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Article

Integrated Transcriptome and Phytohormone Analysis Reveal the Central Role of Auxin in Early Salt Stress Response of Pomegranate Roots

by
Haixia Tang
1,†,
Huaikun Ji
2,†,
Yanqiu Kong
2,
Jia Liu
3,
Qikui Wu
2,* and
Yanlei Yin
1,*
1
Shandong Institute of Pomology, Tai’an 271099, China
2
College of Forestry, Shandong Agricultural University, Tai’an 271018, China
3
Taian Academy of Agricultural Sciences, Tai’an 271099, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(12), 2735; https://doi.org/10.3390/agronomy15122735
Submission received: 10 November 2025 / Revised: 25 November 2025 / Accepted: 25 November 2025 / Published: 27 November 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

Soil salinization is one of the most severe abiotic stresses that restricts agricultural productivity worldwide. Pomegranate exhibits relative tolerance to salinity, yet the early response mechanisms in roots remain unclear. In this study, the physiological and transcriptional responses of pomegranate roots to salinity stress were systematically investigated. Salinity stress significantly induced the accumulation of total soluble sugar and proline by up to 8% and 67%, and enhanced the activities of superoxide dismutase (68%) and peroxidase (31%), indicating the activation of osmotic adjustment and antioxidant defense systems. A total of 7548 and 7462 genes were differentially expressed under 100 mM and 200 mM NaCl treatments, respectively. Functional annotation highlighted the critical roles of pathways involved in stress response and plant hormone signal transduction. Comprehensive transcriptional reprogramming was observed in the auxin pathway, involving biosynthesis (YUCCA), transport (PIN and AUX1), and signaling components (TIR1, AUX/IAA, ARF, and GH3). Hormone quantification and RT-qPCR validation confirmed the regulatory functions of auxin through sophisticated hormonal crosstalk. These findings revealed the pivotal role of auxin as a central hub in coordinating the early salinity stress response in pomegranate roots and provided crucial insights and candidate gene resources for enhancing salt tolerance in woody fruit species.

1. Introduction

Soil salinization poses a serious and increasingly severe threat to global agriculture and food security [1]. More than 800 million hectares of land worldwide are affected by salinity, an area that is still expanding due to irrigation practices, climate change, and other human activities [2,3,4]. Salinization sharply reduces crop yields through imposing ion stress, osmotic stress, and secondary oxidative damage, and threatens the sustainability of agricultural systems and the livelihoods of millions of people [5,6]. The disruption of ion and osmotic homeostasis often leads to the overproduction of reactive oxygen species (ROS), causing oxidative damage to cellular components [7]. Therefore, exploring strategies to mitigate the impact of salt damage is of vital importance for ensuring future food production.
Punica granatum L. (pomegranate) is a famous tree species whose fruit has high nutrient content, antioxidant ability, and health benefits [8]. It holds significant economic and cultural value in many arid and semi-arid regions [9,10]. Notably, pomegranate has relative higher tolerance to salinity than other fruit crops, making it a suitable candidate as the pioneer fruit tree on marginal lands affected by salinity [10,11]. Unraveling the molecular mechanism responding to salinity stress is therefore essential not only for the genetic improvement of pomegranate but also for providing valuable insights and genetic resources to enhance salt tolerance in other sensitive fruit tree species.
Previous studies in higher plants have elucidated a series of comprehensive physiological, biochemical, and molecular mechanisms in responding to salinity stress, which include maintaining ion homeostasis through Na+ excretion and vacuolar compartmentalization, synthesizing compatible solutes like proline and glycine betaine, and enhancing antioxidant systems to scavenge reactive oxygen species [12,13,14,15]. These responses are coordinated by a complex signaling network, in which plant hormones such auxin (IAA), abscisic acid (ABA), and jasmonic acid (JA) are recognized as hub factors, integrating environmental cues into coordinated transcriptional reprogramming [16,17]. The response mechanisms for salinity stress have been constructed in many plants, such as chrysanthemum, grapevine, barley, and rose [18,19,20,21]. In pomegranate, previous studies have investigated the expression trends of genes related to ABA, Ca2+-related, and MAPK signal transduction during a relatively long observation period, i.e., 0, 3, and 6 days after salt treatment in roots and leaves [9]. Our previous study reported that significant and rapid changes were seen in pomegranate leaves at 24 and 48 h after salinity stress at low and high concentrations [22]. However, the change patterns shortly after salinity stress in pomegranate roots are still unclear.
In this study, the response mechanisms to salinity stress were explored in the roots of ‘Taishanhong’ pomegranate. The objectives of our study were to (1) characterize the dynamic transcriptome changes under different salt concentrations and time points; (2) identify the key biological processes and metabolic pathways involved; and (3) pinpoint critical candidate genes governing salt tolerance. Unlike previous studies focusing on longer-term response, this study captures early transcriptional and hormonal dynamics in pomegranate roots, which could provide basic research and gene resources for exploring the rapid regulatory mechanism underlying salt tolerance.

