Rice seeds (cv. ZJZ17) were surface-sterilized with 1% sodium hypochlorite for 5 min, rinsed three times with distilled water, and incubated in darkness at 28°C for 48 h. Uniformly germinated seeds were transferred to hydroponic containers with rice-specific nutrient solution under controlled conditions (14 h/day at 28°C, 10 h/night at 25°C, 70 ± 5% relative humidity) (Liu et al., 2019). White LED lamps provided a photosynthetic photon flux density (PPFD) of 150 μmol m⁻² s⁻¹. Laser diodes (LDs; Hangzhou Canruo Xingchen Intelligent Co., Ltd.) with adjustable light quality and intensity supplemented the background illumination. Seeds with consistent germination were sown and placed in a white light incubator with the addition of different laser treatments cultured until the seedlings reached the three-leaf stage (15-days-old seedlings). Thereafter, the seedlings were placed in an incubator with white light only and salt stress was applied with 180 mM NaCl. To determine the optimal red-to-blue light ratio, seedlings were exposed to six treatments: natural light (CK), red LD (R, 660 nm), blue LD (B, 450 nm), red:blue ratios of 3:1 (R3B1), 1:1 (R1B1), and 1:3 (R1B3). Following identification of the optimal ratio (R3B1), seedlings were pretreated with LD for 15 days until the three-leaf stage prior to salt treatment, with light intensities of 5, 10, or 15 μmol m⁻² s⁻¹ PPFD (Qi et al., 2025). After 3 days of salt stress (180 mM NaCl), samples were collected for analysis. Salt stress continued for 4 additional days (7 days total), followed by 14 days of recovery in normal rice nutrient solution without salt. Phenological parameters (plant height, stem thickness, root length, fresh weight, dry weight, root surface area, and root tip count) were measured for 30 randomly selected plants per treatment. Root activity was assessed using a root activity assay kit.

Fresh leaf tissue (10 mg) was ground in 1 mL of 95% ethanol, macerated for 30 min, and centrifuged. Absorbance of the supernatant was measured at 665 nm, 649 nm, and 470 nm using a microplate reader to quantify chlorophyll a, chlorophyll b, and carotenoids. Photosynthetic parameters, including net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO₂ concentration (Ci), and stomatal conductance (Gs), were measured using the LI-6800 portable photosynthesis system (LI-COR, Lincoln, NE, USA) according to our previous study (Ahmed et al., 2024). SPAD values were determined using a SPAD-502PLUS (Konica, Japan). Measurements were conducted on 10 plants per treatment, with three replicates per plant (Adil et al., 2020).

Samples were rinsed with deionized water, de-greened at 105°C for 30 min, and dried at 70°C to constant weight. For elemental analysis, 0.03 g of ground sample was digested in 6 mL of mixed acid (HNO₃:HClO₄, 4:1 v/v) using a temperature gradient (60°C for 1 h, 120°C for 1 h, 150°C for 1 h, and 190°C until white fumes appeared). The digest was analyzed for P, K, Na, Ca, Mg, and Zn using Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) (Adil et al., 2020). Total nitrogen was quantified by digesting 0.05 g of sample in 5 mL of H₂SO₄ at 190°C for 30 min, followed by 5 mL of 30% H₂O₂ at 280°C for 30 min. The clarified digest was filtered (0.45 μm) and analyzed using a continuous flow analyzer (AA3, Seal Analytical, Norderstedt, Germany).

Leaf and root tissues (0.1 g per treatment) were homogenized in liquid nitrogen, centrifuged, and analyzed for antioxidant enzyme activities (SOD, POD, CAT, APX) using assay kits (Grace Biotechnology, Suzhou, China). ROS accumulation was assessed by measuring malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels with colorimetric assay kits (Grace Instrument Biotechnology, Suzhou, China). Antioxidant enzyme activities, MDA and H₂O₂ contents were obtained according to the mass of the samples following the calculations in the kit. Proline content was quantified using a proline assay kit (Boxbio, Beijing, China). H₂O₂ was visualized using 3,3’-diaminobenzidine (DAB) staining, followed by chlorophyll removal with 95% ethanol at 80°C and fixation for imaging.

