Arabidopsis (Columbia-0) and the Columbia-0 seed line that constitutively expresses the intracellular Ca²⁺ indicator aequorin are preserved in our laboratory. Mutants of pgsip7 (Salk_016683C), pgsip8 (Salk_037413C), and sos2 (Salk_063265C) were purchased from AraShare. The moca1 mutant and p35S::AtMOCA1 were provided by Professor Zhenming Pei. Chlamydomonas reinhardtii, Physcomitrella patens, and Oryza sativa were preserved in our laboratory, and transgenic plants were constructed for this experiment. A growth chamber with controlled conditions (25 °C day/22 °C night, 16 h light/8 h dark, 110 µmol m^-2 s^-1 white light) was used for cultivating plants.

The green cotyledon ratio (RGC) can reflect the degree of stress.  Green Cotyledon Ratio=Green Cotyledon Plants/Total Plants×100%. Arabidopsis seeds were planted on 1/2 MS medium with varying doses of sorbitol (0–400 mM) and NaCl (0–150 mM) and photographed ten days later. The concentration of 60 mM NaCl was selected as the threshold based on preliminary gradient experiments, as this was the minimal concentration that induced significant phenotypic differences between wild-type and salt-sensitive mutants without causing non-specific inhibition. Arabidopsis thaliana’s root length was determined using ImageJ (Zhou et al., 2011).

The Arabidopsis MOCA1 protein sequence was used as a reference, and the conserved domains in the MOCA1 protein sequence were used as the criterion. Download the whole proteome sequences of Chlamydomonas reinhardtii (v5.6), Physcomitrella patens (v3.3), and Oryza sativa (v7.0) from the Phytozome v13 database (https://phytozome-next.jgi.doe.gov/), and use BioEdit to establish a local protein library and BLAST-P to search for potential homologous genes. The TAIR database (https://www.arabidopsis.org/) provided the MOCA1 protein sequence of Arabidopsis.

Information on members of the Arabidopsis GT8 subfamily is provided by the TAIR website. The MAFFT (https://mafft.cbrc.jp/alignment/server/) was applied to align protein sequences using the FFT-NS-I algorithm (Katoh and Standley, 2013). The amino acid sequence scoring matrix parameter was BLOSUM62. The neighbor-joining (NJ) method was then used to perform phylogenetic analysis on the aligned sequences using MEGA X with 1000 bootstrap replicates (Kumar et al., 2018). Transcriptome data of GT8 subfamily members were obtained from the ePlant (http://bar.utoronto.ca/eplant_soybean/). The FPKM values were normalized, and HemI was utilized to create a heat map of expression (Deng et al., 2014). The alpha helices of the MOCA1, PGSIP7, and PGSIP8 protein structures were predicted by the Araemnon (http://aramemnon.botanik.uni-koeln.de). The protein tertiary structure was predicted by the SIWSS-Model and optimized by ChimeraX 1.3. Ensembl (https://plants.ensembl.org/index.html) is the source of information about MOCA1 homologous genes. The sequences were aligned using MAFFT, and further visualized using Jalview software.

Total RNA was obtained with TransZol (TransGen Biotech, Beijing, China), and cDNA was prepared by EasyScript^® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The primers used were designed using Primer 5.0 according to standard primer design principles (Supplementary Table S1). Using the 2× Perfect SYBR Green PCR Mix (TransGen Biotech, Beijing, China) and the QuantStudio 3 PCR System (Life Technologies, Singapore), qRT-PCR was carried out rigorously, adhering to the manufacturer’s instructions. Relative gene transcript levels were measured as 2^−⊿⊿Ct, and ACTIN served as an internal control (Li et al., 2019).

