Potassium (K) is one of the most essential macronutrients for plant growth and development, and it has essential physiological functions, such as plant osmoregulation, photosynthesis, protein synthesis, ion homeostasis, and enzyme activation (Kanai et al., 2011; Hafsi et al., 2014). Four forms of K exist in the soil, namely, exchangeable K, soluble K, lattice K, and fixed K. Only soluble K can be taken up by plants from the soil. The K concentration in the soil ranges from 0.1 to 6.0 mmol L⁻¹ (Zeng et al., 2015). Too high or too low K concentrations in soil affect plant growth; in many regions, K concentrations are lower than 0.3 mmol L⁻¹, and K deficiency limits plant growth (Schroeder et al., 1994). Under low-K conditions, the most common phenomena include stunted growth of plants, yellowing of leaf margins, and yield reduction (Hasanuzzaman et al., 2018). Excessive use of potassium fertilizer causes high-K stress, and excessive application of potassium fertilizer in soil causes soil and water pollution, reducing the productivity of crops. However, plants initiate a series of physiological processes as well as molecular and metabolite mechanisms to adapt to different levels of K stress. K deficiency and excess conditions are typical abiotic stress forms that induce a series of biological responses. Under different K stress conditions, reactive oxygen species (ROS) and phytohormones are affected (Ashley et al., 2006; Amtmann et al., 2008). Plant responses to different K conditions are also due to various complex gene regulatory networks that cause widespread changes in gene expression and metabolite contents (Liang et al., 2013).

Transcriptomes comprehensively and efficiently reveal gene expression, thereby allowing elucidation of the plant molecular mechanism response to different K stresses. In plants, many studies have focused on K uptake, loading, and transport mechanisms. Some related genes have been studied, such as the high-affinity K transporter/uptake transporter (HAK/KUP/KT) family, including AtHAK1/5, PpHAK2, AtHAK5, HvHAK1, OsHAK1, AtKUP3, AtKUP1, and OsHKT2, as well as shaker-like K channels (AKT), including OsAKT1 and AtAKT1/5 (Kim et al., 1998; Banuelos et al., 2002; Xu et al., 2006; Fulgenzi et al., 2008; Jung et al., 2009; Pyo et al., 2010; Kim et al., 2012; Oomen et al., 2012; Wu et al., 2019; Wang et al., 2021). These genes play a vital role in plants’ responses to different K conditions. Transcriptomic analysis of the response of Arabidopsis, rice, maize, soybean, sugarcane, and wild barley to K deficiency conditions has indicated that genes involved in metabolism, signal transduction, and ion transport are altered at the transcript level (Armengaud et al., 2004; Ma et al., 2012; Wang et al., 2012a; Zeng et al., 2014).

Metabolomics, known as qualitative and quantitative analysis of cellular metabolites, has become an important complementary tool for the study of plant functional genomics and systems biology (Weckwerth, 2003). Metabonomic analysis reflects the synthesis, decomposition, or transformation rules of some objects, all metabolites, or some metabolites in the tissue or cell (Hall, 2011). Abiotic stress causes changes in the expression of metabolic products, resulting in metabolite disorders in vivo (Meena et al., 2017). Many studies have reported the changes in small-molecule compounds in response to mineral nutritional stress. Sung et al. (2015) reported metabolic responses to deficiencies in nitrogen (N), phosphorus (P), and K, and they demonstrated that the lack of these elements decreases energy production and amino acid metabolism in tomato leaves and roots. Low-K stress increases monosaccharide, disaccharide, polysaccharide, and putrescine contents in barley (Zhao et al., 2021). The contents of putrescine, aconitate, citrate, malate, and fumarate increased in sunflower under low-K stress (Cui et al., 2019). Citric acid, arginine, and asparagine contents are upregulated under K deficiency in rapeseed leaves (Hu et al., 2021). The levels of glutamic acid and aspartic acid are decreased in peanut under low-K conditions, whereas the levels of histidine, lysine, and arginine are increased in peanut under low-K conditions (Patel et al., 2022). The amino acid contents are increased in both K-sensitive and K-tolerant genotypes of wheat roots under K starvation (Zhao et al., 2020).

