Abstract Background Soil salinization represents the most prevalent abiotic stress, severely impacting a severe impact on plant growth and crop yield. Consequently, delving into the mechanism through which exogenous substances enhance plant salt tolerance holds significant importance for the stabilization and augmentation of crop yield. Result In this study, within the context of salt stress, the seedlings of R. soongorica were subjected to exogenous Ca^2+ and NO treatments. The aim was to comprehensively explore the alleviation effects of exogenous Ca^2+ and NO on the high salt stress endured by R. soongorica from the perspectives of physiology and transcriptomics. The experimental results demonstrated that the combined treatment of exogenous Ca^2+ and NO increased the relative water content and free water content of R. soongorica seedlings during salt stress conditions. Simultaneously, it induced a reduction in the leaf sap concentration, leaf water potential, water saturation deficit, and the ratio of bound water to free water. These modifications effectively regulated water metabolism and mitigated physiological drought induced by salt stress. In addition, the concurrent treatment of exogenous Ca^2+ and NO could diminish Na^+ and Cl^− levels in R. soongorica seedlings under salt stress. At the same time, it was effective in elevating the contents of K^+ and Ca^2+, thereby facilitating the adjustment of the ion equilibrium. As a result, this treatment served to relieve the ion toxicity precipitated by salt stress, which is crucial for maintaining the physiological homeostasis and viability of the seedlings. Transcriptional analysis revealed that 65 differentially expressed genes (DEGs) were observable at three distinct stress time points in the context of high salt stress. Additionally, 154 DEGs were detected at three stress time points during the combined treatment. KEGG enrichment analysis revealed that phenylpropanoids biosynthesis, plant hormone signal transduction, MAPK signalling pathway, brassinosteroid biosynthesis and zeatin biosynthesis were significantly enriched under high salt stress and exogenous Ca^2+ and NO compound treatment. Furthermore, WGCNA uncovered that multiple genes, including ADK, SBT, F-box protein, MYB, ZIP, PAL, METTL, and LRR, were implicated in the adaptive and mitigating mechanisms associated with the combined treatment of exogenous Ca^2+ and NO in modulating high salt stress within R. soongorica seedlings. Conclusion The outcomes of this study are highly conducive to disclosing the mechanism through which the combined treatment of exogenous Ca^2+ and NO ameliorates the salt tolerance of R. soongorica from both physiological and transcriptional aspects. It also paves a solid theoretical groundwork for the employment of biotechnology in the breeding of R. soongorica, thereby offering valuable insights and a scientific basis for further research and practical applications in enhancing the plant's ability to withstand salt stress and for the development of more salt-tolerant varieties of R. soongorica. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11355-w. Keywords: Reaumuria soongorica, Calcium, Nitric oxide, Salt stress, Physiology, Transcriptomics Introduction Salt stress, which is an abiotic stressor, has a direct and significant impact on plant growth and development. As the salt concentration in the soil steadily and continuously rises, plants are consequently subjected to high levels of salt stress. Studies have demonstrated that high salt stress can induce a reduction in plant photosynthesis, the suppression of protein synthesis, and the generation of reactive oxygen species (ROS). These consequences subsequently give rise to oxidative stress, ion toxicity, and malnutrition, ultimately culminating in detrimental effects on plant growth [[32]1]. In addition, the accumulation of salt results in a soil acid–base imbalance that reduces permeability reduces land production efficiency and destroys the soil's ecological balance [[33]2]. At present, the issue of global land salinization is growing increasingly severe. It acts as a significant impediment to the growth and development of plants, thereby disrupting the stability of the ecosystem [[34]3]. This result, in turn, triggers a cascade of ecological and environmental problems. These include the acceleration of land desertification, exacerbation of soil erosion, a steep decline in forest cover, and a problematic reduction in biodiversity [[35]4]. Salinization increases the difficulty of effectively using land resources, leading to the degradation or loss of soil ecosystem functions, severely restricting the sustainable development of human agricultural production and the ecological environment [[36]5]. Therefore, enhancing the salt tolerance of plants holds profound and far-reaching significance when it comes to the rational utilization of salinized land. A high-salt environment greatly harms plant growth [[37]6]. Scholars have reported that the logic and sensible application of plant growth regulators are effective methods for improving the salt resistance of plants [[38]7]. Calcium ions (Ca^2+) play an essential role as the second messenger in plant growth, development, and stress response. Under abiotic stress, exogenous Ca^2+ can alleviate the stress environment's inhibitory influence on plant cell growth and development. It actively contributes to the integrity of the plant cell structure, thereby ensuring the cells' ability to carry out their normal functions [[39]8]. When plants are under salt stress, Ca^2+ channel proteins, important salt signal receptors, can rapidly sense and sharply increase the intracellular Ca^2+ concentration [[40]9]. When the Ca^2+ concentration required by plants under normal conditions is applied to plants under salt stress, plants lack Ca^2+. Adding a high amount of exogenous Ca^2+ can alleviate plant deficiency symptoms, thus maintaining the basic structure and function of the plasma membrane and ensuring the standard transmission of the Ca^2+ signalling system under salt stress [[41]10]. Ca^2+ is also a necessary factor for photosynthetic oxygen release. Exogenous Ca^2+ can significantly increase plants' chlorophyll content and net photosynthetic rate under salt stress [[42]11]. Nitric oxide (NO), a crucial signalling molecule within the realm of plants, assumes a pivotal and critical role in the plant's response mechanisms to both biotic and abiotic stresses [[43]12]. Many reports have investigated the fact that NO has been shown to alleviate seeds' germination rate and germination index under salt stress and promote the absorption of beneficial nutrients by plants [[44]13, [45]14]. In addition, NO can reverse the effects of salt stress on plant photosynthesis [[46]15] and reduce salt stress damage by increasing the contents of proline, betaine and soluble sugars in plants [[47]16] and regulating antioxidant metabolic pathways [[48]17, [49]18]. Relevant studies have documented that nitric oxide (NO) partakes in the positive modulation of abscisic acid (ABA) accumulation in rice (Oryza sativa) and maize (Zea mays) when subjected to salt stress [[50]19]. NO can also regulate the adaptability of perennial ryegrass (Festuca arundinacea) to salt stress by reducing Na^+ accumulation and improving growth and photochemical efficiency [[51]20]. Ca^2+ and NO have emerged as prevalent substances with the capacity to enhance plant salt tolerance. Intriguingly, prior investigations have indicated that the homeostasis of Ca^2+ within plant cells is under the regulatory influence of NO. NO achieves this by stimulating intracellular Ca^2+-permeable channels as well as plasma membrane channels, thereby facilitating the generation of Ca^2+ in plant cells [[52]21]. Therefore, the combined treatment using Ca^2+ and NO is likely to exhibit a more favorable effect in alleviating the abiotic stress endured by plants than the application of a single substance. Nevertheless, to date, there has been a lack of reported research focusing on enhancing the abiotic stress tolerance of plants through the specific combination of Ca^2+ and NO. Reaumuria soongarica is a highly xerophytic salt-secreting shrub of the genus Reaumuria in Tamaricaceae. It has strong drought resistance, sand fixation, and water and soil conservation capabilities, and it plays a significant role in ecological protection and construction in desert and grassland areas [[53]22]. Moreover, the leaves of R. soongorica are rich in protein, fat, and