Comparative transcriptome analysis revealed the molecular mechanism response to salt stress at the seedling stage in hexaploid triticale (× Triticosecale Wittmack)

Abiotic stress and ABA-inducible Group 4 LEA from Brassica napus plays a key role in salt and drought tolerance

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.

The development of a salt-tolerant hexaploid triticale cultivar offers an economical and efficient solution for utilizing marginal land. Understanding how hexaploid triticales respond to salt stress is essential if this goal is to be achieved. A genome-wide association study (GWAS), along with transcriptome and proteome analyses, were used in the present study to determine the molecular responses to salt stress in hexaploid triticale. In total, 81 marker-trait associations for 10 salt-tolerance traits were identified in 153 hexaploid triticale accessions, explaining 0.71% to 56.98% of the phenotypic variation, and 54 GWAS-associated genes were uncovered. A total of 67, 88, and 688 differential expression genes were co-expressed at both the transcriptomic and proteomic levels after 4, 12, and 18 h of salt stress, respectively. Among these differentially expressed genes, six appeared in the coincident expression trends for both the transcriptomic and proteomic levels at the seed germination stage. A total of nine common KEGG pathways were enriched at both the transcriptomic and proteomic levels at 4, 12, and 18 h. After integrating GWAS-target genes with transcriptomics and proteomics approaches that the candidate gene late embryogenesis abundant protein 14 (LEA14) was up-regulated at the transcriptomic and proteomic levels. LEA14 contained important stress-responsive cis-acting regulatory elements that could be dynamically regulated by the binding of transcription factors (TFs). This suggested that LEA14 was a key gene associated with salt tolerance in hexaploid triticale and could respond quickly to salt stress. This study improved understanding about the potential molecular mechanisms associated with hexaploid triticale salt tolerance and contributed to the breeding of salt-tolerant germplasms and the utilization of saline soils.

Late embryogenesis abundant (LEA) proteins are essential to the ability of resurrection plants and orthodox seeds to protect the subcellular milieu against irreversible damage associated with desiccation. In this work, we investigated the structure and function of six LEA proteins expressed during desiccation in the monocot resurrection species Xerophyta schlechteri (XsLEAs). In silico analyses suggested that XsLEAs are hydrophilic proteins with variable intrinsically disordered protein (IDP) properties. Circular dichroism (CD) analysis indicated that these proteins are mostly unstructured in water but acquire secondary structure in hydrophobic solution, suggesting that structural dynamics may play a role in their function in the subcellular environment. The protective property of XsLEAs was demonstrated by their ability to preserve the activity of the enzyme lactate dehydrogenase (LDH) against desiccation, heat and oxidative stress, as well as growth of Escherichia coli upon exposure to osmotic and salt stress. Subcellular localization analysis indicated that XsLEA recombinant proteins are differentially distributed in the cytoplasm, membranes and nucleus of Nicotiana benthamiana leaves. Interestingly, a LEA_1 family protein (XsLEA1-8), showing the highest disorder-to-order propensity and protective ability in vitro and in vivo, was also able to enhance salt and drought stress tolerance in Arabidopsis thaliana. Together, our results suggest that the structural plasticity of XsLEAs is essential for their protective activity to avoid damage of various subcellular components caused by water deficit stress. XsLEA1-8 constitutes a potential model protein for engineering structural stability in vitro and improvement of water-deficit stress tolerance in plants.

