Effects of Exogenous Potassium (K+) Application on the Antioxidant Enzymes Activities in Leaves of Tamarix ramosissima under NaCl Stress

Saline soil is a worldwide distributed resource that seriously harms plants’ growth and development. NaCl is the most widely distributed salt in saline soil. As a typical representative of halophytes, Tamarix ramosissima Lcdcb (T. ramosissima) is commonly grown in salinized soil, and halophytes have different abilities to retain more K+ under salt stress conditions. Halophytes can adapt to different salt environments by improving the scavenging activity of reactive oxygen species (ROS) by absorbing and transporting potassium (K+). In this study, electron microscope observation, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents determination, primary antioxidant enzyme activity determination and transcriptome sequencing analysis were carried out on the leaves of T. ramosissima under NaCl stress at 0 h, 48 h and 168 h. The results showed that H2O2 and MDA contents increased in the 200 mM NaCl + 10 mM KCl and 200 mM NaCl groups, but the content increased the most in the 200 mM NaCl group at 168 h. In addition, the leaves of T. ramosissima in the 200 mM NaCl + 10 mM KCl group had the most salt secretion, and its superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were all higher than those of the 200 mM NaCl group and significantly higher than those of the control group. According to the results of transcriptome sequencing, it was found that the expression of 39 genes related to antioxidant enzyme activity changed significantly at the transcriptional level. Among them, 15 genes related to antioxidant enzyme activities were upregulated, and 24 genes related to antioxidant enzyme activities were downregulated in the leaves of T. ramosissima when exogenous potassium (K+) was applied under NaCl stress for 48 h; when exogenous potassium (K+) was used for 168 h under NaCl stress, 21 antioxidant enzyme activity-related genes were upregulated, and 18 antioxidant enzyme activity-related genes were downregulated in T. ramosissima leaves. Based on the changes of expression levels at different treatment times, 10 key candidates differentially expressed genes (DEGs) (Unigene0050462, Unigene0014843, Unigene0046159, Unigene0046160, Unigene0008032, Unigene0048033, Unigene0004890, Unigene0015109, Unigene0020552 and Unigene0048538) for antioxidant enzyme activities were further screened. They played an important role in applying exogenous potassium (K+) for 48 h and 168 h to the leaves of T. ramosissima in response to NaCl stress. Their expression levels were dominated by upregulation, which enhanced the activity of antioxidant enzymes, and helped T. ramosissima mitigate NaCl poison and resist NaCl stress. Particularly, Unigene0048538 in glutathione S-transferase (GST) activity had the largest log2 fold-change in the comparison groups of 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h and 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h. Its expression level was upregulated and played an important role in NaCl toxicity. At the same time, the results of the phylogenetic tree analysis showed that Unigene0048538 had the closest genetic distance to Prunus persica in the evolutionary relationship. In summary, with the increase of exogenous potassium (K+) application time under NaCl stress, T. ramosissima can resist high NaCl stress by enhancing antioxidant enzymes’ activity and maintaining the growth of T. ramosissima. Still, it is not enough to completely eliminate NaCl poison. This study provides a theoretical basis for the molecular mechanism of salt tolerance and K+ mitigation of NaCl poison by the representative halophyte T. ramosissima in response to NaCl stress.

