Soil salinization has become an increasingly serious global problem. It is estimated that more than 833 million hectares (8.7% of the Earth’s surface) are salinized worldwide with an annual increase of 10% (FAO, 2021). Soil salinization is projected to extend to more than 50% of the arable land by 2050 (Jamil et al., 2011). There are various reasons for soil salinization, including low rainfall, weathering of indigenous rocks, and inappropriate irrigation and fertilization during the cultivation process (Shrivastava and Kumar, 2015). Saline soils are known to suppress plant growth and development, which in turn severely affects crop yields in agricultural production (Yuan et al., 2016; van Zelm et al., 2020). Tomato (Solanum lycopersicum L.) is one of the most grown and valuable vegetable crops in the world, ranking the first among vegetable crops with an annual production of 186 million tons globally (FAO, 2022). Although tomato is thought to be moderately tolerant to salt stress, tomato yield and quality are severely affected by high salinity (Bonarota et al., 2022). The development of salt-tolerant tomato crops is therefore an important goal of plant breeding.

Salt stress can damage plant growth and development in many ways. High salt concentration in the soil modifies the structure of soil porosity and, in turn, hydraulic conductivity. This results in low water potential and nutrient availability, causing osmotic stress and eventually leading to metabolic toxicity and physiological disorders that affect plant growth and development (Tester and Davenport, 2003; Hasanuzzaman and Fujita, 2022; 2023). The rapid accumulation of reactive oxygen species (ROS) frequently occurs during salt stress, which induces oxidative stress, causes damage to cellular macromolecules like proteins and DNA, and destabilizes membranes and organelles (Kesawat et al., 2023). Furthermore, salt stress also decreases stomatal conductance and inhibits photosynthesis (Lawlor and Cornic, 2002; Chaves et al., 2009; Sayyad-Amin et al., 2016; Kesawat et al., 2023). All these negative effects impair most plant growth phases, from seed germination, vegetative growth, flowering and fruiting and eventually overall yield.

Along with the development of multi-omics technology, extensive studies have applied transcriptomics, proteomics, metabolomics or the combined analysis with biochemical and physiological characteristics to investigate the molecular mechanism underlying plant salt tolerance. Based on current understanding, plants adapt various mechanisms, including activation of osmotic adjustment, regulation of ion transport and homeostasis, clearance of reactive oxygen species, regulation of plant hormone signaling, modulation of cytoskeletal dynamics and the cell wall composition, to negate the adverse effects and survive at salinity condition (Wang et al., 2011; Hasanuzzaman and Fujita, 2022; Balasubramaniam et al., 2023). More importantly, regulation of gene expression is the integral part that activates and coordinates all these regulatory pathways.

Gene expression is regulated at transcriptional level mainly exerted by transcription factors and post-transcriptional events involving RNA processing, maturation, transport and turn-over (Zhao et al., 2017; Zhang et al., 2019). Alternative splicing (AS) is the main step during RNA processing to regulate gene expression and proteome diversity. As AS can generate multiple transcripts from a single RNA precursor via exon skipping, intron retention, and selection of alternative donor site or acceptor site as well as other intricate forms of splicing (Keren et al., 2010), AS eventually cause differential expression of the corresponding gene and modulate gene function via altering a protein domain or affecting the stability of the spliced transcript and the corresponding protein. Previous studies have demonstrated that high salinity stress can promote the occurrence of alternative splicing of stress-responsive genes and affect the expression of the genes coding spliceosome components in Arabidopsis (Ding et al., 2014b; Feng et al., 2015; Gu et al., 2018), rice (Yu et al., 2021; Jian et al., 2022), wheat (Liu et al., 2018), Barley (Fu et al., 2019), Date Palm (Xu et al., 2021), grapevine (Jin et al., 2021), cotton (Zhu et al., 2018), Opisthopappus (Han et al., 2024), etc. However, the alternative splicing events in tomato root under salt stress remains to be resolved.