Leaf tissues from seedlings at the three-leaf stage, treated with LD (5, 10, or 15 μmol m⁻² s⁻¹ PPFD) and subjected to 3 days of salt stress, were collected for transcriptome analysis. Each treatment was sampled as a mixed group of 10 rice seedlings as a biological replication, and each treatment was subjected to three sets of biological replications. Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China), and libraries were sequenced on the Illumina NovaSeq X Plus (Novogene, Shanghai, China). Clean reads were mapped to the Oryza sativa Japonica Group reference genome (IRGSP-1.0) using HISAT2 v2.2.1. Gene expression was quantified as Fragments Per Kilobase of transcript per Million mapped reads (FPKM) using StringTie v2.1.4. Differentially expressed genes (DEGs) were identified with |log₂(fold change)| ≥ 1 and adjusted p-value ≤ 0.05 (Benjamini-Hochberg correction) (Robinson et al., 2010). Gene Ontology (GO) and KEGG pathway enrichment analyses were performed using clusterProfiler v4.0 (padj < 0.05) (Qi et al., 2025).

Rice seedlings were irradiated with laser LD (15 μmol m⁻² s⁻¹ PPFD) until the three-leaf stage and then subjected to salt stress treatment (180 mM NaCl) for 3 days. Non-LD treated seedlings served as controls. Each treatment was sampled in a mixed group of 10 rice seedlings as a biological replication, and each treatment was subjected to six sets of biological replications. Leaf samples from each treatment group were collected and analyzed using untargeted metabolomics via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Metabolite extraction involved grinding an appropriate sample mass in a suitable volume of extraction solvent, followed by ultrasonication. The mixture was allowed to stand, centrifuged, and the supernatant was vacuum-dried (Want et al., 2006). The residue was reconstituted in an appropriate volume of extraction solvent for LC-MS/MS analysis. Raw data, acquired using MassLynx V4.2, were processed with Progenesis QI software (Guo et al., 2024). Statistical analysis, metabolite classification, annotation, and functional interpretation were conducted on the BMKCloud platform (www.biocloud.net). Six biological replicates were included for each treatment.

Total RNA from samples used for transcriptome sequencing was extracted according to the RNA extraction kit manufacturer’s instructions. First-strand cDNA was synthesized by the Prime Script™ RT kit (TaKaRa) and used as a template for expression assays. A SYBR^®Premix Ex Taq™ II (TaKaRa) with CFX96 Real-Time PCR Detection System (Bio-Rad) was used to perform qRT-PCR to check the expression of the genes of selected. The rice Ubiquitin gene was used as an internal reference and relative transcriptional level was calculated using the 2^-ΔΔCt method. Three biological replicates and three technical replicates were performed for each sample. The primer sequences were listed in Supplementary Table S1 .

Data are presented as mean ± standard error of the mean (SEM). For pairwise comparisons between two datasets, student’s t-test was employed. For comparisons involving three or more datasets, one-way analysis of variance (ANOVA) was performed using Tukey’s HSD test. One-way ANOVA followed by Duncan’s multiple range test was performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, USA) and Statistix 8.1 (Analytical Software, Tallahassee, USA). Statistical significance was set at p < 0.05.

Phenotypic assessments were conducted on rice seedlings irradiated with LD until the three-leaf stage and subjected to 3 days of salt stress ( Supplementary Figure S1A ). Using the optimal R3B1 ratio, seedlings were exposed to red-blue LD at intensities of 5, 10, or 15 μmol m⁻² s⁻¹ PPFD (designated L5, L10, and L15, respectively) to evaluate salt tolerance. Compared to the non-irradiated control (L0), the L5, L10, and L15 groups showed significant increases in plant height, stem diameter, fresh weight, and dry weight ( Figures 1A–E ). Root morphology was also enhanced in LD-treated groups, with longer primary roots, greater total root length, larger root surface area, higher root tip counts, and increased root dry weight, particularly in the L10 and L15 groups ( Figures 1F–J ). After prolonged salt stress (7 days), control seedlings (L0) exhibited wilting and severe damage, whereas LD-treated seedlings (L5, L10, L15) displayed greater resilience, with more pronounced recovery after 7 days in normal rice nutrient solution ( Figure 2A ). Survival rates were significantly higher in LD-treated groups compared to L0, with L10 and L15 outperforming L5 ( Figure 2B ). These results show that laser treatment promotes both shoot and root development of rice seedlings, especially at intensities of 10 or 15 μmol m^-² s^-¹ PPFD.