Apple (Malus domestica) is one of the most important fruits in the world, and apple production and consumption are the highest in China. K fertilizer plays a key role in apple growth and ripening. When K deficiency occurs in apple trees, the middle and lower leaves of new shoots turn yellow. In severe cases, the leaves gradually show brown withered spots, resulting in a curly scorched appearance, and new shoots stop growing early, forming small flower buds and small fruits with a color difference and a decline in quality (Chang et al., 2014). High-K stress causes the occurrence of apple bitter pox, which reduces the absorption of cations, such as calcium and magnesium, by plants, thus affecting the yield of plants. Excessive application of potassium fertilizer causes soil environmental pollution and water pollution. Many studies have investigated the molecular mechanisms that occur under K deficiency in model plants, such as Arabidopsis, rice, and maize (Armengaud et al., 2004; Ma et al., 2012; Wang et al., 2012a). However, these mechanisms have rarely been reported in apple, especially under high-potassium stress. In the present study, we investigated the molecular response mechanism and metabolite changes of apple to low-potassium and high-potassium stresses, and we provided a theoretical basis for further study on the response mechanism of apple to potassium.

K plays essential roles in many physiological and biochemical processes in the plants, such as ion homeostasis, enzyme activation, osmoregulation, and protein synthesis (Kanai et al., 2007; Ma et al., 2020). K stress affects the normal growth of plants; the scarcity of K slows plant growth, reduces plant height, reduces stem diameter (Tester and Blatt, 1989), and decreases photosynthesis (Kanai et al., 2011). The plant height, stem diameter, plant DW, and photosynthesis were decreased under different LK and HK stresses in apple ( Tables 1 , 2 ). Trankner et al. (2018) revealed that K deficiency also reduces photosynthetic CO₂ fixation, as well as the transportation and consumption of photoassimilates, thus damaging plant membranes and chlorophyll under low-K conditions. In the present research, the SPDA value and photosynthetic characteristics decreased under LK stress in apple ( Table 2 ). Xu et al. (2020) reported that an adequate supply of K increased the photoassimilate transportation rate from apple leaves to roots as well as increased nutrient use efficiency by influencing photosynthesis. The SPAD value of apple plants was slightly increased under HK stress, while the photosynthetic index decreased to a lesser extent under HK stress compared to LK stress ( Table 2 ); it means that apple seedlings under low-potassium stress are more damaged than those under high-potassium stress. In the present study, RNA-Seq and KEGG enrichment analysis indicated that a different potassium environment had an effect on plant photosynthesis. Combined transcriptome metabolome analysis showed that the DEGs and DAMs were associated with biological processes, such as carbohydrate metabolism and photosynthesis. Hafsi et al. (2014) revealed that K stress limits plant leaf growth, which may be due to sugar starvation in stems and leaves. In the present study, genes involved in the TCA cycle, such as CS, IDH, and TCA, were downregulated in apple leaves under LK ( Figure 5 ), which may induce apple plant growth restriction.

Plants under K stress conditions increase ROS production, resulting in oxidative stress (Hernandez et al., 2012). The accumulation of higher K in plant cells restores oxidative stress by increasing the activity of antioxidant enzymes, such as glutathione reductase (GPX), dehydroascorbate reductase (DHAR), ascorbate peroxidase (APX), CAT, SOD, and POD (García-Martí et al., 2019). The H₂O₂ content, SOD activity, and POD activity were affected by different K stresses in apple seedlings. The SOD and POD activities were significantly increased to combat different K conditions ( Figures 1B–D ). Under HK stress, the enzyme activities increased more significantly than under LK stress, which indicated that LK had a greater effect on apple plants. A previously metabolome analysis has revealed that the glutathione content is increased in roots in low-K-tolerant KN9204 wheat but not in low-K-sensitive BN207 wheat (Zhao et al., 2020). Under low potassium, the content of S-(methyl)glutathione in apple leaves and roots was significantly increased in the present study ( Table S4 ). Thus, these findings indicated that glutathione is an important metabolite for plant adaptation to K deficiency.