Expression of eight LEA genes enhanced desiccation tolerance in yeast, including two LEA_2 genes encoding atypical, stably folded proteins. The recombinant proteins showed enzyme, but not membrane protection during drying. To screen for possible functions of late embryogenesis abundant (LEA) proteins in cellular stress tolerance, 15 candidate genes from six Arabidopsis thaliana LEA protein families were expressed in Saccharomyces cerevisiae as a genetically amenable eukaryotic model organism. Desiccation stress experiments showed that eight of the 15 LEA proteins significantly enhanced yeast survival. While none of the proteins belonging to the LEA_1, LEA_5 or AtM families provided protection to yeast cells, two of three LEA_2 proteins, all three LEA_4 proteins and three of four dehydrins were effective. However, no significantly enhanced tolerance toward freezing, salt, osmotic or oxidative stress was observed. While most LEA proteins are highly hydrophilic and intrinsically disordered, LEA_2 proteins are "atypical", since they are more hydrophobic and possess a stable folded structure in solution. Because nothing was known about the functional properties of LEA_2 proteins, we expressed the three Arabidopsis proteins LEA1, LEA26 and LEA27 in Escherichia coli. The bacteria expressed all three proteins in inclusion bodies from which they could be purified and refolded. Correct folding was ascertained by Fourier transform Infrared (FTIR) spectroscopy. None of the proteins was able to stabilize liposomes during freezing or drying, but they were all able to protect the enzyme lactate dehydrogenase (LDH) from inactivation during freezing. Significantly, only LEA1 and LEA27, which also protected yeast cells during drying, were able to stabilize LDH during desiccation and subsequent rehydration.

ABSTRACTPresent findings hypothesize that salt-tolerant and -sensitive oilseed plants are expected to exhibit deviant patterns of growth through lipolytic events in seedling cotyledons. It reports the growth response and different lipolytic mechanisms operating during oil body (OB) mobilization in the seedling cotyledons of salt-tolerant (DRSH 1) and salt-sensitive (PSH 1962) varieties of sunflower (Helianthus annuus L.). Salt tolerance or sensitivity to 120 mM NaCl correlates with high proteolytic degradation of OB membrane proteins, particularly oleosins, whereas salt-sensitive seedling cotyledons exhibit negligible proteolytic activity, thereby retaining OB membrane integrity for a longer time. High lipoxygenase (LOX) activity and its further upregulation by salt stress are the unique features of salt-sensitive sunflower seedlings. Salt-tolerant seedling cotyledons exhibit noteworthy modulation of phospholipase-D (PLD) activity by salt stress. Salt-sensitive seedling cotyledons exhibit higher lipase activity than salt-sensitive ones and enzyme activity is downregulated by salt stress. Salt-sensitive variety exhibits higher lipid accumulation and faster lipid mobilization with seedling development than salt-tolerant variety. Accumulation of oleic and linoleic acid in the seedling cotyledons of salt-tolerant and sensitive varieties exhibits differential sensitivity to salt stress. Novel detection of hexanoic acid (6:0) is a noteworthy feature as a response to salt stress in salt-sensitive variety. These findings, thus, provide new information on long-distance salt stress sensing mechanisms at seedling stage of plant development.

Label-free quantitative proteomic analysis of drought stress-responsive late embryogenesis abundant proteins in the seedling leaves of two wheat (Triticum aestivum L.) genotypes

Most crops are sensitive to salt stress, but their degree of susceptibility varies among species and cultivars. In order to understand the salt stress adaptability of Brassica napus to salt stress, we collected the phenotypic data of 505 B. napus accessions at the germination stage under 150 or 215 mM sodium chloride (NaCl) and at the seedling stage under 215 mM NaCl. Genome-wide association studies (GWAS) of 16 salt tolerance coefficients (STCs) were applied to investigate the genetic basis of salt stress tolerance of B. napus. In this study, we mapped 31 salts stress-related QTLs and identified 177 and 228 candidate genes related to salt stress tolerance were detected at germination and seedling stages, respectively. Overexpression of two candidate genes, BnCKX5 and BnERF3 overexpression, were found to increase the sensitivity to salt and mannitol stresses at the germination stage. This study demonstrated that it is a feasible method to dissect the genetic basis of salt stress tolerance at germination and seedling stages in B. napus by GWAS, which provides valuable loci for improving the salt stress tolerance of B. napus. Moreover, these candidate genes are rich genetic resources for the following exploration of molecular mechanisms in adaptation to salt stress in B. napus.

