Salt stress, as one of the major abiotic stressors limiting crop growth and productivity, is commonly encountered in fruit tree cultivation systems worldwide. The global extent of saline-alkali land reaches approximately 954 million hectares, with China accounting for around 36.7 million hectares-nearly 3.8% of the total-and 12.3 million hectares of which possess significant potential for agricultural development¹. In the Loess Plateau of northwest China, a core production region for high-quality and distinctive peaches, soil salinization poses a particularly severe challenge to sustainable agriculture. The loessal soils, widely distributed across this region, typically exhibit pH values exceeding 8.0, leading to reduced availability of essential nutrients and increased risk of sodium ion toxicity, both of which impose significant physiological stress on fruit tree root systems. In practical production systems, it is precisely this unique soil environment that renders improper rootstock selection highly susceptible to inducing tree chlorosis, diminished tree vigor, reduced yield and quality, and even catastrophic orchard-wide failure.

Prunus davidiana Carr., a deciduous small tree of the genus Prunus (Rosaceae), is widely distributed across North, Northwest, and Southwest China. As a heliophilous species, it exhibits tolerance to cold, drought, and saline-alkali conditions, with well-developed root systems and strong sprouting capacity². Owing to its vigorous growth, high productivity, and superior fruit quality, this species is extensively used as a grafting rootstock for various stone fruit crops in northwestern China³. Prunus persica L., commonly referred to as the hairy peach, is native to northern and central China and has been widely cultivated across diverse regions. This species exhibits cold and drought tolerance but is sensitive to waterlogging⁴. In long-term production practices, it has been observed that peach trees grafted onto hairy peach rootstocks are prone to developing chlorosis after fruiting, leading to reduced yields. In contrast, those grafted onto mountain peach rootstocks exhibit normal growth and fruiting performance, making them widely adopted in commercial orchard systems. Most fruit trees are propagated through grafting; the selection of rootstocks with high salt-alkali tolerance is critical for improving the salt-alkali resistance of fruit trees. Zhao et al.⁵ conducted a comparative study on the salt-alkali tolerance of peach trees across different habitats and found that mountain peach rootstocks exhibit strong tolerance. Salt stress severely disrupts physiological activities and molecular processes in fruit trees through mechanisms including ion toxicity, osmotic stress, and oxidative stress. Liu et al.⁶ reported that salt stress causes significant changes in plant chlorophyll content and photosynthesis performance. Zhai et al.⁷ further demonstrated that saline-alkali stress significantly inhibits the growth and development of 'M9-T337' seedlings; reduces photosynthetic performance; induces ion accumulation, thereby disrupting the osmotic adjustment system, endogenous hormone system, antioxidant system. Transcriptomics further revealed that the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Actinidia chinensis Planch. under 4% salt stress are mainly enriched in glycine metabolism, serine and threonine metabolism, glutathione metabolism, and pyruvate metabolism⁸. Chen et al.⁹ reported that under salt stress, 1,968 and 4,700 (DEGs) were identified in grapevines following short-term and long-term salt treatments, respectively, when compared to the control group. The study highlighted significant enrichment of key metabolic pathways associated with salt stress responses, including flavonoid biosynthesis, starch and sucrose biosynthesis, phenylpropanoid biosynthesis, and terpenoid biosynthesis. Although mountain peach is widely recognized as a tree species with strong salt-alkali tolerance, research into the underlying physiological and molecular mechanisms of this trait remains limited; its comparative advantages over hairy peach rootstocks in terms of stress responses and regulatory pathways have not yet been clearly elucidated.

Therefore, this study employed mountain peach and hairy peach as experimental materials to systematically compare their physiological responses and transcriptomic regulatory networks under salt stress. The aim was to elucidate the intrinsic mechanisms underlying their differential salt tolerance through integrated analyses of leaf anatomical structure, ion homeostasis, antioxidant defense systems, and differential gene expression regulation. These findings not only provide a robust theoretical foundation for understanding the physiological and molecular basis of the superior salt tolerance in mountain peach but also offer valuable insights for the development of salt-tolerant rootstocks in peach breeding programs.