2. Materials and Methods

2.1. Plant Materials and Salt Treatment

The two-year-old pomegranate seedlings used in this study were planted in the pomegranate experimental base of the Shandong Fruit Research Institute, Tai’an, Shandong (36°10′15″ N, 117°09′25″ E), where they grew in plastic containers with a diameter of 260 mm and 3.4 kg of humus and vermiculite (1:1, v/v). After three months of growth, seedlings with similar growth conditions were selected for salt treatment. NaCl solutions at three concentrations, namely, 100 mM, 200 mM, and 300 mM, were used for salinity stress treatment, with water as the control group. A total of 800 mL NaCl solution with different salt concentrations was poured into each container. After salt treatments, the root tissues were collected from each seedling at 0 h (initial), 6 h, 12 h, 24 h, and 48 h, according to previous studies [9,22]. There were three biological replicates for each sampling, and each replicate consisted of three individual plants mixed. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for further study.

2.2. Physiological Indicator Measurement

A total of four physiological indicators were selected for salt-tolerant performance [22,23]. The contents of total soluble sugar were measured by anthrone colorimetry using glucose as standard. Proline contents were determined by acid ninhydrin colorimetry with L-proline as standard. The activities of superoxide dismutase (SOD) and peroxidase (POD) were measured by the nitrogen blue tetrazole method and the guaiacol method, using the SPECTRONIC 200 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at 470 nm and 560 nm, respectively, with quantification based on standard curves.

2.3. Transcriptome Analysis

2.3.1. Sample Collection and RNA Extraction

The samples at 6, 12, and 24 h after salt treatment with two concentrations of 100 mM (L) and 200 mM (H) were selected for transcriptome analysis with the sample at 0 h being the control group (C). All the root samples of pomegranate seedlings were collected for total RNA extraction with the Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). After quality, purity, and integrity assessments, the total RNA samples were used for cDNA library construction. A total of 21 cDNA libraries were sequenced on a NovASeq 4000 Sequencing platform (Illumina, San Diego, CA, USA), and 150 bp paired-end reads were generated.
The acquired clean reads were aligned using HISAT2 v2.1.0 to the pomegranate genome database (GCF_007655135.1) to comprehend their functionalities [24]. The gene expression level was assessed by quantifying the reads using the fragments per kilobase of exon model per million mapped fragments (FPKM) methodology. The differentially expressed genes (DEGs) between different germplasms were identified if their log2|Fold Change|was over 2 with a p-value < 0.05. Gene Ontology (GO) annotation and Kyoto En-cyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to gain further insights into the functions of DEGs.

2.3.2. RT-qPCR Analysis

RT-qPCR experiments were performed for verifying the transcriptome data and analyzing the expression patterns of key DEGs using the SYBR Green qPCR kit (Accurate Biology, Changsha, China), and a CFX96 Real-time System was used for all procedures. PgGADPH served as an internal control during the 2−ΔΔCT analysis of the expression patterns. Each sample was biologically replicated three times. The primers of twelve DEGs were designed and are listed in Supplementary Table S1.