Rice seedlings treated with a 3:1 red-to-blue light ratio showed significantly higher chlorophyll content compared to other ratios ( Supplementary Figures S1B–D ). Survival rates after 7 days of recovery in normal rice nutrient solution further indicated that the R3B1 ratio conferred superior salt tolerance ( Supplementary Figure S1E ). Additionally, LD-treated seedlings retained greener leaves, as confirmed by higher SPAD values after 7 days of salt stress ( Figure 2C ). Photosynthetic pigment analysis revealed elevated levels of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in the L5, L10, and L15 groups compared to L0 ( Figures 2D–G ), underscoring the protective effect of LD irradiation on photosynthetic capacity under salt stress.

Photosynthetic results demonstrated that LD treatments significantly improved Pn, Tr, Ci, and Gs in rice seedlings under salt stress ( Figure 3 ). Salt stress significantly reduced Pn in the non-LD treated group (L0), but LD treatments at 5, 10, and 15 μmol m⁻² s⁻¹ PPFD (L5, L10, and L15, respectively) significantly mitigated this decline ( Figure 3A ). Compared to L0, Tr increased by 29.6%, 40.5%, and 50.4% in L5, L10, and L15, respectively ( Figure 3B ). Similarly, Ci rose by 12.2%, 15.7%, and 23.5% in L5, L10, and L15 compared to L0 ( Figure 3C ). Although salt stress reduced Gs relative to non-stressed conditions, LD-treated groups exhibited higher Gs than L0 under salt stress, with the effect being more pronounced at higher intensities ( Figure 3D ). These findings indicate that LD irradiation enhances photosynthetic performance under salt stress, facilitating partial recovery of seedling growth by improving CO₂ assimilation and stomatal function.

In summary, LD partially mitigated the damage of salt stress on rice seedlings by increasing the chlorophyll and carotenoid contents and enhancing the photosynthetic efficiency.

These results revealed that N content in the leaves increased in the L5, L10, and L15 groups relative to the L0, suggesting that the laser treatment restored some of the salt stress-induced reduction in N content ( Figure 4A ). Similarly, root activity assays demonstrated enhanced root vigor in LD-treated groups, with laser treatment partially restoring the decline in root activity caused by salt stress ( Figure 4B ). These phenomena indicate that LD treatment enhanced salt tolerance in rice seedlings to a certain extent. The ratio of Na⁺/K⁺ content in plant tissues is another crucial indicator of salt tolerance in crops. Salt stress significantly increased Na⁺ levels and decreased K⁺ levels in rice seedling leaves and roots compared to normal conditions. However, laser treatment reduced this Na⁺ accumulation and restored K⁺ levels under stress relative to the control (L0) group ( Figures 4C–F ). Consequently, salt stress markedly raised the Na⁺/K⁺ ratio, while laser treatment significantly mitigated this increase ( Figures 4G, H ). A high pH environment disrupts the balance of mineral ion uptake in plants. Compared to the L0 group, the P content in the shoot of the L5, L10, and L15 groups increased by 14%, 30%, and 35%, respectively ( Supplementary Figure S2A ). Relative to the L0 group, the L5, L10, and L15 groups showed P content in the roots of the increases of 9%, 18%, and 30%, respectively ( Supplementary Figure S2B ). Statistical analysis further indicated that laser treatment effectively restored the absorption of essential elements, including Ca, Mg, Zn, and Mn, in both shoots and roots of rice seedlings under salt stress ( Supplementary Figures S2C–J ).

