the continuous threat of salt stress. More critically, previous studies have revealed that OsMADS56 also contributes to grain size, thermotolerance, and photoperiodic flowering in rice (Ryu et al., 2009; Zhan et al., 2022; He et al., 2024). Thus, OsMADS56 provides a promising target for increasing productivity, stress tolerance, and adaptability of rice globally. The rice cultivar Xiushui134 (Oryza sativa L. ssp. Japonica) was used as WT for physiological experiments and genetic transformation. The knockout mutants of OsMADS56 were generated by CRISPR/Cas9 system, and OsMADS56 overexpressing lines were driven by Ubiquitin promoter. The osmads56 mutant in Zhonghua11 (ZH11) background was purchased from the WIMI Biotechnology Co., Ltd (Changzhou, China) (Lu et al., 2017). Plants were grown in the growth chamber or glasshouse with a 14 h : 10 h, 30°C : 25°C, light : dark cycle (300 μmol photons m−2 s−1). Relative humidity was controlled at 60% humidity. The RNA-seq data and the DEGs dataset were collected from our previous studies (Shang et al., 2022; Wei et al., 2024). The method of eQTL analysis was conducted in the same way as described in Wei et al. (2024). For PAV-eGene, eQTL with lead SNP located within 2 kb upstream and downstream of the gene were classified as cis, while the others were considered as trans. To analyze seed germination under salt treatment, roughly 90 seeds of osmads56 mutants, OsMADS56 overexpressing lines, and corresponding WT (three replicates per genotype) were randomly placed in ½-strength Murashige & Skoog medium (½ MS medium) supplemented with 150 mM NaCl. Seeds were considered to have germinated when the radicle or germ reaches a length of c. 1 mm. The germination rates were recorded daily. For phenotype analysis at post-germination, the uniformly germinated seeds were grown on ½ MS medium containing 150 mM NaCl at 28°C. Then, the shoot length, root length, and fresh weight were measured to assess the salt inhibition. For salinity stress testing, 15-d-old seedlings grown in ½ MS medium were treated with 150 mM NaCl solution for 3–5 d. After 7-d recovery, seedlings were photographed and the survival rate was determined. Seeds and seedlings grown in ½ MS medium were used as the control. For salt stress response by quantitative real-time polymerase chain reaction, the WT (XS134) was exposed to 150 mM NaCl, and the samples were, collected at different time points. Nitroblue tetrazolium staining and 3,3′-diaminobenzidine staining were used for detecting O2− and H2O2, respectively, as described previously (Zhang et al., 2014). The activities of CAT, APX, and SOD were determined as reported previously (Foyer & Noctor, 2005). To analyze the promoter activity of OsMADS56, a 3.5-kb region upstream of the translation start codon of OsMADS56 was cloned into pCXGUS-P vector (Chen et al., 2009) to create pOsMADS56::GUS construct. Then, the resulting construct was transformed into rice calli of XS134 by an Agrobacterium tumefaciens-mediated method (Hiei et al., 1994). Rice samples were incubated in GUS staining solution at 37°C in the dark for 12 h, and chlorophyll was removed using 75% ethanol. Sixteen-day-old seedlings were treated with 150 mM NaCl for 6 h, while seedlings without NaCl treatment were considered as a control. Whole plants were then sampled for RNA sequencing. TRIzol (Life technologies, Carlsbad, CA, USA) was used for extracting total RNA. For transcriptome analysis, cDNA libraries were constructed according to standard Illumina protocols and sequenced using the Illumina HiSeq 4000 system. Differentially expressed genes were defined as those with a twofold expression difference and a P-value < 0.05. The extracted RNA was reverse transcribed with First Strand cDNA Synthesis Kit (Thermo, Waltham, MA, USA). Quantitative real-time polymerase chain reaction was performed with the SYBR Premix Ex Taq (Takara, Otsu, Shiga, Japan) following the operation manual, and the rice OsActin1 gene was used as endogenous control. Data from three biological replicates and three technical repetitions were collected. A list of primers is shown in Table S2. We thank Prof. Shaokui Wang (South China Agricultural University, Guangdong) for kindly providing the near isogenic lines NIL-GL10 and NIL-gl10. This work was supported by grants from STI2030-Major Projects (2023ZD04076), The National Natural Science Foundation of China (32188102, 32301882), Innovation Program of Chinese Academy of Agricultural Sciences, Youth Innovation of Chinese Academy of Agricultural Sciences (Y2023QC36), and the Special Project for Public Welfare Research Institute of Fujian Province (2021R1027005). None declared. LS, QQ and YZ designed the research. YC, YL, YZ, HW, YP, HH, HQ and XC performed the experiments. LY, ZZ, XZ, TW, WH, XL, CS, QY and XY analysed the data. LS, LC, YL and FW revised the manuscript. All authors reviewed and approved the final manuscript. YC, YL, HW and YP contributed equally to this work. All study data are included in the article and Supporting Information. The DEGs dataset from this article can be found in the PlantTFDB (https://planttfdb.gao-lab.org/). RNA-seq raw data have been deposited in the NCBI SRA database with bioproject no. PRJNA1009219. Fig. S1 Manhattan plot of presence–absence variation-eQTL c. 22 candidate genes under salt stress condition. Fig. S2 Scatter plot of pearson's correction coefficient of 21 candidate genes between the Fragments per Kilobase of transcript per Million mapped reads and salt stress condition and survival rate. Fig. S3 Identification of candidate genes. Fig. S4 Structure and 1-kb presence–absence variations of OsMADS56 in several major salt-tolerant varieties. Fig. S5 OsMADS56 was responsive to NaCl. Fig. S6 Development of osmads56 mutants through CRISPR/Cas9-mediated two different target sites and the acquisition of OsMADS56 overexpression lines. Fig. S7 Phenotypes and germination rate of the wild-type, overexpressing transgenic plants (OE-2), and knockout plants (Cas9-72) for OsMADS56 under ½ MS and 150 mM NaCl treatment. Fig. S8 Statistical analysis of shoot length, primary root length, and fresh weight of the OsMADS56 overexpression lines and osmads56 mutants after salt stress treatment. Fig. S9 Response and the corresponding survival rate of the osmads56 mutants in ZH11 background to 5-d NaCl treatment followed by a 7-d recovery. Fig. S10 Changes in levels of salt-responsive genes in OsMADS56 overexpression lines, osmads56 mutants, and wild-type plants under salt treatment. Fig. S11 Relationship between OsMADS56 and salt tolerance genes (STG5 and MKK10.2). Table S1 List of 22 candidate genes that overlapped with differentially expressed genes and dynamic presence–absence variation-eGenes under salt stress condition and transcription factor database. Table S2 Primers used in this study. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.