Effects of Myo -Inositol on NaCl Stress in Tamarix ramosissima : Insights from Transcriptomics and Metabolomics

: NaCl stress adversely affects plant growth. Tamarix ramosissima Ledeb ( T. ramosissima ), a halophyte, thrives in saline-alkali areas. Myo -inositol, a lipid-soluble compound, is crucial for stress response, but its role in mitigating NaCl damage remains underexplored. We analyzed transcriptome sequencing and metabolites in T. ramosissima roots under NaCl stress at various intervals (0 h, 48 h, and 168 h). We identiﬁed ten Myo -inositol oxygenase-related genes. Nine of these genes, linked to metabolic pathways involving Myo -inositol, showed differential expression. Myo -inositol accumulation increased over time, suggesting its role as an osmotic regulator and reactive oxygen species (ROS) scavenger. This accumulation likely shields T. ramosissima from NaCl-induced osmotic and oxidative damage. Notably, Unigene0002140 and Unigene0095980 , associated with Myo -inositol oxygenase, appear to regulate Myo -inositol accumulation and correlate signiﬁcantly with its levels. We hypothesize they are key genes in controlling Myo -inositol levels, warranting further study. This research illuminates the role of Myo -inositol oxygenase-related genes in T. ramosissima roots combating NaCl stress, offering insights for selecting salt-tolerant tree species.


Introduction
In recent years, the saline soil area has expanded due to climate change and human activities. More than 3% of the world's soil resources are affected by salinity [1]. It is estimated that by 2050, more than 50% of the world's arable land will be affected by salinization [2,3], posing a serious environmental problem that threatens the development of sustainable agriculture, forestry, and future food security. Therefore, the efficient utilization of saline soil has become an urgent issue to be resolved [4]. Importantly, NaCl is one of the most widely distributed salts in saline soils, and it can have negative effects on various physiological and biochemical processes at different stages of plant growth and development, ultimately leading to NaCl stress [5,6].
NaCl stress is one of the most significant types of abiotic stress, posing a severe threat to the development of agriculture and forestry [7,8]. NaCl stress is a complex mechanism that affects almost all growth physiological pathways during plant growth and development [9,10]. NaCl stress can cause membrane disintegration, toxic metabolite production, inhibition of photosynthesis, generation of reactive oxygen species (ROS), and weakened nutrient acquisition capacity in plants, leading to cell and whole plant death [11][12][13][14]. Therefore, NaCl stress is a major factor limiting plant growth [15]. Notably, the plant organ that first senses stress signals under NaCl stress is the root, which is also the most directly damaged [16][17][18]. Under normal growth conditions, plant roots can absorb water and nutrients to maintain cellular homeostasis [19]. Still, NaCl stress inhibits root respiration and disrupts root metabolism, leading to internal root dysfunction, ultimately destroying this balance and affecting plant growth [20]. Inositol, a type of cyclohexanehexol, is a prevalent small molecule found throughout the biological realm. It boasts a notably stable structural and chemical profile. Of its nine isomers, Myo-inositol stands out as the most biologically active [21,22]. Myo-inositol is a class of small molecule compounds that are essential for life growth and development [23,24]. It also acts as the structural cornerstone for various lipid signaling molecules that modulate complex cellular pathways, encompassing stress responses and the production of ascorbic acid [25]. Notably, inositol signaling is postulated to be pivotal in diverse facets of plant development and resilience [26]. Myo-inositol and its derivative metabolites are widely present in all eukaryotes. They play significant roles in stress responses and developmental processes, functioning as both lipids and soluble compounds [24,26,27]. Inositol phosphatases, vital in the synthesis and breakdown of Myoinositol and its derivatives, can remove the phosphate from the inositol ring, influencing inositol signaling [26]. Studies by Eisenberg and Kindl [28] have shown that the synthesis pathway of inositol includes the following steps: D-Glucose is phosphorylated to generate D-Glucose-6-phosphate by hexokinase (HK). D-Glucose-6-phosphate is then cyclized to form Myo-inositol-1-phosphate by Myo-inositol-1-phosphate synthase (MIPS). Myo-inositol-1-phosphate is subsequently dephosphorylated by inositol monophosphatase (IMP) to produce inositol finally. Particularly, the cyclization process of D-Glucose-6-phosphate is irreversible, and MIPS plays a crucial role in this step [29]. Furthermore, inositol acts as a compatible solute to balance cell expansion and can enhance various basic metabolisms to adapt to NaCl stress [30]. Moreover, it serves as an osmoprotectant and ROS scavenger to protect plants from osmotic stress and oxidative damage. It also functions as a signaling molecule, regulating many plant metabolic pathways and enhancing salt tolerance [31]. Hu et al. noted that external Myo-inositol under NaCl stress increases sugar levels, enhances antioxidant activity, reduces Na + absorption, and removes ROS in Malus hupehensis Rehd [32].
Tamarix ramosissima Ledeb (T. ramosissima) is a salt-excreting halophyte that has developed efficient abiotic stress tolerance systems to adapt to adverse environments and cope with abiotic stress [33], such as root salt avoidance and salt gland secretion [34,35]. However, research indicates that T. ramosissima normally grows under NaCl concentrations below 100 mM, while concentrations above 200 mM affect its regular growth [36]. Consequently, this study integrates transcriptomics and metabolomics to delve into the role of Myo-inositol in the roots of T. ramosissima under NaCl stress at a molecular level. Our objective is to pinpoint key genes and metabolic pathways. This research will lay a scientific and theoretical foundation and provide genetic resources for the exploration of NaCl stress resistance and enhancement of salt tolerance in Tamarix plants.