2.4. Hormone Content Measurement

The contents of hormone in pomegranate roots under salinity stress were determined by liquid chromatography–mass spectrometry (LC-MS) according to a previous study with modifications [25]. The LC separation was performed on a HYPESIL Gold column C18 column using a Sciex ExionLC™ AC system. During the LC process, mobile phase A was water with 0.1% formic acid (v/v) and mobile phase B was acetonitrile containing 0.1% formic acid (v/v). The separation was performed using the following gradient: 90% A at 0–0.2 min, maintained until 8 min, shifted to 10% A at 8.1 min, and held until 10 min. The flow rate was 0.3 mL min−1, the column temperature was 35 °C, and the injection column was 5 μL. The MS data were collected using a Sciex Quadrupole 4500 system equipped with an electrospray ionization (ESI) source operated in both positive and negative ion modes. The ion source parameters were set as follows: ionspray voltage, ±5500 V/4500 V; source temperature, 550 °C; curtain gas, 30 psi; collision gas, 9 psi. Quantification was achieved using multiple reaction monitoring (MRM) mode with optimized potential and collision energy for each hormone.

2.5. Data Analysis

Values of different indicators are expressed as mean ± SD (n = 3). One-way analysis of variance (ANOVA) with repeated measures was used to test the changes in each index separately. Pearson correlation coefficients were used to examine relationships between transcriptional levels and gene expression levels. All statistics were performed using SPSS R27.0.1.0 (IBM, Armonk, NY, USA). For all parameters measured, α was set at 0.05 and p values less than α were considered significant.

3. Results

3.1. Physiological Responses of Pomegranate Roots to Salinity Stress

To investigate the physiological adaptation of pomegranate roots to salinity stress, we assessed the dynamic changes in total soluble sugar content, proline content, SOD, and POD activity at four time points (6 h, 12 h, 24 h, and 48 h) after different salt treatments with NaCl concentrations of 0 mM (CK), 100 mM, 200 mM, and 300 mM. The content of total soluble sugar, a key osmolyte, showed a steady upward trend with the increase in salt concentration and the extension of treatment time (Figure 1a). Moreover, soluble sugar levels in all salt treatment groups were significantly higher than those in the CK group at 6 h and continued to accumulate over time, stabilizing between 24 h and 48 h at a 4% and 8% increase for 200 mM and 300 mM treatments. Proline had a more rapid accumulation pattern with a pronounced biosynthesis burst between 6 h and 12 h under 200 mM and 300 mM salt treatments and showed peaks with a 54% and 67% increase at 24 h, respectively (Figure 1b). Concurrently, the activities of SOD and POD were significantly induced in a concentration- and time-dependent manner (Figure 1c,d). SOD activity, responsible for eliminating superoxide anions, increased rapidly and peaked at 24 h under 300 mM salt treatment, being 68% higher than that of the CK group. POD activity, in contrast, exhibited greater sustainability, maintaining high levels even at the late stage (48 h), with its peak activity being 31% higher than that of the CK group. Given these robust physiological responses observed under moderate stress across the early to mid-stage, we selected root samples from the 100 mM and 200 mM treatments at time points 6 h, 12 h, and 24 h, for transcriptome analysis.

3.2. Analysis of Transcriptome Profile in Response to Salinity Stress

3.2.1. Global Analysis of RNA-Seq Data

To further explore the molecular mechanism of pomegranate response to salinity stress, twenty-one cDNA libraries were constructed and the raw data was deposited at the NCBI Sequence Read Archive (SRA) under accession numbers SRR75169~SRR75189. An average 28,900,864 (97.29%) of clean reads was obtained after removing the adaptor and low-quality reads (Table 1), and the average percentages of Q20 and Q30 base rates were 97.08% and 92.25%, respectively. The average GC content was 48.36%, and the rate of total mapping ranged from 92.51% to 96.54%, which indicated that the data obtained by transcriptome sequencing were of high quality and could be used for further analysis.