In this study, we investigated the transcriptomic response of tomato root to salt stress, focusing on the global dynamics of transcriptome reprogramming and AS changes during the initial 12 hours under salt exposure. We found a large number of early response differentially expressed (DE) genes induced by salt stress while simultaneously elevating AS events of both DE and non-DE genes. Our findings provide a comprehensive understanding of tomato root response to salt stress and highlights the vital role of AS in tomato’s adaptation to salt stress.

Salinity is one of the most significant environmental factors adversely affecting crop growth, development and yield. Tomato is moderately sensitive to salinity stress with seedlings especially susceptible due to its sensitive osmotic potential which is readily disrupted by salt stress during growth (Cuartero et al., 2006; Tanveer et al., 2019). Consequently, understanding of the underlying mechanisms of salinity tolerance will contribute to the breeding of salt tolerant tomato cultivars. To explore the gene regulatory network of tomato to salt stress, we investigated the early transcriptional responses to salt treatment over a 12-hour period in tomato roots.

The early morphological changes by tomato to salt stress ( Figure 1 ) is consistent with the view that salt-specific signaling pathways are rapidly triggered in plant roots during the very early stages of salt stress (Galvan-Ampudia et al., 2013; Choi et al., 2014). The response of plant roots to salt stress involves complex regulation of gene expression at multiple levels, including at transcription, post-transcription, translation, post-translation, and metabolism, which eventually result in phenotypic changes (Barkla et al., 2013; van Zelm et al., 2020). Here we show that transcriptomic analysis of tomato roots under salt stress revealed a considerable and dynamic expression of transcripts in tomato roots during the early 12-hour treatment process of salt exposure. The gene expression patterns of continuously up or down-regulation, peak expression at 1 or 3 hours, and reduction at the first 3 hours were highly pronounced ( Figure 2 ). The GO terms enriched in those clusters revealed the important changes of key biological processes, such as hormone signaling, cell cycle, amino acid metabolism and response to oxidative stress, during the 12-hour salt treatment.

This study revealed that amino acid metabolism was greatly enhanced at the early response of tomato root to salt treatment ( Figure 2 ; Supplementary Table S7 ; Supplementary Figure S1 ). Amino acid metabolism is involved in various strategies during plant adaption to abiotic stress conditions (Huang and Jander, 2017; Hildebrandt, 2018; Batista-Silva et al., 2019; Reshi et al., 2023). Accumulation of free amino acids have been generally observed in diverse plants under various abiotic stress (Hildebrandt et al., 2015; Huang and Jander, 2017) and the enhancement of amino acid biosynthesis and amino acid transmembrane transport have been reported to improve plant tolerance to salt stress (Batista-Silva et al., 2019; Shohan et al., 2019). While some amino acids like proline are known to be potential ROS scavengers to protect plant cell from oxidative damage (Hayat et al., 2012), several amino acids, such as phenylalanine, tyrosine and tryptophan, arginine, methionine and lysine, act as precursors for the synthesis of nitrogenous secondary metabolites and signaling molecules (Tzin and Galili, 2010; Batista-Silva et al., 2019; Heinemann and Hildebrandt, 2021). Therefore, enhancement of amino acid metabolism is likely to be an important adaptive strategies to eliminate the adverse effects of salt stress in tomato root. On the other hand, it’s known that the high levels of ROS concentration can affect amino acid metabolism, specially the site-specific chemical modification of amino acids such as arginine, lysine, proline, threonine and tryptophan, which cause increased vulnerability to proteolytic degradation (Moller et al., 2007). In this study, most of the DEGs involved in response to ROS and oxidative stress exhibited a continuously up-regulated expression pattern, suggesting the continuous accumulation of ROS throughout treatment of salt stress ( Figure 2 ; Supplementary Table S7 , cluster 9). Thus, the ROS accumulation induced by salt stress may contribute to the considerable changes of amino acid metabolic and catabolic processes under salt tress.