In conclusion, the laser balanced ion homeostasis by reducing Na⁺ accumulation and elevating K⁺ content in rice seedlings under salt stress, while enhancing root vigor and mineral element uptake to effectively mitigate ion toxicity.

A substantial reduction in antioxidant enzyme activity was noticed in rice plants under salt induced stress condition. However, laser treatment enhanced the activities of antioxidant enzymes in leaves and roots under salt stress conditions. The leaves of L5, L10, and L15 pretreated rice seedlings showed significantly improved SOD activity of 24%, 56%, and 63% compared with the L0 control group, while root SOD activity was significantly improved by 27%, 41%, and 16% compared with the L0, respectively ( Figures 5A, B ). In addition, after salt stress, the POD activity of seedlings in the laser treatment groups increased by 22%, 24%, and 62%, respectively, compared with the L0, and the POD activity of the root system increased by 6%, 22%, and 32%, respectively ( Figures 5C, D ). The CAT activity of seedling leaves in the L5, L10, and L15 laser pretreatment groups was 20%, 63%, and 54% higher than that in the L0, while the CAT activity of the root system was 31%, 10%, and 22% higher than that in the L0, respectively ( Figures 5E, F ). Under salt stress, the APX activity in the leaves of the laser-treated groups increased by 3%, 12%, and 14%, respectively, while the APX activity in the root system increased by 21%, 64%, and 72% compared with the L0 ( Figures 5G, H ).

Salt stress typically induces oxidative damage in rice, leading to the accumulation of reactive ROS. To determine whether elevated ROS levels corresponded to increased cellular damage, we measured H₂O₂ and MDA contents across treatment groups. Under normal conditions, laser pretreatment had no significant effect on H₂O₂ levels in leaves or roots ( Figure 5I ). However, after salt treatment, laser-pretreated seedlings displayed lighter 3,3’-diaminobenzidine (DAB) staining in both tissues, indicating reduced oxidative damage compared to non-laser-treated controls ( Figure 5J ). The quantitative test results for H₂O₂ were consistent with the staining observations ( Figures 5K, L ). LD pretreatment significantly reduced the MDA content in rice seedling leaves and roots under salt stress ( Figures 5M, N ). Notably, LD pretreatment also significantly increased proline levels in rice seedlings ( Figures 5O, P ). These findings demonstrate that LD treatment reduces MDA content and H₂O₂ accumulation while increasing activity of key antioxidant enzymes, thus significantly enhancing the cellular protection mechanisms and antioxidant stress capacity of rice seedlings.

In conclusion, laser treatment increased antioxidant enzyme activities and proline content and decreased MDA and H₂O₂ accumulation in rice seedlings under salt stress. Therefore, laser may support higher cytoprotective and antioxidant tolerance capacity of rice under salt stress.