3.2.2. Identification of DEGs Among Different Comparison Combinations

To identify the DEGs that were significantly regulated among different groups, the expression level of genes was constructed. Compared to the C0h, there were 3737 (2904 upregulated and 833 downregulated), 6038 (4749 upregulated and 1289 downregulated), and 5136 (4045 upregulated and 1091 downregulated) DEGs identified in L6h, L12h, and L24h, respectively, at different times after a low concentration of salinity stress (Figure 2a; Table S2). For the high concentration of salinity stress, a total of 1406 (942 and 464), 5547 (3847 and 1700), and 5626 (4298 and 1328) DEGs were found in H6h, H12h, and H24h, respectively. Three comparison groups between 100 mM and 200 mM salinity stress treatments at the same time were constructed, namely, L6h vs. H6h, L12h vs. H12h, and L24h and H24h. Among these groups, 356, 1017, and 1570 DEGs were identified, respectively, with more downregulated genes in the high-concentration salinity stress treatment.
Furthermore, Venn diagrams were constructed in three comparison combinations (Figure 2b). Compared to the C0h, there were 7548 and 7462 DEGs found to be differentially expressed in at least one period after 100 mM and 200 mM salt treatments, respectively, while 2491 and 820 DEGs were identified in the whole observed stage after salt treatment, respectively. A total of 2497 DEGs were identified to be differentially expressed between the samples under two salt treatments at least in one stage, and 40 DEGs were found at all three time points.

3.2.3. GO Annotation Analysis of DEGs Among Different Treatments

To further explore the biological functions of DEGs under salinity stress treatments, GO annotation analysis was performed (Figure 3). Among the top metabolic pathways mapped to DEGs, the biological process (BP) category was mainly enriched in cellular process (GO: 0009987), metabolic process (GO: 0008152), response to stimulus (GO: 0050896), biological regulation (GO: 0065007), etc. Binding (GO:0005488) accounted for most in the molecular function (MF) category, followed by catalytic activity (GO: 0003824). On the other hand, in the cellular component category, cell (GO:0005623), cell part (GO: 0044464), organelle (GO:0043226), membrane (GO:0016020), membrane part (GO:0044425), and organelle part (GO:0044446) were the main mapped pathways. The enrichment-related terms, such as response to stimulus and biological regulation, implied a potential central role for phytohormones in mediating the transcriptional changes under salinity stress.

3.2.4. KEGG Enrichment Analysis of DEGs Among Different Treatments

To comprehensively understand the biological pathways responding to salinity stress treatments, KEGG pathway enrichment analysis of identified DEGs was performed (Figure 4 and Figure S1). Compared to the control group, the DEGs identified in the low-/high-concentration salt treatments were significantly enriched in plant hormone signal transduction (map04075) and MAPK signaling pathway–plant (map04016). These findings indicated that hormone signaling integrated with stress-activated MAPK signaling to orchestrate the response to salinity stress in pomegranate.

3.2.5. Analysis of Key Genes Involved in Auxin Biosynthesis and Signal Transduction

Understanding the auxin regulatory pathway could reveal the response mechanism of pomegranate to salinity stress. A total of 39 genes encoding key components involved in auxin biosynthesis, transportation, and signal transduction were identified and their expression patterns were performed (Figure 5; Table S3). Among the biosynthesis genes, two TAA1 homologs showed somewhat downward trends under salinity stress, and one of them was particularly suppressed across all treatment time points and concentrations. In contrast, three identified YUCCA genes were markedly upregulated. For auxin transport, seven PIN and four AUX1 genes were found. Most of these transporters were strongly induced, suggesting a coordinated role in redistributing auxin under stress. The expressions of two TIR1 receptor genes were consistently upregulated, implying the activation of a potent negative feedback mechanism to fine-tune auxin signaling. There were nine genes identified as AUX/IAA, most of which showed up-trends under salt treatments. Concurrently, five ARF genes were mainly downregulated, which was consistent with this feedback inhibition. Moreover, five GH3 genes, which are classic early auxin response markers and involved in auxin homeostasis, were overwhelmingly upregulated. This confirmed the successful activation of the auxin signaling pathway and highlighted a mechanism for modulating free auxin levels through conjugation under salinity stress. In summary, salinity stress triggered a comprehensive transcriptional reshuffling of the auxin pathway in pomegranate roots, which may play an active and complex role in mediating the stress response.