Experimental Materials and NaCl Treatment
For this experiment, cuttings of T. ramosissima with similar growth and five-month age were selected and cultured in hydroponic boxes (twenty-four holes) with 1/2 Hoagland's nutrient solution. The nutrient solution was replaced every three days (cultivation environment: temperature 26 ± 2 • C, relative humidity: 40%~55%). After two months, the plants were ready for use. Every experiment had a control and a treatment group, with eight plants in each. This was repeated three times. Plants cultured in 1/2 Hoagland's nutrient solution served as the control group, while those cultured in 1/2 Hoagland's nutrient solution supplemented with 200 mM NaCl formed the treatment group. The nutrient solution was replaced every three days. Root samples were collected at 0 h, 48 h, and 168 h after treatment and were used for transcriptome sequencing and metabolite detection.

Transcriptome Sequencing and Differentially Expressed Genes Screening
We selected root samples from treated T. ramosissima and sent them to biotechnology company. (GENE Denovo, Guangzhou, China) for transcriptome sequencing. RNA was isolated using the Invitrogen kit (Beinuo Bio, Shanghai, China). Following isolation, mRNA was enriched with oligo (dT) cellulose and then segmented to an average length of 200 nt with a fragmentation buffer from New England Biolabs (#E7530). Utilizing random hexamer primers, the first-strand cDNA synthesis occurred, succeeded by the creation of the second-strand cDNA using DNA polymerase I and RNase H. Subsequent steps included the purification of the cDNA fragments, the addition of a terminal "A" base, and their ligation to Illumina adapters. Size selection was conducted on an agarose gel, from which appropriate fragments were isolated and subjected to PCR amplification. The sequenced reads of these fragments were generated on the Illumina HiSeq system by biotechnology company Company (GENE Denovo, Guangzhou, China) [37].
The raw data obtained from transcriptome sequencing was submitted to the Short Reads Archive (SRA) database of the National Center for Biotechnology Information (NCBI) with an SRP number of SRP356215. We conducted DEG screening on the obtained data [38] and annotated them to the Gene Ontology (GO) [39] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [40] for enrichment analysis.

Metabolite Extraction, Detection and Differential Metabolite Screening
T. ramosissima root samples, after treatment, were submitted to a biotechnology company (GENE Denovo, Guangzhou, China) for metabolites extraction and metabolites detection, then analyzed via liquid chromatography-mass spectrometry (LC-MS). The LC-MS analysis employed a Vanquish UHPLC system from Thermo Fisher, Bremen, Germany, which was integrated with an Orbitrap Q Exactive TM HF-X mass spectrometer. These samples were channeled into a Hypersil Gold column measuring 100 × 2.1 mm with a 1.9 µm particle size, applying a linear gradient over 17 min at a flow rate of 0.2 mL/min.
For positive polarity mode, the eluents used were eluent A, which is 0.1% FA in water, and eluent B, methanol. Conversely, in the negative polarity mode, we utilized eluent A, which is 5 mM ammonium acetate at pH 9.0, and eluent B, methanol. We followed a solvent gradient pattern: starting with 2% B for 1.5 min, ramping up to 100% B over 12 min, holding at 100% B for 14 min, dropping to 2% B by the 14.1-min mark, and then maintaining 2% B for the remainder up to 17 min.
The Q Exactive TM HF-X spectrometer operated in both positive and negative modes, set with a 3.2 kV spray voltage, a capillary temperature at 320 • C, and gas flows of 40 arb for sheath and 10 arb for auxiliary [41]. We performed differential metabolite screening and p-value testing on the acquired data and annotated the obtained differential metabolites to the KEGG database (www.kegg.jp/kegg/pathway.html accessed on 16 June 2023) [42].