To explore the molecular mechanism of laser pretreatment in improving the salt tolerance of rice seedlings, this study conducted transcriptome sequencing analysis on rice leaves in different laser pretreatment groups (L5, L10, L15) and untreated control group (L0) under salt stress. The results of PCA and heatmap of DEG expression patterns showed that laser pretreatment of different intensities significantly affected the transcription level of rice seedlings under salt stress ( Figures 6A, B ). The Venn diagram shows that there are 17,173 differentially expressed genes (DEGs) shared between the L5, L10, and L15 groups ( Figure 6C ). Statistical analysis of DEGs revealed that the number of DEGs increased with higher laser pretreatment intensities, with a particularly notable rise in the number of upregulated genes ( Figure 6D ). Gene Ontology analysis indicated that the upregulated DEGs were primarily enriched in terms related to photosynthesis, photosystem II, oxidoreductase activity, carbohydrate metabolic process, and ion transport ( Supplementary Figure S3A ), whereas the downregulated DEGs were mainly associated with microtubule binding, cellular carbohydrate metabolic process, response to stimulus, and tubulin binding ( Supplementary Figure S3B ). KEGG pathway analysis showed that the upregulated DEGs in the L5, L10, and L15 groups were predominantly involved in carbon fixation in photosynthetic organisms, photosynthesis, starch and sucrose metabolism, carbon metabolism, nitrogen metabolism, carotenoid biosynthesis, flavonoid biosynthesis, glutathione metabolism, and phenylpropanoid biosynthesis ( Figure 6E ). In contrast, the downregulated DEGs were primarily enriched in plant-pathogen interaction, motor proteins, phenylpropanoid biosynthesis, fatty acid metabolism, plant hormone signal transduction, and amino sugar and nucleotide sugar metabolism ( Figure 6F ). To elucidate the effects of red and blue LD pretreatment on chloroplast function in rice seedlings, this study focused on analyzing the expression patterns of photosystem- and chloroplast-related DEGs. The results showed that the expression levels of multiple photosynthesis-related genes in the L5, L10, and L15 groups were significantly higher than those in the L0 group under salt stress. These genes included the photosystem I gene OsPsaO and Photosystem II polypeptide genes OsPsbR1/2/3, OsPsbS1/2 and OsPsbP. Additionally, the expression of genes related to the light-trapping complexes of PSI and PSII was significantly elevated, including the light-harvesting-like proteins ONE-HELIX PROTEIN1 (OHP1) and OHP2) associated with the stabilization of photosystem II, the light-harvesting chlorophyll a/binding proteins Lhca1-6 associated with photosystem I (PSI), and the photosystem II (PSII)-associated protein Lhcb1-7 ( Supplementary Figure S4A ). Given that photosynthetic pigments are upstream components of light signal transduction, this study further analyzed the expression of photoreceptor-related genes. Heatmap analysis revealed that RBLD pretreatment upregulated the expression of cryptochrome genes (OsCRY1, OsCRY2, OsCRY3), which perceive blue light, while downregulating the expression of phytochrome genes (OsPhyA, OsPhyB, OsPhyC), which perceive red/far-red light. Concurrently, the expression of phytochrome-interacting factor-like (PIL) genes (OsPIL12, OsPIL13, OsPIL14) was upregulated ( Supplementary Figure S4B ).

Because the scavenging of ROS is an important mechanism for plants to withstand oxidative stress and improve salt tolerance, the differences in the expression of genes related to ROS scavenging enzymes were analyzed after laser treatment under salt stress. Key genes in the salt overly sensitive (SOS) pathway were upregulated in the L5, L10 and L15 groups, including OsSOS1/OsNHX7 (Sodium/hydrogen exchanger 7), OsSOS2/OsCIPK24 (CBL-interacting protein kinase 24) and OsSOS3/OsCBL4 (Calcineurin B-like protein 4). Additionally, most of the key genes regulating the absorption, transport, excretion, and distribution of Na⁺ and K⁺ under salt stress were significantly upregulated in the L5, L10, and L15 groups, such as vacuolar membrane Na⁺/H⁺ antiporter genes (OsNHX1, OsNHX2, OsNHX3, OsNHX5), high-affinity K⁺ transporter genes (OsHAK), and plasma membrane Na⁺/K⁺ transporter-related genes (OsHKT1, OsHKT2), indicating that LD preconditioning contributed to the maintenance of salt-stressed ionic homeostasis in rice seedlings under salt stress ( Supplementary Figure S4C ). To investigate the response of the antioxidant system under salt stress, the expression of genes related to ROS scavenging enzymes was analyzed. The results showed that after laser pretreatment, the expression of most antioxidants genes such as OsSODC, OsPER, OsAPX and OsCAT were significantly upregulated ( Supplementary Figure S4D ). In addition, qRT-PCR results showed that laser treatment under salt stress resulted in elevated expression levels of most photosynthesis-related, salt-response-related, and antioxidant-activity-related genes, which is consistent with the results of the transcriptome data ( Supplementary Figure S5 ). In summary, laser pretreatment may synergistically enhance the tolerance of rice seedlings to salt stress by activating photosynthesis-related genes, ion homeostasis regulatory genes, and up-regulating the expression of antioxidant enzyme system genes.