trace elements, making this species a good forage in desert areas, where it can be used to graze sheep and camels. Therefore, R. soongorica is the primary species used in constructing forage shrubs and cultivating degraded grasslands [[54]23]. The results highlighted that R. soongorica exhibits a relatively broad niche width across diverse salinity gradients as a characteristic salt-secreting plant. It can mitigate the adverse impacts of salt damage through the mechanisms of leaf salt secretion and the modulation of reactive oxygen species. However, drought and saline-alkali land are the main factors affecting the distribution of the R. soongorica community [[55]24]. Research has shown that exogenous regulatory substances, including hydrogen sulfide, can slow the damage caused by high salt stress in R. soongorica seedlings by regulating the metabolism of reactive oxygen species [[56]25]. Our previous preliminary experimental results revealed that the alleviation effect of Ca^2+ and NO compound treatment on high salt stress in R. soongorica was significantly more significant than that of a single treatment with either Ca^2+ or NO [[57]26]. Further in-depth research has demonstrated that the exogenous application of Ca^2+ and NO effectively attenuates the inhibitory influence of high salt stress on the growth of R. soongorica seedlings [[58]26]. This attenuation is achieved by modulating the intricate processes of reactive oxygen species metabolism, carbon, and nitrogen metabolism. Transcriptomics can detect the overall transcriptional activity of any species at the single nucleotide level, especially non-model species such as R. soongorica. This technology can analyze gene expression levels more accurately and identify new transcripts and specific genes. Ying et al. [[59]27] used the transcriptome to identify transcription factors differentially expressed under salt stress in Glycyrrhiza infata, including MYB, bZIP and NAC family. In Rhododendron simii, it was found that RsWRKY40 is referred to salt tolerance by transcriptome analysis [[60]28]. Therefore, the Illumina NovaSeq 6000 platform was used in this study to sequence the transcriptomes of R. soongorica seedlings treated with exogenous Ca^2+ and NO under high salt stress. When integrated with the variation characteristics of phenotypic parameters, water metabolism, and ion content, the results served as a valuable source of basic data. This data was essential for comprehensively elucidating the molecular mechanism underlying the synergistic regulation of exogenous Ca^2+ and NO in R. soongorica seedlings to relieve high salt stress. Results Effects of exogenous Ca^2+ and NO compound treatment on the growth parameters of R. soongarica seeds under high salt stress The results presented in Fig. [61]1 have been published in our previous paper [[62]26]. We will better explain the growth parameters here to reveal the relationship between gene expression and growth traits. As shown in Fig. [63]1A, the growth differences of R. soongorica under various stresses can be observed. Beginning on the 9th day, compared with the control, high salt stress significantly reduced the plant height of R. soongorica seedlings (P < 0.05), exogenous Ca^2+ and NO alleviated salt stress, and the plant height was significantly higher in the high salt treatment than in the control (Fig. [64]1B). Moreover, it was observed that root length exhibited a higher degree of sensitivity to salt stress. In comparison with the control group (CK), salt stress notably diminished the root length of R. soongorica seedlings. This reduction became particularly conspicuous on the third day of treatment, registering a decrease of 3.88%. However, the exogenous application of Ca^2+ and NO served to either decelerate or even reverse this inhibitory effect, thereby demonstrating these exogenous substances' potential to safeguard the seedlings' root growth and overall health under saline conditions (Fig. [65]1C). The changes in the stem diameter of R. soongorica seedlings with different stress times, high salt stress and exogenous Ca^2+ and NO treatment were similar to those in plant height and root length (Fig. [66]1D). As the stress duration lengthened, the aboveground fresh weight of R. soongorica seedlings manifested a gradually increased. However, salt stress initiated adverse impacts on the ninth day of treatment. Notably, applying Ca^2+ and NO led to a significant augmentation in the aboveground fresh weight under salt stress conditions, with an increase of 1.70% (Fig. [67]1E). High salt stress significantly reduced the aboveground dry weight of R. soongorica seedlings. Moreover, beginning on the 9th day of treatment, applying Ca^2+ and NO highly increased the aboveground dry weight under salt stress. Still, both were lower than the control at the same time (Fig. [68]1F). On the 3rd day of stress, the fresh weight of the underground part of R. soongorica seedlings under high salt stress was not different from that of the control (P > 0.05). On the 9th day of stress, the fresh weight of the underground part of R. soongorica seedlings under high salt stress was significantly lower than that of the control. Similarly, beginning on the 9th day of treatment, the application of Ca^2+ and NO significantly increased the fresh weight of the underground part under salt stress (Fig. [69]1G). Beginning from the 9th day of treatment, it was evident that salt stress exerted a significant negative impact, leading to a notable reduction in the dry weight of the underground parts of R. soongorica seedlings. After the treatment with exogenous Ca^2+ and NO, a significant augmentation was observed in the dry weight of the underground part of the plants when subjected to salt stress. Nevertheless, it is essential to note that both values remained lower than those of the control group during the identical period (Fig. [70]1H). Fig. 1. [71]Fig. 1 [72]Open in a new tab Effect of exogenous Ca^2+ and NO compound treatment on growth parameters of Reaumuria soongarica seedlings under high salt stress. Different capital letters indicate that the difference between various treatments at the same sampling time is significant, and different lowercase letters indicate that the difference between different times at the same treatment is substantial. The same is below Effects of exogenous Ca^2+ and NO compound treatment on the water metabolism of R. soongarica seeds under high salt stress Figure [73]2 presents a vivid illustration of the alterations in the water metabolism of R. soongorica seedlings in response to diverse treatment regimens. Compared with the control, high salt stress significantly reduced the relative water content of R. soongorica seedlings (P < 0.05). Compared with salt stress, the external application of Ca^2+ and NO significantly increased the relative water content under salt stress. Still, both were lower than those in the control during the same period (Fig. [74]2A). In comparison with the condition under the control treatment, it was found that the water saturation deficit under high salt stress exhibited a significant elevation. Fig. 2. [75]Fig. 2 [76]Open in a new tab Effect of exogenous Ca^2+ and NO compound treatment on water metabolism of R. soongarica seedlings under high salt stress Conversely, when exogenous Ca^2+ and NO treatment were applied, the water saturation deficit under salt stress was significantly reduced. However, it should be emphasized that the water saturation deficit under high salt stress remained notably more prominent than that under the control treatment during the same time frame, as depicted in Fig. [77]2B. The change in the bound water content was related to the stress period. At the 3-day mark, the content of bound water under high salt stress was marginally higher than that under the control treatment (CK). Additionally, the content of bound water under exogenous treatment was slightly more significant than that observed under salt stress. However, at fifteen days, a considerable divergence was noted. The content of bound water under high salt stress was substantially lower than that under CK. In contrast, the content of bound water under the combined treatment with Ca^2+ and NO was remarkably more significant than both that under high salt stress and the control, as clearly illustrated in Fig. [78]2C. Compared with the control, the free water content under high salt stress was significantly lower; however, after the combined treatment with Ca^2+ and NO, the free water content of R. soongorica