Identification and sequence analysis of LpSOS1
Based on the full-length sequence from the transcriptome data, we cloned an LpSOS1 (accession no. OM650676) open reading frame (ORF) of 3,486 bp (Supplementary Fig. 1), encoding 1,162 amino acids. Conserved domain analysis using NCBI Conserved Domain Search showed that the protein encoded by LpSOS1 contained the conserved region NhaP of the Na+/H+ antiporter family. Analysis using ProtParam software determined that the molecular formula of LpSOS1 was C5849H9233N1543O1670S39, the relative molecular weight was 129.14 kDa, and the theoretical isoelectric point (Pi) was 6.59. A BLAST search of the amino acid sequence of LpSOS1 revealed that this protein belonged to the phi class of SOS1 proteins. Amino acid alignment analysis showed that the LpSOS1 protein contains a conserved Na+/H+ antiporter domain spanning the NhaP transmembrane region and catabolite gene activator protein-effector domain (CAP_ED) binding sites. The secondary structure of the LpSOS1 protein features 12 obvious transmembrane structures in its N-terminal region, according to the TMHMM server (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) (Supplementary Fig. 2). The amino acid sequence of LpSOS1 displayed 65.44–67.63% sequence identity with OsSOS1 (Oryza sativa, ATU90113.1), VvSOS1 (Vitis vinifera, NP_001268140.1), AsSOS1 (Aegilops speltoides, CAX83736.1), and TmSOS1 (Triticum monococcum, CAX83735.1) (Fig. 1).
Phylogenetic analysis
The SOS1 family protein sequences were downloaded from the NCBI database. A phylogenetic tree (Fig. 2) based on amino acid sequences was constructed using MEGA 7.0 software based on the neighbour-joining method. LpSOS1 was found to be closely related to PpSOS1 (CAM96566.1), CnoSOS1 (CAD20320.1), IsSOS1 (AGB06353), OsSOS1 (AAW33875.1), AlSOS1 (Aeluropus littoralis, AEV89922.1), and other homologous proteins in closely related plants. Interestingly, LpSOS1 occupied a single branch during the evolutionary process of SOS1 proteins.
Spatial And Temporal Expression Analyses
To investigate the tissue-specific expression of LpSOS1 under salt stress, the plants were watered with 200 mM NaCl solution for 7 d. In the absence of NaCl, LpSOS1 expression was very low in all tissues but was higher in the stems than in the leaf and root tissues. Salt-stressed plants showed significantly increased LpSOS1 transcription in all tissues, with higher expression levels in roots than in stems and leaves (Fig. 3).
Subcellular Localisation Of Lpsos1
The LpSOS1 gene was inserted into the pCAMBIA2300 plant expression vector and fused with GFP to observe the localisation of the LpSOS1 protein in cells. Using tobacco grown for approximately 4 weeks, the pCAMBIA2300-LpSOS1-GFP expression vector and pCAMBIA2300-GFP empty vector were transformed into tobacco leaves, and their localisation was observed (Fig. 4). When the empty vector was transfected, fluorescence signals were detected in the nucleus, cell membrane, and cytoplasm. Furthermore, upon transfection of pCAMBIA2300-LpSOS1-GFP, the signals were detected in the cytoplasm. Thus, colocalisation with marker proteins revealed that the LpSOS1 fusion is associated with the membranes and cytoplasm.
Identification of Arabidopsis loss-of-function mutants and development of transgenic Arabidopsis plants
To explore whether LpSOS1 is involved in the Arabidopsis salt stress response, we purchased Arabidopsis SOS1-1 mutant seeds with T-DNA insertions from the Arabidopsis Mutant Seed Bank Resource Center. Genomic DNA from WT A. thaliana and SOS1-1 mutants was extracted using the cetyltrimethylammonium ammonium bromide method, while the three-primer method was used for PCR amplification of the genomic DNA (Fig. 5a), followed by identification of homozygous mutant lines. The primer RP + LBb1.3 could amplify only the mutant plant DNA (Fig. 5b) and not the WT DNA; at the same time, the LP + RP primer could amplify only the WT DNA template. These results indicated that the mutant strain was homozygous.
To explore the biological function of LpSOS1 in response to abiotic stress, the ORF fragment of LpSOS1 was cloned into the vector pCAMBIA3301 and transformed using the Agrobacterium-mediated inflorescence dip method. Selection of Arabidopsis transgenic seeds was carried out on 1/2 MS medium with Basta. Transgenic plants were selfed for three generations to obtain homozygotes, and six of these plants were evaluated using RT‒PCR. None of the PCR products obtained from the transgenic plants were present in the Arabidopsis SOS1-1 mutant plants (Fig. 5c). The expression levels of LpSOS1 in six vigorously growing transgenic Arabidopsis lines were measured using qRT‒PCR (Fig. 5d). The transgenic line with the highest relative expression was selected for further experiments.