Effects of salt stress on Na⁺, K⁺, Cl⁻ concentrations and Na⁺/K⁺ ratio in mountain peach and hairy peach rootstocks

The effects of different salt stress treatments on the ion contents of the rootstocks of mountain peach and hairy peach are shown in Fig. 1. Salt stress significantly decreased Na⁺ concentration in mountain peach rootstocks, Compared with the control (CK), treatments T1-T3 induced significant reductions of 31.81%, 36.36%, and 31.81%, respectively. In contrast, Na⁺ concentration in hairy peach rootstocks increased gradually, with the highest level observed under T3 treatment (an 82.00% increase relative to CK, Fig. 1A). Under salt stress, the K⁺ concentration of both peach rootstock varieties exhibited a trend of first increasing, then decreasing, and then increasing again. Specifically, the K⁺ concentration in mountain peach rootstocks was highest under T3 treatment, showing a 14.72% increase relative to the CK. For hairy peach rootstocks, the K⁺ concentration under T1 treatment was significantly higher than that of the CK, with an increase of 34.41% (Fig. 1B). As salt stress concentration increased, the Cl⁻ concentration of both peach rootstock varieties increased significantly. For mountain peach rootstocks, the Cl⁻ concentration peaked under T2 treatment, showing a 33.09% increase relative to the CK, In contrast, the Cl⁻ concentration of hairy peach rootstocks reached its maximum under T3 treatment (Fig. 1C). Salt stress decreased the Na⁺/K⁺ ratio in mountain peach rootstocks while increasing it in hairy peach rootstocks. Notably, the Na⁺/K⁺ ratio for hairy peach rootstocks reached its maximum under T3 treatment (Fig. 1D).

Transcriptome sequencing was performed on eight samples from two peach rootstock varieties using the DNBSEQ platform. After raw read filtering, a total of 1,069,964,526 clean reads and 156.62 G clean bases were obtained. The GC content was approximately 45%, and the Q30 base percentage exceeded 96% in all samples, indicating that the transcriptome sequencing data was of high quality and reliability, and thus suitable for downstream analyses (Supplementary Table S2).

As shown in Fig. 5A and Fig. 5B, the first two principal components (PC1 and PC2) explained 78.3% of the total variance. Data points for the same material under different treatments were distributed in distinct clusters, indicating significant changes in gene expression between treated groups and CK following salt stress. Additionally, the three replicates of each treatment clustered closely together, demonstrating good reproducibility. As shown in (Fig. 5B), correlation analysis across the 24 samples further confirmed strong correlations between biological replicates of the salt stress treatments, validating that the sequencing data were highly reliable and suitable for subsequent analyses.

The ST_CK vs ST_T2 comparison yielded the largest number of DEGs, with a total of 3,317 DEGs including 2,816 upregulated genes and 501 downregulated genes. The second largest number of DEGs was observed in the mountain peach T3 treatment, with 1,222 DEGs comprising 350 upregulated genes and 872 downregulated genes. For the peach rootstock hairy peach, the T3 treatment resulted in the largest number of DEGs compared to the CK, with a total of 11,625 DEGs including 5,448 upregulated genes and 6,117 downregulated genes. Among different rootstock cultivars subjected to the same treatment, the T3 treatment induced the largest number of DEGs, with a total of 11,646 DEGs including 5,489 upregulated genes and 6,157 downregulated genes. Among the DEGs, 256 DEGs were common to the three stress treatments (ST_CK vs ST_T1, ST_CK vs ST_T2, and ST_CK vs ST_T3) of mountain peach (Fig. 5C). For hairy peach, 1,196 DEGs were shared across its three stress treatments (MT_CK vs MT_T1, MT_CK vs MT_T2, and MT_CK vs MT_T3) (Fig. 5D). Additionally, 863 DEGs were common to the four cultivar comparison groups (MT_CK vs ST_CK, MT_T1 vs ST_T1, MT_T2 vs ST_T2, and MT_T3 vs ST_T3) under respective treatments (Fig. 5E).