seedlings was markedly greater than that under high salt stress. Still, both were lower than the controls at the same time (Fig. [79]2D). Compared with the control group, high salt stress led to a strikingly significant increase in the bound water/free water ratio of R. soongorica seedlings. In addition, at 0 and 3 days, the bound water/free water ratio in the combined treatment with exogenous Ca^2+ and NO was significantly greater under salt stress (Fig. [80]2E). High salt stress significantly increased the leaf water potential of R. soongorica. In contrast, the leaf water potential of R. soongorica significantly decreased after the combined treatment with exogenous Ca^2+ and NO. Still, both were considerably greater than the control at the same stress time (Fig. [81]2F). The variation tendency of the leaf sap concentration was in harmony with that of the leaf water potential. High salt stress brought about a notably significant elevation in the leaf sap concentration of R. soongorica. Specifically, it attained a peak of 13.89% at the 9-day mark. In contrast, compared with high salt stress, the leaf sap concentration of R. soongorica exhibited a significant decline following the combined treatment with exogenous Ca^2+ and NO, as vividly depicted in Fig. [82]2G. Effects of exogenous Ca^2+ and NO compound treatment on the ion content of R. soongarica seedlings under high salt stress The change in the ion content of R. soongorica seedlings was related to stress duration (Fig. [83]3). Compared with that in control, the content of Na^+ in the high-salt stress treatment significantly increased (P < 0.05) and reached a maximum value of 1.04 mmol·g^−1 at 9 days. Compared to the content of Na^+ in the high-salt stress treatment, a significant reduction was observed in the content of Na^+ in the mixed treatment involving exogenous Ca^2+ and NO. However, it is essential to note that both values were greater than those in the control during the same period, as shown in Fig. [84]3A. The content of Cl^− under high salt stress was significantly greater than that under the control. The Cl^− content under the combination of exogenous Ca^2+ and NO was considerably lower than that under the control treatment at the same time under salt stress (Fig. [85]3B). The content of K^+ under high salt stress was markedly lower than that in the control group. In contrast, the content of K^+ in the mixture of exogenous Ca^2+ and NO was more excellent and significantly more remarkable than that under salt stress. Still, both were considerably lower than the controls at the same stress time (Fig. [86]3C). Fig. 3. [87]Fig. 3 [88]Open in a new tab Effect of exogenous Ca^2+ and NO compound treatment on ion content of R. soongarica seedlings under high salt stress Moreover, compared to the content of Ca^2+ under the control treatment (CK), the content of Ca^2+ under high salt stress was conspicuously lower. Additionally, the content of Na^+ under the combined treatment of exogenous Ca^2+ and NO was remarkably more excellent than that under high salt stress and the control treatment during the same period (Fig. [89]3D). Compared with that under CK, the ratio of K^+/Na^+ under high salt stress significantly decreased, and the ratio of K^+/Na^+ under high salt stress increased dramatically under the combination treatment of exogenous Ca^2+ and NO. Still, both were simultaneously lower than those under CK (Fig. [90]3E). Compared to the control, the ratio of Ca^2+/Na + under high salt stress was substantially lower. Notably, the ratio of K^+/Na^+ under high salt stress was significantly greater under the combined treatment with exogenous Ca^2+ and NO. Except for the treatment with exogenous Ca^2+ and NO for 9 days, the ratios at the other time points were lower than those in the control during the same period, as illustrated in Fig. [91]3F. RNA quality inspection and sequencing results The outcomes of the RNA quality inspection for the samples demonstrated that the total RNA of each of the 27 samples exhibited a high level of purity and integrity. The transcriptome sequencing results revealed that the number of clean bases in each sample was more significant than 6.14 GB, with an error rate of 0.0245% ~ 0.0284%, a range that falls below 0.03%; the percentages of Q20 and q30 bases were 96.81% and 91.12%, respectively; and the percentage range of the GC content was 44.16% ~ 45.02% (Table [92]1). Further comparison of the clean reads revealed that the percentage of total mapped reads among the total reads of all the samples was more incredible than 68.71% (Table S1), indicating that the data quality of this study was high and could be used for subsequent analyses. Table 1. Summary of transcriptome sequencing data and assembly results Sample Raw reads Clean reads Clean bases Error rate(%) (%) (%) GC content(%) Mapped ratio CK_3d1 45,649,668 43,755,730 6,464,800,865 0.0246 98.16 94.63 44.24 77.67% CK_3d2 54,100,314 52,425,812 7,524,715,100 0.0247 98.16 94.66 44.74 81.11% CK_3d3 44,929,982 43,758,824 6,531,141,668 0.0247 98.13 94.44 44.62 74.47% NaCl_3d1 63,029,446 61,306,382 9,074,215,069 0.0246 98.17 94.63 44.58 75.87% NaCl_3d2 53,174,234 51,806,866 7,680,210,818 0.0245 98.2 94.68 44.56 76.83% NaCl_3d3 42,568,254 41,431,022 6,170,216,953 0.0246 98.17 94.62 44.57 75.95% NaCl_NO_Ca_3d1 45,706,120 43,898,408 6,491,092,812 0.0246 98.14 94.63 44.27 78.62% NaCl_NO_Ca_3d2 42,707,004 41,413,062 6,143,443,528 0.0245 98.21 94.75 44.42 77.77% NaCl_NO_Ca_3d3 50,769,362 49,167,166 7,291,081,641 0.0246 98.17 94.66 44.38 78.60% CK_9d1 45,429,250 44,423,352 6,478,856,652 0.0272 97.23 92.14 44.62 75.55% CK_9d2 48,324,828 47,558,544 6,984,370,493 0.0275 97.13 91.84 44.69 74.57% CK_9d3 52,921,884 51,877,042 7,609,771,601 0.0275 97.15 91.9 44.78 75.69% NaCl_9d1 51,644,358 49,915,190 7,337,219,084 0.0281 96.87 91.4 44.54 76.35% NaCl_9d2 49,783,208 47,890,586 7,049,235,868 0.0278 96.98 91.68 44.58 75.02% NaCl_9d3 44,694,318 42,997,176 6,316,979,096 0.0281 96.86 91.37 44.6 77.37% NaCl_NO_Ca_9d1 44,518,638 43,661,780 6,403,023,652 0.0281 96.91 91.35 44.76 74.85% NaCl_NO_Ca_9d2 44,036,892 43,217,464 6,305,146,741 0.0277 97.06 91.66 44.74 76.61% NaCl_NO_Ca_9d3 47,790,872 46,878,778 6,774,015,070 0.028 96.96 91.47 44.72 76.72% CK_15d1 44,519,022 43,984,444 6,413,961,286 0.0269 97.36 92.37 44.73 77.61% CK_15d2 45,347,670 44,534,818 6,526,020,373 0.0283 96.85 91.2 44.68 74.53% CK_15d3 44,124,900 43,162,314 6,274,833,254 0.0276 97.11 91.83 44.65 75.54% NaCl_15d1 69,446,722 67,406,450 9,818,215,434 0.0278 97.01 91.68 44.76 76.56% NaCl_15d2 48,369,396 47,459,988 6,891,385,695 0.0284 96.81 91.12 44.84 76.13% NaCl_15d3 43,292,360 42,572,696 6,219,598,480 0.0275 97.11 91.84 44.91 76.60% NaCl_NO_Ca_15d1 51,183,178 50,405,450 7,374,251,215 0.0279 97 91.49 44.86 75.63% NaCl_NO_Ca_15d2 50,812,204 50,073,672 7,339,278,207 0.0276 97.1 91.75 44.8 77.07% NaCl_NO_Ca_15d3 56,511,812 55,553,608 8,147,523,329 0.0276 97.1 91.75 44.74 74.87% [93]Open in a new tab Transcriptome assembly and transcriptional functional annotation The transcriptome assembly results revealed that the number of unigenes was 109,434, the number of transcripts was 202,755, and the average length of N50 was 1732 bp. Further analysis showed that 64.8% of the total BUSCO sequences were unigene sequences with the expected size, and 87.3% of the total BUSCO sequences were script sequences with the expected length, indicating that the unigene and script obtained in this study had high assembly integrity (Table S2). Statistical results revealed that unigene and transcript sequences with a 200–500 bp sequence length accounted for 40% and 34% of the total, respectively. The number of unigene and transcript sequences with a more than 4500 bp sequence length was the lowest, accounting for only 2.00% of the total (Fig. [94]4A and B). The BLAST search results revealed that the number of genes annotated to the NR database was the greatest, at 41,824, followed by those annotated to the GO database, at 3361 (Fig. [95]4C, Table S2). In addition, Venn diagram analysis revealed that 8362 genes, accounting for 19.56%, were annotated to the GO, KEGG, eggNOG, Pfam, NR, and Swiss-Prot databases. A total of 2285 genes were annotated only to the NR database, the highest number of individually annotated genes among all the databases (Fig. [96]4D). Fig. 4. [97]Fig. 4 [98]Open in a new tab Unigene and transcript assembly and database annotation. A: The length and quantity distribution of unigene; B: The length and quantity distribution of transcripts; C: Statistics of annotation results of 6 primary database functions; D: Venn diagram analysis of 6 database annotation results Analysis and identification of DEGs The number of DEGs in R. soongorica seedlings under high salt stress for different durations and under combined treatment with exogenous Ca^2+ and NO was significantly different. Compared with those at 3 d and 15 d under salt stress, the number of upregulated DEGs identified at 9 d was the lowest, at only 72. Similarly, after exogenous Ca^2+ and NO treatment under salt stress, the number of upregulated DEGs identified on the 9th day was still the lowest, with 183 genes. In addition, compared with those under high salt stress, the number of up-and downregulated genes decreased slightly after exogenous Ca^2+ and NO compound treatment for 15 d, but the number of up-and downregulated genes increased significantly on Days 3 and 9; for example, the number of upregulated genes increased threefold-fold on Day 3 (Fig. [99]5A, Table S3). In the analysis of the specific genes expressed at the three treatment time points and intuitively identify the common and unique DEGs between salt stress and exogenous Ca^2+ and NO treatment under salt stress, Venn diagram analysis revealed that there were only 30 genes in the six comparison groups (Fig. [100]5B, Figure S1). In addition, the study of all DEGs under high salt stress revealed 65 DEGs at three different stress time points, and the number of unique genes was most significant at 15 d, accounting for 77.09% (Fig. [101]5C). Venn diagram analysis of the effects of exogenous Ca^2+ and NO compound treatment revealed that there were 154 genes at the three stress time points. Among the unique genes, the number was most significant at 15 d and lowest at 3 d (Fig. [102]5D). Fig. 5. [103]Fig. 5 [104]Open in a new tab Statistics of DEGs under different treatments and Venn plot analysis. A unigene quantity statistics under different treatments; B: Venn plot analysis of DEGs under different treatments; C: Venn plot analysis of DEGs under salt treatment; D: Venn plot analysis of DEGs under the combined treatment of exogenous Ca^2+ and NO DEG functional enrichment analysis GO enrichment analysis The results revealed that, under high salt stress, the number of genes associated with metabolic processes and cellular processes was the greatest. The number of genes enriched with cell and membrane parts was the greatest among the cell components. Regarding biological processes, the number of genes associated with catalytic activities and binding was the most significant (Fig. [105]6A, C and E). When subjected to exogenous analysis, most GO terms were related to high salt stress (Fig. [106]6B, D and F). Still, the two groups differed in the number of enriched terms. At 3 and 9 d, the number of GO terms enriched with exogenous Ca^2+ and NO was approximately twice that enriched with high-salt stress. However, the two groups had no significant difference in enriched terms at 15 d. Fig. 6. [107]Fig. 6 [108]Open in a new tab GO enrichment analysis of DEGs under high salt stress and combined treatment for 3 d, 9 d and 15 d KEGG enrichment analysis We performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the DEGs of different comparison groups. We selected the top 20 pathways with the lowest Q values for display (Fig. [109]7). Compared with the control, the enrichment pathways of R. soongorica under high salt stress were mainly flavonoid biosynthesis, glucosinolate biosynthesis, monoamide biosynthesis, tryptophan metabolism and so on. Under the combined treatment of exogenous Ca^2+ and NO, the main enrichment pathways were anthocyanin biosynthesis; flavonoid biosynthesis; diphenyl-like, diarylheptane and gingeroid biosynthesis; diarylheptanoid and gingerol biosynthesis; and limonene and pinene degradation (Fig. [110]7B). Among them, phenylpropanoid biosynthesis, plant hormone signal transduction and the MAPK signalling pathway were significantly enriched under high salt stress and exogenous Ca^2+ and NO compound treatment. On the 9th day of treatment, the pathways associated with the accumulation of rhodopseudae under high salt stress were mainly glucosinolate biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, glutathione metabolism and alpha-linolenic acid metabolism (Fig. [111]7C). Under the combined treatment with exogenous Ca^2+ and NO, the main enriched pathways were thiamine metabolism, glycosphingolipid biosynthesis ganglion series, glycolytic glycan degradation and alpha-linolenic acid metabolism (Fig. [112]7D). The MAPK signalling pathway and plant and plant-pathogen interactions are shared among these pathways. At 15 d, the pathways enriched under high salt stress were anthocyanin biosynthesis, exopolysaccharide biosynthesis, flavonoid biosynthesis, brassinosteroid biosynthesis and so on. Under the combined treatment of exogenous Ca^2+ and NO, the main enrichment pathways were exopolysaccharide biosynthesis, brassinosteroid biosynthesis, zeatin biosynthesis and vitamin B6 metabolism (Fig. [113]7F). Among them, the MAPK signalling pathway, plant pathway, zeatin biosynthesis pathway and brassinosteroid biosynthesis pathway are common. Fig. 7. [114]Fig. 7 [115]Open in a new tab KEGG enrichment analysis of DEGs on days 3, 9 and 15 under salt stress and combined treatment Coexpression network analysis of high salt stress combined with exogenous Ca^2+ and NO to alleviate high salt stress Construction of the gene coexpression module WGCNA represents a widely utilized approach for constructing a gene coexpression network. In this study, WGCNA was carried out on 27 samples obtained at three distinct stress time points during the imposition of high salt stress and the combined treatment of exogenous Ca^2+ and NO (Fig. [116]8). The results obtained from the analysis disclosed that the gene expression information of each one of the samples was partitioned into 25 distinct gene coexpression modules, as illustrated in Fig. [117]8A. The number of genes encompassed within each gene coexpression module exhibited a pronounced disparity. The turquoise, grey, blue, brown, and yellow modules harbored a relatively more significant number of genes. In contrast, the dark grey, turquoise, dark green, and light yellow modules incorporated a comparatively smaller number of genes (Fig. [118]8B, Table S4). Fig. 8. [119]Fig. 8 [120]Open in a new tab WGCNA module division, gene number analysis, correlation analysis between modules, and phenotypic characteristics. A Hierarchical clustering analysis of coexpression genes; B: Analysis of the number of genes within the coexpression module; C: Heat map analysis of the correlation between coexpression modules and trait Gene coexpression module construction and its association analysis with the phenotype In the present study, 25 gene modules were employed to conduct an association analysis with the phenotypic traits that had been ascertained in the preceding phase, as depicted in Fig. [121]1. The outcomes of this analysis unveiled that seven phenotypic traits exhibited a significantly negative correlation with the tan module. The correlation coefficients in this regard ranged from −0.726 to −0.447. Moreover, it was observed that all of the phenotypic traits were notably and positively correlated with the royal blue module. This finding implies that the royal blue module was highly associated with the alterations in the phenotypic characteristics of R. soongorica under high salt stress conditions and the combined treatment of exogenous Ca^2+ and NO. The genes encompassed within this module were principally engaged in the active promotion and positive modulation of the growth of R. soongorica. Analogously, the grey and light yellow modules also significantly and positively correlated with most phenotypic traits. The correlation coefficients within this range were between 0.312 and 0.599. The correlations between other modules and phenotypic characteristics were not high (Fig. [122]8C). Consequently, in the ensuing analysis, this research endeavoured to probe into the gene functions of these four modules. Specifically, it aimed to elucidate their responses to salt stress and the alleviating impact of the combined treatment of exogenous Ca^2+ and NO. Functional enrichment analysis of four related modules The GO annotation analysis of the four gene modules significantly related to phenotype revealed that the molecular functions of the genes in the four gene modules were catalytic activity, transport activity, binding, transcriptional