ROS produced by xanthine dehydrogenase 1 (AtXDH1) in Arabidopsis were detected by nitroblue tetrazolium (NBT) and 3,3’-diaminobenzidine (DAB) staining. The Arabidopsis roots were immersed in NBT solution (0.1%, pH 7.8) and vacuum infiltrated for 20 minutes to allow the solution to fully penetrate the root tissue, incubated in the dark for 15 minutes, and washed with 95% ethanol to remove chlorophyll. The production of superoxide anions was assessed by observing the distribution of blue precipitates under a microscope. The roots of Arabidopsis were immersed in DAB solution (1 mg/mL, pH 3.8) and vacuum infiltrated for 20 minutes, incubated in the dark for 45 minutes, and decolorized with 95% ethanol. Hydrogen peroxide production was assessed by observing the distribution of brown precipitates under a microscope. For quantitative analysis, grayscale analysis was performed using ImageJ software (National Institutes of Health, USA). The mean gray value of the stained area was measured to quantify ROS accumulation levels. Briefly, the color images were converted to 8-bit grayscale, and the mean gray value was calculated after subtracting the background. Each experiment was repeated three times, with no less than 10 roots selected per group.

Arabidopsis that expresses aequorin was used to assess the cellular free Ca²⁺ concentration ([Ca²⁺]_i) (Knight et al., 1991; Yuan et al., 2014). In a 150 mm × 150 mm petri dish, 3.3 mL of 10 mM coelenterazine was equally sprayed on nine-day-old seedlings and left in the dark for 12 hours. A ChemiPro HT system equipped with a cryocooled, back-illuminated CCD (charge-coupled device) camera and a light-tight box was used for imaging. WinView/32 (Roper) controls the camera. Treat seedlings with either water or 200 mM NaCl, and take photos every four minutes to record fluorescence values. The seedlings were then exposed to strong light for one minute and photographed for two minutes to record chlorophyll spontaneous fluorescence. The experiments were conducted in the dark. Luminous images were analyzed using MetaMorph.

The target fragments were amplified from cDNA libraries of different plant materials. The homologous genes of the MOCA1 gene in Chlamydomonas reinhardtii, Physcomitrella patens, and Oryza sativa are Cre_10g422450v5, Pp3c2_32110, Pp3c1_6500, and loc_Os02g41520.1, respectively. A complete circular plasmid was constructed by ligating the target sequence to the linear vector, leveraging the complementarity between the restriction enzymes at both ends of the sequence and those at both ends of the linear vector. After transforming the recombinant plasmid into Agrobacterium GV3101, we applied the floral dip method to transform the Arabidopsis mutant moca1. T0 seeds were planted on 1/2 MS medium (HygB⁺) and transplanted after growing four true leaves. Plants that grew normally on the antibiotic-containing selective medium were T1 generation positive transgenic plants.

The principle of GUS staining is that under appropriate reaction conditions, β-glucuronidase (GUS) could react with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) to produce insoluble blue substances, 5,5’-dibromo-4,4’-dichloroindigo, which are visible under a microscope. The 1500 bp sequence before the ATG of the CreMOCA1 gene’s start codon was selected to construct the proCreMOCA1::GUS vector and further obtain transgenic Arabidopsis plants. Seven-day-old seedlings of proCreMOCA1::GUS transgenic Arabidopsis were exposed to NaCl (0, 50, and 100 mM) for 6 hours and then completely immersed in the GUS staining solution. After incubation at 37 °C in the dark for 2 hours and decoloring with 70% ethanol, the activity sites of GUS showed blue.

Statistics were analyzed using SPSS. Statistical significance was evaluated using two-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001. Results were expressed as mean ± s.d. Each experiment was conducted three times independently.

