Effect of N-Carbonyloctanoyl homoserine lactone on salt tolerance of Malus robusta seedlings

【Objective】The aim of this study was to investigate the effects of exogenous N-carbonyloctanoyl homoserine lactone (3OC8- HSL) on salt tolerance of Malus robusta seedlings by evaluating growth performance, physiological parameters, and the expression of salt- responsive genes under salt stress.【Methods】A greenhouse pot experiment was conducted with four treatment groups: Including the control group (CK, 0 mmol · L- 1 NaCl + water), the 3OC8-HSL treatment group (AC, 0 mmol · L- 1 NaCl + 10 μmol·L-1 3OC8-HSL), salt stress group (Salt, 200 mmol·L-1 NaCl + water), and 3OC8-HSL + salt stress group (AS, 200 mmol· L- 1 NaCl + 10 μmol· L- 1 3OC8-HSL). Each treatment was applied to the seedlings for 45 days. 3OC8-HSL solution (10 μmol · L- 1 ) was drench-applied to the root zone on days 1, 7, and 14, while salt stress was imposed by watering with 200 mmol·L-1 NaCl every 3 days. The growth parameters (plant height and fresh mass) were measured at the end of the experiment. The leaf chlorophyll content was determined (total chlorophyll, as well as chlorophyll a and b) as an indicator for photosynthetic status. The malondialdehyde (MDA) content in seedling roots was quantified to assess lipid peroxidation (oxidative stress damage). The leaf Na + and K+ concentrations were measured, and the Na + /K+ ratio was calculated to evaluate ionic homeostasis under each treatment. For molecular analysis, the root samples from salt-stressed seedlings with and without 3OC8-HSL were subjected to RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) between the AS treatment and Salt treatment were identified using DESeq2 (standard: |log2 fold change|＞1 and adjusted p-value＜0.05). The gene Ontology (GO) enrichment analysis was performed to characterize the biological processes enriched among these DEGs. Furthermore, four DEGs associated with stress responses were selected for validation by quantitative real- time PCR (RT- qPCR): MdPAL, MdGST, MdHKT1, and MdLRR- RLK. 【Results】The Growth and Physiological Responses: Under non-saline conditions, the exogenous 3OC8- HSL markedly promoted the growth of Malus robusta seedlings. The treated seedlings exhibited a 23.74% increase in plant height and a 17.23% increase in fresh mass compared with the untreated controls, along with a slight but significant rise in the leaf chlorophyll content (+4.06%). These results indicated that 3OC8- HSL itself had a growth- promoting effect even without stress. Under salt stress (200 mmol · L- 1 NaCl), 3OC8-HSL application substantially improved seedling performance and stress tolerance. The 3OC8-HSL-treated salt-stressed seedlings grew taller (+11.61% plant height) and heavier (+26.38% fresh mass than those under salt stress alone. Notably, 3OC8- HSL alleviated salt- induced damage: the treated seedlings maintained significantly higher chlorophyll levels (total chlorophyll + 36.98% vs salt control), indicating better photosynthetic capacity under stress. In addition, 3OC8-HSL reduced oxidative damage, as evidenced by a 36.20% reduction in root MDA accumulation under salt stress relative to untreated seedlings. This lower MDA level suggested that 3OC8-HSL would enhance the antioxidant defense, thereby protecting cellular membranes from peroxidation damage caused by salt stress. The Ion Homeostasis: 3OC8-HSL also improved ionic balance in Malus robusta under salinity. In the salt-stressed seedlings, 3OC8-HSL treatment led to significantly lower Na+ accumulation and higher K+ retention in plant tissues. Specifically, the Na + content in the 3OC8-HSL-treated seedlings was 11.01% lower than that in the untreated salt- stressed controls, while the K+ content was 19.46% higher. Consequently, The Na + /K+ ratio, a key indicator of ion homeostasis under salt stress, was reduced by 22.17% in the 3OC8-HSL group compared with the salt treatment alone. This improvement in Na + /K+ homeostasis reflected a better maintenance of ionic equilibrium, which is crucial for salt tolerance. The results suggested that 3OC8-HSL would help seedlings limit sodium uptake or transport and/ or enhance potassium uptake, thereby mitigating ion toxicity under high salinity. Consistently, the treated seedlings showed healthier morphology and less salt injury than the untreated ones (greener leaves, less wilting), as observed qualitatively during the experiment. The Gene Expression and Pathway Activation: The transcriptome profiling of roots under salt stress revealed significant molecular changes due to 3OC8-HSL. A total of 560 genes were differentially expressed in the 3OC8-HSL-treated vs. untreated salt-stressed roots, with 177 genes upregulated and 383 downregulated (adjusted P＜0.05). The GO enrichment analysis of these DEGs indicated that 3OC8-HSL activated multiple stress-responsive pathways. Many of the upregulated genes were associated with secondary metabolite biosynthesis and defense-related processes. In particular, biological process categories such as phenylpropanoid biosynthetic and metabolic pathways (involved in the synthesis of lignin and other phenolics), glutathione metabolism, response to wounding, and cuticle development were significantly enriched among the 3OC8- HSL-induced genes. These results implied that 3OC8-HSL would trigger a broad reprogramming of the plant’s stress response at the molecular level, enhancing both structural and chemical defenses under the salt stress. Importantly, several key salt-tolerance genes were strongly upregulated by 3OC8-HSL treatment. For instance, transcripts of the MdPAL (phenylalanine ammonia-lyase, a rate-limiting enzyme in the phenylpropanoid pathway leading to lignin synthesis) increased 4.6-fold in the 3OC8-HSL-treated roots (after 7 days of salt stress) compared with the untreated controls. The MdHKT1, encoding a high-affinity K+ transporter involved in Na+ /K+ homeostasis, was upregulated 9.6-fold, and the MdLRRRLK, encoding a leucine-rich repeat receptor-like kinase implicated in stress signal perception, rose by 8.4-fold (both after 7 days). Additionally, the MdGST (glutathione S-transferase, involved in detoxification and ROS scavenging via the glutathione pathway) showed a 6.4- fold induction at 24 hours after treatment. The robust induction of these genes, which would play roles in lignin biosynthesis (cell wall fortification), antioxidant activity, ionic transport, and stress signaling, would underscore the multi-faceted mechanism by which 3OC8-HSL would enhance salt tolerance.【Conclusion】This study demonstrated that the exogenous 3OC8-HSL improved the salt tolerance of Malus robusta seedlings by promoting growth, enhancing photosynthetic capacity, maintaining Na + /K+ homeostasis, and reducing oxidative damage. The transcriptome and RT-qPCR analysis showed that the 3OC8-HSL upregulated the expression of the MdPAL, MdGST, MdHKT1, and MdLRR-RLK, and activated pathways related to phenylpropanoid metabolism and glutathione metabolism under the salt stress. These results would provide a theoretical basis for the application of AHL signals in improving salt resistance of apple rootstocks.