Tissue culture seedlings of M. hupehensis were cultured on Murashige and Skoog (MS) medium containing 0.5 mg/L indole-3-butyric acid (IBA) and 0.5 mg/L 6-benzylaminopurine (6-BA). The plants were sub-cultured every 3 weeks for a period of 60 d to promote rooting, and then transplanted into soil as described by Zheng et al. (2018). The M. hupehensis plants were maintained at 23 ± 2°C under a 16-h light/8-h dark cycle with a light intensity of 150 μmol m⁻² s⁻¹. After 14 days, seedlings exhibiting similar growth status were selected for subsequent treatment involving K⁺ deficiency and exogenous Res.

A total of 240 M. hupehensis plants were evenly divided into six groups. Group I was irrigated with Hoagland nutrient solution (40 μM K⁺) and served as the control. Group II was irrigated with K⁺ deficiency nutrient solution (4 μM K⁺). Groups III to VI received the same treatment as Group II, but with the addition of exogenous Res (Sangon, Shanghai, China) at concentrations of 10, 50, 100, or 500 μM, respectively. Res was dissolved in ethanol to a final concentration of 10 mM and stored at -20°C. The plants were sprayed and irrigated with Res every 2 days. After 33 days of treatment, the phenotype of the apple seedlings was photographed, and measurements were taken for chlorosis rate, plant height, fresh weight, and dry weight. Each experiment was repeated three times for both technical and biological replicates.

After 33 days of treatment, the chlorosis rate was determined by calculating the number of etiolated or dead plants as a percentage of the total number of seedlings in each group. Ten seedlings were randomly selected from each group to measure plant height (the distance from the base of the seedling stem to the top bud growth point), fresh weight (seedlings were washed with distilled water, dried with filter paper, and weighed), and dry weight (seedlings were treated at 100°C for 30 minutes, then placed in a breathable paper bag and dried at 65°C until a constant weight was reached). Each experiment was independently repeated three times.

After 33 days of K deficiency and exogenous Res treatment, 20 apple plants from each group were randomly selected to determine the chlorophyll content and basic photosynthetic parameters. Four leaves from each seedling were measured. The chlorophyll content was measured using a SPAD-502 Plus (Konica Minolta, Tokyo, Japan) under light conditions. Photosynthesis rate, transpiration rate, and stomatal conductance were measured using the CIRAS-3 portable photosynthetic system (PP Systems, Amesbury, United States). The light intensity was set to 800 μmol·m^-2·s^-1, with 50% humidity and temperature of 23°C. Each experiment was independently repeated three times.

After 33 days of K⁺ deficiency and exogenous Res treatment, 10 apple plants from each group were randomly selected for root scanning. The roots were cleaned and placed in a sample tray containing a small amount of deionized water. The root phenotype, root surface area, root length, root tip number, and root diameter were analyzed and determined using an LA-2400 root scanner (Regenting Trumentsing, United States). Each experiment included three biological replicates.

Twenty apple seedlings were randomly selected from each group to detect ROS levels, including O₂ ^- and H₂O₂. O₂ ^- was detected with Nitroblue tetrazolium reagent (NBT; Sangon, Shanghai, China) and H₂O₂ was detected with 3,3-diaminobenzidine reagent (DAB; Sangon, Shanghai, China) in leaves, as described by Zheng et al. (2017). Additionally, roots were immersed in 5-(and 6)-chlorom-ethyl-2′-7′-dichlorodihy- drofluoresc-ein diacetate acetyl ester (CM-H2DCFDA) (Thermo Fisher, MA, USA) solution for 30 min and rinsed with distilled water for another 30 minutes. The fluorescence of roots was observed and recorded with a live plant fluorescence detector (Vilber Bio Imaging, Paris, France). Malondialdehyde (MDA) content and POD, SOD, and CAT activities were measured as described by Sun et al. (2022a). Each experiment was independently repeated three times.

After 33 days of K⁺ deficiency and exogenous Res treatment, fresh leaves (0.5 g) from each group were used to detect electrolyte leakage and osmolytes. Electrolyte leakage was measured as described by Ahmad et al. (2016). The osmolytes, including proline, soluble sugar, and soluble protein, were detected using extraction kits (Grace, Suzhou, China) following the manufacturer’s instructions. Each experiment was repeated three times.