3.2.6. Verification of the Key Gene Expressions Under Salinity Stress

To further verify the transcriptome results and analyze the function of the key genes, six genes involved in auxin biosynthesis, transportation, and signal transduction were selected to measure the expression patterns under different salt treatments (Figure 6). The expression trends of key genes were similar between transcriptome results and RT-qPCR analysis, with the correlations ranging from 0.927 (p < 0.01) to 0.989 (p < 0.01). This high reproducibility confirms the reliability of our transcriptomic data in profiling the salt tolerance response in pomegranate. The key auxin-related genes showed upward change trends under salinity stress, which was associated with the hormonal regulatory network in responding to the environmental signal of salinity. Furthermore, the expression levels of key genes under low salt treatments were higher than these under high salt treatments, indicating that the high salinity may compromise the ability to sustain a regulated hormonal response in the roots of pomegranate. Collectively, these results not only confirm the reliability of our transcriptional dataset but also pinpoint specific components of the auxin pathway as key regulators in the fine-tuning of growth and defense under saline conditions, offering potential targets for future genetic improvement of salt tolerance.

3.3. Analysis of Phytohormone Contents in Response to Salinity Stress

To explore the hormonal regulation underlying the salinity stress response, the contents of key phytohormones in the roots of pomegranate were measured (Table 2; Figure S2). The content of IAA showed up–down change trends under both low and high salt treatments, which suggested that auxin played an active role in responding to salinity stress signaling. ABA content decreased at 6 h and then continuously increased under a low concentration of salinity stress, exhibiting a fluctuating pattern with valley level at 24 h under high salt treatments. JA levels showed a massive and sustained induction, especially under high salinity stress, indicating a potent and lasting activation of the JA-mediated defense pathway. The change in TZ content oscillated during the treatment period. The content of GA1 and GA3 was significantly induced at 24 h after salt treatment, suggesting a potential preparation for recovery growth after the initial stress shock. SA levels were systematically reduced at 24 h under both low and high salinity stresses, and this widespread downregulation may represent a strategic resource reallocation to favor the ABA/JA-dominated abiotic stress defense pathways over SA-mediated biotic stress responses. Collectively, a sophisticated strategy was employed responding to salinity stress in pomegranate roots, among which auxin played a hub and active role in the coordinated network.