Based on salt tolerance assays and transcriptome analysis, which revealed that rice seedlings under salt stress exhibited enhanced salt tolerance and more pronounced differential responses in L15 compared to L5 and L10, we conducted a metabolomic study using LC-MS to investigate the effects of laser treatment on salt stress tolerance. This study compared seedlings from the laser-treated group (L15) and the control group (L0) under salt stress conditions. PCA revealed distinct metabolic profiles between groups, confirming the reproducibility among biological replicates of L0 and L15 ( Figure 7A ). To effectively visualize patterns of relative metabolite abundance changes, we plotted hierarchical clustered heat maps, demonstrating significant metabolic alterations in the laser-treated group under salt stress ( Figure 7B ). Compared with L0, the levels of 3054 metabolites were elevated, whereas the levels of 2083 metabolites were reduced in rice seedlings from the L15 treatment group ( Figure 7C ). KEGG pathway enrichment analysis indicated that differentially abundant metabolites (DAMs) were significantly enriched in phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, phenylpropanoid biosynthesis, glutathione metabolism, and starch and sucrose metabolism pathways ( Figure 7D ). To identify the key metabolites that laser regulate the tolerance of rice seedlings to salt stress, we performed expression profiling of differential metabolites of key pathways. Heatmap analysis revealed significantly increased abundance in laser-treated groups for metabolites associated with phenylpropanoid biosynthesis (L-phenylalanine, trans-cinnamic acid, L-tyrosine, chlorogenic acid, coniferyl alcohol), carbon fixation in photosynthetic organisms (sedoheptulose 1,7-bisphosphate), photorespiration (L-glutamate), glutathione metabolism (L-glutamate, dehydroascorbic acid), flavonoid biosynthesis (hesperetin, chlorogenic acid, pelargonidin chloride, delphinidin), indole alkaloid biosynthesis (dialdehyde), and starch and sucrose metabolism (CDP-glucose, sucrose). Importantly, phenylalanine metabolism, glutathione metabolism, and starch and sucrose metabolism pathways exhibited active responses related to energy homeostasis under salt stress. Conversely, metabolites showing decreased accumulation in L15 versus L0 included those associated with anthocyanin biosynthesis (malvidin-3-(p-coumaroyl)-rutinoside-5-glucoside, delphinidin 3-glucoside 5-(caffeoyl-glucoside)), D-amino acid metabolism (D-lysine, L-arginine, L-aspartic acid), and pentose phosphate pathway (2-dehydro-3-deoxy-D-gluconate, α-D-ribose 1-phosphate) ( Figure 7E ).

To investigate the intricate interplay between DEGs and DAMs in laser-treated rice seedlings under salt stress, we performed a comprehensive co-expression network analysis. PCA revealed significant differences in both transcript and metabolome ( Figure 8A ). Notably, metabolite module-gene module correlation chord diagrams confirmed strong positive correlations between numerous DEGs and DAMs ( Figure 8B ). Integrated analysis of DEGs and DAMs identified 83 co-regulated pathways shared between the transcriptomic and metabolomic datasets ( Figure 8B ). Specifically, KEGG analysis of the common pathways revealed that differences were significantly enriched in the pathways of carbon fixation, starch and sucrose metabolism, porphyrin and chlorophyll metabolism, phenylpropanoid biosynthesis, carbon metabolism and photosynthetic organisms, glutathione metabolism, and flavonoid biosynthesis ( Figure 8C ). Light affects the accumulation of phenylpropanoid compounds in plants, which play a role in salt tolerance in rice. Key metabolites in the phenylpropane metabolic pathway, coumaric acid, tyrosine, caffeic acid, 5-Hydroxyconiferylalcohol and sinapyl alcohol, accumulated more in L15 ( Figure 8D ). The expression patterns of key genes in the phenylalanine metabolic pathway, including phenylalanine/tyrosine ammonia-lyase (OsPTAL), 4-coumarate: CoA ligase (Os4CL), cinnamoyl-CoA reductase (OsCCR), shikimate O-hydroxycinnamoyltransferase (OsHCT), catechol-O-methyltransferase (OsCOMT) and cinnamyl-alcohol dehydrogenase (OsCAD), exhibited coordinated trends with the abundance of corresponding metabolites ( Figure 8E ).