Mineral element contents were measured using 5 g apple plant tissue after 33 days of K⁺ deficiency and exogenous Res treatment. The samples were dried for 30 min at 105°C and then baked continuously at 80°C for 72 h. Subsequently, 0.5 g of the dried seedlings was ground into powder and digested with 12 mL of a mixture of HNO₃ and HClO₄ (5:1 ratio), and then diluted with deionized water to 25 mL for the detection of mineral elements. The concentrations of K, Na, Ca, phosphorus (P), Mg, iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) were determined by inductively coupled plasma emission spectrometry (ICP–ES) (PerkinElmer, Waltham, United States) as described by Su et al. (2020). Each experiment was independently repeated three times.

The real-time net K⁺ flux of apple plant roots from each group was measured using non-invasive micro-test techniques (NMT) (Xuyue Company, Beijing, China) as described by Jing et al. (2021). Prior to the test, the root tips were soaked in test solution (10 mM KCl, 0.1mM CaCl₂, pH6.0) for 8 minutes and the K⁺ flux sensor was calibrated with two concentration correction fluids (1.5 mM KCl, 0.1mM CaCl2, pH6.0; 15 mM KCl, 0.1mM CaCl2, pH6.0). During the measurement, the K⁺ flux sensor was positioned adjacent to the root tip under a microscope to measure the net flux. The K⁺ flux data was recorded using the imFluxes V2.0 software. Each plant was tested three times, with five plants tested per treatment group.

Total RNA from each treatment was extracted using the RNA prep pure Plant Plus kit (Tiangen, Beijing, China). The total RNA was then reversely transcribed into single-stranded cDNA using the SPARKscript II RT Plus Kit (SparkJade, Jinan, China). Real-time quantitative PCR assays were performed using the LightCycler QLR 480 II system (Roche, Rotkreuz, Switzerland). Primers used for qPCR are listed in Supplementary Table S1 . Apple Actin (accession number: MDP0000774288) was used as the internal control. Candidate genes were identified through a comprehensive review and comparison of literature related to potassium deficiency stress. The relative expression levels were calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001). Each experiment was independently repeated three times.

All data were analyzed using Microsoft Excel 2019 software, and statistical computations were carried out using SPSS software via One-way ANOVA, followed by Tukey’s honestly significant difference (HSD) test (IBM, Armonk, NY, USA). Differences were considered statistically significant at P < 0.05. Bar charts was created using GraphPad Prism 9.5.

In this study, M. hupehensis was used to determine the optimal concentration of Res and explore its effects on the growth of apple plants under K⁺ deficiency stress. As shown in Supplementary Figure S1 , under K⁺ deficiency stress, the leaves of apple seedlings showed high chlorosis rates (86%) and impaired root development. However, this was alleviated when different concentrations of Res were applied ( Supplementary Figures S1A, B ). At low concentrations of Res (10, 50, 100 µM), the chlorosis rates of the apple seedlings significantly decreased from 86% to 55.7%, 37%, and 67.3%, respectively. Conversely, at a high concentration of Res (500 µM), the chlorosis rate was 97.6%, showing no significant difference from the group without Res under K⁺ deficiency stress ( Supplementary Figure S1C ). In addition, the fresh weight of apple plants, which was severely affected by K⁺ deficiency stress, significantly improved with low concentrations of Res, particularly at 50 µM ( Supplementary Figure S1D ). In conclusion, the treatment with 50 µM exogenous Res exhibited the best phenotype and was selected for further research. When transplanted into the soil, the seedlings still exhibited high chlorosis rates under K⁺ deficiency stress. However, these rates significantly decreased from 83.6% to 31.6% after the application of 50 µM Res, with the leaves remaining green ( Figures 1A, B ). Furthermore, plant height, fresh weight, and dry weight were all remarkably affected under K⁺ deficiency stress for 33 days. Plant height decreased from 11.1 cm to 6.7 cm under K⁺ deficiency stress but recovered to 9.9 cm after treatment with 50 µM Res ( Figure 1C ). Fresh weight and dry weight decreased from 5.92 g and 0.63 g to 4.43 g and 0.39 g, respectively, under K⁺ deficiency stress, but were restored to 5.46 g and 0.50 g after treatment with 50 µM Res ( Figures 1D, E ). These results indicate that exogenous Res can protect apple plants from K⁺ deficiency stress.

