Arbuscular mycorrhizal fungi regulate ion homeostasis and the AsA-GSH cycle to enhance saline-alkaline tolerance in apple rootstock M9-T337

【Objective】Salt- alkali stress, involving elevated sodium (Na + ) levels and high pH conditions, poses a significant threat to crop growth and productivity in saline-alkali soils, which are widespread in many regions of the world. Finding effective strategies to mitigate the harmful effects of such stress on crops is crucial for sustainable agricultural development. This study aimed to investigate the effects of arbuscular mycorrhizal fungi (AMF) inoculation on the growth, physiological responses, and salt-alkali stress tolerance of apple rootstock M9-T337. The primary objective was to explore how AMF inoculation enhances salt- alkali tolerance, particularly by regulating ion homeostasis and modulating the ascorbate- glutathione (AsA-GSH) cycle in plants subjected to saline- alkali stress. The study provides a deeper understanding of the mechanisms underlying AMF- mediated tolerance in apple rootstocks, which are important for fruit production in marginal environmental conditions.【Methods】The experiment was conducted using one- year- old M9-T337 apple rootstock seedlings, which were transplanted into pots containing a sterilized substrate composed of soil, perlite, and vermiculite (3∶1∶1). These pots were sterilized in an autoclave at 121 ℃ for 2 hours to eliminate any microbial contaminants. The seedlings were subject to three treatments: (1) a control group receiving clean water irrigation (CK), (2) a saline-alkali stress group irrigated with 300 mL of a solution of 200 mmol· L- 1 NaCl∶NaHCO2 (1∶1, pH 8.36) (SA) every 6 days, and (3) a saline-alkali stress group treated with AMF inoculation (SA+AMF). The AMF inoculum used was Claroideoglomus etunicatum (Ce, BGC G203C), with a spore count of 40 spores· g- 1 . A total of 90 seedlings were used, with 10 seedlings per treatment and three biological replicates for each treatment. The AMF inoculum was added to the substrate after transplantation, and after 45 days. The seedlings were harvested 30 days after the onset of saline-alkali stress to measure growth parameters, ion content, and the antioxidant system response.【Results】The results revealed that saline-alkali stress severely inhibited the growth of M9-T337 seedlings, as evidenced by significant reductions in biomass, stem thickness, leaf area, plant height increment, and leaf relative water content. However, AMF inoculation alleviated these negative effects. Specifically, the shoot and root biomass of AMF-treated seedlings increased by 35.7% and 28.6%, respectively, compared to those under saline-alkali stress alone. Additionally, stem thickness, leaf area, plant height increment, and leaf relative water content were significantly higher in the AMF-treated seedlings, demonstrating the positive impact of AMF on plant growth under stress conditions. The physiological responses of AMF-treated seedlings were also significantly enhanced, as indicated by substantial increases in antioxidant enzyme activities. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased by 30.45%, 38.87%, and 19.35%, respectively, compared to the saline- alkali stressed group. This increase in antioxidant enzyme activities resulted in a reduction in hydrogen peroxide (H2O2) and superoxide anion (O2 - · ) accumulation by 18.09% and 27.03%, respectively, thus mitigating the oxidative damage caused by salt-alkali stress. In terms of ion homeostasis, AMF inoculation had a significant effect on the ion content in the seedlings. The AMF-treated seedlings exhibited a marked reduction in the sodium ion (Na+ ) content in the leaves by 18.02%, and a decrease in the sodium-to-potassium (Na+ / K+ ) ratio by 28.96%, suggesting that AMF helped to maintain an optimal ion balance under saline-alkali conditions. The potassium ion (K+ ) content in the leaves of AMF-treated seedlings also increased by 15.40%, indicating that AMF promoted K+ uptake and enhanced its transport to the leaves. These changes in ion balance are critical for reducing the toxic effects of Na+ accumulation in the plant tissues. AMF likely facilitated the efflux of Na+ from the root system and its compartmentalization into vacuoles, and improved the selective uptake of K+ . Gene expression analysis further confirmed the role of AMF in regulating ion transport. Key genes involved in Na + exclusion (MdSOS2), Na + sequestration (MdNHX2), and K+ uptake (MdGORK1) were upregulated in AMF-treated seedlings, contributing to improved ionic balance under stress conditions. Moreover, AMF inoculation enhanced the activity of the AsA-GSH cycle, which plays a vital role in plant responses to oxidative stress. The activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in AMF-treated seedlings increased by 48.89% and 21.38%, respectively, leading to a more efficient AsA-GSH cycle, which might help to scavenge reactive oxygen species (ROS) and reduce oxidative damage. The increased antioxidant capacity in the AMF- treated seedlings was associated with a lower level of oxidative stress markers, such as H2O2 and O2 - · , in the plant tissues. These findings suggest that AMF inoculation not only improves ion homeostasis but also enhances the plant’s ability to cope with oxidative stress under saline-alkali conditions.【Conclusion】 The findings of this study demonstrate that AMF inoculation significantly enhanced the salt-alkali tolerance of apple rootstock M9-T337 by improving both ion homeostasis and antioxidant capacity. AMF treatment effectively reduced Na+ accumulation in the leaves, enhanced K+ uptake, and lowered the Na+ / K+ ratio, thereby mitigating ion toxicity. Additionally, AMF inoculation enhanced the AsA-GSH cycle, improving the plant’s antioxidant defense system and reducing oxidative damage caused by salt-alkali stress. These results suggest that AMF can be a powerful tool to enhance the growth and stress tolerance of apple rootstocks in saline-alkali soils, offering potential applications in sustainable fruit production. The insights gained from this study also provide a theoretical basis for the practical use of mycorrhizal symbiosis technology to improve the resilience of crops in saline-alkali soils.