Quantitative Real-Time PCR Validation of Candidate Genes
We randomly selected ten candidate genes to verify the accuracy of the transcriptome sequencing results. We used the RNAprep pure kit (Tiangeng, Beijing, China) to extract total RNA from the root samples of T. ramosissima and synthesized cDNA using the Prime Script™DEG II1st Strandc DNA Synthesis Kit (Takara, Beijing, China). Primers were designed using Primer Premier 5 software (Supplementary Table S1). The samples were analyzed using the SYBR Green Realtime PCR Master Mix (TOYBO, Jinan China) on the ABI ViiA™ 7 Real-time PCR system (ABI, California, CA, USA). Each gene was biologically replicated three times, using Tubulin as an internal reference gene. The relative expression levels were calculated using the 2 −∆∆Ct method [43].

Prediction of Pfam Protein Structure Domains
We compared the protein sequences of the candidate genes in the Pfam database [44] to obtain annotations of the structural domains.

Experiment Processing
We used Microsoft Excel (Microsoft, Washington, DC, USA) to perform data calculation and processing. This included calculating the mean, standard deviation of the raw data,

Analysis of Myo-Inositol in the Roots of T. ramosissima under NaCl Stress
The metabolites in the roots of T. ramosissima were detected and analyzed by LC-MS under NaCl stress at 0 h, 48 h, and 168 h. The results indicated that Myo-inositol was annotated to eight KEGG pathways (Table 1): metabolic pathways (ko01100), microbial metabolism in diverse environments (ko01120), biosynthesis of antibiotics (ko01130), ABC transporters (ko02010), Galactose metabolism (ko00052), ascorbate and aldarate metabolism (ko00053), Inositol phosphate metabolism (ko00562), phosphatidylinositol signaling system (ko04070). The Myo-inositol content (Log 2 fold-change) exhibited a slight decrease under NaCl stress at 48 h. However, with the increase of NaCl stress time, the Myo-inositol content (Log 2 fold-change) demonstrated a significant increase trend under NaCl stress at 168 h ( Figure 1).

Analysis of Myo-Inositol Oxygenase-Related Genes in the Roots of T. ramosissima under NaCl Stress
Based on the roots of T. ramosissima under NaCl stress at 48 h and 168 h, we identifie ten Myo-inositol oxygenase-related genes ( Table 2). With the exception of Unigene000616 which was not annotated to any KEGG pathway, the remaining nine genes were all linke to three KEGG pathways: metabolic pathways (ko01100), ascorbate and aldarate metabo lism (ko00053), and Inositol phosphate metabolism (ko00562). Moreover, judging by the expression levels (Supplementary Figure S1), three genes (Unigene000214 Unigene0032727, and Unigene0056095) initially demonstrated a decrease in expression which was later followed by an increase under NaCl stress at the checkpoint of the 48 and 168 h marks. There were five genes (Unigene0006163, Unigene000616 Unigene0006165, Unigene0032727, and Unigene0067118) whose expression levels first in

Analysis of Myo-Inositol Oxygenase-Related Genes in the Roots of T. ramosissima under NaCl Stress
Based on the roots of T. ramosissima under NaCl stress at 48 h and 168 h, we identified ten Myo-inositol oxygenase-related genes ( Table 2). With the exception of Unigene0006165, which was not annotated to any KEGG pathway, the remaining nine genes were all linked to three KEGG pathways: metabolic pathways (ko01100), ascorbate and aldarate metabolism (ko00053), and Inositol phosphate metabolism (ko00562). Moreover, judging by their expression levels (Supplementary Figure S1), three genes (Unigene0002140, Unigene0032727, and Unigene0056095) initially demonstrated a decrease in expression, which was later followed by an increase under NaCl stress at the checkpoint of the 48 h and 168 h marks. There were five genes (Unigene0006163, Unigene0006164, Unigene0006165, Unigene0032727, and Unigene0067118) whose expression levels first increased and then decreased at 48 h and 168 h under NaCl stress. Significantly, these genes were considerably upregulated at 48 h under NaCl stress compared to the control group. It's also worth noting that Unigene0095980 did not exhibit any change in expression levels under NaCl stress at both 0 h and 48 h but was markedly upregulated at 168 h.

Pfam A Protein Structure Domain Analysis of Myo-Inositol Oxygenase-Related Genes in the Roots of T. ramosissima
We aligned the protein sequences of the ten Myo-inositol oxygenase-related genes, identified in the roots of T. ramosissima under NaCl stress, to the PfamA database and analyzed them (Supplementary Table S2). The results revealed that the predicted structural domains of the Unigene-encoded protein sequences by the HMM model of Myo-inositol oxygenase-related genes are from position 1 to 319. The HMM length al 249, the bit score ranging from 34.5 to 417.7, hmm acc as PF05153.15, clan as CL0237, and PfamA_definition as Myo-inositol oxygenase.