regulation of plant stomata and water utilization, ion balance control, enzyme activations, etc. [6,12]. ROS generated by salt stress typically leads to lipid peroxidation and induces potassium (K + ) loss from cells by activating potassium (K + ) efflux channels [13,14]. It has been reported that improving the nutritional status of potassium in plants can significantly reduce the production of reactive oxygen species [6,15].
Tamarix ramosissima Lcdcb (T. ramosissima) is a halophyte that excretes salts. Its salt glands secrete salt directly to the leaf surface. Its salt glands are mainly distributed on stems and leaves, which can effectively discharge excess salt in cells [16]. During its long-term survival and evolution, T. ramosissima has been highly tolerant to various abiotic stresses such as salt, drought and high temperature. This suggests that it has developed efficient abiotic stress tolerance systems to adapt to adverse environments [17]. Salt glands are an important morphological feature of T. ramosissima to adapt to the saline-alkali environment and one of its main salt secretion mechanisms [18]. In addition, different halophytes have different abilities to retain more potassium (K + ) under salt stress conditions [19]. Previous studies have found that NaCl stress below 100 mM can promote the growth of T. ramosissima, while NaCl stress above 200 mM inhibits the growth of T. ramosissima [20]. Additionally, the addition of exogenous 10 mM KCl can effectively alleviate the growth toxicity of Alternanthera philoxeroides (Mart. Griseb) under drought stress, increasing the potassium (K + ) enrichment level in plants [21]. It was found that overexpression of ApKUP4 in Arabidopsis thaliana significantly enhanced the K + enrichment level and ROS scavenging ability of transgenic plants under NaCl stress conditions, thereby improving the tolerance of transgenic plants to NaCl stress [22]. In this study, the physiological and transcriptional data of T. ramosissima were combined to reveal the changes in salt secretion and antioxidant enzyme activities in the leaves of T. ramosissima in response to NaCl stress by exogenous potassium (K + ) application and to explore critical genes and their potential regulatory networks and provide a scientific theoretical basis and the genetic resources for studying the damage-relieving effect of exogenous potassium (K + ) on Tamarix plants under NaCl stress.

Plant Materials
The experiment was completed at the key laboratory, School of Forestry, Nanjing Forestry University from October 2019 to May 2021, and T. ramosissima seedlings were provided by Dongying Experimental Base of Shandong Academy of Forestry. We selected 5-month-old T. ramosissima seedlings with similar growth and transferred them to a 24-hole hydroponic box (40 cm × 30 cm × 16 cm) filled with 1/2 Hoagland's nutrient solution. The culture medium was replaced every 3 days and placed in a greenhouse with a temperature of 26 ± 2 • C (day) and maintained at a relative humidity of 40% to 55%.

NaCl Treatment and NaCl + KCl Treatment
The control group and treatment groups were set in each experiment, with 8 strains in each group, and the experiment was repeated three times in total. They were cultured with 1/2 Hoagland's nutrient solution as the control group. We added 1/2 Hoagland's nutrient solution of 200 mM NaCl and 1/2 Hoagland's nutrient solution of 200 mM NaCl + 10 mM KCl as the treatment group, changing the culture medium every 3 days. Then, the T. ramosissima root samples were collected at 0 h, 48 h and 168 h after treatment, immediately placed in liquid nitrogen for treatment and then transferred to a −80 • C refrigerator for storage for later use.

Analysis of Leaf Salt Secretion under Different Treatments of T. ramosissima
We collected leaf materials from the control and treatment groups at 0 h, 48 h and 168 h, respectively, and used a JSZ6S stereo microscope (Jiangnan, Nanjing, China) to observe the distribution of salt secretion on the leaf surface for analysis.

Determination and Analysis of Antioxidant Enzyme Activity
The leaves of 8 plants were randomly selected at 48 h and 168 h after treatment, and each treatment had 3 biological replicates to measure H 2 O 2 and MDA contents, SOD, POD and CAT activities [23]. The antioxidant enzyme activity was studied via the physiological mechanism of alleviation of T. ramosissima, by exogenous potassium (K + ) application under NaCl stress.

Transcriptome Sequencing and Differentially Expressed Gene Screening
The leaf samples of T. ramosissima were treated with liquid nitrogen and sent to Guangzhou GENE Denovo Company for 3-generation high-throughput transcriptome sequencing. Referring to Chen's transcriptome sequencing method [23], the obtained raw sequencing data were submitted to the National Center for Biotechnology Information (NCBI) Short Reads Archive (SRA) database, and the SRP number was SRP356215. The reads count data obtained by sequencing were analyzed using DESeq2 [24] to obtain the final correct FDR value (FDR value is the p-value after BH correction), and a corrected p < 0.05 was considered to be significantly enriched. Based on the differential analysis results, we screened out genes as significantly DEGs according to the criteria of FDR < 0.05 and |log2FC| > 1. Finally, the DEGs were used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses using the GO database and release 93.0, respectively [25,26].