Since exogenous Res could alleviate the chlorosis phenotype of plants under K⁺ deficiency stress, we measured chlorophyll content and photosynthetic parameters. As shown in Figure 1F , the chlorophyll content of the apple seedlings sharply decreased from 52.53 SPAD to 40.18 SPAD under K⁺ deficiency stress. However, the application of exogenous Res maintained the chlorophyll content at 50.28 SPAD. Moreover, the photosynthetic parameters, including net photosynthesis rate, stomatal conductance, and transpiration rate, followed a similar trend to chlorophyll content. All values were significantly inhibited under K⁺ deficiency stress but increased with the application of exogenous Res ( Figures 1G-I ), particularly the net photosynthesis rate. Under K⁺ deficiency stress, the net photosynthesis rate decreased significantly from 2 to 0.3 µmol·m^-2 s^-1 but recovered to 1.45 µmol·m^-2 s^-1 when exogenous Res was applied ( Figure 1G ). Furthermore, K⁺ deficiency stress remarkably decreased stomatal conductance and transpiration rate in apple seedlings by 75.17% and 50.95%, respectively. These values increased by 35.22% and 20.49%, respectively, after treatment with exogenous Res ( Figures 1H, I ). These results indicate that exogenous Res can protect chlorophyll content and the photosynthetic system under K⁺ deficiency stress, thereby maintaining the normal green color of apple leaves.

The root system of the apple plants was examined using a root scanner to investigate the impact of exogenous Res under K⁺ deficiency stress ( Figure 2A ). The root surface area significantly decreased from 44.28 cm² to 30.21 cm² under K⁺ deficiency stress for 33 days, but recovered to 37.49 cm² with the application of exogenous Res ( Figure 2B ). Similarly, root diameter significantly decreased by 17.2% under K⁺ deficiency stress but returned to normal levels when exogenous Res was applied ( Figure 2C ). In addition, the changes in root length and root tip number followed a similar pattern. Root length and root tip number significantly reduced from 13.49 cm and 399 to 8.53 cm and 214 cm, respectively, under K⁺ deficiency stress. The application of exogenous Res partially alleviated these reductions ( Figures 2D, E ).

K⁺ deficiency stress causes oxidative damage to plants. Therefore, oxidative damage and antioxidant enzyme activities in apple plants were determined after K⁺ deficiency stress and exogenous Res treatment. The staining results of O₂ ^- and H₂O₂ revealed that the leaves of apple plants were seriously damaged by K⁺ deficiency stress. However, exogenous Res significantly reduces the levels of O₂ ^- and H₂O₂ ( Figure 3A ). The trend in MDA content mirrored that of O₂ ^- and H₂O₂ under K⁺ deficiency stress and exogenous Res treatment. The MDA content in the leaves under K⁺ deficiency stress increased from 1.22 to 3.99 nmol/g but decreased to 2.33 nmol/g with the application of exogenous Res ( Figure 3B ). In addition, the activities of antioxidant enzymes (SOD, POD, and CAT) in apple leaves were measured and showed a similar trend ( Figures 3C-E ). SOD, POD, and CAT activities significantly decreased from 71.23, 35.75, and 1749 U/g to 26, 16, and 1426 U/g, respectively, under K⁺ deficiency stress. However, when exogenous Res was applied, the activities of these enzymes were returned to normal levels compared to the control group.

ROS staining and antioxidant enzyme activities were also examined in apple roots. The staining results for ROS in roots showed a trend similar to that in leaves, with exogenous Res reducing ROS content in plant roots under K⁺ deficiency stress ( Figure 4A ). The MDA content in roots under K⁺ deficiency stress increased from 17.71 to 48.5 nmol/g but decreased to 18.3 nmol/g under exogenous Res application ( Figure 4B ). The trend of SOD and POD activities in roots differed from those in leaves. SOD and POD activities significantly increased from 168.9 and 42.5 U/g to 611.5 and 258.2 U/g under K⁺ deficiency stress but decreased to 328.5 and 178.1 U/g with the application of exogenous Res ( Figures 4C, D ). However, the trend in CAT activity was similar to that in leaves. CAT activity significantly decreased from 1482.7 to 350.8 U/g under K⁺ deficiency stress but increased twofold compared to the group without Res under K⁺ deficiency stress when exogenous Res was applied ( Figure 4E ). These results indicate that Res enhances the activities of SOD, POD, and CAT in leaves and CAT in roots of apple plants under K⁺ deficiency stress, thereby removing ROS.