4. Discussion

Soil salinity imposes osmotic and ionic stress that disrupts cellular homeostasis and metabolism, leading to growth inhibition and oxidative damage in plants. Understanding the sophisticated regulatory network that plants have evolved to cope with salinity stress is crucial for developing salt-tolerant crops [26,27]. In this study, we revealed a dynamic and complex response mechanism in pomegranate roots under varying salt concentration and exposure durations.
Physiological analysis demonstrates that pomegranate roots mount a rapid and integrated defense against salinity stress, characterized by osmotic adjustment through the accumulation of total soluble sugar and proline, coupled with a robust activation of the antioxidant enzymes SOD and POD. Soluble sugar acts as an osmotic regulator that maintains cell turgor and stabilizes membrane integrity under osmotic stress [28,29]. Similarly, proline not only contributes to osmotic balance but also scavenges ROS and protects enzyme structures, as has been widely reported in plants such as Arabidopsis, cotton, and poplar [30,31,32]. The significant increases in SOD and POD activities further indicate an efficient ROS detoxification system in pomegranate roots, mitigating oxidative damage and maintaining redox homeostasis [32,33]. This coordinated enhancement of osmotic and antioxidant systems constitutes an essential component of early salt tolerance, effectively mitigating initial oxidative damage and maintaining cellular function [34,35].
The transcriptome profiling revealed dynamic and complex transcriptional reprogramming underlying these physiological adaptations. There were fewer DEGs induced by high salinity stress than low concentration treatment at the early stage (6 h), and this suppression of the initial transcriptional response may be related to the general limitation of cellular metabolic activities caused by severe osmotic stress [36]. However, as the stress persisted, a pronounced transcriptional activation occurred from 12 h to 24 h, indicating a strategic shift from initial stress shock absorption to active transcriptional reprogramming for adaptation, a phased response model also observed in poplar, rice, and alfalfa under salt conditions [37,38]. Furthermore, GO annotation analysis indicated that DEGs related to rapid signal transduction processes, such as response to stimulus and biological regulation, were highly enriched in the early stage of stress, while more identified DEGs were associated with metabolism and cellular components in the later stage. The response to salinity stress in pomegranate roots gradually shifted from initial signal perception to systemic physiological and structural remodeling, and this phased response mechanism ensured that plants could efficiently allocate resources based on the duration and intensity of stress, prioritizing survival [38,39]. KEGG enrichment demonstrated that plant hormone signal transduction and the MAPK signaling pathway were significantly enriched, suggesting the core role of hormone coordination in the response of pomegranate roots to salinity stress. Our data particularly highlighted the crucial role of auxin as a hub signal in integrating the stress response. The transcriptome profile and gene expression pattern analysis showed that most genes involved in auxin biosynthesis (YUCCA), transport (PIN and AUX1), and signal perception (TIR1, AUX/IAA, and GH3) had undergone significant transcriptional reprogramming. The comprehensive activation suggested that pomegranate roots actively adjusted the internal distribution of auxin and the intensity of signals to cope with salinity stress. It is particularly noteworthy that the widespread upregulation of AUX/IAA repressor genes and the downregulation of ARF may reveal a powerful negative feedback regulatory mechanism [40,41,42]. This mechanism likely serves to fine-tune and attenuate auxin signal after its initial activation, preventing excessive resource expenditure and maintaining cellular homeostasis, a crucial aspect of the growth–defense balance under stress [43,44].
The hormonal quantification further illuminated a sophisticated crosstalk network orchestrated by auxin in response to salinity stress. The level of JA showed continuous and intense accumulation under high salinity stress, especially in the later stage (24 h), which indicated that pomegranate initiated a strong JA-mediated defense program. The explosive growth of JA was temporally connected with the early rapid response of auxin, suggesting a synergistic effect between auxin and JA [44,45]. Different from the continuous rise in JA, the change in ABA was complex with a wave trend, and this dynamic change reflected the characteristics of ABA as a primary stress signal molecule [46,47]. The interaction between auxin and ABA may jointly regulate the developmental plasticity of the root system, for example, by inhibiting the growth of the main root and promoting possible lateral root formation to optimize the root structure and adapt to the stress environment [48]. Moreover, GA was induced in the later stage of stress. The comprehensive change in the hormonal patterns clearly depicted the strategy of pomegranate under salinity stress: auxin and ABA coordinated to respond initially and adjust growth in the early stage; JA strongly activated defense pathways and prepared for possible recovery (through GA) from the middle to the late stage. The systematic downregulation of the SA level further supported the idea that pomegranate prioritized ABA/JA-dominated non-biological stress defense in resource allocation. Collectively, these results depict a hierarchical hormone interaction network in pomegranate roots, with auxin acting as a central coordinator that links early osmotic adjustment, ROS detoxification, and subsequent transcriptional and hormonal reprogramming.
Compared with other fruit crops such as grape and citrus, pomegranate exhibited a particularly rapid activation of auxin-related genes and early accumulation of JA, which may underlie its relatively strong salt tolerance. The combined enhancement of osmolyte metabolism, antioxidant enzyme activity, and hormonal regulation suggests that pomegranate has evolved an efficient multi-tiered defense system integrating metabolic and transcriptional plasticity. Future studies focusing on protein–protein interactions and post-translational regulation within auxin-JA signaling will further elucidate the molecular framework underlying salt tolerance in pomegranate.