Validation of Real-Time Quantitative Reverse Transcription PCR (qRT-PCR)
Ten DEGs (Unigene0104732, Unigene0083695, Unigene0069097, Unigene0024962, Uni-gene0007135, Unigene0088781, Unigene0028215, Unigene0082586, Unigene0003066 and Uni-gene0090596) were randomly selected for quantitative reverse transcription PCR (qRT-PCR) to verify the accuracy of RNA-seq results. We extracted total RNA from leaf material using Omega kit (Beinuo Bio, Shanghai, China) from Omega Bio-Tek. The primers were reverse-transcribed into 1-strand cDNA using PrimerScript TM RT Master Mix (Perfect Real Time) kit from TaKaRa Company (Bao Bio, Dalian, China) of Bao Bioengineering Co., Ltd. as a template. DEGs-specific expression primers were designed (Supplemental Table S1), and the PowerUp TM SYBR Green Master mix reagent from Thermo Fisher Scientific Co., Ltd. (Thermo Fisher, Shanghai, China) was used for the samples, the qRT-PCR detection was performed on the ABI ViiA™ 7 real-time PCR system (ABI, Carlsbad, CA, USA) of Applied Biosystems, and each candidate genes was subjected to 4 technical replicates for a total of 3 biological replicates. Using actin as the internal reference gene, the relative expression level was calculated by the 2 −∆∆Ct method [23].

Analysis of H2O2 and MDA Content in T. ramosissima Leaves under Different Treatments
In this study (Figure 2), within 168 h, the H2O2 content did not change significantly in the control group; the H2O2 content in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed an increasing trend over time. At 48 h and 168 h, there were no significant changes in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups compared with the control group, but the H2O2 content of the 200 mM NaCl group was higher than that of the 200 mM NaCl + 10 mM KCl group. Within 168 h, the MDA content in the control group showed a slowly increasing trend, but there was no significant change. The MDA content in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed an increasing trend over time. At 48 h, the MDA content of the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups had no significant changes compared with that of the control group. At 168 h, the MDA content of the 200 mM NaCl group was higher than that of the control

Analysis of H 2 O 2 and MDA Content in T. ramosissima Leaves under Different Treatments
In this study (Figure 2), within 168 h, the H 2 O 2 content did not change significantly in the control group; the H 2 O 2 content in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed an increasing trend over time. At 48 h and 168 h, there were no significant changes in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups compared with the control group, but the H 2 O 2 content of the 200 mM NaCl group was higher than that of the 200 mM NaCl + 10 mM KCl group. Within 168 h, the MDA content in the control group showed a slowly increasing trend, but there was no significant change. The MDA content in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed an increasing trend over time. At 48 h, the MDA content of the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups had no significant changes compared with that of the control group. At 168 h, the MDA content of the 200 mM NaCl group was higher than that of the control group, and there was a significant difference. Still, the MDA content of the 200 mM NaCl + 10 mM KCl group was slightly increased compared to the control group, but there was no significant difference. group, and there was a significant difference. Still, the MDA content of the 200 mM NaCl + 10 mM KCl group was slightly increased compared to the control group, but there was no significant difference.
In conclusion, the 200 mM NaCl group had the highest H2O2 content and MDA content within 168 h.

Antioxidant Enzyme Activity Analysis of T. ramosissima Leaves under Different Treatments
The SOD activity in the control group did not change significantly within 168 h; the SOD activity in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed an increasing trend with time. Among them, the SOD activity of the 200 mM NaCl + 10 mM KCl group increased the most. The SOD activity of the groups increased more than that of the control group at 168 h, with a significant difference. The 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups also significantly differed in SOD activity at 168 h. The POD activity in the control group did not change significantly within 168 h. The POD activity in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed a slowly increasing trend within 168 h. Among them, the POD activity increased the most in the 200 mM NaCl + 10 mM KCl group. Significantly, at 48 h and 168 h, the POD activity of the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups significantly increased compared with that of the control group. The CAT activity in the control group did not change significantly within 168 h; the CAT activity showed an upward trend in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups over time. Among them, the 200 mM NaCl + 10 mM KCl group increased the most in CAT activity. At 48 h and 168 h, the CAT activity of the 200 mM NaCl group was higher than that of the control group, but there was no significant difference. However, the CAT activity in the 200 mM NaCl + 10 mM KCl group was higher than that in the control group, and there was a significant difference. Notably, within 168 h, the activities of the three antioxidant enzymes of the 200 mM NaCl + 10 mM KCl group were higher than those in the 200 mM NaCl group and were significantly higher than those in the control group ( Figure 3). In conclusion, the 200 mM NaCl group had the highest H 2 O 2 content and MDA content within 168 h.