regulation activity, structural molecular activity, and molecular function regulation; cell components included cells, organelles, cell parts and membrane components; and biological processes included metabolic processes, multiple biological processes, cellular processes, responses to stimuli and biological regulation (Fig. [123]9). Fig. 9. [124]Fig. 9 [125]Open in a new tab GO enrichment analysis of significant correlation modules. A module grey; B: module royal blue; C: module light yellow; D: module tan. The same is below KEGG annotation analysis revealed that the genes in the grey module were enriched mainly in phenylalanine metabolism and phenylpropane biosynthesis. The genes in the royal blue module were primarily annotated to be involved in thiamine metabolism, cysteine and methionine metabolism, ether lipid metabolism and biosynthesis in various plant secondary organisms. The genes in the light yellow module were annotated as being involved in oxidative phosphorylation and proteasome, starch and sucrose metabolism. The genes in the tan module were mainly annotated as being involved in tyrosine metabolism, sesquiterpene and triterpene biosynthesis, and amino sugar and nucleotide sugar metabolism (Fig. [126]10). Fig. 10. [127]Fig. 10 [128]Open in a new tab KEGG enrichment analysis of significant correlation modules Visual analysis of the hub genes in the gene module To mine hub genes related to phenotypic traits, this study performed gene network visualization and gene connectivity analysis on the top 30 genes according to the weights of the grey, blue, light yellow and yellow–brown modules (Fig. [129]11, Table [130]2). The hub gene of the grey module was TRINITY_DN4274_c0_g1 (phenylalanine ammonia-lyase 1). The core genes of the royal blue module included TRINITY_DN10257_c0_g1 (adenylate kinase 1, ADK), TRINITY_DN21718_c0_g1 (subtilisin-like protein, SBT), TRINITY_DN21963_c0_g1 (F-box protein), TRINITY_DN23412_c0_g1 (MYB family transcription factor) and four zinc transporter genes (ZIP, TRINITY_DN26057_c0_g1, TRINITY_DN28219_c0_g1, TRINITY_DN5840_c0_g3, TRINITY_DN5858_c0_g1). The hub gene of the light yellow module is TRINITY_DN15020_c0_g1 (NADH dehydrogenase subunit 1). The core genes of the tan module are TRINITY_DN4596_c0_g1 (wall-associated receptor kinase-like 2), TRINITY_DN46548_c0_g2 (glutathione S-transferase zeta class, GLU), TRINITY_dn4927_c0_g1 (RING-H2 finger protein), TRINITY_dn86020_c0_g1 (methyltransferase erase like protein, METTL), and several receptor kinases rich in leucine repeats (LRR, TRINITY_DN11359_c0_g1, TRINITY_DN13516_c0_g1, TRINITY_DN1680_c2_g1, TRINITY_DN17563_c0_g1, TRINITY_DN18427_c0_g1, TRINITY_DN23283_c0_g1, TRINITY_DN35211_c0_g1, TRINITY_DN4512_c1_g1, TRINITY_DN5554_c0_g1, TRINITY_DN62171_c0_g1, TRINITY_DN74. Fig. 11. [131]Fig. 11 [132]Open in a new tab Visual analysis of significant related modules. A module grey; B: module royal blue; C: module light yellow; D: module tan Table 2. Candidate genes of mitigation of salt stress in Reaumuria soongarica by exogenous Ca^2+ and NO Compound Treatment Work Name Gene Id Description 15 d 9 d 3 d NaCl_NO_Ca^2+ vs CK NaCl vs CK NaCl_NO_Ca^2+ vs CK NaCl vs CK NaCl_NO_Ca^2+ vs CK NaCl vs CK ADK TRINITY_DN10257_c0_g1 Adenylate kinase 1, chloroplastic-like up up up up up down SBT TRINITY_DN21718_c0_g1 Subtilisin-like protease SBT3.13 up down up down up up F-box gene TRINITY_DN21963_c0_g1 F-box protein up up up down up up MYB TRINITY_DN23412_c0_g1 Myb family transcription factor EFM up down up up up up ZIP TRINITY_DN26057_c0_g1 Zinc transporter 1-like up down up down up down ZIP TRINITY_DN28219_c0_g1 Zinc transporter 4 up down up down up down ZIP TRINITY_DN5840_c0_g3 Zinc transporter 1 up down up down up down ZIP TRINITY_DN5858_c0_g1 Zinc transporter 1-like up down up down up down PAL TRINITY_DN4274_c0_g1 Phenylalanine ammonia-lyase 1 up up up up down up E3 ligase gene TRINITY_dn4927_c0_g1 RING-H2 finger protein ATL29 down down down down down down METL protein TRINITY_DN86020_c0_g1 Methyltransferase-like protein 7A down down down down down down LRR-like proteins TRINITY_DN11359_c0_g1 Receptor-like protein 6 down down down down down down LRR-like proteins TRINITY_DN13516_c0_g1 Receptor-like protein 7 down down down down down down LRR-like proteins TRINITY_DN1680_c2_g1 Receptor-like protein 33 down down down down down down LRR-like proteins TRINITY_DN18427_c0_g1 Receptor-like protein 7 down down down down down down LRR-like proteins TRINITY_DN23283_c0_g1 Receptor like protein 30-like down down down down down down LRR-like proteins TRINITY_DN4512_c1_g1 Receptor like protein 30-like down down down down down down LRR-like proteins TRINITY_DN62171_c0_g1 Receptor-like protein 12 down down down down down down LRR-like proteins TRINITY_DN7470_c0_g1 Receptor like protein 30-like down down down down down down LRR-like proteins TRINITY_DN7504_c0_g2 Receptor like protein 30-like down down down down down down LRR-like proteins TRINITY_DN4596_c0_g1 Wall-associated receptor kinase-like 1 down down down down down down [133]Open in a new tab qRT‒PCR validation of the transcriptome analysis To further verify the authenticity and reliability of the transcriptome data, based on the results of WGCNA, 12 DEGs were selected from four key modules (royal blue, grey, light yellow and tan) for qRT‒PCR experiments (Table S5). The results revealed that the variation trend of the relative gene expression level calculated via qRT‒PCR was consistent with that determined via RNA-seq, and the correlation coefficient was very high (R^2 = 0.8994), indicating that the transcriptome data were reliable (Fig. [134]12). Fig. 12. [135]Fig. 12 [136]Open in a new tab Correlation analysis of RNA-seq. and qRT-PCR analysis results Discussion Effects of exogenous Ca^2+ and NO on the water metabolism of R. soongorica seedlings under high salt stress Changes in plant water metabolism are a direct response to plant stress. Halophytes can usually maintain high water content under salt stress [[137]29]. This study revealed that, compared with the control, high salt stress significantly reduced the relative water content of R. soongorica seedlings. In contrast, the external application of Ca^2+ and NO significantly increased the water content under salt stress, similar to the findings in other plant species [[138]30]. R. soongorica can also experience physiological drought under salt stress, and combined treatment with Ca^2+ and NO can reduce the degree of cell water loss, but it cannot be eliminated. Arad and Richmond [[139]31] reported that adding NaCl to the root medium of barley plants (Hordeum vulgare L.) markedly increased leaf RNase activity in parallel with an increase in leaf water saturation deficit. The results of this study are consistent with these findings. However, the exogenous Ca^2+ and NO treatments significantly reduced the water saturation deficit. In addition, under salt stress, the free water content of R. soongorica decreased significantly, the bound water content did not change substantially, and the bound water/free water ratio increased significantly. After exogenous Ca^2+ and NO combination treatment, the three parameters significantly increased, indicating that the ability of the combination of Ca^2+ and NO to mitigate high salt stress in R. soongorica is very complex and that the changes only in free water and bound water contents cannot be fully explained. Relevant studies reported that the leaf water potential of tomato (Solanum lycopersicum) seedlings tended to decrease with increasing salt concentration [[140]32]. In contrast, the changes in the SAP concentration and leaf water potential of R. soongorica were the same; they significantly increased under salt stress and decreased after exogenous Ca^2+ and NO treatment, indicating that this may be a specific response of different species to salt stress. Comprehensive analysis revealed that salt stress reduced the relative water content, free water content, leaf juice concentration and water potential (both increased) and increased the water saturation deficit and bound water/free water ratio in R. soongorica. The combined treatment of Ca^2+ and NO alleviated the salt stress of R. soongorica seedlings by regulating water-saving metabolism. Effects of exogenous Ca^2+ and NO compound treatment on the ion absorption of R. soongorica seedlings under high salt stress Under salt stress, plants accumulate excessive Na^+ and Cl^−, resulting in osmotic ionic, and nutritional imbalances. Osmotic stress is caused by