5. Conclusions

In this study, the response mechanism of pomegranate roots to salinity stress was investigated through different salt concentrations and stress durations. After salt treatments with three concentrations, the osmolyte contents and antioxidant enzyme activities increased rapidly during a short period (6–48 h) after salinity stress. Furthermore, the transcriptome profile revealed dynamic and complex transcriptional reprogramming, with phytohormone signaling, particularly the auxin pathway, playing a central role. Six candidate genes, involved in auxin biosynthesis (YUCCA), transport (PIN and AUX1), and signaling components (TIR1, AUX/IAA, ARF, and GH3), showed significant expression changes, indicating an active role for auxin in mediating the salinity stress response. Our multi-level analysis, integrating physiological, transcriptional, and hormonal data, provided the basis for exploring the early molecular mechanism of response to salinity stress and the genetic improvement of salinity tolerance in pomegranate.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15122735/s1, Figure S1: KEGG enrichment analysis of DEGs identified among different treatment groups at the same time points; Figure S2: LC-MS chromatogram of detected hormones; Table S1: All RT-qPCR primer sequences used in this experiment; Table S2: List of DEGs identified among different salt treatments; Table S3: List of genes involved in auxin biosynthesis, transportation, and signal transduction.

Author Contributions

Conceptualization, Q.W. and Y.Y.; methodology, H.T. and H.J.; software, H.T. and H.J.; validation, Q.W. and Y.Y.; formal analysis, H.T., H.J., Y.K. and J.L.; investigation, H.T., H.J., Y.K. and J.L.; resources, Y.Y. and J.L.; data curation, H.T., H.J., Y.K., J.L. and Q.W.; writing—original draft preparation, H.T. and H.J.; writing—review and editing, Q.W.; visualization, H.T., H.J. and Y.K.; supervision, Q.W. and Y.Y.; project administration, Y.Y.; funding acquisition, Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Project of Shandong Province (2022TZXD009, 2023TZXD088) and the Shandong Province Fruit Industry Technology System Project (SDAIT-06-23).

Data Availability Statement

The original contributions presented in the study are included in the Article/Supplementary Materials; further inquiries can be directed to the corresponding authors.