<p indent=0mm>The deterioration of soil salinization has caused great harm to modern agricultural production in the world. Sorghum is not only one of five main crops, but also an outstanding salt tolerant crop. The screening and identification of sorghum salt toletion of sorghum salt tolerance will be very important to the development and utilization of salinized land, increasing grain yield and maintaining sustainable agricultural development. In this experiment, 110 sorghum landraces were selected to test salt tolerance at germination <sc>(200 mmol L^-1</sc> NaCl treated) and seedling stages <sc>(100 mmol L^-1</sc> NaCl treated). The two germination indicators including the germination potential and the germination rate, as well as seven seedling indicators, including the relative chlorophyll content (SPAD), seedling length, root length, seedling fresh weight, seedling dry weight, root fresh weight, root dry weight were measured. The relative values of various indicators under salt stress were calculated, showing that the relative germination potential and relative germination rate of 110 sorghum landraces were 0<bold>–</bold>98.89% and 23.65<bold>%–</bold>101.79%, and The seven seedling indicators were 59.53%<bold>–</bold>99.91%, 52.47%<bold>–</bold>95.23%, 47.87%<bold>–</bold>100.14%, 27.43%<bold>–</bold>95.28%, 30.48%<bold>–</bold>98.26%, 21.62%<bold>–</bold>100.34%, 31.46%<bold>–</bold>102.13%, respectively. Combined the membership function value analysis, principal component analysis with cluster analysis, the salt tolephyll content (SPAD), seedling length, root length, seedling fresh weight, seedling dry weight, root fresh weight, root dry weight were measured. The relative values of various indicators under salt stress were calculated, showing that the relative germination potential and relative germination rate of 110 sorghum landraces were 0<bold>–</bold>98.89% and 23.65<bold>%–</bold>101.79%, and The seven seedling indicators were 59.53%<bold>–</bold>99.91%, 52.47%<bold>–</bold>95.23%, 47.87%<bold>–</bold>100.14%, 27.43%<bold>–</bold>95.28%, 30.48%<bold>–</bold>98.26%, 21.62%<bold>–</bold>100.34%, 31.46%<bold>–</bold>102.13%, respectively. Combined the membership function value analysis, principal component analysis with cluster analysis, the salt tolerance ability of the 110 landraces at germination stage and seedling stage was comprehensively evaluated and the 110 landraces were clustered into four groups. A batch of sorghum landraces with salt tolerance at germination and seedling stages were identified, especially ten landraces, including Chaoyangbangchui (00003011) from Inner Mongolia and the Baidazimao (00001081) from Beijing showed high salt tolerance, which can be used in further research. There was no significant correlation in salt toleghum landraces with salt tolerance at germination and seedling stages were identified, especially ten landraces, including Chaoyangbangchui (00003011) from Inner Mongolia and the Baidazimao (00001081) from Beijing showed high salt tolerance, which can be used in further research. There was no significant correlation in salt tolerance between the germination stage and the seedling stage. Principal component analysis results indicated that seedling dry weight and root fresh weight can be used as indicators for the evaluation of salt tolerance of a large number of sorghum landraces at seedling stage.

Salt stress is a major constraint in rice production worldwide. Salt stress is estimated to cause annual losses of 30–50% in rice production. Discovering and deploying salt-resistance genes are the most effective ways to control salt stress. We performed a genome-wide association study (GWAS) to detect QTLs related to salt tolerance at the seedling stage using the japonica-multiparent advanced generation intercross (MAGIC) population. Four QTLs (qDTS1-1, qDTS1-2, qDTS2, and qDTS9) associated with salt tolerance were identified on chromosomes 1, 2, and 9. Among these QTLs, a novel QTL, qDTS1-2, was located between flanking SNPs (1354576 and id1028360) on chromosome 1, with the largest −log10(P) value of 5.81 and a total phenotypic variance of 15.2%. RNA-seq analysis revealed that among the seven differentially expressed genes (DEGs) commonly identified in both P6 and JM298 showing salt tolerance, two upregulated genes, Os01g0963600 (ASR transcription factor) and Os01g0975300 (OsMYB48), related to salt and drought tolerance, were also involved in the target region of qDTS1-2. The results of this study can provide insights into further understanding of salt tolerance mechanisms and developing DNA markers for marker-assisted selection (MAS) breeding to improve the salt tolerance of cultivars in rice breeding programs.