GO enrichment analysis of the three comparison groups in mountain peach (ST_T1 vs ST_CK, ST_T2 vs ST_CK, and ST_T3 vs ST_CK) revealed that: for cellular component (CC), the most significantly enriched terms were "integral component of membrane" and "intrinsic component of membrane". Molecular function (MF), dominant terms included "carbon-oxygen lyase activity" and "terpene synthase activity". Biological process (BP), key enriched terms were "amine metabolic process" and "defense response". In hairy peach (MT_T1 vs MT_CK, MT_T2 vs MT_CK, and MT_T3 vs MT_CK), GO enrichment analysis showed that for CC, "integral component of membrane" and "intrinsic component of membrane" were also significantly enriched MF, the main terms were "tetrapyrrole binding" and "heme binding". BP, the most prominent terms were "response to stress" and "defense response". Furthermore, comparative GO enrichment analysis across the four inter-cultivar comparison groups (MT_CK vs ST_CK, MT_T1 vs ST_T1, MT_T2 vs ST_T2, and MT_T3 vs ST_T3)-representing the two rootstock cultivars under identical treatment conditions-revealed distinct functional patterns. CC, the primary enriched terms were "external encapsulating structure" and "cell wall". MF, key terms included "tetrapyrrole binding" and "heme binding"; and for BP, the dominant terms were "response to stress" and "defense response".

Analysis of the top 20 significantly enriched KEGG pathways for DEGs in mountain peach and hairy peach showed the following For mountain peach (ST_T1 vs ST_CK, ST_T2 vs ST_CK, and ST_T3 vs ST_CK), 150 out of 256 DEGs were enriched in 47 pathways, with alpha-Linolenic acid metabolism, Biosynthesis of secondary metabolites, and Metabolic pathways being significantly enriched. For hairy peach (comparison groups: MT_T1 vs MT_CK, MT_T2 vs MT_CK, and MT_T3 vs MT_CK), 526 out of 1,196 DEGs were enriched in 90 pathways, with Biosynthesis of secondary metabolites, Phenylpropanoid biosynthesis, and Cyanoamino acid metabolism being significantly enriched. For the four cultivar comparison groups between the two peach rootstock cultivars under the same treatment (MT_CK vs ST_CK, MT_T1 vs ST_T1, MT_T2 vs ST_T2, and MT_T3 vs ST_T3), 276 out of 863 DEGs were enriched in 76 pathways, with Biosynthesis of secondary metabolites, Sesquiterpenoid and triterpenoid biosynthesis, and Plant-pathogen interaction being significantly enriched.

KEGG enrichment analysis revealed that mountain peach and hairy peach were co-significantly enriched in the alpha-Linolenic acid metabolism and Phenylpropanoid biosynthesis pathways. In the Phenylpropanoid biosynthesis pathway, hairy peach exhibited 18 significantly downregulated genes, substantially more than the 4 in mountain peach, Among these, CCR2, PRXA21, and PRX44 were identified as significantly co-enriched genes (Fig. 7A). In the alpha-Linolenic acid metabolism pathway, hairy peach showed enrichment of 7 DEGs, with 1 upregulated (OPR2) and 6 downregulated, whereas mountain peach displayed enrichment of 6 DEGs, including one upregulated (ADH) and 5 downregulated genes (Fig. 7B). Mountain peach specifically enriched the Glutathione metabolism, Nitrogen metabolism, and Pyruvate metabolism pathways. Meanwhile, hairy peach was uniquely enriched in the Flavonoid biosynthesis and Cutin, suberin, and wax biosynthesis pathways (Supplementary Table S3).