The MOCA1 gene belongs to the glucuronosyltransferase subfamily 8 (GT8), which contains 34 members, divided into the GUX, GolS, GAUT, and GATL branches (Yin et al., 2010). Among them, MOCA1 is in the GUX branch, while PGSIP7 and PGSIP8 are not in these four branches, which are separate branches (Figure 1A). The PGSIP gene is an alpha-glucosyltransferase involved in initiating starch synthesis. Before being discovered as a salt sensor, the MOCA1 gene was annotated as PGSIP6 and serves a major part in low manganese resistance in Arabidopsis (Bian et al., 2018), which means that it may have multiple functions as a sensor. PGSIP7 and PGSIP8 are evolutionarily closer to MOCA1 than other members, so they may have similar functions. The transcriptome databases’ (Zhang et al., 2020a) cluster analysis showed that the expression levels of this gene family’s members differ across tissues (Figure 1B). The GAUT1 gene has the highest expression in all tissues, while the GolS7 gene has the lowest expression. The MOCA1 gene was highest expressed in the roots, the PGSIP7 gene was highest in the roots and endosperm, and the PGSIP8 gene was highest expressed in the siliques. Each gene showed differential expression in Arabidopsis tissues, indicating that genes in this family have different functions and may be redundant.

Transcriptome sequencing data revealed that PGSIP7, PGSIP8, and MOCA1 gene expression levels in various tissues of Arabidopsis are shown in Figure 2A. Compared with the other two genes, the MOCA1 gene has higher expression in various tissues, especially in seeds, embryos, and endosperm, suggesting that the MOCA1 gene is crucial for germination. In roots, all three genes were highly expressed. Further investigation of these three genes’ expression under different abiotic stresses, including salt, cold, and drought, revealed that the MOCA1 gene is sensitive to common abiotic stresses, the PGSIP7 gene has high expression of nutrient deficiency, irradiation, and ozone, and the PGSIP8 gene has high expression of nutrient deficiency, irradiation, and oxidation. The three genes’ expression varies in tissues and abiotic stress. MOCA1 is the first salt-sensing gene discovered, and the PGSIP7 and PGSIP8 genes also have certain expressions under salt stress. Figure 2B illustrates that the MOCA1 protein sequence is the longest, while the PGSIP8 sequence is the shortest. Both MOCA1 and PGSIP7 proteins contain six helices, while PGSIP8 only contains five helices. Tertiary structure prediction of the protein sequences showed that their tertiary structures were highly similar, both consisting of two chains, A and B. The two subunits were tightly combined and contained multiple alpha helices, indicating that they may have similar functions.

Arabidopsis T-DNA insertion mutants were identified by the tri-primer method, and the primer information is shown in Supplementary Table 2. The amplification product in Col-0 had only long fragments and no short fragments, while the three mutants had only short fragments and no long fragments, proving that all three mutants were homozygous mutants (Supplementary Figure 1A). According to information analysis in the TAIR database, it was found that the T-DNA insertion site of the pgsip7 mutant was located in the third intron region of the gene, while the T-DNA insertion site of the pgsip8 mutant was located in the first intron region of the gene, and the moca1 mutant had a loss of a 12 bp fragment in the ninth exon region. Further experiments at the transcriptional level showed a significant decrease in gene expression in the mutants compared to the wild type (Supplementary Figure 1B).

Under varying concentrations of salt stress, the three mutants exhibited different extents of growth inhibition. The roots of plants were first inhibited by excessive Na⁺ under salt stress. When measuring root length, it was found that the root length of moca1, pgsip7, and pgsip8 mutants was significantly inhibited under 60 mM salt concentration (Figures 3A, B). RGC index testing found that moca1 and pgsip7 were more salt-sensitive than the pgsip8 mutant (Figure 3C). All three mutants were insensitive to osmotic stress and potassium stress (Figures 3D-F). When subjected to salt stress, plants will quickly respond to changes in their surroundings and regulate gene expression through intracellular signal transduction, among which ROS signal plays a very important role (Hasanuzzaman and Fujita, 2022; Kesawat et al., 2023; Zhou et al., 2024). The accumulation of ROS in the roots of the three mutants varied significantly under different salt stress concentrations (Figures 3G-I). Superoxide anions and hydrogen peroxide are accumulated in the roots of the moca1 mutant, which increases with increasing salt concentration. It may be that the moca1 mutant is insensitive to Na⁺, the normal ROS signal transduction pathway is affected, and the expression of related genes is suppressed, causing the ROS produced by stress not to be promptly cleared. Superoxide anions in the roots of the pgsip7 mutant also accumulated with the increase of salt concentration. In contrast, superoxide anions in the roots of wild-type and pgsip8 mutants did not accumulate with increasing salt concentration but were constantly cleared to maintain normal growth. ROS plays an important regulatory role as a signal molecule. Insensitivity to ROS signaling in moca1 and pgsip7 mutants will inhibit growth and development.