LpSOS1 expression conferred higher salt tolerance to transgenic SOS1 mutant plants
To test whether LpSOS1-transgenic plants could complement the salt sensitivity of the SOS1 mutant, the growth (expressed as the germination ratio and biomass) of two LpSOS1-transgenic lines, the SOS1 mutant and the WT, was analysed and compared under NaCl treatment. Seeds of T3 homozygous transgenic lines of the WT and mutant transgenic lines obtained by means of screening were sown on 1/2 MS medium with or without 100 mM NaCl, with nontransgenic lines used as controls for salt tolerance. It was found that all tested seeds could germinate under normal conditions. However, comparative analysis showed that only 13% of the seeds of the SOS1 mutant germinated on the 7th day after treatment with 100 mM NaCl, while 79% of the atsos1-LpSOS1 seeds germinated. The germination rate of the WT seeds was 44%, whereas that of the WT-LpSOS1 seeds was 80%. The T3 generation seeds of the SOS1 deletion mutant, atsos1-LpSOS1, WT, and WT-LpSOS1 were transferred to 1/2 MS medium containing 0, 50, 75, and 100 mM NaCl and then allowed to grow for 15 d, followed by measurement of their root lengths and fresh weights. Compared to the non-saline conditions, NaCl treatment inhibited the growth of all plants. However, the growth of the atsos1-LpSOS1 and WT-LpSOS1 plants under salt stress was better than that of SOS1 mutant and WT plants, thereby indicating that LpSOS1 expression made plants more salt-tolerant. As shown in Fig. 6, with an increase in salt concentration, the root length and fresh weight of the SOS1 mutant and WT plants decreased significantly, while LpSOS1-transgenic plants showed little change. Compared to those observed in the SOS1 deletion mutant under treatment with 100 mM NaCl, the root lengths of the atsos1-LpSOS1 and WT-LpSOS1 plants were 2.8- and 2.9-fold greater, respectively (Fig. 6). These results indicated that LpSOS1 expression confers salt tolerance to transgenic plants.
LpSOS1 expression maintains Na+ and K+ homeostasis in plants
WT, WT-LpSOS1, atsos1, and atsos1-LpSOS1 plants were sown in 1/2 MS medium for 5 d and then transferred to soil for four weeks for normal growth. Then, the experimental group was treated with 200 mM NaCl for two weeks. As shown in Fig. 7a, compared to the control group, the mutant atsos1 displayed the greatest inhibitory effect, with wilted leaves, chlorosis, and cessation of plant growth. However, the plants of the two transgenic lines, WT-LpSOS1 and atsos1-LpSOS1, grew well without wilting. The SOS1 deletion mutant plants almost died after two weeks of salt stress, whereas the plants overexpressing LpSOS1 grew well. These results further suggested that LpSOS1 expression confers salt tolerance to transgenic plants.
Nutrient imbalance is one of the most deleterious effects of salinity on the physiology and biochemical processes of plant cells. Thus, we determined the Na+, K+, and Na+/K+ ratios in all the lines (Fig. 7b–d). Under control conditions, the Na+, K+, and Na+/K+ ratios of WT, atsos1, and transgenic plants transformed with LpSOS1 were not significantly different; however, there was a significant increase in the Na+ content of the lines under NaCl treatment, with atsos1 displaying the highest levels, followed by WT plants, and transgenic atsos1-LpSOS1 supplementing the index to a level close to that of the WT. Compared to the WT plants, transgenic WT-LpSOS1 plants displayed a significant decrease in the relative content (P < 0.05). These results suggested that LpSOS1 expression can alleviate the inhibitory effect of salt stress on plant growth by reducing Na+ levels and maintaining K+ homeostasis in plants.
LpSOS1-transgenic plants displayed less MDA accumulation and higher SOD, POD, and CAT activities under salt stress
MDA levels as well as SOD, POD, and CAT activities were determined in the eight lines (two WT, two WT-LpSOS1, two atsos1, and two atsos1-LpSOS1 lines) that were subjected to salt stress (Fig. 8). With no salt stress, there was no significant difference in the physiological indices. Salt stress, on the other hand, increased the MDA content in four of the lines, with the level in atsos1 being significantly higher than that in the WT (P < 0.05). In both the WT and the deletion mutant atsos1, the MDA content in the shoots of the plants decreased significantly after being transformed with LpSOS1; the content was 34.3% lower in the WT-LpSOS1 line than in the WT line. Compared to the atsos1 line, the atsos1-LpSOS1 line displayed a 52% decrease. The above results indicated that the cell membrane integrity and stability of the transgenic line were higher under stress conditions, and the degree of membrane lipid peroxidation was lower. Simultaneously, the plasma membrane suffered less damage. Therefore, transgenic lines can adapt better to adverse environments.
Under control conditions, there were no significant differences in SOD, POD, and CAT activities in the shoots of WT, atsos1, transgenic WT-LpSOS1, and atsos1-LpSOS1 seedlings (P > 0.05). However, with NaCl treatment, there was a significant increase in SOD, POD, and CAT activities of the transgenic WT-LpSOS1 and atsos1-LpSOS1 lines compared to those of the WT and atsos1 lines (P < 0.05). This indicated that the transgenic SOS1-overexpressing plants had relatively less accumulation of reactive oxygen species (ROS), higher activity of antioxidant enzymes, and stronger ROS scavenging and antioxidant capacity upon being subjected to stress, leading to their enhanced salt tolerance.
Expression analysis of salt stress-related genes in LpSOS1-transgenic Arabidopsis
Because the overexpression of LpSOS1 in Arabidopsis led to salt tolerance, we selected several salt stress-related genes, including salt hypersensitivity 2 (AtSOS2), salt hypersensitivity 3 (AtSOS3), Na+/H+ antiporter agent 1 (AtNHX1), SOS3-like calcium binding protein 8 (AtCIPK8), and high-affinity K+ transporter 1;1 (AtHKT1;1), which serve as markers for monitoring salt stress response pathways in Arabidopsis, and examined the expression levels of these genes using qRT‒PCR in samples from WT, SOS1 mutants, and transgenic plants subjected to both normal and NaCl treatment conditions. All six genes were upregulated in the WT, SOS1 mutants, and transgenic lines after salt treatment, but the expression levels were dramatically higher in the transgenic plants than in the WT plants and SOS1 mutants (Fig. 9). These results suggested that LpSOS1 participates in responses to salt stress by regulating the expression of stress-related genes during plant growth and development.