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.

As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1-silenced peppers and CaLEA1-overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus-induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl-treated leaves. CaLEA1-OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild-type plants because of enhanced stomatal closure and increased expression of stress-responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA-mediated cell signaling.

Maize (Zea mays) is a crop of significant global importance, yet its productivity is considerably hindered by salt stress. In this study, we investigated two maize cultivars, one exhibiting high salt tolerance (ST) and the other showing salt sensitivity (SS) at the seedling stage. The ST cultivar demonstrated superior seedling survival rates, higher relative water content, and lower electrolyte leakage and malondialdehyde levels in its leaves after both 3-day and 7-day salt treatments, when compared to the SS cultivar. To explore the molecular basis of these differences, we performed comparative transcriptome sequencing under varying salt treatment durations. A total of 980 differentially expressed genes (DEGs) were identified. Gene ontology (GO) functional enrichment analysis of DEGs indicated that the oxidation-reduction process, phosphorylation, plasma membrane, transferase activity, metal ion binding, kinase activity, protein kinase activity and oxidoreductase activity process is deeply involved in the response of ST and SS maize varieties to salt stress. Further analysis highlighted differences in the regulatory patterns of transcription factors encoded by the DEGs between the ST and SS cultivars. Notably, transcription factor families such as AP2/ERF, bZIP, MYB, and WRKY were found to play crucial roles in the salt stress regulatory network of maize. These findings provide valuable insights into the molecular mechanisms underlying salt stress tolerance in maize seedlings.

Raising crops for dry and saline lands: Challenges and the way forward.

BackgroundSoil salinity poses a serious threat to cotton production worldwide by impairing growth, yield, and fiber quality. Salt stress disrupts key morphological, physiological, and biochemical processes in cotton plants, leading to considerable reductions in productivity. Therefore, identifying salt-tolerant cotton genotypes is essential for improving crop performance in saline environments.MethodsIn this study, fifty-one cotton genotypes were evaluated for their response to salinity stress at the seedling stage. Plants were grown in hydroponic culture under controlled glasshouse conditions and subjected to 200 mM NaCl to simulate salt stress. The experiment followed a completely randomized design (CRD) with three replications, and data were analyzed using two-way analysis of variance (ANOVA) and multivariate approaches, including principal component analysis (PCA), heatmap analysis, and the multi-trait genotype-ideotype distance index (MGIDI).ResultsANOVA showed significant variation among genotypes for all traits. Salt stress caused significant reductions in growth traits, including shoot and root length, fresh and dry biomass, water relation traits, gaseous exchange traits and photosynthetic pigments. In contrast, excised leaf water loss (ELWL), sodium (Na+ )accumulation in roots and shoots, oxidative stress markers like hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), osmolytes including proline, glycine betaine (GB), and saponin, and antioxidant enzyme activities like superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) increased, while potassium contents (K+) and sodium to potassium ratio (K⁺/Na+) decreased. Under control conditions, PCA showed little variation, whereas under salt stress, it explained 64.8% of the variance and separated growth- from stress-related traits. Heatmap analysis confirmed these patterns and grouped genotypes into three clusters based on ion homeostasis and oxidative stress traits. MGIDI index integrated all traits into a single score and identified superior genotypes like G2 (NIAB-868), G22 (NIA-Noori), G32 (FH-530), G3 (NIAB-878-B), G49 (FH-911), G28 (FH-416), G33 (FH-534), and G39 (FH-546).ConclusionThese findings suggest that multivariate and multi-trait screening at the seedling stage is a useful method for identifying cotton germplasm with salt tolerance, providing a foundation for breeding programs and further field evaluation that may contribute to stable yields under saline conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07354-4.