Phytohormones are active substances that widely exist in plants to regulate their physiological metabolism, affect plant development, affect plant growth, and play a regulatory role in stress conditions. Different potassium environments influence phytohormones in plants, such as brassinosteroids, IAA, ABA, and jasmonic acid (JA) (Ahanger et al., 2018; Ahanger et al., 2020; Yang et al., 2020). After 15 days of different K stress treatments, ABA and IAA contents increased in both apple leaves and roots ( Figures 1E, F ). The concentration of ABA in peanut leaves also increases under low-K stress (Patel et al., 2022). Therefore, considering the importance of phytohormones in plant growth, these findings indicated that ABA, JA, SA, and other phytohormones are important molecules in plant resistance to K stress.

Potassium uptake and absorption are mainly accomplished through potassium transporters and potassium channels in the plasma membrane (Ashley et al., 2006). K transporters and channels play vital roles in translocation and cell growth in various plants (Wang and Wu, 2013). Under the condition of K deficiency, the expression levels of HAK1 and HAK5 in maize are upregulated, and AtHAK5, OsHAK1, and HvHAK1 are also induced by K-limited conditions (Santa-María et al., 1997; Banuelos et al., 2002; Ahn et al., 2004; Gierth et al., 2005; Fulgenzi et al., 2008; Qin et al., 2019). In addition, transcriptome analysis of rice roots under LK stress has revealed that the OsHAK1, OsHAK7, OsHAK11, and OsHKT2;1 genes are upregulated; in addition, potassium channel genes, such as OsAKT1, OsAKT2/3, and OsKCO1, are also increased in response to low-K conditions (Ma et al., 2012). In tobacco seedlings, the KUP3 K transporter and the SKOR K channel are increased under LK stress (Lu et al., 2015). In apple seedlings, the AKT (MD15G1178200) and HAK (MD11G1302600, MD11G1303100, MD11G1302900, MD13G1133200, and MD16G1143900) genes were upregulated in roots under KL. Moreover, the KAT (MD05G1284400) and KUP (MD01G1165900 and MD07G1232700) genes were upregulated in apple leaves, whereas HAK (MD03G1283600, MD10G1204500, MD11G1302600, and MD11G1303100) was increased in apple roots ( Table 3 ). These results were consistent with previous studies, showing that a common regulatory mechanism exists across plant species whereby the transcription of genes encoding K transporters and channels increases, which may be an efficient strategy to increase potassium uptake in plants under a K-deficient environment. Therefore, these findings suggested that apple AKT, HAK, KUP, and HAK are key genes involved in potassium channels and transporters, which play an important role in coping with low- and high-potassium stresses in apple seedlings.

Under potassium-deficient conditions, the amino acid content increases in cotton (Wang et al., 2012b) and in roots of the K-tolerant genotype of wheat (Zhao et al., 2020), and lysine, histidine, and arginine accumulated in peanut leaves and roots (Patel et al., 2022). In addition, the citric acid, arginine, and asparagine contents increase under LK in rapeseed (Hu et al., 2021), and most amino acids increase in tomato roots under LK (Sung et al., 2015). Armengaud et al. (2009) found that the selective reduction of acidic amino acids contributes to maintaining charge balance in response to potassium-deficient conditions. Under high-N conditions, most amino acids decreased in apple leaves, and under HK conditions, most amino acids decrease in apple (Sun et al., 2021). In plants, the accumulation of free amino acids has been reported under N and P deficiency conditions (Hernández et al., 2007; Pant et al., 2015; Mo et al., 2019; Ding et al., 2021). In this study, we found that the synthesis of most amino acids was increased in apple leaves under LK stress, especially in ornithine and arginine ( Figure 5 ; Table S4 ). These results show that the deficiency of macronutrients affects the accumulation of amino acids in plants.