Analysis of the Ascorbate and Aldarate Metabolism Pathway in the Roots of T. ramosissima under NaCl Stress
Based on the analysis of the ascorbate and aldarate metabolism pathway (Table 3), in the control group-0 h vs. 200 mM NaCl-48 h (CK-0 h vs. N-48 h) comparison group, there are thirty-one differentially expressed genes (DEGs) and one differentially annotated metabolite in the ascorbate and aldarate metabolism pathway. Within them, nineteen DEGs were upregulated, and twelve DEGs were downregulated. In the 200 mM NaCl-48 h vs. 200 mM NaCl-168 h (Na-48 h vs. N-168 h) comparison group, there were thirty-four DEGs and two differentially annotated metabolites in the ascorbate and aldarate metabolism pathway. Among them, thirteen DEGs were upregulated, and twenty-one DEGs were downregulated. In the control group-0 h vs. 200 mM NaCl-168 h (CK-0 h vs. N-168 h) comparison group, there were sixteen DEGs and four differentially annotated metabolites in the ascorbate and aldarate metabolism pathway, and the differentially annotated metabolites in ascorbate and aldarate metabolism showed significant differences (p < 0.05). Amid them, there were eight upregulated and eight downregulated DEGs.  Figure S2 show that in the root of T. ramosissima under NaCl stress at 48 h and 168 h, the expression levels of eight DEGs (Unigene0066846, Unigene0037768, Unigene0105617, Unigene0002140, Unigene0032727, Uni-gene0105238, Unigene0102888, and Unigene0064911) first decreased and then increased. Notably, the expression levels of seven DEGs (Unigene0056095, Unigene0105480, Uni-gene0021103, Unigene0021104, Unigene0017008, Unigene0095536, and Unigene0105664) consistently increased. In addition, Unigene0095980 showed no change in expression levels under NaCl stress at 0 h and 48 h, but its expression level was significantly increased under NaCl stress at 168 h.

Analysis of Differential Metabolites in the Ascorbate and Aldarate Metabolism Pathway
From studying the T. ramosissima roots subjected to NaCl stress for 48 and 168 h, we identified a total of four differential metabolites linked to the ascorbate and aldarate metabolism pathway ( Figure 2). In the comparison group of CK-0 h vs. N-48 h, one differential metabolite (L-Threonate) was annotated, and it was degraded. In the comparison group of N-48 h vs. N-168 h, two differential metabolites (Myo-inositol and L-Gulono-1,4-lactone) were annotated, and both were accumulating. In the comparison group of CK-0 h vs. N-168 h, four differential metabolites (Myo-inositol, 4-lactone, L-Gulono-1, L-Threonate, and 2-Oxoglutarate) were annotated. Among them, Myo-inositol and L-Gulono-1,4-lactone were accumulating, while L-Threonate and 2-Oxoglutarate were degrading (Supplementary Table S4

Pearson Correlation Analysis of Differentially Expressed Genes and Their Related Differential Metabolites in the Ascorbate and Aldarate Metabolism Pathway
According to the absolute Pearson correlation coefficient |Corr| > 0.8 and p < 0.05 requirement, we performed Pearson correlation analysis on the DEGs and their related differential metabolites in the ascorbate and aldarate metabolism pathway (Figure 2).

Analysis of in Inositol Phosphate Metabolism Pathway in the Roots of T. ramosissima under NaCl Stress
Based on the analysis of the inositol phosphate metabolism pathway (Table 4), in the CK-0 h vs. N-48 h comparison group, there are thirty DEGs in the inositol phosphate metabolism pathway. Within them, eighteen DEGs were upregulated, and twelve DEGs were downregulated. However, no differentially expressed metabolites were annotated to the inositol phosphate metabolism pathway. In the N-48 h vs. N-168 h comparison group, there were twenty-seven DEGs and two differentially annotated metabolites in the inositol phosphate metabolism pathway. Among them, nine DEGs were upregulated, and twelve DEGs were downregulated. In the CK-0 h vs. N-168 h comparison group, there were twelve DEGs and one differentially annotated metabolite in the inositol phosphate metabolism pathway. Amid them, there were four upregulated and wight downregulated Figure 3. Pearson correlation analysis of DEGs and their related differential metabolites in the ascorbate and aldarate metabolism pathway. (T. ramosissima roots under NaCl stress at 48 h and 168 h annotated DEGs and their related differential metabolites in the ascorbate and aldarate metabolism pathway Pearson correlation analysis. Note: p ≥ 0.05 is not marked; 0.001 < p < 0.01 is marked as **; p ≤ 0.001 is marked as ***).