the rapid increase in salt concentration around roots in the early stage of salt stress, and the massive accumulation of Na^+ and Cl^− in the later stage leads to nutritional imbalance, which leads to ionic toxicity [[141]33]. Plants are characterized by reducing water absorption by roots, metabolic dysfunction, inhibition of photosynthesis, and excessive accumulation of osmotic adjustment substances [[142]34]. Na^+ has an ion hydration radius similar to that of K^+. Excessive Na^+ in plants affects the plants' absorption of K + . Under high salt concentrations, plants are subjected to the dual stresses of Na^+ toxicity and K^+ deficiency, thereby inhibiting enzymatic reactions [[143]35, [144]36]. Under salt stress, the exogenous application of Ca^2+ can improve the ion homeostasis and antioxidant defence of rice (Oryza sativa) seedlings and increase the tolerance of rice to salt stress [[145]37]. The results revealed that the contents of Na^+ and Cl^− in R. soongorica seedlings significantly increased under salt stress and that the contents of Na^+ and Cl^− in R. soongorica seedlings significantly decreased after the combination treatment of exogenous Ca^2+ and NO, indicating that the application of Ca^2+ and NO can hinder the absorption of Na^+ and Cl^− by plants, thereby reducing the degree of salt stress damage. This study also revealed that the contents of K^+ and Ca^2+ under high salt stress were significantly lower than those under the control conditions because R. soongorica absorbed too much Na^+ during salt treatment, which inhibited K^+ and Ca^2+. The contents of K^+ and Ca^2+ increased significantly after the combination treatment with exogenous Ca^2+ and NO, which was due to the exogenous supply of Ca^2+, and the increase in K^+ was due to the alleviation of ion toxicity caused by salt stress. In addition, the changes in the K^+/Na^+ and K^+/Na^+ ratios revealed that the ion balance destroyed by salt stress could be partially recovered after the combined treatment with Ca^2+ and NO. The combined treatment of exogenous Ca^2+ and NO effectively alleviated the ion toxicity caused by salt stress in R. soongorica and had a pronounced mitigating effect. Differences in the number of DEGs under different durations of stress At present, transcriptomics has been widely used in the study of plant salt resistance, which has revealed the functions of plant metabolic pathways and related genes, such as in wheat (Triticum aestivum) [[146]38, [147]39], cotton (Gossypium spp.) [[148]40], banana (Musa paradisiaca) [[149]41] and tomato [[150]42]. In addition, transcriptomics is widely used to study the mitigating effects of exogenous mitigators on plant salt stress, such as the increase in the salt resistance of Nitraria tangutorum seedlings caused by exogenous jasmonic acid [[151]43] and the mitigating effect of exogenous calcium on millet (Setaria italica) salt stress [[152]44]. In this study, R. soongorica seedlings subjected to different durations of stress were collected for high salt stress and exogenous Ca^2+ and NO compound treatment to alleviate salt stress via transcriptome analysis. The results revealed that, compared with those under high salt stress, the number of up-and downregulated genes under Ca^2+ and NO compound treatment at 3 and 9 d significantly increased, indicating that the stress duration was shorter and that the effects of Ca^2+ and NO compound treatment may be alleviated by increasing gene expression. Compared with those under high salt stress, the number of up- and downregulated genes decreased slightly after treatment with exogenous Ca^2+ and NO for 15 d, which showed that R. soongorica had been adapted to salt stress, so the number of genes mobilized by exogenous Ca^2+ and NO was reduced. Therefore, the number of genes expressed under the combined treatment of exogenous Ca^2+ and NO was not significantly different from that under high salt stress at 15 d. Functional enrichment of DEGs This study revealed that the GO terms and pathways for both classes of DEG (extraneous Ca^2+ and NO compounds vs. Salt stress and Salt stress vs. CK) enrichment were essentially similar, which indicated that exogenous Ca^2+ and NO did not affect the expression of many genes with new biological functions, mainly by altering the expression level of salt-tolerant genes or promoting the expression of related genes, to increase the salt tolerance of R. soongorica seedlings. The results of the GO analysis were rich and concentrated. The terms associated with significant enrichment after exogenous Ca^2+ and NO combination treatment were the same as those related to high salt stress, including cell part, membrane part, metabolic process, cell process, catalytic activity and binding. The difference was that the number of DEGs associated with the same terms differed after treatment for 3 d and after treatment for 9 d. The number of DEGs associated with exogenous Ca^2+ and NO combination treatment was approximately twice that related to high salt stress. These results were consistent with the number of DEGs identified, indicating that the combination of exogenous Ca^2+ and NO alleviated the stress of R. soongorica, which was significantly correlated with the duration of stress. The results of KEGG analysis were rich in anthocyanin biosynthesis; flavonoid biosynthesis; diphenyl-like, diarylheptane and gingeroid biosynthesis; diarylheptanoid and gingerol biosynthesis; limonene and pinene degradation; exopolysaccharide biosynthesis and other pathways, all of which were significantly enriched under the combined treatment of exogenous Ca^2+ and NO. In addition, the pathways closely related to salt stress, such as phenylpropanoid biosynthesis, plant hormone signal transduction, the MAPK signalling pathway, brassinosteroid biosynthesis, zeatin biosynthesis and vitamin B6 metabolism, were significantly enriched under high salt stress and exogenous Ca^2+ and NO compound treatment. The involvement of plant hormones in regulating plant salt tolerance has been widely reported [[153]45]. For example, under salt stress, the content of cytokinin in cotton rapidly decreases [[154]46]. The results of this study revealed the differential expression of genes in response to salt stress and the combination of exogenous Ca^2+ and NO to alleviate multiple salt stress-induced hormone signalling pathways, including plant hormone signal transduction, brassinosteroid biosynthesis and zeatin biosynthesis, reflecting the complexity of the hormone signals involved in regulating the plant salt stress response. The MAPK signalling cascade pathway is essential in plant stress signal transduction. By predicting the interaction protein network of MAPK pathway genes that are differentially expressed in response to salt stress, multiple interactions, such as between MAPK2 and MAPK4, which positively regulate the salt tolerance of A. thaliana, were identified [[155]47]. In this study, the pathway with significant enrichment in KEGG was the MAPK signalling pathway, indicating that the combination of exogenous Ca^2+ and NO alleviated the salt stress of R. soongorica, which was accompanied by the differential expression of genes related to the MAPK signalling pathway. The molecular mechanism of treatment with exogenous Ca^2+ and NO compounds on the salt stress of R. soongorica seedlings The tolerance of plants to salt stress and the combined treatment of exogenous Ca^2+ and NO to alleviate salt damage is achieved mainly by regulating the expression levels of related genes. Adenylate kinase (ADK) is a phosphotransferase that plays a vital role in maintaining the regular content of nucleotides in cells and energy metabolism activities and then regulates the corresponding stress of plants [[156]48, [157]49]. Peterson et al. [[158]50] treated the roots and stems of maize with two different ratios of Ca^2+/Na^+ salt solutions. The results showed that ADK, which regulates adenylate metabolism, has an essential but complex relationship with plant salt stress. In this study, an adenosine monophosphate kinase gene (TRINITY_DN10257_c0_g1) was differentially expressed under different treatments, indicating that the combination of exogenous Ca^2+ and NO could increase the tolerance of R. soongorica to salt stress by regulating the differential expression of ADK. In this study, the SBT gene (TRINITY_DN21718_c0_g1) was shown to regulate exogenous Ca^2+ and NO compound treatment to alleviate the salt stress of R. soongorica. As the second most prominent member of the serine protease