Antioxidant Enzyme Activity Analysis of T. ramosissima Leaves under Different Treatments
The SOD activity in the control group did not change significantly within 168 h; the SOD activity in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed an increasing trend with time. Among them, the SOD activity of the 200 mM NaCl + 10 mM KCl group increased the most. The SOD activity of the groups increased more than that of the control group at 168 h, with a significant difference. The 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups also significantly differed in SOD activity at 168 h. The POD activity in the control group did not change significantly within 168 h. The POD activity in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups showed a slowly increasing trend within 168 h. Among them, the POD activity increased the most in the 200 mM NaCl + 10 mM KCl group. Significantly, at 48 h and 168 h, the POD activity of the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups significantly increased compared with that of the control group. The CAT activity in the control group did not change significantly within 168 h; the CAT activity showed an upward trend in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups over time. Among them, the 200 mM NaCl + 10 mM KCl group increased the most in CAT activity. At 48 h and 168 h, the CAT activity of the 200 mM NaCl group was higher than that of the control group, but there was no significant difference. However, the CAT activity in the 200 mM NaCl + 10 mM KCl group was higher than that in the control group, and there was a significant difference. Notably, within 168 h, the activities of the three antioxidant enzymes of the 200 mM NaCl + 10 mM KCl group were higher than those in the 200 mM NaCl group and were significantly higher than those in the control group ( Figure 3).    comparison group, 974 DEGs were changed, 727 DEGs were upregulated, and 247 DEG were downregulated in the top 20 KEGG Pathways. There were 18 KEGG pathways cla sified as Metabolism, 1 of them belonged to Environmental Information Processing, an 1 of them belonged to Genetic Information Processing (Figure 4).  To summarize, T. ramosissima leaves were exposed to exogenous potassium for 48 h and 168 h under NaCl stress. With the increased time, a large number of DEGs were involved in the Metabolism-related KEGG pathway, and the number of upregulated DEGs significantly increased. This indicated that the upregulated genes in the leaves of T. ramosissima were mainly regulated, and related DEGs were involved in the KEGG pathway and resisted NaCl injury. showed that the leaves of T. ramosissima were exposed to exogenous potassium (K + ) for 48 h and 168 h under NaCl stress. With the increase of time, its antioxidant mechanism was affected by exogenous potassium (K + ) via upregulating the expression of antioxidant genes to initiate corresponding physiological responses to alleviate the damage caused by NaCl ( Figure S2).     Figure S1). Therefore, using BLAST, the protein amino acid sequence of Unigene0048538 was selected for alignment in the National Center for Biotechnology Information (NCBI). In this study, 21 homologous gene species were selected (Table 3), and using MEGA software (MEGA Software, Pennsylvania, USA), the phylogenetic tree was constructed by combining the amino acid sequence of Unigene0048538 protein of T. ramosissima with the amino acid sequences of these 21 homologous gene species ( Figure 6). The results showed that Unigene0048538 had the closest genetic distance to Prunus persica in the evolutionary relationship.   Figure S1). Therefore, using BLAST, the protein amino acid sequence of Unigene0048538 was selected for alignment in the National Center for Biotechnology Information (NCBI). In this study, 21 homologous gene species were selected (Table 3), and using MEGA software (MEGA Software, Pennsylvania, USA), the phylogenetic tree was constructed by combining the amino acid sequence of Unigene0048538 protein of T. ramosissima with the amino acid sequences of these 21 homologous gene species ( Figure 6). The results showed that Unigene0048538 had the closest genetic distance to Prunus persica in the evolutionary relationship.   Figure 6. Phylogenetic tree analysis of T. ramosissima GST and other species GST. Phylogenetic tree analysis of Unigene0048538 protein amino acid sequence and protein amino acid sequence of other 21 species of T. ramosissima.

Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) Validation
This study selected 10 DEGs (Supplemental Table S2) for qRT-PCR verification. The results showed that these qRT-PCR verification results were completely consistent with the expression trend of the transcriptome sequencing analysis results (Supplemental Figure S1). The results showed that the transcriptome data obtained in this study were accurate and reliable and could provide gene resources and transcriptional level basis for studying the mechanism of exogenous potassium (K + ) application under NaCl stress in T. ramosissima to alleviate salt toxicity and salt tolerance.