Analysis of in Inositol Phosphate Metabolism Pathway in the Roots of T. ramosissima under NaCl Stress
Based on the analysis of the inositol phosphate metabolism pathway (Table 4), in the CK-0 h vs. N-48 h comparison group, there are thirty DEGs in the inositol phosphate metabolism pathway. Within them, eighteen DEGs were upregulated, and twelve DEGs were downregulated. However, no differentially expressed metabolites were annotated to the inositol phosphate metabolism pathway. In the N-48 h vs. N-168 h comparison group, there were twenty-seven DEGs and two differentially annotated metabolites in the inositol phosphate metabolism pathway. Among them, nine DEGs were upregulated, and twelve DEGs were downregulated. In the CK-0 h vs. N-168 h comparison group, there were twelve DEGs and one differentially annotated metabolite in the inositol phosphate metabolism pathway. Amid them, there were four upregulated and wight downregulated DEGs.

Analysis of Differentially Expressed Genes in the Inositol Phosphate Metabolism Pathway
There are thirty-six DEGs annotated to the Inositol phosphate metabolism pathway in the roots of T. ramosissima under NaCl stress (Supplementary Table S5). As shown in Supplementary Figure S4, the expression levels of eight DEGs (Unigene0032727, Unigene0002140, Unigene0097496, Unigene0023711, Unigene0069542, Unigene0068974, Unigene0077925, and Unigene0037080) first decreased and then increased. The expression levels of three DEGs (Unigene0056095, Unigene0101311, and Unigene0105664) have been consistently increasing. Notably, Unigene0026986 and Unigene0095980 did not change expression levels under NaCl stress at 0 h and 48 h, but their expression levels significantly increased under NaCl stress at 168 h.

Analysis of Differential Metabolites in the Inositol Phosphate Metabolism Pathway
There are two differentially expressed metabolites annotated to the Inositol phosphate metabolism pathway in the roots of T. ramosissima under NaCl stress (Supplementary Table S6

Pearson Correlation Analysis of Differentially Expressed Genes and Their Related Differential Metabolites in the Inositol Phosphate Metabolism Pathway
Using the criteria of an absolute Pearson correlation coefficient |Corr| greater than 0.8 and a p-value less than 0.05, we analyzed the DEGs in the Inositol phosphate metabolism pathway and associated differential metabolites ( Figure 4). From Figure 5, we can know that in the comparison group of Na-48 h vs. N-168 h, four DEGs (Unigene0038696, Unigene0090825, Unigene0069542, and Unigene0037080) are located downstream of D-Glucose 6-phosphate, modulate its degradation. Unigene0038696 and Unigene0090825 positively regulate the degradation of D-Glucose 6-phosphate, with no significant correlation.  : accumulation of differential metabolites; : degradation of differential metabolites; black genes in the box: decrease in the expression levels of DEGs; red genes in the box: increase in the expression levels of differentially expressed genes).

Pearson Correlation Analysis of Differentially Expressed Genes and Their Related Differential Metabolites in the Ascorbate and Aldarate Metabolism Pathway
According to the absolute Pearson correlation coefficient |Corr| > 0.8 and p < 0.05 requirement, we performed Pearson correlation analysis on the DEGs and their related differential metabolites in the ascorbate and aldarate metabolism pathway (  : accumulation of differential metabolites; : degradation of differential metabolites; black genes in the box: decrease in the expression levels of DEGs; red genes in the box: increase in the expression levels of differentially expressed genes).

Pearson Correlation Analysis of Differentially Expressed Genes and Their Related Differential Metabolites in the Ascorbate and Aldarate Metabolism Pathway
According to the absolute Pearson correlation coefficient |Corr| > 0.8 and p < 0.05 requirement, we performed Pearson correlation analysis on the DEGs and their related differential metabolites in the ascorbate and aldarate metabolism pathway ( Figure 2). The results show (Figure 3