family, SBT has a wide variety and diverse functions. This protein is induced by plant salt stress [[159]51, [160]52]. Most SBT genes can respond to plant salt stress [[161]53]. F-box protein is widely involved in abiotic and abiotic stresses in plants and is regulated by a significant stress gene [[162]52]. For example, Kumar and Kirti [[163]54] reported that F-box protein is related to salt stress in peanuts (Arachis hypogaea). The interacting proteins of the salt-response factor OsGRF7 include an F-BOX-containing protein (OsFBO13), which interacts to regulate salt stress in rice [[164]55]. MYB transcription factors are involved in the Arabidopsis salt stress response and negatively regulate the response to salt stress by activating ABA signalling [[165]56]. In the MYB family, MYB3, MYB4, MYB13, and MYB59 were all involved in the wheat salt stress response, simultaneously acting on several downstream target genes associated with salt stress [[166]57]. In this study, an F-box gene (TRINITY_DN21963_c0_g1) related to the phenotypic changes after salt stress and exogenous Ca^2+ and NO combination treatment was obtained, indicating that the F-box gene may also play an active role in alleviating salt stress via Ca^2+ and NO combination. MYB, as a large family, actively responds to plant abiotic stress. For example, the SlMYB102 gene is induced by salt stress and improves the salt tolerance of tomato salt tolerance by regulating ROS-scavenging enzyme accumulation [[167]58]. In this study, a MYB gene (TRINITY_DN23412_c0_g1), which is involved in the process by which Ca^2+ and NO compounds alleviate salt stress in R. soongorica, was also identified, and its specific regulatory mechanism needs to be further verified. On the other hand, four ZIP genes (TRINITY_DN26057_c0_g1, TRINITY_DN28219_c0_g1, TRINITY_DN5840_c0_g3, TRINITY_DN5858_c0_g1) were identified via coexpression analysis. These results indicate that these genes combine exogenous Ca^2+ and NO to alleviate the growth inhibition caused by salt stress and subsequently increase the salt tolerance of R. soongorica. Under high salt stress, AtZTP29 may cause the UPR in cells by regulating the concentration of zinc in the endoplasmic reticulum, reducing the damage caused by unfolded proteins in the endoplasmic reticulum to cells, and improving the salt tolerance of plants [[168]59]. AtHB23 is a HD-Zip class I transcription factor meditating Arabidopsis adaptation to salt stress by regulating primary and lateral root development [[169]60]. After salt stress, the activity of the VcPAL enzyme in orchids is directly proportional to gene expression [[170]61]. The expression of pal1 and PAL8 in Nelumbo nucifera was significantly upregulated under salt stress, which promoted the accumulation of flavonoids and other secondary metabolites [[171]62]. Under salt stress, the PAL1 gene was overexpressed in pepper (Piper longum) roots, indicating that PAL1 responds positively to salt stress [[172]63]. Wheat research has also achieved similar results [[173]64]. The expression of the PAL gene and the change in PAL activity were closely related to salt stress. In this study, we identified a PAL gene (TRINITY_DN4274_c0_g1), which was differentially expressed when exogenous Ca^2+ and NO compound treatment alleviated salt stress, indicating that the change in the PAL gene expression level is one of the mechanisms by which exogenous Ca^2+ and NO compound treatment alleviates salt stress in R. soongorica. We should continue to determine PAL activity in subsequent studies to verify the role of PAL activity and its gene correlation in enhancing salt stress in R. soongorica. Research has shown that ring-type ZIP plays an important role in the plant response to salt stress [[174]65]. In addition, Guo et al. [[175]66] and Stone and Callis [[176]67] noted that E3 ligases containing rings are involved in the growth and development process and in the regulatory mechanism involved in the response to abiotic stress. In this study, we found that a ring containing the E3 ligase gene (TRINITY_dn4927_c0_g1) plays a role in the exogenous Ca^2+ and NO compound treatment process to alleviate the salt stress of R. soongorica. The biological process of this gene is ubiquitin-dependent protein catabolism, and its KEGG function is enriched in E3 ubiquitin ligases. Previous studies have shown that plants may respond to salt stress by regulating the level of DNA methylation and gene expression [[177]68, [178]69]. In this study, we identified a METL protein (TRINITY_DN86020_c0_g1), which plays a role in alleviating the salt stress of R. soongorica under combined treatment with exogenous Ca^2+ and NO, indicating that the change in the DNA methylation level may also be one of the reasons for the increased ability of R. soongorica to resist salt stress. Jung et al. [[179]70] reported that CaLRR1 was expressed rapidly not only after Capsicum annuum was infected with the anthrax pathogen but also under high salt stress, ABA treatment and other conditions to respond to stress. Lee et al. [[180]71] noted that the OsRLK1 gene in rice can be induced under low temperatures and under salt stress. In this study, 9 LRR-like proteins (TRINITY_DN11359_c0_g1, TRINITY_DN13516_c0_g1, TRINITY_DN1680_c2_g1, TRINITY_DN18427_c0_g1, TRINITY_DN23283_c0_g1, TRINITY_DN4512_c1_g1, TRINITY_DN62171_c0_g1, TRINITY_DN7470_c0_g1, TRINITY_DN7504_c0_g2), and a wall-associated receptor-like kinase (TRINITY_DN4596_c0_g1) were identified. These results indicate that these receptor-like kinases not only participate in the response of R. soongorica to salt stress but also alleviate the damage caused by salt stress by altering the expression levels of these genes under combined treatment with exogenous Ca^2+ and NO. The above analysis revealed that at the transcriptional level, ADK, SBT, F-box protein, MYB, ZIP, PAL, METTL and other related genes are involved in the adaptation and mitigation mechanisms of exogenous Ca^2+ and NO compound treatment in controlling high salt stress in R. soongorica seedlings. Conclusion This study comprehensively analyzed the physiological and transcriptome alterations in R. soongorica seedlings under high salt stress conditions and the combined treatment of exogenous Ca^2+ and NO. High salt stress can induce physiological drought and ion toxicity in R. soongorica. The concurrent application of Ca^2+ and NO can modulate water metabolism in several ways. It achieves this by diminishing the relative and free water content while augmenting the leaf sap concentration, potential saturation deficit, and bound water/free water ratio. Additionally, it reduces the Na^+ and Cl^− contents and elevates the K^+ and Ca^2+ contents, thereby regulating the ion balance and ultimately adjusting the adaptability of R. soongorica seedlings to salt stress. Through the utilization of WGCNA, it was uncovered that genes such as ADK, SBT, F-box protein, MYB, ZIP, PAL, METTL, and LRR are implicated in the adaptive and mitigating mechanisms of the exogenous Ca^2+ and NO combined treatment in the regulation of high salt stress within R. soongorica seedlings. These findings offer valuable insights into the molecular and physiological responses of R. soongorica to salt stress and the potential protective role of exogenous substances, which could pave the way for developing strategies to enhance the plant's salt tolerance and overall survival in saline environments. Materials and methods Experimental materials and salt treatments In this study, a typical salt-secreting plant, R. soongorica, was selected as the test material, and the method in our previous analysis was adopted. In brief, R. soongorica seeds of uniform size were chosen, disinfected with 1% sodium hypochlorite, and then sown in a pot filled with sterilized quartz sand (10 cm × 10 cm × 8.5 cm), with five seeds in each pot. The plants were cultured in an artificial climate chamber at 25 °C/h for 14 h, 22 °C/h for 10 h, a light intensity of 600 μmol^−1 m^−2, and a relative humidity of 60%. The seedlings were irrigated with Hoagland nutrient solution every 5 d. After 90 d of growth, the seedlings with good and consistent growth were selected for experimental treatment. The concentrations of NaCl (marked as N), exogenous NO and Ca^2+ were 400 mmol·L^−1, 0.25 mmol·L^−1 and 20 mmol·L^−1, respectively; the ratio of Ca^2+ to NO was 1:3 (marked as ComT), and these concentrations and ratios were obtained through preliminary test screening. Moreover, an equal volume of deionized water was used as a control (CK), and each treatment was repeated three times. Index determination Determination of water metabolism The relative water content was determined via the drying and weighing method. The specific steps were as follows: 0.5 g of R. soongorica seedling leaves were taken, the fresh weight (W0) was determined, the weighed leaves were placed in distilled water for 24 h, the surface water was dried, the weight (W1) was determined, the saturated leaves were placed in the oven at 105 °C for 30 min, and the dry matter weight (W2) was determined after drying at 65 °C to a constant weight. The water saturation deficit, bound water content and free water content were determined according to the methods of Fujino et al. [[181]72]. The leaf water potential was measured with a dew point water potential meter (psypro, Dianjiang Technology Co., Ltd., Shanghai, China). The leaf water potential before dawn and afternoon was measured at 6:00 and 14:00, respectively. The change in leaf water potential was measured every 2 h from 7:00 to 21:00. The concentration of leaf sap was measured with a hand-held sugar meter (SR-1, Yueda Electromechanical Equipment Co., Ltd., Shandong, and China). During the measurement, the seedlings were wiped clean and placed on the juicer to squeeze out the juice; a small amount of juice was taken from the dropper and dropped into the hand-held sugar meter for measurement. Determination of the ion content The R. soongorica seedlings were washed with distilled water, placed in an oven at 105 °C for 30 min, and dried at 65 °C to a constant weight. The dried samples were ground into powder by a pulverizer and sieved through 100 mesh for ion content determination. Na^+ and K^+ contents were determined via the flame photometer method. Ca^2+ content was determined via atomic absorption spectrometry (AA-1800F, Meixie Instrument Co., Ltd., Shanghai, China). Cl^− content was determined via ion chromatography (pic-10, Puren Instrument, Qingdao, China). Transcriptome analysis RNA extraction To eliminate the differences caused by different growth times, 27 samples of R. soongorica seedlings were collected on days 3rd, 9th and 15th of treatment, frozen in liquid nitrogen and stored at −80 °C for transcriptomic analysis. A total RNA extraction kit (Tiangen, Beijing, China) was used to extract RNA from the collected R. soongorica seedlings. The concentration and purity of the RNA were detected via a NanoDrop 2000, and the integrity of the RNA was detected via agarose gel electrophoresis. The RIN value of the RNA was determined via an Agilent 5300 (total RNA = 1 µg, concentration ≥ 30 ng·µL^−1, RIN > 6.5, OD260/280 between 1.8 and 2.2). Database construction and sequencing After the RNA quality test of the sample was performed, magnetic beads with oligo (DT) and Ploya were used for A-T base pairing, and the mRNA was isolated from the total RNA for the analysis of transcriptome information. The mRNA was randomly broken into small fragments of approximately 300 bp by adding a fragmentation buffer. A small fragment of mRNA was used as a template, random primers were used under reverse transcriptase, and the mRNA was inverted to synthesize single-strand cDNA. Then, the two strands were synthesized to form a stable double-strand structure. An end repair mixture was used to fill the sticky end of the double-stranded cDNA structure into the flat back, and a base at the 3' end was added to connect the Y-shaped connector. After connecting to the adapter, the product was purified and sorted, and the sorted product was amplified via PCR to obtain the final library. After the library passed quality inspection, Shanghai Meiji Biotechnology Co., Ltd. was subjected to sequencing on the Illumina NovaSeq 6000 platform. Bioinformatics credits Trinity software ([182]https://github.com/trinityrnaseq/trinityrnaseq/wiki) was used for assembling the clean data in this study, and translate software ([183]http://hibberdlab.com/transrate/) was used for filtering and optimizing the transcript sequences. Redundant sequences were removed via sequence alignment clustering. BUSCO software ([184]http://busco.ezlab.org) was used to assess transcriptome integrity. The transcripts obtained were subsequently compared with six databases (NR, Swiss-Prot, Pfam, COG, GO and KEGG databases) via the Blastx algorithm to obtain the annotation information in each database, and the annotation situation of each database was statistically analysed. RSEM software ([185]http://deweylab.github.io/RSEM/) was used for quantitative analysis and FPKM conversion. After read counts were obtained, DESeq2 software ([186]http://bioconductor.org/packages/stats/bioc/DESeq2/) was used for differentially expressed gene (DEG) analysis, and the screening criteria were FDR < 0.05 and |log2FC|≥ 1. Goatools software ([187]https://github.com/tanghaibao/GOatools) was subsequently used for GO enrichment analysis, and KOBAS ([188]http://kobas.cbi.pku.edu.cn/home.do) was used for KEGG pathway enrichment analysis. Weighted gene coexpression network analysis (WGCNA) The Meiji bio-cloud platform ([189]https://cloud.majorbio.com/) was used for the WGCNA of 27 samples at three stress time points under high salt stress and exogenous Ca^2+ and NO compound treatment. The minimum number of genes in the module was set to 30, the merging threshold of similar modules was set to 0.25, and the modules with a similarity of 0.8 were merged. The other parameters were set to their defaults. After the coexpression network was generated, Cytoscape 3.9.1 and the Cytonca plug-in were used to draw the network diagram for the key nodes among the top 30 nodes regarding connectivity within the module. qRT‒PCR validation Based on the results of WGCNA, 12 DEGs were selected from four key modules (royal blue, grey, light yellow and tan) for qRT‒PCR further to verify the authenticity and reliability of the transcriptome data. Gene-specific primers were designed with Premier 5.0 software (Table S5). The real-time fluorescent quantitative PCR system was 20 µL in volume, amplified via a two-step method. The samples were pre-denatured at 94 °C for 5 min, denatured at 95 °C for 15 s, and annealed at 60 °C for 30 s for 40 cycles. The relative expression of each gene was calculated via the 2^−ΔΔCT method [[190]73], and Actin was used as the internal reference gene. Data analysis SPSS 22.0 software was used for statistical analysis, single-factor ANOVA was used to analyse and process multiple groups of samples, Duncan's new multiple extreme difference method was used for significant analysis of variance, and Microsoft Excel 2007 was used for drawing and data processing. Supplementary Information [191]Supplementary Material 1.^ (9.9KB, xlsx) [192]Supplementary Material 2.^ (6.5MB, xlsx) [193]Supplementary Material 3.^ (512.1KB, xlsx) [194]Supplementary Material 4.^ (1.1MB, xlsx) [195]Supplementary Material 5.^ (10.8KB, xlsx) [196]Supplementary Material 6.^ (253KB, docx) Authors’ contributions ZHL and PFC conceived and designed the experiment. ZHL, HHL, BBT and XDW performed the experiments. ZHL and HHL analyzed all the data. ZHL wrote the manuscript. PFC revised the manuscript. All of the authors read and approved the final manuscript. Funding This study was funded by the Youth Doctoral Support Project for Universities in Gansu Province (2024QB-073). Data availability The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA1136819. Declarations Ethics approval and consent to participate Experimental research and studies on plants in this study, including the collection of plant material, comply with the institutional, national, and international guidelines and legislation. Seeds of R. soongarica were collected in Laohukou, Wuwei, Gansu Province, China (102°58′ E, 38°44′ N; elevation 1315–1375 m), in late October 2019. This sampling area's average annual temperature, rainfall, and evaporation are 7.5 °C, 110 mm and 2, 646 mm, respectively. The seeds (voucher number: 063–2) were identified by Dr. X. Liu at the Institute of the Gansu Minqin National Studies Station for Desert Steppe Ecosystems (MSDSE). Seed samples were deposited at the Herbarium of the Scientific Research Experimental Station of the Longqu Seed Orchard, Gansu Province Academy of Qilian Water Resource Conservation Forests Research, in Zhangye. Plant materials were collected per the Technical Regulations for the Seed Collection of Rare and Endangered Wild Plants of the People's Republic of China (LYT2590-2016). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References