Discussion
Tamarix plants are halophytes that excrete salt, and the salt gland is one of the important morphological characteristics of the T. ramosissima ability to adapt to the salinealkali environment [27]. Its salt glands secrete salt directly to the leaf surface, which can effectively remove excess salt in cells and reduce the damage of ion poisoning to plants [16]. This study's electron microscopy analysis result showed that leaf salt secretion increased with the prolongation of NaCl stress time. Particularly, the addition of exogenous potassium (K + ) increased the rate of salt secretion of T. ramosissima, suggesting that T. ramosissima could alleviate and respond to the toxicity of salt stress through the phenomenon of salt secretion. In particular, adding exogenous potassium (K + ) could speed up the mitigation and adaptation of T. ramosissima to prolonged high-salt stress. Tamarix plants have evolved a series of complex mechanisms to resist salt stress during the long-term adaptation process to the environment. Plants' salt stress responses are complex processes

Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) Validation
This study selected 10 DEGs (Supplemental Table S2) for qRT-PCR verification. The results showed that these qRT-PCR verification results were completely consistent with the expression trend of the transcriptome sequencing analysis results (Supplemental Figure S1). The results showed that the transcriptome data obtained in this study were accurate and reliable and could provide gene resources and transcriptional level basis for studying the mechanism of exogenous potassium (K + ) application under NaCl stress in T. ramosissima to alleviate salt toxicity and salt tolerance.