The color change of plant leaves serves as an intuitive indicator of salt tolerance, primarily regulated by chlorophyll content. Under salt stress, a reduction in chlorophyll levels leads to leaf yellowing and wilting. When stress intensity exceeds a critical threshold, it can result in plant death^{10, 11}. Aazami et al.¹² reported that the levels of chlorophyll a and b in grape leaves decline with increasing salt stress intensity. This study revealed that the leaves of both mountain peach and hairy peach under CK treatment exhibited a bright green color, with chlorophyll a content in mountain peach slightly higher than that in hairy peach, indicating a greater inherent stability of the photosynthetic system in mountain peach As salt concentration increased, hairy peach leaves progressively developed from marginal yellowing to extensive chlorosis (Fig. 4). Although only slight scorching was observed at the leaf tips and margins of mountain peach, with the main leaf area remaining green, this phenotypic difference aligns with changes in chlorophyll content: the chlorophyll a/b ratio of hairy peach exhibited a decreasing trend under intensified salt stress, whereas that of mountain peach initially increased before stabilizing. These findings suggest that mountain peach mitigates salt stress through enhanced photosynthetic activity, demonstrating greater salt tolerance compared to hairy peach.

Salt stress impairs plant growth and physiological functions through multiple mechanisms, including salt ion toxicity, osmotic stress, and ionic imbalance in the rhizosphere¹³. Therefore, the capacity of plants to maintain intracellular ionic homeostasis under saline conditions is critical for mitigating salt-induced damage¹⁴. The present study demonstrated that, with increasing salt stress intensity, the accumulation of Na⁺ and Cl⁻ in mountain peach was significantly lower than thoes in hairy peach, while the K⁺/Na⁺ ratio was notably higher. Similar findings were reported by Wu et al.¹⁵ in hairy peach, suggesting that the capacity for selective ion uptake and compartmentalization in mountain peach likely constitutes the key physiological basis for its superior salt tolerance. The mechanism underlying such ionic balance differences may be attributed to divergent structural responses in leaves of the two peach rootstocks cultivars under salt stress. A higher palisade-to-spongy mesophyll ratio, along with the presence of leaf trichomes, cuticular wax, and specialized stomatal traits, is associated with enhanced adaptability to adverse environmental conditions. Wu et al.¹⁶ demonstrated that under 6.0 g·L⁻¹ NaCl treatment, the leaf epidermal cell layer, cuticle, and spongy mesophyll of two cherry cultivars exhibited significant thickening, whereas the palisade mesophyll showed reduced thickness. Liu et al.¹⁷ reported that abiotic stress induced increased leaf thickness, higher stomatal density, and cuticular thickening in mountain peach. In this study, with the increase of salt concentration, the thicknesses of palisade tissue, spongy tissue and leveas of both mountain peach and hairy peach gradually increased. However, the CTR of mountain peach leaves first decreased and then increased with the enhancement of salt stress, while that of hairy peach leaves gradually decreased (Fig. 4). The compact mesophyll structure of mountain peach may restrict ion transport, enables efficient ion compartmentalization, and thereby enhances its salt tolerance.

Ionic and osmotic stresses induced by salt-alkali stress trigger metabolic disturbances and the toxic accumulation of reactive oxygen species (ROS) in plants, leading to cellular oxidative damage and thereby severely impairing normal growth and development in fruit trees^{18, 19}. Plants typically induce the expression of antioxidant enzymes to scavenge ROS-induced cellular damage²⁰. Studies have demonstrated that the activities of POD and SOD in melon exhibit varying degrees of elevation under salt stress of different concentrations²¹. Zong et al.²² reported that the POD activity of Pyrus betulifolia Batal. (yellow-fruited genotype) seedlings shows an upward trend under salt stress. Our study revealed that with increasing salt stress concentration, SOD and POD activities in mountain peach initially increased and then declined, whereas those in hairy peach exhibited a progressive elevation. Mountain peach demonstrates a concentration-dependent tolerance threshold to salt stress, while hairy peach maintains sustained enzymatic activity and stress tolerance within the tested concentration range. NOX serves as a key component in the initiation of adaptive signaling pathways in plants; however, its uncontrolled activity can trigger oxidative damage^{23, 24}. Under salt stress induction, NOX activity in mountain peach leaves progressively declined, whereas in hairy peach, NOX activity increased concomitantly with escalating stress intensity. These findings suggest that mountain peach adopts an active avoidance strategy to minimize oxidative damage under salinity, while hairy peach depends on a passive resource-allocation mechanism to maintain tolerance. Taken together, the data demonstrate that mountain peach possesses superior salt tolerance compared to hairy peach.