Pearson Correlation Analysis of Differentially Expressed Genes and Their Related Differential Metabolites in the Inositol Phosphate Metabolism Pathway
Using the criteria of an absolute Pearson correlation coefficient |Corr| greater than 0.8 and a p-value less than 0.05, we analyzed the DEGs in the Inositol phosphate metabolism pathway and associated differential metabolites (Figure 4). From Figure 5, we can know that in the comparison group of Na-48 h vs. N-168 h, four DEGs (Unigene0038696, Uni-gene0090825, Unigene0069542, and Unigene0037080) are located downstream of D-Glucose 6-phosphate, modulate its degradation. Unigene0038696 and Unigene0090825 positively regulate the degradation of D-Glucose 6-phosphate, with no significant correlation. Uni-gene0069542 and Unigene0037080 negatively regulate the degradation of D-Glucose 6phosphate, and Unigene0037080 has a significant correlation with D-Glucose 6-phosphate. In the comparison group of 200 mM NaCl-48 h vs. 200 mM NaCl-168 h, five DEGs (Uni-gene0095469, Unigene0006164, Unigene0032727, Unigene0002140, and Unigene0095980) are located downstream of Myo-inositol, regulating its accumulation. Two DEGs (Unigene0066145 and Unigene0041348), both up and downstream, regulate the accumulation of Myo-inositol. Among them, Unigene0066145, Unigene0041348, Unigene0095469, and Unigene0006164 negatively regulate the accumulation of Myo-inositol, with no significant correlation. Uni-gene0032727, Unigene0002140, and Unigene0095980 positively regulate the accumulation of Myo-inositol. In particular, Unigene0002140 and Unigene0095980 positively regulate the accumulation of Myo-inositol, and they have a significant correlation. In the CK-0 h vs. N-168 h comparison group, two DEGs (Unigene0056095 and Unigene0095980) are located downstream of Myo-inositol, regulating its accumulation. Among them, Unigene0056095 negatively regulates the accumulation of Myo-inositol. Notably, Unigene0095980 positively regulates the accumulation of Myo-inositol and has a significant correlation.
Among them, Unigene0056095 negatively regulates the accumulation of Myo-inositol. Notably, Unigene0095980 positively regulates the accumulation of Myo-inositol and has a significant correlation. Figure 5. Pearson correlation analysis of DEGs and their related differential metabolites in the inositol phosphate metabolism pathway. (T. ramosissima roots under NaCl stress at 48 h and 168 h annotated DEGs and their related differential metabolites in the inositol phosphate metabolism pathway Pearson correlation analysis. Note: p ≥ 0.05 is not marked; 0.01 < p < 0.05 is marked as *; 0.001 < p < 0.01 is marked as **; p ≤ 0.001 is marked as ***).

Quantitative Real-Time PCR Validation of Candidate Genes in the Ascorbate and Aldarate Metabolism Pathway
In this study, we randomly selected 10 candidate genes for qRT-PCR to validate the reliability of the transcriptome sequencing data obtained. The findings indicate that the trends in gene expression from the qRT-PCR validation align with those from the transcriptome sequencing analysis (Supplementary Figure S6). This confirms the accuracy and reliability of the transcriptome data obtained in this study. It can, therefore, offer a scientific theoretical foundation and gene resources for identifying candidate genes in T. ramosissima roots that enhance salt tolerance and mitigate damage from NaCl stress.

Discussion
NaCl stress adversely affects plants by inducing osmotic stress, ion toxicity, nutritional imbalance, and oxidative stress, which can retard plant growth and even cause death [45][46][47]. In addition, excessive accumulation of ROS is a secondary stress that further impairs plant performance [48]. It causes oxidative damage to plants, affecting their growth and development [49]. When plants are subjected to NaCl stress, the integrity of the plant cell membrane is disrupted, ions become imbalanced, and ultimately ROS is produced. Excessive accumulation of ROS can cause oxidative stress, leading to cell death [50]. As the plants of the Tamarix genus have evolved under adverse conditions for a long time, they have developed a strong ability to self-regulate [33]. In our research, we delved into the relationship between differentially expressed genes (DEGs) and associated differential metabolites in the ascorbate and aldarate metabolism pathway in T. ramosissima roots subjected to 48 and 168 h of NaCl stress. This analysis provides valuable insights Figure 5. Pearson correlation analysis of DEGs and their related differential metabolites in the inositol phosphate metabolism pathway. (T. ramosissima roots under NaCl stress at 48 h and 168 h annotated DEGs and their related differential metabolites in the inositol phosphate metabolism pathway Pearson correlation analysis. Note: p ≥ 0.05 is not marked; 0.01 < p < 0.05 is marked as *; 0.001 < p < 0.01 is marked as **; p ≤ 0.001 is marked as ***).

Quantitative Real-Time PCR Validation of Candidate Genes in the Ascorbate and Aldarate Metabolism Pathway
In this study, we randomly selected 10 candidate genes for qRT-PCR to validate the reliability of the transcriptome sequencing data obtained. The findings indicate that the trends in gene expression from the qRT-PCR validation align with those from the transcriptome sequencing analysis (Supplementary Figure S6). This confirms the accuracy and reliability of the transcriptome data obtained in this study. It can, therefore, offer a scientific theoretical foundation and gene resources for identifying candidate genes in T. ramosissima roots that enhance salt tolerance and mitigate damage from NaCl stress.