Upon salt stress, multiple genes, including HKT1, are involved in the stress response to alleviate adverse impacts (Gholizadeh et al., 2024; Liu et al., 2024). Among them, the SOS signal transduction pathway, MAPK signal transduction pathway, and ABA signal transduction pathway have been studied in depth. We selected some key genes in these pathways for testing (Figure 4) (Ndathe et al., 2022; Zhang et al., 2023b; Liu et al., 2024). SOS1 encodes a plasma membrane Na⁺/H⁺ antiporter. In wild-type plants, SOS1 expression remained relatively stable across salt concentrations. In the moca1 mutant, SOS1 expression was significantly suppressed, consistent with its salt-hypersensitive phenotype. The pgsip7 mutant showed decreased SOS1 expression under salt stress, whereas the pgsip8 mutant exhibited elevated SOS1 expression with increasing salt concentration. SOS2, a protein kinase that interacts with SOS3 to activate SOS1, showed moderate induction in wild-type plants under salt stress. However, SOS2 expression was severely suppressed in both pgsip7 and moca1 mutants, particularly at 100 mM NaCl. CAX1 (Cation Exchanger 1), a vacuolar Ca²⁺/H⁺ antiporter, was significantly upregulated in wild-type plants under salt stress, peaking at 100 mM NaCl. By contrast, CAX1 expression in moca1 and pgsip7 mutants displayed a transient upregulation followed by downregulation, indicating disrupted calcium signaling in these mutants. MAPK5 (Mitogen-Activated Protein Kinase 5), a key component of the MAPK cascade, showed a moderate up-regulation in wild-type plants at 60 mM NaCl. Both moca1 and pgsip7 mutants exhibited severe suppression of MAPK5 expression under salt stress, particularly at 100 mM NaCl. The pgsip8 mutant also showed reduced MAPK5 expression relative to wild type, but the suppression was less severe than in moca1 and pgsip7. RD29A is sensitive to salt (NaCl and KCl) and non-ionic hyperosmotic stresses (Justamante et al., 2025; Zhou et al., 2025). Compared with wild type, the expression of the RD29A gene in moca1 and pgsip7 mutants exhibited severe suppression with increasing salt concentration, implying that they might be incapable of responding to salt stress signals, resulting in reduced tolerance in high-salt environments. HKT1 (High-Affinity K⁺ Transporter 1), responsible for xylem Na⁺ unloading and Na⁺/K⁺ homeostasis, showed relatively minor expression variations across genotypes. Wild-type and pgsip8 plants exhibited slight HKT1 downregulation under salt stress, while pgsip7 and moca1 mutants showed slight upregulation at 100 mM NaCl. Moca1 and pgsip7 mutants displayed highly correlated expression patterns, whereas pgsip8 showed distinct responses. This suggests that PGSIP7 and MOCA1 may function in a common regulatory module, distinct from PGSIP8. Collectively, these findings indicate that PGSIP7 positively regulates salt stress responses, potentially through modulation of the SOS signaling pathway.

In order to further confirm that the PGSIP7 gene may be involved in the transport of Na⁺, F2 generation hybrid double mutant plants pgsip7 sos2 and pgsip8 sos2 were treated with different salt concentration gradients (Figure 5). Quantification of cotyledon greening showed that at 60 mM NaCl all lines remained ≥ 70% green, whereas at 100 mM the ratio for pgsip7 sos2 fell to 24%—significantly below Col-0 (86%) and pgsip8 sos2 (71%)—indicating lower salt tolerance. No difference was detected between the pgsip8 sos2 double mutant and the wild type. The sos2 single mutant attained only 34% green cotyledons and failed to expand; pgsip8 sos2 partially rescued this phenotype, suggesting that the PGSIP8 gene is not required for the SOS response, whereas PGSIP7 may participate in Na⁺ transport within the SOS signaling pathway.