Transcriptome analysis further elucidated the molecular mechanisms underlying salt tolerance divergence among peach rootstock cultivars. In this study, transcriptomic profiling identified distinct metabolic pathways in mountain peach and hairy peach under salt stress, with phenylalanine metabolism and α-linolenic acid metabolism emerging as shared core pathways. Notably, phenylalanine metabolism functions as a central hub of plant secondary metabolism, giving rise to downstream products such as lignin and phenolic compounds that play critical roles in cell wall fortification, stress resistance responses, and flavor compound biosynthesis^{25, 26}. Within the phenylpropanoid biosynthesis pathway, hairy peach harbored 18 downregulated genes, whereas mountain peach contained only 4 downregulated genes. The co-downregulated genes CCR2, PRXA21, and PRX44 suggest that lignin polymerization and peroxidase-mediated cell wall remodeling play critical roles in the salt stress response²⁷. α-Linolenic acid is a key unsaturated fatty acid in plants, which is involved in the transduction of stress resistance signals and the establishment of antioxidant defense systems^{28, 29}. In this study, 6 and 7 DEGs were identified to be enriched in the α-linolenic acid metabolic pathway in mountain peach and hairy peach, respectively. Notably, the ADH gene in mountain peach and OPR2 gene in hairy peach were both upregulated (Fig. 6). These findings suggest that mountain peach and hairy peach synergistically enhance salt tolerance through mechanisms that involve activating the jasmonic acid (JA) signaling pathway and modulating the phenylalanine metabolic branch, thereby triggering systemic defense responses and antioxidant^{30, 31}. Furthermore, the enrichment of mountain peach-specific pathways-glutathione metabolism, nitrogen metabolism, and pyruvate metabolism-constitutes a coordinated network that integrates antioxidant defense, nitrogen assimilation, and energy supply³². By contrast, the enrichment of hairy peach-specific pathways-including flavonoid biosynthesis, cutin/suberin/wax biosynthesis, and plant-pathogen interaction-reveals a complex defense strategy that relies on physical barrier reinforcement, secondary metabolite accumulation, and stress signal transduction. This fundamental divergence in pathway enrichment patterns explains the intrinsic molecular mechanism underlying the stronger salt tolerance of mountain peach relative to hairy peach.

To summarize, under salt stress, mountain peach primarily maintains a high K⁺/Na⁺ ratio and chlorophyll a/b ratio, coupled with a biphasic pattern of antioxidant enzyme activity-initially increasing and subsequently declining-thereby establishing an efficient antioxidant defense system. Furthermore, the enhanced leaf compactness and specific activation of the glutathione metabolic pathway in mountain peach, acting through synergistic multi-layered mechanisms, effectively preserve leaf structural and functional integrity, ultimately underpinning its superior salt tolerance compared to hairy peach (Fig. 9). Future studies should focus on validating the molecular functions of salt stress-responsive DEGs and reconstructing their regulatory and protein-protein interaction networks.

This study elucidates how two peach rootstock genotypes achieve efficient adaptation to salt stress through multi-layered coordinated regulatory mechanisms. At the physiological level, mountain peach rootstock maintain growth and suppress chlorosis under salinity by implementing a coordinated strategy characterized by enhanced ion homeostasis regulation and an inducible antioxidant defense system. At the molecular level, mountain peach precisely regulates the phenylpropanoid metabolic pathway and concurrently activates key metabolic routes such as glutathione and nitrogen metabolism, thereby constructing a robust and integrated defense network. Collectively, these findings reveal the mechanistic basis of the superior salt tolerance in mountain peach from both physiological and molecular perspectives, offering substantial theoretical support for the development of salt-tolerant peach cultivars.