Discussion
NaCl stress adversely affects plants by inducing osmotic stress, ion toxicity, nutritional imbalance, and oxidative stress, which can retard plant growth and even cause death [45][46][47]. In addition, excessive accumulation of ROS is a secondary stress that further impairs plant performance [48]. It causes oxidative damage to plants, affecting their growth and development [49]. When plants are subjected to NaCl stress, the integrity of the plant cell membrane is disrupted, ions become imbalanced, and ultimately ROS is produced. Excessive accumulation of ROS can cause oxidative stress, leading to cell death [50]. As the plants of the Tamarix genus have evolved under adverse conditions for a long time, they have developed a strong ability to self-regulate [33]. In our research, we delved into the relationship between differentially expressed genes (DEGs) and associated differential metabolites in the ascorbate and aldarate metabolism pathway in T. ramosissima roots subjected to 48 and 168 h of NaCl stress. This analysis provides valuable insights into enhancing plant salt tolerance and unravels the intricate physiological and molecular underpinnings of T. ramosissima's salt tolerance.

Myo-inositol
Oxygenase is an essential monooxygenase that can catalyze the transformation of Myo-inositol into D-Glucuronic acid (D-GlcUA) [51]. Myo-inositol Oxygenase plays a critical role in balancing the concentration of Myo-inositol, thereby aiding in the normal growth and development of plants [52]. In plants, Myo-inositol Oxygenase catalyzes the formation of D-GlcUA from Myo-inositol and activates D-GlcUA into UDP-GlcUA [53]. Myo-inositol Oxygenase and D-GlcUA in plant cells synthesize ascorbic acid, a crucial free radical scavenger, and antioxidant [54,55]. In rice (Oryza sativa L.), OsMIOX is expressed in the root system, and it can enhance plant tolerance under abiotic stress by reducing oxidative damage [56]. In other research, Malus hupehensis's MhMIOX2 has been shown to enhance the salt tolerance of poplar and Arabidopsis through heterologous expression [57]. Likewise, the heterologous expression of MsMIOX2 in the yeast of Medicago sativa has improved the resistance to salt, salinity, drought, and cold [58]. In this study, we identified ten genes related to Myo-inositol oxygenase. Specifically, three genes (Unigene0002140, Uni-gene0032727, Unigene0056095) exhibited reduced expression at 48 h, followed by an increase at 168 h during NaCl stress. Five genes (Unigene0006163, Unigene0006164, Unigene0006165, Unigene0032727, and Unigene0067118) increased in expression level at 48 h under NaCl stress, and these genes significantly upregulated compared to the control group. Notably, Unigene0095980 showed no change in expression level at 0 h and 48 h under NaCl stress. However, its expression level significantly increased at 168 h under NaCl stress. The results suggest that Myo-inositol oxygenase-related genes are consistently active at 48 h and 168 h under NaCl stress, enhancing plant tolerance against NaCl stress.
In the face of plant adversities, Myo-inositol and its derivatives play a crucial role in enhancing plant tolerance [59]. Myo-inositol serves as an osmoregulatory substance and ROS scavenger to protect plants from osmotic stress and oxidative damage, and as a signaling molecule, it regulates many metabolic pathways within plants, enhancing their salt tolerance [31]. Existing studies have shown that the expression of Myo-inositol transporter MfINT-like in alfalfa is induced by NaCl stress, and the ectopic expression of MfINT-like in transgenic tobacco enhances its tolerance to NaCl stress [60]. In quinoa (Chenopodium quinoa L.), the application of exogenous Myo-inositol under NaCl stress increases the activity of antioxidant enzymes, as well as the content of ascorbic acid and glutathione, improving quinoa's membrane stability, reducing oxidative damage, alleviating NaCl injury, and enhancing its salt tolerance [25]. Importantly, the halophyte ice plant (Mesembryanthemum crystallinum) can alleviate the dehydration effect of salt-stressed ice plant seedlings by decreasing the root Na + /K + ratio and increasing the above-ground Na + /K + ratio under the supply of Myo-inositol [61]. In this study, the content of Myoinositol decreased initially and then increased after 48 h and 168 h of NaCl stress. Among the DEGs in the Na-48 h vs. N-168 h comparison group, six genes (Unigene0095469, Uni-gene0006164, Unigene0032727, Unigene0056095, Unigene0002140, and Unigene0095980) were found to be involved in the Ascorbate and aldarate metabolism pathway and Inositol phosphate metabolism pathway, regulating the accumulation of Myo-inositol downstream. Specifically, Unigene0002140 and Unigene0095980 positively regulated the accumulation of Myo-inositol and showed a significant correlation. In the CK-0 h vs. N-168 h comparison group, two DEGs (Unigene0056095 and Unigene0095980) were found to be involved in the same metabolic pathways and regulated the accumulation of Myo-inositol. Unigene0056095 negatively regulated Myo-inositol accumulation, while Unigene0095980 positively regulated it with significant correlation. Therefore, Unigene0002140 and Unigene0095980, as genes related to Myo-inositol oxygenase, play an important role in regulating Myo-inositol.
The results indicate that Myo-inositol, as a precursor for the synthesis of ascorbic acid, indirectly promotes the antioxidant defense mechanism in plants. It acts as a compatible solute to maintain cell turgor and prevent cell dehydration under high salt conditions. Overall, Myo-inositol, along with genes associated with Myo-inositol oxygenase, actively engages in both the ascorbate and aldarate metabolism pathway and the inositol phosphate metabolism pathway. This engagement responds to NaCl stress by clearing excessive ROS accumulation, preserving cellular homeostasis, and enhancing the salt tolerance of T. ramosissima. L-gulono-1,4-lactone serves as a direct precursor of ascorbic acid in plant cells, and it is a substrate of L-gulono-1,4-lactone oxidoreductase, which catalyzes the final step of ascorbic acid biosynthesis [62][63][64]. Ascorbic acid plays essential metabolic and antioxidative roles in living organisms [65][66][67]. In tobacco, wild-type (wt) plants had elevated ascorbic acid levels when fed L-gulono-1,4-lactone [68]. In tomato seedlings, exogenous ascorbic acid increased resistance to NaCl stress and reduced lipid peroxidation, promoting the survival rate of tomato seedlings [69]. In this study, in the Na-48 h vs. N-168 h comparison group, Unigene0064911 was upstream of L-Gulono-1,4-lactone and positively regulated the accumulation of L-Gulono-1,4-lactone, showing a significant correlation. Unigene0007112, Unigene0007113, Unigene0068803, and Unigene0105480 were downstream of L-Gulono-1,4-lactone and regulated its accumulation. In addition, Unigene0007112 and Unigene0007113 negatively regulated the accumulation of L-Gulono-1,4-lactone, with Uni-gene0007112 showing a significant correlation with L-Gulono-1,4-lactone. Unigene0068803 and Unigene0105480 positively regulated the accumulation of L-Gulono-1,4-lactone. In the CK-0 h vs. N-168 h comparison group, Unigene0105480 was downstream of L-Gulono-1,4lactone and regulated its accumulation. Moreover, the content of L-Gulono-1,4-lactone in the roots of T. ramosissima decreased first and then increased under NaCl stress at 48 h and 168 h, suggesting that L-Gulono-1,4-lactone was involved in increasing ascorbic acid content and alleviating NaCl toxicity. Importantly, although Unigene0105480 actively participated in regulating the accumulation of L-Gulono-1,4-lactone, its correlation with L-Gulono-1,4-lactone was not significant and required further validation. Additionally, other research has shown that under 200 mM NaCl stress, the ascorbate and aldarate metabolism pathway plays an important role in promoting and protecting the growth of cotton [70]. In this study, the roots of T. ramosissima under NaCl stress at 48 h and 168 h had 43 DEGs and four differentially regulated metabolites in the ascorbate and aldarate metabolism pathway responding to NaCl stress and mitigating the detrimental effects of NaCl stress on the growth of T. ramosissima.