Discussion
Tamarix plants are halophytes that excrete salt, and the salt gland is one of the important morphological characteristics of the T. ramosissima ability to adapt to the saline-alkali environment [27]. Its salt glands secrete salt directly to the leaf surface, which can effectively remove excess salt in cells and reduce the damage of ion poisoning to plants [16]. This study's electron microscopy analysis result showed that leaf salt secretion increased with the prolongation of NaCl stress time. Particularly, the addition of exogenous potassium (K + ) increased the rate of salt secretion of T. ramosissima, suggesting that T. ramosissima could alleviate and respond to the toxicity of salt stress through the phenomenon of salt secretion. In particular, adding exogenous potassium (K + ) could speed up the mitigation and adaptation of T. ramosissima to prolonged high-salt stress. Tamarix plants have evolved a series of complex mechanisms to resist salt stress during the long-term adaptation process to the environment. Plants' salt stress responses are complex processes involving and regulated by multiple genes at the molecular level. There are few reports on the molecular mechanism of applying exogenous potassium (K + ) in T. ramosissima under NaCl stress. Studies have shown that using potassium (K + ) fertilizer under salt stress conditions can improve plant growth [6]. Therefore, the transcriptome sequencing analysis of the leaves of T. ramosissima under NaCl stress with an exogenous potassium (K + ) application can help to reveal the response of Tamarix plants to the salt stress environment and the mechanism of potassium (K + ) alleviating salt toxicity.
Plants are usually subjected to various adverse factors during the growth process, which produces a large amount of ROS in the plant. The main types of ROS are H 2 O 2 , OH − , O 2− , etc. Excessive accumulation of ROS can lead to oxidative stress, damage biological macromolecules, biofilms and other structures, and even lead to cell death in severe cases. The increase in the content of various antioxidant enzymes is an important physiological mechanism for plants to adapt to the salt environment. Plants scavenge reactive oxygen species through changes in the activity of the protective enzyme system, prevent membrane lipid peroxidation and inhibit the accumulation of MDA content in the membrane. Hence, plants of the salt-tolerant genotype detoxify by increasing the antioxidant defense system [28][29][30]. In this study, the H 2 O 2 and MDA contents increased in both 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups within 168 h, but the 200 mM NaCl group had the most H 2 O 2 and MDA contents. In addition, SOD, POD and CAT activities all increased in the 200 mM NaCl and 200 mM NaCl + 10 mM KCl groups. In particular, the activities of these three antioxidant enzymes in the (200 mM NaCl + 10 mM KCl)-treated group were higher than those in the (200 mM NaCl)-treated group and were significantly higher than those in the control group. In order to explore the response of DEGs related to the antioxidant system in T. ramosissima to NaCl stress and the changes in expression levels of adding exogenous potassium (K + ) to mitigate NaCl poison, in this study, the leaves of T. ramosissima were exposed to exogenous potassium (K + ) under NaCl stress, and the expressions of 39 ROS scavenging-related DEGs were significantly changed. Among them, the application of exogenous potassium (K + ) for 48 h under NaCl stress of T. ramosissima resulted in the upregulation of 15 genes and the downregulation of 24 genes; the application of exogenous potassium (K + ) for 168 h under NaCl stress resulted in the upregulation of 21 genes and the downregulation of 18 genes. These DEGs have regulatory effects on the physiological mechanism of salt tolerance and alleviation of NaCl poison in T. ramosissima.
Maintaining the integrity of the membrane system is the key to a plant's salt resistance. In the long-term evolution process, plants have correspondingly formed two major reactiveoxygen-species-scavenging systems, enzymatic systems and nonenzymatic systems. SOD, CAT and POD are important enzymes for ROS metabolism in plants, which can slow down or resist the damage of adversity to plants to a certain extent [31]. Under salt stress, plants can use antioxidant enzymes such as SOD, POD and CAT to reduce or remove ROS damage [32]. In this study, three DEGs (Unigene0033269, Unigene0050462 and Unigene0082550) in SOD activity, three DEGs (Unigene0014843, Unigene0049353 and Unigene0094375) in POD activity and two DEGs (Unigene0046159 and Unigene0046160) in CAT activity were upregulated in the 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h comparison group; one DEG (Unigene0050462) in SOD activity, three DEGs (Unigene0013825, Unigene0014843 and Unigene0029752) in POD activity and three DEGs (Unigene0046159, Unigene0046160 and Uni-gene0087092) in CAT activity were upregulated in the 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h comparison group. Among them, four DEGs, including Unigene0050462 in SOD activity, Unigene0014843 in POD activity and Unigene0046159 and Unigene0046160 in CAT activity were upregulated by exogenous potassium (K + ) for 48 h and 168 h under NaCl stress. This suggests that these DEGs are affected by exogenous potassium (K + ) and play a role in alleviating NaCl poison. As in previous studies, the upregulation of the expression levels of these three types of enzyme-related genes will promote the enhancement of enzyme activity, enhance the ability to scavenge ROS, and thus improve the resistance of plants to abiotic stress [33]. Ascorbate peroxidase (APX) and glutathione peroxidase (GPX) can protect cells by catalyzing H 2 O 2 under plant stress and are the key enzymes for scavenging ROS. Studies have shown that APX family genes contribute to plant tolerance mechanisms during salt stress [34]. The overexpression of ascorbate peroxidase in tobacco chloroplasts enhances tolerance to salt stress and deficiency [35]. Moreover, potassium (K + ) application can improve the scavenging system's ability to scavenge ROS, especially the activities such as GPX [6,36]. NtPHGPX in tobacco can enhance salinity tolerance by scavenging ROS accumulation and redox balance in the body membrane [37]. It has also been reported that LePHGPX can inhibit salt, heat shock and Bax protein-induced cell death, thereby protecting plant tissues from various stresses [38]. In this study, two DEGs (Unigene0008032 and Unigene0048033) in APX activity were upregulated in the 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h comparison group; There were three DEGs in APX activity (Unigene0008032, Unigene0008513 and Unigene0048033), and one DEG in GPX activity up-regulated in the 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h comparison group (Unigene0035407). This indicated that both APX and GPX were involved in plant response to salt stress. With the increase of exogenous potassium (K + ) time addition, more APX-and GPX-related DEGs resisted NaCl stress by upregulating their expression levels. In APX activity, Unigene0008032 and Unigene0048033 were upregulated under NaCl for 48 h and 168 h, indicating that these two DEGs played a role in resistance to salt stress in T. ramosissima. Glutathione S-transferase (GST) protects plants from different abiotic stresses and adversities, and its main functions are detoxification and antioxidant defense [39]. Studies have shown that the expression of related genes in GST activity is increased, the chloroplast antioxidant defense system is enhanced, and the salt tolerance ability of plants is improved [40]. GST-activity-related genes are sensitive to a high concentration of salt (≥200 mM) stress. Still, the related genes in GST activity are not sensitive to low and medium concentrations of salt (≤100 mM) stress [41], and the expression level of related genes in GST activity is upregulated under salt stress, which can improve the tolerance of plants under salt stress [42]. Five DEGs in GST activity in this study (Unigene0004890, Unigene0015109, Unigene0020552, Unigene0048538 and Unigene0069058) were upregulated in the 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h comparison group; ten DEGs in GST activity (Unigene0001041, Unigene0004890, Unigene0012650, Unigene0015109, Unigene0020552, Unigene0048538, Unigene0064942, Unigene0069060, Unigene0082147 and Unigene0098941) were upregulated in 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl level comparison group. In particular, four DEGs in GST activity including Unigene0004890, Unigene0015109, Unigene0020552 and Unigene0048538 showed upregulated expression levels under NaCl application of exogenous potassium (K + ) for 48 h and 168 h. These results indicated that with the increase of exogenous potassium (K + ) addition time under NaCl stress, a large number of genes in GST activity were activated, and the expression level was dominated by the upregulation, which resisted NaCl stress and maintained plant growth. Plant photosynthesis is reduced under high salt stress, and the reduction in photosynthetic pigment content exposes plants to excessive light energy, which accelerates the production of ROS and causes damage to cell membranes. The activities of CAT, APX and GR are insufficient to exceed the production of H 2 O 2 [43]. It has been reported that the expression of the tea tree CsGR2 gene is downregulated in response to salt stress [44], which is similar to the results of this study. In this study, Unigene0075696 and Unigene0098587 in GR activity were downregulated in the 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h and 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h comparison groups. It is inferred from this that adding exogenous potassium (K + ) alleviated the NaCl poison to a certain extent under high NaCl stress but could not completely eliminate the NaCl poison. Therefore, the GR activity was decreased.