Acknowledgments

We are deeply indebted to the entire research team for their support during this research work.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The change trends of physiological indicators under different salt treatments. (a) Total soluble sugar content. (b) Proline content. (c) SOD activity. (d) POD activity. Different lowercase letters indicate statistically significant differences among treatments (p < 0.05).
Figure 1. The change trends of physiological indicators under different salt treatments. (a) Total soluble sugar content. (b) Proline content. (c) SOD activity. (d) POD activity. Different lowercase letters indicate statistically significant differences among treatments (p < 0.05).
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Figure 2. Identification and distribution of DEGs among different treatment groups. (a) Distribution of upregulated and downregulated DEGs among different samples. (b) Venn diagram of DEGs among different samples.
Figure 2. Identification and distribution of DEGs among different treatment groups. (a) Distribution of upregulated and downregulated DEGs among different samples. (b) Venn diagram of DEGs among different samples.
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Figure 3. GO annotation analysis of DEGs identified among different treatment groups.
Figure 3. GO annotation analysis of DEGs identified among different treatment groups.
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Figure 4. KEGG enrichment analysis of DEGs identified among different treatment groups.
Figure 4. KEGG enrichment analysis of DEGs identified among different treatment groups.
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Figure 5. Expression patterns of key genes associated with auxin biosynthesis and signal transduction based on transcription profiles.
Figure 5. Expression patterns of key genes associated with auxin biosynthesis and signal transduction based on transcription profiles.
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Figure 6. Expression patterns of six selected genes based on RT-qPCR performance. ** indicates a significant correlation at the p < 0.01 level.
Figure 6. Expression patterns of six selected genes based on RT-qPCR performance. ** indicates a significant correlation at the p < 0.01 level.
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Table 1. Statistical analysis of transcriptome sequencing data.
Table 1. Statistical analysis of transcriptome sequencing data.
SampleClean ReadsQ20/%Q30/%GC Content/%Mapping Rate/%
C0h28,322,85497.0592.1246.3196.14
L6h28,909,25697.1692.4247.5993.68
L12h25,092,08597.3192.7449.9395.00
L24h28,933,69597.1292.3349.0594.07
H6h29,527,12197.4092.8746.9995.80
H12h30,247,13496.8691.7849.4594.85
H24h31,273,90596.6891.4849.2395.21
Mean28,900,86497.0892.2548.3694.97
SD3,438,4600.340.681.610.94
Table 2. Content patterns of hormones (ng g−1) under different salt treatments.
Table 2. Content patterns of hormones (ng g−1) under different salt treatments.
HormoneC0hL6hL12hL24hH6hH12hH24h
IAA1.38 ± 0.02 c2.36 ± 0.23 bc2.86 ± 0.01 ab1.89 ± 0.06 c4.17 ± 0.21 a3.19 ± 0.21 ab2.36 ± 0.24 bc
ABA15.81 ± 0.17 a4.25 ± 0.19 d5.90 ± 0.05 cd7.61 ± 0.20 c16.11 ± 0.43 a10.84 ± 0.29 b14.29 ± 0.27 a
JA80.52 ± 2.61 c36.12 ± 1.30 d64.51 ± 1.01 c78.84 ± 5.35 c88.11 ± 0.64 c168.50 ± 2.46 b184.12 ± 9.37 a
TZ1.28 ± 0.11 bc0.48 ± 0.03 cd2.38 ± 0.18 a1.12 ± 0.13 c1.89 ± 0.17 ab0.28 ± 0.03 d1.24 ± 0.05 bc
GA113.13 ± 0.25 b10.29 ± 0.51 c11.45 ± 0.26 c20.96 ± 0.86 a8.95 ± 0.32 c10.56 ± 0.53 c21.15 ± 1.80 a
GA31.34 ± 0.01 c1.73 ± 0.04 a1.43 ± 0.02 c3.13 ± 0.10 a1.05 ± 0.04 d1.07 ± 0.08 d2.92 ± 0.12 a
SA263.99 ± 4.91 a214.99 ± 4.57 b254.25 ± 1.20 a109.38 ± 9.99 d183.63 ± 4.61 c237.85 ± 7.49 ab122.59 ± 2.95 d
Note: Different lowercase letters indicate statistically significant differences among treatments (p < 0.05).
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Tang, H.; Ji, H.; Kong, Y.; Liu, J.; Wu, Q.; Yin, Y. Integrated Transcriptome and Phytohormone Analysis Reveal the Central Role of Auxin in Early Salt Stress Response of Pomegranate Roots. Agronomy 2025, 15, 2735. https://doi.org/10.3390/agronomy15122735

AMA Style

Tang H, Ji H, Kong Y, Liu J, Wu Q, Yin Y. Integrated Transcriptome and Phytohormone Analysis Reveal the Central Role of Auxin in Early Salt Stress Response of Pomegranate Roots. Agronomy. 2025; 15(12):2735. https://doi.org/10.3390/agronomy15122735

Chicago/Turabian Style

Tang, Haixia, Huaikun Ji, Yanqiu Kong, Jia Liu, Qikui Wu, and Yanlei Yin. 2025. "Integrated Transcriptome and Phytohormone Analysis Reveal the Central Role of Auxin in Early Salt Stress Response of Pomegranate Roots" Agronomy 15, no. 12: 2735. https://doi.org/10.3390/agronomy15122735

APA Style

Tang, H., Ji, H., Kong, Y., Liu, J., Wu, Q., & Yin, Y. (2025). Integrated Transcriptome and Phytohormone Analysis Reveal the Central Role of Auxin in Early Salt Stress Response of Pomegranate Roots. Agronomy, 15(12), 2735. https://doi.org/10.3390/agronomy15122735

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