Plant hormones play vital roles in maintaining plant growth and enable plants to survive under conditions of salt stress (Ryu and Cho, 2015; Yu et al., 2020). It was reported that tomatoes could adapt to salt stress by dynamically regulating their hormone levels to establish new hormone balance (Wang et al., 2023a). The levels of ABA, SA, and JA and their respective signal transduction pathways were reported to be significantly increased, while decrease in the levels of GA and IAA were observed during the early response to salt stress (Wang et al., 2023a). In the current study, we also observed the dynamic regulation of plant hormone signaling transduction. Regulation of ABA and auxin-mediated signaling pathways were found to be significantly pronounced throughout the early response to salt stress ( Figure 5 ; Supplementary Tables S7 , S11 ). ABA is the primary hormone that promotes plant salt tolerance (Sah et al., 2016; Vishwakarma et al., 2017; Pye et al., 2018) where auxins promote plant growth (Zhang et al., 2022). The majority of genes involved in both hormone pathways appeared in expression clusters exhibiting continuous down-regulation or enhanced expression within the first 6 hours. This suggests that the regulation of salt tolerance and growth are closely intertwined. In contrast to a previous study (Wang et al., 2023a), significant regulation of the SA-mediated signaling pathway were not detected. On the other hand, response to JA was found to be prominent throughout the early response to salt stress ( Supplementary Table S7 ). A previous study by Abouelsaad and Renault (2018) found that activation of JA signaling pathway enhanced tomato salt tolerance, aligning with our current result.

Cytokinin is another important hormone that modulates plant development and tolerance to various environmental stimuli (Mandal et al., 2022; Papon and Courdavault, 2022; Yin et al., 2023) by regulating cell cycle and differentiation, promoting antioxidant systems, impeding plant senescence, and cross-talking with stress-related phytohormones (Liu et al., 2020; Mandal et al., 2022). While defective cytokinin signaling mitigates high salinity in Arabidopsis via regulation of the lipid and flavonoid gene-to-metabolite networks, enhancement of cytokinin content was reported to improve tomato salt tolerance in tomato (Zizkova et al., 2015). We noticed that the pathway of response to cytokinin was only enriched in the cluster of down-regulated DEGs ( Figure 2 , cluster 10; Supplementary Table S7 ), suggesting that cytokinin-mediated signaling pathway was suppressed during the early response of tomato root to salt stress.

Transcription factors (TFs) play a central role to regulate the expression of the genes responsible for plant stress tolerance. Numerous TFs from the families like bZIP, NAC, WRKY, MADS, MYB, ZF, HSF and bHLH families are involved in conferring salt tolerance in various crop species (Duan et al., 2019; Zang et al., 2019; Li et al., 2020; Guo et al., 2021; Li et al., 2021; Wang et al., 2021a, Wang et al., 2021b; Liu et al., 2023; Rosca et al., 2023; Sukumaran et al., 2023; Ye et al., 2023; Wang et al., 2023b), including in tomato (Pan et al., 2010, Pan et al., 2012; Wang et al., 2013; Klay et al., 2014; Campos et al., 2016; Bai et al., 2018; Klay et al., 2018; Li et al., 2018; Waseem et al., 2019; Zhang et al., 2020; Guo et al., 2021; Qian et al., 2021; Chen et al., 2022) In this study, more than 700 TFs were found to be differentially expressed in tomato roots under salt stress ( Supplementary Table S13 ) with members from MYB, ZF, bHLH and AP2/ERF gene families being the most abundant. Among these salt responsive TFs, several of them were previously reported to modulate tomato salt tolerance. For example, a R1-MYB type TF coding gene, SlARS1 (Gene ID 101257705), that was reported to affect ABA-mediated stomatal conductance under salt stress (Campos et al., 2016), was found to be significantly induced under salt stress especially at 3-hour salt induction ( Supplementary Table S13 ); SlWRKY13 previously proved to be negative regulator of tomato salt tolerance (Birhanu et al., 2020) was among the decreased WRKY TF group in this study. We also observed significant down-regulation of several AP2/ERF family TFs ( Supplementary Table S13 ), such as SlERF.B1 (Gene ID 543867) (Wang Y. et al., 2022) and SlERF.B3 (Gene ID 108511945) (Klay et al., 2014) that negatively regulate tomato salt tolerance. Given the key roles of transcription factors in regulation of salt response in plants, the salt-sensitive TFs identified in this study deserve further investigation in the future.