The MOCA1 gene is the first salt-sensing gene found in Arabidopsis thaliana, and its gene function may have been obtained through gradual evolution. The evolutionary analysis from lower to higher plants is shown in Figure 6A. The homologous gene of the MOCA1 gene first appeared in Chlamydomonas reinhardtii, a single-celled aquatic plant. As terrestrial plants emerged, the MOCA1 gene appeared in Physcomitrella patens. We speculate that the MOCA1 gene may have early incomplete salt-sensing ability at this time. As organisms on earth have experienced species explosion, plants continue to evolve and develop. The homologous genes of the MOCA1 gene generally appear in higher plants, including Oryza sativa, potato, Nicotiana tabacum, and Arabidopsis, among others, giving them salt-sensing ability. The MOCA1 gene evolves continuously as plants adapt to surroundings, from a single species at lower levels to multiple species at higher levels, showing rapid expansion.

During evolution, species may gradually change from one gene to multiple genes to adjust to their surroundings (Ebadi et al., 2023). The internal core segment of a gene generally does not mutate during evolution, but one or more amino acid sites will change as the environment changes (Dunham and Beltrao, 2021). The MOCA1 gene is a key gene for salt sensing, and the evolution of key sites is particularly important in the process of adapting plants from sea to land. MOCA1 homologous genes from lower to higher plants and from marine to terrestrial plants were selected for protein sequence comparison (Figure 6B). It was found that these homologous genes all contained a conserved domain and were α-helical regions. The amino acids in this domain change from hydrophilic amino acids such as serine and glycine to hydrophobic amino acids such as valine and alanine. The higher the plant, the more hydrophobic amino acids, and the lower the affinity for water molecules. This is crucial for the formation of alpha-helix regions and may make plants increasingly sensitive to Na⁺. From Chlamydomonas reinhardtii to higher plants, Arabidopsis and Oryza sativa, the hydrophobicity of amino acids in conserved domains increases, which is very important for plants to avoid adverse environments.

We constructed transgenic Arabidopsis plants by cloning the homologous genes Cre_10g422450v5, Pp3c2_32110, Pp3c1_6500, and loc_Os02g41520.1 of the MOCA1 genes in Chlamydomonas reinhardtii, Physcomitrella patens, and Oryza sativa. Comparing the root length and RGC index of the plants under different salt treatments, it was found that the five restorer lines had different degrees of resistance to salt stress, especially significant compared with the moca1 mutant (Figures 7A, B). In order to know whether they have resistance to osmotic stress, they were treated with different concentrations of sorbitol (Figures 7C, D). The findings demonstrated no discernible differences in green cotyledons and root development compared with the wild type, revealing that the MOCA1 homologous gene overexpression is insensitive to osmotic stress but has certain tolerance to salt stress. Remarkably, overexpression of the homologous gene CreMOCA1 in Chlamydomonas reinhardtii can also restore root development, and the two homologous genes in Physcomitrella patens have differences in tolerance to salt stress.

Plants respond rapidly after being exposed to salt stress, including the efflux of Na⁺, the MAPK cascade reaction, and the storage by vacuoles (Waheed et al., 2024; Liu et al., 2025b). We analyzed salt-responsive gene expression to investigate whether the MOCA1 homologous gene has the function of improving salt stress response (Figure 7E). The findings demonstrated that the expression of the Na⁺/H⁺ antiporter SOS1 gene in the moca1 mutant decreased under different concentrations of salt stress, and its expression also decreased in transgenic plants of the Chlamydomonas reinhardtii MOCA1 homologous gene. However, its expression in the MOCA1 transgenic plants of Physcomitrella patens increased significantly under salt stress, and its expression in the Arabidopsis MOCA1 gene restorer line plants was significantly higher compared to others. RD29A is a gene that responds to salt stress, and its overexpression can significantly improve the salt resistance of plants. Under salt stress, the RD29A gene in Pp1MOCA1 and AtMOCA1 plants increased significantly compared with other plants. In short, salt-responsive gene expression in transgenic plants with MOCA1 homologues was increased compared with the moca1 mutant under salt stress, which is helpful to enhance salt tolerance.