Conclusions
Myo-inositol is pivotal to the growth and development of plants. Serving as an osmotic regulator and a ROS scavenger, inositol safeguards plants against osmotic stress and oxidative harm, bolstering their resilience to stress. In our research, after exposing T. ramosissima roots to 48 h and 168 h of NaCl stress, numerous genes associated with Myo-inositol oxygenase were identified to be involved in the ascorbate and aldarate metabolism pathway, as well as the inositol phosphate metabolism pathway. These genes effectively modulated the rise of metabolites like L-Gulono-1,4-lactone and Myo-inositol. Such metabolites are instrumental in fostering the growth, development, and defense mechanisms of T. ramosissima against NaCl stress, ensuring its regular progression. Specifically, these metabolites positively influence the accumulation of Myo-inositol within the aforementioned metabolic pathways and are closely correlated with Myo-inositol. Among these, Unigene0002140 and Unigene0095980 stand out as potential key genes associated with Myo-inositol, meriting further investigation.  Funding: This work was jointly supported by grants from "Research on Key Techniques for Boosting Carbon Sequestration and Cultivating Energy Crops and Forests in Coastal Agroforestry Systems" (BE2022305).

Data Availability Statement:
We uploaded our data to the NCBI database with an SRP number of SRP356215.

Conflicts of Interest:
The authors declare no conflict of interest.