Electrolyte leakage was measured in the leaves and roots of apple plants treated with K⁺ deficiency and exogenous Res after 33 days. Under K⁺ deficiency stress, leaf electrolyte leakage increased from 35.4% to 64.4%, but decreased to 44.7% after exogenous Res application. Similarly, root electrolyte leakage increased from 46.4% to 68.8%, and then decreased to 56.8% following exogenous Res treatment ( Figure 5A ).

The osmolyte contents in the leaves and roots were also measured under K⁺ deficiency stress and exogenous Res treatment. Proline content in leaves and roots significantly increased from 105.3 and 199.8 µg/g to 390.9 and 343.6 µg/g under K⁺ deficiency stress, but decreased to 226.8 and 225.9 µg/g after exogenous Res treatment ( Figure 5B ). Soluble sugar content in both leaves and roots changed significantly, especially in leaves, which significantly increased from 86.1 to 227.2 mg/g and decreased to 123.2 mg/g under K⁺ deficiency stress, while there was no significant change in roots after exogenous Res treatment ( Figure 5C ). The soluble protein content in leaves increased under K⁺ deficiency stress and returned to normal levels after exogenous Res treatment, whereas root protein content did not change significantly before or after treatment ( Figure 5D ).

We measured the macronutrient and micronutrient contents in apple plants under K⁺ deficiency and after exogenous Res treatment. K⁺ content decreased from 15.01 to 4.48 mg/g under K⁺ deficiency stress and increased to 7.89 mg/g after exogenous Res treatment. The trend for Mg content was similar to that of K. Conversely, Na and Ca contents increased from 0.83 and 3.64 mg/g to 3.01 and 5.84 mg/g, respectively, under K⁺ deficiency stress, then decreased to 1.71 and 4.6 mg/g after exogenous Res treatment. There was no significant change in P content ( Figure 6A ). For micronutrients, Fe and Zn contents doubled under K⁺ deficiency stress, but Fe content did not decrease after exogenous Res treatment, while Zn content was significantly decreased. There was no significant change in Mn content under K⁺ deficiency stress, and Cu content increased twofold with exogenous Res application ( Figure 6B ). We also examined the K:Na ratio, an important physiological indicator of plant stress tolerance. Under K⁺ deficiency stress, the K:Na ratio decreased 12.2-fold compared to the control. However, this ratio increased from 1.49 to 4.61 after exogenous Res treatment ( Figure 6C ).

Additionally, we examined K⁺ flux in apple roots under K⁺ deficiency stress and exogenous Res treatment. The positive values of this experiment indicate ion efflux, while the negative values indicate ion influx. As shown in Figure 6D , the average of K⁺ flux in apple roots was 73.85 pmol·cm^-2·s^-1 under control condition and it turned into influx with a flux of -397.74 pmol·cm^-2·s^-1 under K⁺ deficiency stress. When exogenous Res was applied, K⁺ flux decreased to -1178.33 pmol·cm^-2·s^-1 compared to K⁺ deficiency stress alone. These results indicate that K⁺ influx increases under K⁺ deficiency stress and that exogenous Res significantly enhances K⁺ influx under these conditions ( Figure 6D ).

As shown in Figure 7 , we examined the expression levels of nine genes related to K⁺ deficiency response and K⁺ transport in the leaves of M. hupehensis, including six K⁺ transporter genes (MdAKT1, MdHAK5, MdHKT1, MdNHX1, MdTPK1, MdGORK1), tow related kinase genes (MdCIPK23, MdCAM1), and one Ca²⁺-sensor gene (MdCBL9). Among these, the expression levels of MdAKT1, MdHAK5, MdNHX1, and MdTPK1 were significantly up-regulated under K⁺ deficiency stress and further up-regulated after exogenous Res treatment. The expression level of MdHKT1 showed no change under K⁺ deficiency stress but increased significantly when exogenous Res was applied. Conversely, the expression of MdGORK1 was significantly down-regulated under K⁺ deficiency stress and further down-regulated after exogenous Res treatment. Regarding the three kinase genes, the expressions of MdCIPK23 and MdCBL9 were up-regulated under K⁺ deficiency stress. However, MdCIPK23 was further up-regulated, while MdCBL9 was down-regulated after exogenous Res treatment. There was no significant change in the expression of MdCAM1. These results indicate that Res influences K⁺ transport and alleviates K⁺ deficiency stress by modulating the expression of specific K⁺ transporter and kinase genes.