Salt sensing in Arabidopsis is due to the combination of external Na⁺ with GIPC sphingolipid anions located on the plasma membrane, which opens the calcium ion channel, and a large amount of extracellular Ca²⁺ influx to trigger intracellular Na⁺ efflux or vacuole storage. Therefore, the detection of calcium ion signals is crucial for salt-sensing research. Ca²⁺ imaging based on aequorin bioluminescence can well detect the changes of intracellular calcium concentration. In transgenic plants aequorin can recombine and can exhibit Ca²⁺ increases resulting from salt, dryness, cold, and oxidative stress (Shen et al., 2025). We detected the calcium ion signals of different transgenic plants treated with 200 mM NaCl (Figure 8). Within 2 minutes after being stimulated by Na⁺, the wild-type plants’ Ca²⁺ changed dramatically. On the contrary, there was no significant change in Ca²⁺ after the moca1 mutant plants were stimulated by exogenous Na⁺, which is consistent with the previous results (Jiang et al., 2019). This suggests that the moca1 mutant exhibits sodium insensitivity, indicating that the sensing of Na⁺ in plants depends on the normal expression of the MOCA1 gene. CreMOCA1 restorer lines also had no signal detected, while Pp1MOCA1 detected a calcium ion signal, but the signal was weak. AtMOCA1 completely restored calcium ion signaling, which may be due to the fact that the plant’s MOCA1 gene was highly expressed. Although OsMOCA1 was also detected, the signal was weak compared to AtMOCA1 plants. It is worth noting that among the two homologous genes of MOCA1 in Physcomitrella patens, Pp2MOCA1 has almost no calcium ion signal detected. We speculate that it may be distantly related to the MOCA1 gene and does not yet possess a complete salt-sensing function.

As a signal hub, ROS plays a very important role in signal transmission, so understanding how ROS changes in plant root tips is crucial (Kesawat et al., 2023; Jiang et al., 2025). Under varying salt stress concentrations, significant differences in ROS content were observed in the roots of wild-type plants, the moca1 mutant, and multiple MOCA1 homologous transgenic lines (Figure 9). The moca1 mutant shows the accumulation of ROS in roots, which increases with increasing salt concentration. This may be related to its insensitivity to Na⁺, which affects the normal ROS signal transduction pathway, suppresses the expression of related genes, and fails to clear superoxide anions and hydrogen peroxide produced by stress in time. Further analysis found that the accumulation of ROS in the roots of all transgenic plants was considerably lower than in the moca1 mutant. AtMOCA1 has the strongest clearance ability due to the overexpression of Arabidopsis thaliana’s own genes. Although CreMOCA1 has little signal in calcium ion detection, it still has the ability to clear excess ROS. MOCA1 homologous transgenic Arabidopsis can clear excess ROS, thereby alleviating the damage to roots caused by oxidative stress due to high salinity.

β-glucuronidase (GUS) could react with X-Gluc to produce a blue substance under certain circumstances. The substance is insoluble in the transgenic nucleus and visible under a microscope (He et al., 2020). To investigate whether the early MOCA1 homologous gene’s promoter has certain salt-sensing ability and specific tissue expression localization, we established transgenic plants with fusion expression of proCreMOCA1 and GUS. It was found that the CreMOCA1 gene was mainly expressed in root tips and leaves of Arabidopsis (Figure 10). As the salt content rises, the blue color of the leaves becomes darker, indicating that the rise in salt concentration induces the CreMOCA1 gene to elevate expression.