Conclusions
T. ramosissima leaves showed salt secretion in the 200 mM NaCl + 10 mM KCl and 200 mM NaCl groups, and the H 2 O 2 and MDA contents and activities of SOD, POD and CAT increased. Among them, the H 2 O 2 and MDA contents of the 200 mM NaCl group was the highest at 168 h. Notably, the leaves in the 200 mM NaCl + 10 mM KCl group had the most salt secretion, and the activities of SOD, POD and CAT were all higher than those in the 200 mM NaCl group, which were significantly higher than those in the control group. According to the transcriptome sequencing of T. ramosissima leaves in response to exogenous potassium (K + ) application under NaCl stress, it was found that the expression of 39 DEGs related to antioxidant enzyme activities changed significantly at the transcriptional level. Fifteen DEGs related to antioxidant enzyme activities were upregulated, and twentyfour DEGs related to antioxidant enzyme activities were downregulated in the leaves of T. ramosissima under NaCl stress for 48 h. Twenty-one DEGs related to antioxidant enzyme activities were upregulated, and eighteen DEGs related to antioxidant enzyme activities were downregulated when exogenous potassium (K + ) was applied to tamarind leaves under NaCl stress for 168 h. A further screening was conducted to obtain 10 key candidate DEGs (Unigene0050462, Unigene0014843, Unigene0046159, Unigene0046160, Unigene0008032, Unigene0048033, Unigene0004890, Unigene0015109, Unigene0020552 and Unigene0048538) for antioxidant enzyme activities. In response to NaCl stress, they played an important role in 48 h and 168 h of exogenous potassium (K + ) application in T. ramosissima leaves. They mainly upregulated the expression level, enhanced the activity of antioxidant enzymes and helped T. ramosissima mitigate NaCl poison. Particularly, Unigene0048538 in GST activity had the largest log 2 fold-change in the comparison groups of 200 mM NaCl-48 h vs. 200 mM NaCl + 10 mM KCl-48 h and 200 mM NaCl-168 h vs. 200 mM NaCl + 10 mM KCl-168 h. Its expression level was upregulated and played an important role in NaCl toxicity. At the same time, the results of the phylogenetic tree analysis showed that Unigene0048538 had the closest genetic distance to Prunus persica in the evolutionary relationship.
In conclusion, with the increase of the exogenous potassium (K + ) application time under NaCl stress, T. ramosissima can resist high NaCl stress by enhancing the activity of antioxidant enzymes to maintain the growth of T. ramosissima. Still, it is not enough to completely eliminate the toxicity of NaCl. This study provides a scientific basis for further research on the salt tolerance and K + alleviation molecular mechanism of exogenous potassium (K + ) in T. ramosissima under NaCl stress.