Alternative splicing (AS) is an important post-transcriptional mechanism that regulates plant growth and development and is prevalent during stress (Kelemen et al., 2013; Jabre et al., 2019; Punzo et al., 2020; Rosenkranz et al., 2022). In tomato, around 65% of the annotated protein-coding genes possess multiple transcript isoforms (Clark et al., 2019). Alternative splicing changes have been reported in tomato plants grown under phytotron vs greenhouse conditions (Wang et al., 2017), in inflorescences of cultivated and wild tomato species (Zhou et al., 2022), during fruit development regulation (Sun and Xiao, 2015; Wang et al., 2016), pollen responses to heat stress (Keller et al., 2017), tomato responses to drought stress (Lee et al., 2020), water deficit stress (Ruggiero et al., 2022), low nitrate stress (Ruggiero et al., 2022), phosphate starvation (Tian et al., 2021), and response to the fungal infection by Trichoderma harzianum (De Palma et al., 2019). Based on these studies, differential alternative splicing (DAS) was found to be tissue-specific, developmental stage-related or stress-responsive condition. As there is limited understanding regarding the involvement of alternative splicing in tomato’s response to salt stress, we investigated the changes of AS events during early response of tomato root to salt stress in this stduy ( Figure 3A , 4B ). A total of 46,115 AS events, including A5SS, A3SS, RI, MXE and SE, were detected in the tomato root transcriptome ( Figure 7 ; Supplementary Table S14 ), revealing a comprehensive and dynamic alteration in AS patterns in tomato roots during early responses to salt stress. An integrated genome-wide study (Clark et al., 2019) reported that RI was the prevalent AS event (18.9%) followed by alternative A5SS and A3SS, while SE was the least AS type, accounting for only 6%, among total 369,911 AS events in tomato. By contrast, SE was the most abundant AS event in tomato root under salt stress ( Figure 7 ; Supplementary Table S14 ), suggesting that the alternative splicing pattern of SE might be susceptible to salt stress in tomato root. The dominance of SE in AS events was also previously reported in tomato root and shoot during phosphate starvation (Tian et al., 2021) and in date palm seedlings under salt stress (Xu et al., 2021), suggesting that AS patterns are not constant, but may change depending on the abiotic condition. On the other hand, RI event was reported to be the most frequent event induced by salt stress in Arabidiopsis (Ding et al., 2014b), wheat (Liu et al., 2018), cotton (Zhu et al., 2018) and Barley (Fu et al., 2019), while A3SS was the mostly affected AS events in rice by salt stress (Fu et al., 2019). Given that the differences in AS profiles are related to tissue type, stress condition and genotype (Gan et al., 2011; Vitulo et al., 2014; Martín et al., 2021; Zhou et al., 2022), the differences on the alternative splicing preference induced by salt may contribute to the evolutionary adaptation process in tomato.