Carbohydrate metabolism plays core roles in plant metabolism, providing energy for plant growth and development, and it acts as a bridge in the communication of proteins, lipids, and metabolism (Rolland et al., 2006). The increased content of soluble sugars, including glucose, sucrose, and fructose, in plants is a typical response to different stresses (Armengaud et al., 2004; Rosa et al., 2009; Carvalhais et al., 2011; Wang et al., 2012b; Tang et al., 2015). Zeng et al. (2018) found that most sugars are significantly upregulated under a K-deficient environment in barley roots and leaves. Under LK and HK stresses, the glucose and glycerate-3P contents also increased in apple, suggesting that increased accumulation of sugar may be one of the physiological characteristics for different K stress adaptations in plants. Sugar and potassium have a common function in regulating osmotic potential. We also identified DEGs involved in carbohydrate metabolism, especially those related to glycolysis and the TCA cycle, which were differentially expressed in response to different K stresses. Glycolysis is a process of glucose breakdown to form pyruvate (Fernie et al., 2004). Changes in the levels of gene transcripts in the glycolytic pathway, such as phosphofruckinase-1 (PFK), aldolase (AD), pyruvate-phosphate kinase (PPDK), and phosphoenolpyruvate carboxylase kinase (PPCK), were found under LK and HK conditions in apple. PFK catalyzes a reversible reaction in glycolysis and regulates the glycolysis pathway (Schaeffer et al., 1996; Mustroph et al., 2013). It has been reported that the PFK gene is upregulated in barley under LK (Ye et al., 2022). In this study, the expression of PFK (MD01G107500 and MD07G1144100) was increased in apple leaves, which may have induced an increase the glycerate-3P content in apple leaves under LK ( Figure 5 ). Li et al. (2018) revealed that the TCA cycle of two soybean genotypes is inhibited in leaves and roots under low-N stress. We have previously reported that the TCA cycle is also decreased under N-deficient stress in apple leaves (Sun et al., 2021). The DEGs involved in the TCA cycle, namely, fumarase (FUM), isocitrate dehydrogenase (IDH), and citrate synthase (CS), were also downregulated in apple leaves under LK ( Figure 5 ), indicating that LK stress caused greater damage to apple leaves. Carbohydrate metabolism enzymes, particularly those involved in glycolysis and the TCA cycle, may be indispensable for plant survival under low nutrient conditions (Zeng et al., 2015).

Combined KEGG enrichment analysis of these pathways showed that the biosynthesis of flavonoids was a significantly enriched pathway under different K stresses ( Figure 6 ). Flavonoids are secondary metabolites with low molecular weights, and they are widely found in plant communities and are closely related to the UV protection, flower color formation, plant growth regulation, and pathogen resistance. Many studies have found that flavonoids are related to macronutrients in plants, such as N, P, and K. In rapeseed, nitrogen deficiency enhances ANS and DFR gene expression (Koeslin-Findeklee et al., 2015). The expression of the PAL5, CHS2, F3’H, and F3’5’H genes is significantly increased in tomato leaves under N deficiency stress (Løvdal et al., 2010). Luo et al. (2019) reported that flavonoids are significantly decreased under low P in maize. In the present study, the DEGs and DAMs involved in the flavonoid pathway also changed in apple under different K conditions ( Figure 6 ). The level of naringenin chalcone in apple leaves and roots was increased under LK conditions. Moreover, the phenylalanine content decreased in apple leaves under different K conditions. The PAL gene in apple leaves was upregulated under LK stress but downregulated under HK conditions. The C4H, 4CL, CHS, F3H, ANS, CHI, and DFR genes were upregulated under LK stress, but the UGT gene was downregulated under LK stress. Under HK conditions, the PAL, UGT, CHI (MD01G1118300), and DFR (MD11G1229100) genes were downregulated in leaves. Together, these results indicated that the flavonoid pathway plays an important role in the apple response to different potassium stresses.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

BZ and QW designed the experiments. TS prepared the plant samples. TS, JKZ, and QZ conducted the experiments and analyzed the data. TS, BZ, and QW wrote the manuscript, with the help of XL, ML, YY, and JZ. All authors contributed to the article and approved the submitted version.