Salt-induced AS of non-differentially expressed genes may contribute to the transcriptome reprogramming for salt tolerance of tomato root. Interestingly, except for unclassified genes, differentially alternative splicing induced by salt stress in tomato root were largely detected in the gene families of serine/threonine-protein kinase, PPR-containing protein, and E3 ubiquitin ligase ( Figure 8D ). Serine/threonine-protein kinases are key enzymes that reversibly phosphorylate the OH group of serine or threonine residues at the post-translational level. The network of serine/threonine kinases in plant cells is considered a central unit to accept and convert signaling information from sensing receptors of various stimulus and phytohormones and in turn guide responsive changes in gene expression, metabolism, plant growth and development (Hardie, 1999). One of the most representative serine/threonine-protein kinases belong to the SnRK2 family, which are involved in the ABA-dependent signaling pathway to regulate plant development and plant responses to diverse abiotic stresses (Kulik et al., 2011). Ubiquitin E3 ligases are major players that catalyze the covalent attachment of ubiquitin to target proteins (Mazzucotelli et al., 2006; Kelley, 2018). Ubiquitination of substrates is a dynamically regulated process and can generate diverse functional outcomes like potential degradation or activation of target proteins and changes in subcellular localization (Kelley, 2018). E3 ubiquitin ligases are thus well-known to be central regulators of many plants molecular processes, including plant hormone biosynthesis, signaling transduction and response to various stress conditions (Wang S. et al., 2022). AS susceptibility of serine/threonine kinases and E3 ubiquitin ligases in tomato root under salt stress poses as an additional complication in understanding the relationship between hormone signaling transduction and salt-responsive gene regulation. Pentatricopeptide (PPR) proteins are characterized by tandem arrays of a degenerate 35-amino-acid sequence motifs (Lurin et al., 2004). They are a large family of modular RNA-binding proteins with essential roles in organelle biogenesis, RNA editing, mRNA maturation and thus involved in many diverse biological processes during plant growth, development and stress acclimation (Barkan and Small, 2014). A previous genome-wide analysis revealed that the tomato genome has 471 PPR-coding genes (Ding et al., 2014a). In this study, extensive AS occurred in PPR-coding genes under salt stress as 96 out of the 471 PPR-coding genes were found to be differentially alternative spliced ( Figure 8D ; Supplementary Table S17 ). The dynamic AS changes of PPR-coding genes may also contribute to the gene regulation and transcriptome reprogramming under salt stress.

We also observed that some genes, such as the genes coding CPR1-like F-box protein and HsfA2 in Figure 9 , were abnormally spliced under salt. These observations indicate that AS modulation in response to salt stress is more complicated than previously envisioned and that modulation of alternative splicing deserves more attention in future studies. The genes and their transcripts identified in the present study can be targeted for the improvement of tomato salt tolerance.

Analysis on the differential expression of spliceosome pathway-associated proteins ( Figure 10 ) revealed the potential roles of specific groups of AS-associated proteins in regulating tomato root response to salt stress. Expression of many genes coding key component assembled in spliceosome machinery, such as small nuclear ribonucleoprotein complexes (snRNPs), U1, U2, U4, U5, and U6, was found to be affected in tomato root under salt stress. Except for core components of spliceosome machinery, the expression of trans-factors, including serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoprotein (hnRNP), were also significantly regulated under salt stress ( Figure 10 ; Supplementary Table S21 ). In addition to differential expression, alternative splicing in some of AS-related genes, such as SR-like splicing factors ( Figure 8D ; Supplementary Table S17 ), was also observed, which adds an additional complication of AS regulation during salt stress. It is reported that the salt-responsive regulation of SR gene isoforms may result in inaccurate identification of splicing sites and destabilization of the spliceosome complex (Albaqami et al., 2019; Xu et al., 2021; Laloum et al., 2023). Therefore, future studies on the AS events of splicing-related proteins will provide new insights on how genes are regulated in salt-stressed tomato.

Collectively, this study provides a comprehensive view of transcriptome changes and highlights the key role of AS in tomato root response to salt stress. A large number of DEGs and DAS genes involved in diverse metabolic pathways, such as hormone signaling transduction, DNA transcription, RNA binding and processing, were identified. The findings in this study expand our current understanding of transcriptional and post-transcriptional regulation in the response of tomato roots to salinity stress and provide an important gene resource for developing salt-tolerant tomato plants.

The sequencing data are available at https://www.ncbi.nlm.nih.gov/sra/ at NCBI with accession number PRJNA1110838.

JG: Data curation, Investigation, Writing – original draft. YQ: Data curation, Formal analysis, Writing – original draft. YT: Investigation, Writing – original draft. LZ: Data curation, Writing – review & editing. TWO: Validation, Writing – review & editing. YY: Conceptualization, Methodology, Writing – original draft. LT: Conceptualization, Data curation, Supervision, Writing – original draft, Writing – review & editing.